Halophilic bacterium

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 391 - 394
Gramella echinicola gen . nov., sp . nov., a novel halophilic bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius; Nedashkovskaya OI et al.; A novel marine bacterium, strain KMM 6050(T), was isolated from the sea urchin Strongylocentrotus intermedius, which inhabits the Sea of Japan . The strain studied was strictly aerobic, heterotrophic, yellow-orange-pigmented, motile by gliding, Gram-negative and oxidase-, catalase-, beta-galactosidase- and alkaline phosphatase-positive . The results of 16S rRNA gene sequence analysis showed that strain KMM 6050(T) occupies a distinct lineage within the family Flavobacteriaceae and is most closely related to the species Mesonia algae and Salegentibacter salegens (sequence similarity of 92.5-92.6 %) . The DNA G+C content of KMM 6050(T) was 39.6 mol% . The major respiratory quinone was MK-6 . The predominant fatty acids were i15 : 0, a15 : 0, 15 : 0, i16 : 1, i16 : 0, i16 : 0 3-OH and i17 : 0 3-OH . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, the novel bacterium has been assigned to the genus Gramella gen . nov., as Gramella echinicola sp . nov . The type strain is KMM 6050(T) (=KCTC 12278(T)=NBRC 100593(T)=LMG 22585(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 379 - 383
Idiomarina seosinensis sp . nov., isolated from hypersaline water of a solar saltern in Korea; Choi DH et al.; A halophilic gamma-proteobacterium, designated CL-SP19(T), was isolated from hypersaline water from a solar saltern located in Seosin, Korea . Analysis of the 16S rRNA gene sequence revealed an affiliation with the genus Idiomarina . The sequence similarities between CL-SP19(T) and type strains of the genus Idiomarina ranged from 95.9 to 96.9 % . Cells were straight or slightly curved rods and were motile by means of a single polar flagellum . The major fatty acids were C(15 : 0) iso (17.1 %) and C(17 : 0) iso (15.2 %) . Three fatty acids, C(19 : 0)omega8c cyclo (3.5 %), C(14 : 1)omega5c (1.4 %) and C(18 : 3)omega6c (1.2 %), were found in minor quantities, but uniquely in CL-SP19(T) among Idiomarina species . The DNA G+C content was 45.0 mol% . On the basis of its physiology, fatty acid composition and 16S rRNA gene sequence, strain CL-SP19(T) could be assigned to the genus Idiomarina but distinguished from the recognized species of the genus . Strain CL-SP19(T), therefore, represents a novel species, for which the name Idiomarina seosinensis sp . nov . is proposed, with CL-SP19(T) (=KCTC 12296(T)=JCM 12526(T)) as the type strain.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 369 - 373
Sphingopyxis flavimaris sp . nov., isolated from sea water of the Yellow Sea in Korea; Yoon JH et al.; A Gram-negative, motile, yellow-pigmented, slightly halophilic bacterial strain, SW-151(T), was isolated from sea water of the Yellow Sea in Korea, and subjected to a polyphasic taxonomic study . The isolate grew optimally at 30 degrees C and in the presence of 2-3 % NaCl . Strain SW-151(T) was characterized chemotaxonomically as having Q-10 as the predominant respiratory lipoquinone and C(18 : 1)omega7c, C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH and C(17 : 1)omega6c as the major fatty acids . Sphingoglycolipid, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine were the major polar lipids . The DNA G+C content was 58 mol% . 16S rRNA gene sequence analysis showed that strain SW-151(T) joins the evolutionary radiation enclosed by the genus Sphingopyxis . Similarities between the 16S rRNA gene sequences of strain SW-151(T) and the type strains of Sphingopyxis species ranged from 92.3 to 94.3 %, which is low enough to categorize strain SW-151(T) as a species distinct from previously described Sphingopyxis species . On the basis of phenotypic properties and phylogenetic distinctiveness, strain SW-151(T) (=KCTC 12232(T)=DSM 16223(T)) should be classified as a novel Sphingopyxis species, for which the name Sphingopyxis flavimaris sp . nov . is proposed.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 315 - 320
Isolation of Lentibacillus salicampi strains and Lentibacillus juripiscarius sp . nov . from fish sauce in Thailand; Namwong S et al.; Eight strains of aerobic, spore-forming, Gram-positive, moderately halophilic bacteria were isolated from sauce (nam-pla and bu-du) produced in Thailand by the fermentation of fish . They grew optimally in the presence of 10 % NaCl, at 37 degrees C and pH 7.0 . A diagnostic diamino acid, meso-diaminopimelic acid, was present in the cell-wall peptidoglycan . The predominant menaquinone was MK-7 . The major cellular fatty acids were anteiso-C(15 : 0) and iso-C(16 : 0) . Phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid were found to be the major polar lipid components . The DNA G+C content was 42-43 mol% . These bacteria were further divided into two groups based on phenotypic characteristics and DNA-DNA similarities . Three strains of Group I were highly affiliated to the type strain of Lentibacillus salicampi in terms of phenotypic characterization and DNA-DNA similarities (96-102 %); accordingly, they were identified as strains of L . salicampi . A representative strain of Group II, strain IS40-3(T), was most closely related to L . salicampi in terms of 16S rRNA-based phylogenetic analysis, although five strains of Group II could be distinguished from L . salicampi by means of several phenotypic properties, low 16S rRNA gene sequence similarity (95.2 %) and low DNA-DNA similarities (12-32 %) . Therefore, the Group II strains should be included in a novel species of the genus Lentibacillus, for which the name Lentibacillus juripiscarius sp . nov . is proposed . The type strain is IS40-3(T) (=JCM 12147(T)=PCU 229(T)=TISTR 1535(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 239 - 243
Salinimonas chungwhensis gen . nov., sp . nov., a moderately halophilic bacterium from a solar saltern in Korea; Jeon CO et al.; A halophilic non-spore-forming bacterium of the gamma-Proteobacteria, designated strain BH030046(T), was isolated from a solar saltern in Korea . Cells were Gram-negative, chemoheterotrophic, short rod-shaped and motile with a polar flagellum . Comparative 16S rRNA gene sequence analysis revealed that strain BH030046(T) formed a distinct lineage in the family Alteromonadaceae and was distinguished from its closest related genera Alteromonas (91.4-94.8 %), Aestuariibacter (92.1-93.5 %) and Glaciecola (92.1-93.5 %) on the basis of low 16S rRNA gene sequence similarities . Physiological and biochemical data also showed that the isolate was different from members of these three genera . The predominant cellular fatty acids were C(16 : 0) and C(18 : 1)omega7c . DNA G+C content was 48 mol% and the major isoprenoid quinone was Q-8 . The strain grew optimally at 30-35 degrees C, pH 7.0-8.0 and 2-5 % NaCl . On the basis of physiological and molecular properties, strain BH030046(T) (=KCTC 12239(T)=DSM 16280(T)) represents a novel genus and species in the family Alteromonadaceae, for which the name Salinimonas chungwhensis gen . nov., sp . nov . is proposed.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 171 - 176
Psychrobacter alimentarius sp . nov., isolated from squid jeotgal, a traditional Korean fermented seafood; Yoon JH et al.; Two Gram-negative, non-motile, non-spore-forming, moderately halophilic strains, JG-100(T) and JG-102, were isolated from squid jeotgal, a traditional Korean fermented seafood . The two strains grew optimally at 30 degrees C and in the presence of 2-3 % (v/w) NaCl . Strains JG-100(T) and JG-102 were characterized chemotaxonomically; they both had ubiquinone-8 as the predominant respiratory lipoquinone and C(18 : 1)omega9c as the major fatty acid . Their DNA G+C content was 44 mol% . Strains JG-100(T) and JG-102 showed 1 bp difference in their 16S rRNA gene sequences and a mean DNA-DNA relatedness level of 88 % . Phylogenetic analysis based on 16S rRNA gene sequences showed that strains JG-100(T) and JG-102 form a distinct phylogenetic lineage within the cluster comprising Psychrobacter species . The 16S rRNA gene sequences of strains JG-100(T) and JG-102 had similarity levels of 95.2-98.4 % to sequences of the type strains of recognized Psychrobacter species . Levels of DNA-DNA relatedness between strains JG-100(T) and JG-102 and the type strains of some phylogenetically related Psychrobacter species were 6-24 % . On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains JG-100(T) and JG-102 should be placed in the genus Psychrobacter as a novel species, for which the name Psychrobacter alimentarius sp . nov . (type strain, JG-100(T)=KCTC 12186(T)=DSM 16065(T)) is proposed.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 165 - 170
Pontibacillus chungwhensis gen . nov., sp . nov., a moderately halophilic Gram-positive bacterium from a solar saltern in Korea; Lim JM et al.; Three moderately halophilic, spore-forming strains, designated BH030062(T), BH030049 and BH030080, were isolated from a solar saltern in Korea . Phylogenetic analyses and comparative 16S rRNA gene sequence studies revealed that the isolates represent a novel distinct monophyletic lineage within the phyletic group classically defined as the genus Bacillus and are most closely related to members of the genera Gracilibacillus (93.7-95.1 % similarity), Virgibacillus (93.5-94.8 %), Halobacillus (94.8-95.9 %), Filobacillus (94.4-94.8 %) and Lentibacillus (93.3-93.7 %) . Strain BH030062(T) was strictly aerobic, rod-shaped, Gram-positive and motile by means of peritrichous flagella . It grew in the presence of 1-15 % (w/v) NaCl and at temperatures of 15-45 degrees C . The cell wall peptidoglycan contained A1gamma-meso-diaminopimelic acid as the diagnostic diamino acid . The predominant cellular fatty acids were iso-C(15 : 0), anteiso-C(15 : 0) and iso-C(16 : 0) . DNA G+C content was about 41 mol% and the major isoprenoid quinone was MK-7 . On the basis of their physiological and molecular properties, the isolates represent a new genus, Pontibacillus gen . nov., and novel species, Pontibacillus chungwhensis sp . nov . The type strain is BH030062(T) (=KCTC 3890(T)=DSM 16287(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 143 - 148
Marinobacter bryozoorum sp . nov . and Marinobacter sediminum sp . nov., novel bacteria from the marine environment; Romanenko LA et al.; Two marine, Gram-negative, aerobic, halophilic strains, designated KMM 3657(T) and KMM 3840(T), were isolated and found to be phylogenetically closely related to each other, showing 96.6 % 16S rRNA gene sequence similarity . Both strains are members of the genus Marinobacter in the gamma-Proteobacteria (94.7-98.0 % 16S rRNA gene sequence similarity) . Strain KMM 3657(T) and Marinobacter lipolyticus SM19(T) were closely related, with 98.0 % sequence similarity . The novel strains shared generic physiological and chemotaxonomic properties with Marinobacter species, but differed in their temperature range for growth, inability to grow in 20 % NaCl and at >43 degrees C, metabolic properties and fatty acid composition . On the basis of phenotypic and phylogenetic analysis data, it is proposed that the strains represent two novel species, Marinobacter bryozoorum sp . nov., with the type strain KMM 3840(T) (=50-11(T)=DSM 15401(T)), and Marinobacter sediminum sp . nov., with the type strain KMM 3657(T) (=R65(T)=DSM 15400(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 105 - 110
Thalassobacter stenotrophicus gen . nov., sp . nov., a novel marine {alpha}-proteobacterium isolated from Mediterranean sea water; Macian MC et al.; A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain) . 16S rRNA gene sequence comparisons showed that the isolate represented a separate branch within the alpha-3 subclass of the Proteobacteria, now included within the order 'Rhodobacterales' . Jannaschia helgolandensis was the closest relative, but their low sequence similarity and other features indicated that they were not related at the genus level . Isolate 5SM22(T) produced bacteriochlorophyll a and grew on solid media as regular salmon-pink colonies . Cells are motile rods, with polar flagella . The DNA G+C content is 59.1 mol% . Morphological, physiological and genotypic differences from related, thus far known genera support the description of Thalassobacter stenotrophicus gen . nov., sp . nov . with strain 5SM22(T) (=CECT 5294(T)=DSM 16310(T)) as the type strain.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 95 - 99
Tenuibacillus multivorans gen . nov., sp . nov., a moderately halophilic bacterium isolated from saline soil in Xin-Jiang, China; Ren PG et al.; Two Gram-positive, rod-shaped, spore-forming and moderately halophilic bacteria (strains 28-1(T), 28-4), isolated from a soil sample from a neutral salt lake in Xin-Jiang, China, were characterized polyphasically . On the basis of fasta (ungapped) analyses of 16S rRNA gene sequences, strains 28-1(T) and 28-4 were shown to belong to the Bacillaceae and to be closely related to Filobacillus milensis DSM 13259(T) (97.0 %) and Bacillus haloalkaliphilus DSM 5271(T) (95.7 %) . 16S rRNA gene sequence similarity with other recognized species was not more than 94.1 % . Phylogenetic, chemotaxonomic, physiological and biochemical data supported the differentiation of these novel strains from F . milensis and B . haloalkaliphilus . Therefore these two previously unidentified strains are considered to represent a new genus and species, for which the name Tenuibacillus multivorans gen . nov., sp . nov . is proposed . The type strain is 28-1(T) (=AS 1.3442(T)=NBRC 100370(T)).

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 71 - 75
Erythrobacter seohaensis sp . nov . and Erythrobacter gaetbuli sp . nov., isolated from a tidal flat of the Yellow Sea in Korea; Yoon JH et al.; Two Gram-negative, non-spore-forming, slightly halophilic strains, SW-135(T) and SW-161(T), which were isolated from a tidal flat of the Yellow Sea in Korea, were subjected to a polyphasic taxonomic study . The two isolates lacked bacteriochlorophyll a and contained ubiquinone-10 (Q-10) as the predominant respiratory lipoquinone and C(18 : 1)omega7c and C(17 : 1)omega6c as the major fatty acids . The DNA G+C contents of strains SW-135(T) and SW-161(T) were 62.2 and 64.5 mol%, respectively . Phylogenetic analyses based on 16S rRNA gene sequences showed that the two strains fall within the radiation of the cluster comprising Erythrobacter species . Strains SW-135(T) and SW-161(T) exhibited a 16S rRNA gene sequence similarity value of 96.9 % and a mean DNA-DNA relatedness level of 12.3 % . Sequence similarities between strains SW-135(T) and SW-161(T) and the type strains of recognized Erythrobacter species ranged from 96.7 to 98.5 % . Levels of DNA-DNA relatedness were low enough to indicate that strains SW-135(T) and SW-161(T) represent members of two species separate from all recognized Erythrobacter species . On the basis of polyphasic taxonomic data, strains SW-135(T) (=KCTC 12228(T)=DSM 16221(T)) and SW-161(T) (=KCTC 12227(T)=DSM 16225(T)) were classified as two novel Erythrobacter species, for which the names Erythrobacter seohaensis sp . nov . and Erythrobacter gaetbuli sp . nov . are proposed, respectively.

J Chromatogr B Analyt Technol Biomed Life Sci, 2005 Feb 5, 815(1-2), 237 - 250
Proteomics of Halophilic archaea; Joo WA et al.; Halophilic archaea is a member of the Halobacteriacea family, the only family in the Halobacteriales order . Most Halophilic archaea require 1.5M NaCl both to grow and retain the structural integrity of the cells . The proteins of these organisms have thus been adapted to be active and stable in the hypersaline condition . Consequently, the unique properties of these biocatalysts have resulted in several novel applications in industrial processes . Halophilic archaea are also to be useful for bioremediation of hypersaline environment . Proteome data have expended enormously with the significant advance recently achieved in two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) . The whole genome sequencing of Halobacterium species NRC-1 was completed and this would also provide tremendous help to analyze the protein mass data from the similar strain Halobacterium salinarum . Proteomics coupled with genomic databases now has become a basic tool to understand or identify the function of genes and proteins . In addition, the bioinformatics approach will facilitate to predict the function of novel proteins of Halophilic archaea . This review will discuss current proteome study of Halophilic archaea and introduce the efficient procedures for screening, predicting, and confirming the function of novel halophilic enzymes.

Yi Chuan, 2004 May, 26(3), 343 - 8
{Analysis of partial sequence of the gene for a novel br protein.}; Xu XW et al.; A strain of halophilic archaeum AB1 was isolated and purified from Aibi Lake located in the north of Xinjiang Uygur Autonomous Region . Partial DNA fragment encoding a bateriorhodopsin (Br) protein as well as 16S rRNA of AB1 was amplified by PCR, and their nucleotide sequences were determined subsequently . On the basis of homology and phylognetic analysis about 16S rRNA gene (16S rDNA), it could be speculated that the strain AB1 is a novel member of the genus Natronococcus . The hydrophathy analysis of Br fragment revealed that the AB1 Br had a transmembrane heptahelical structure similar to that of other Brs . On the other hand, homology alignment using the deduced partial amino acid sequence of Br protein of AB1 with other Br proteins showed that AB1 Br protein is obviously different to others . These facts indicated that the Br in halophilic archaeum AB1 is a new Br protein.

J Zhejiang Univ Sci, 2005 Feb, 6(2), 142 - 6
Isolation and characterization of a novel strain of Natrinema containing a bop gene; Xu XW et al.; A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China . The strain AJ2 requires at least 10% (w/v) NaCl and grows 10% to 30% (optimum at 20%) . Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain AJ2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting AJ2 is a novel member of Natrinema . A bacteriorhodopsin-encoding (bop) gene was subsequently detected in the AJ2 genome using the polymerase chain reaction technique . The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from AJ2 is different from that reported for bacteriorhodopsins . This is the first reported detection of a bop gene in Natrinema.

Microbiology, 2005 Jan, 151(Pt 1), 25 - 33
In vivo analyses of constitutive and regulated promoters in halophilic archaea; Gregor D et al.; The two gvpA promoters P(cA) and P(pA) of Halobacterium salinarum, and the P(mcA) promoter of Haloferax mediterranei were investigated with respect to growth-phase-dependent expression and regulation in Haloferax volcanii transformants using the bgaH reading frame encoding BgaH, an enzyme with beta-galactosidase activity, as reporter . For comparison, the P(fdx) promoter of the ferredoxin gene of Hbt . salinarum and the P(bgaH) promoter of Haloferax lucentense (formerly Haloferax alicantei) were analysed . P(fdx), driving the expression of a house-keeping gene, was highly active during the exponential growth phase, whereas P(bgaH) and the three gvpA promoters yielded the largest activities during the stationary growth phase . Compared to P(fdx), the basal promoter activities of P(pA) and P(mcA) were rather low, and larger activities were only detected in the presence of the endogenous transcriptional activator protein GvpE . The P(cA) promoter does not yield a detectable basal promoter activity and is only active in the presence of the homologous cGvpE . To investigate whether the P(cA)-TATA box and the BRE element were the reason for the lack of the basal P(cA) activity, these elements and also sequences further upstream were substituted with the respective sequences of the stronger P(pA) promoter and investigated in Hfx . volcanii transformants . All these promoter chimera did not yield a detectable basal promoter activity . However, whenever the P(pA)-BRE element was substituted for the P(cA)-BRE, an enhanced cGvpE-mediated activation was observed . The promoter chimeras harbouring P(pA)-BRE plus 5 (or more) bp further upstream also gained activation by the heterologous pGvpE and mcGvpE proteins . The sequence required for the GvpE-mediated activation was determined by a 4 bp scanning mutagenesis with the 45 bp region upstream of P(mcA)-BRE . None of these alterations influenced the basal promoter activity, but the sequence TGAAACGG-n4-TGAACCAA was important for the GvpE-mediated activation of P(mcA).

Biochemistry, 2005 Jan 11, 44(1), 29 - 39
A Two-alpha-Helix Extra Domain Mediates the Halophilic Character of a Plant-Type Ferredoxin from Halophilic Archaea(,); Marg BL et al.; The {2Fe-2S} ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibits a high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 amino acids near its N-terminus which is extremely rich in acidic amino acids . Unfolding studies reveal that HsFdx has an unfolding temperature of approximately 85 degrees C in 4.3 M NaCl, but of only 50 degrees C in low salinity, revealing its halophilic character . The three-dimensional structure of HsFdx was determined by NMR spectroscopy, resulting in a backbone rmsd of 0.6 A for the diamagnetic regions of the protein . Whereas the overall structure of HsFdx is very similar to that of the plant-type ferredoxins, two additional alpha-helices are found in the acidic extra domain . (15)N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility of residues is similar throughout the molecule . Monitoring protein denaturation by NMR did not reveal differences between the core fold and the acidic domain, suggesting a cooperative unfolding of both parts of the molecule . A mutant of the HsFdx in which the acidic domain is replaced with a short loop of the nonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern . The halophilic wild-type protein is readily expressed in large amounts in H . salinarum, but not in Escherichia coli, whereas the mutant ferredoxin could only be overexpressed in E . coli . The salt concentration was also found to play a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the cluster in the mutant protein proceeds efficiently in low salt . These findings suggest that the acidic domain mediates the halophilic character which is reflected in its thermostability, the exclusive expression in H . salinarum, and the ability to efficiently reconstitute the iron-sulfur cluster only at high salt concentrations.

Proteomics . 2005 Jan 3; {Epub ahead of print}
Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii; Karadzic IM et al.; Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking . We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer . In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-{(3-cholamidopropyl)dimethylamino}-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1) . This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.

Proteomics . 2004 Dec 23; {Epub ahead of print}
Analysis of the cytosolic proteome of Halobacterium salinarum and its implication for genome annotation; Tebbe A et al.; The halophilic archaeon Halobacterium salinarum (strain R1, DSM 671) contains 2784 protein-coding genes as derived from the genome sequence . The cytosolic proteome containing 2042 proteins was separated by two-dimensional gel electrophoresis (2-DE) and systematically analyzed by a semi-automatic procedure . A reference map was established taking into account the narrow isoelectric point (pI) distribution of halophilic proteins between 3.5 and 5.5 . Proteins were separated on overlapping gels covering the essential areas of pI and molecular weight . Every silver-stained spot was analyzed resulting in 661 identified proteins out of about 1800 different protein spots using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting (PMF) . There were 94 proteins that were found in multiple spots, indicating post-translational modification . An additional 141 soluble proteins were identified on 2-D gels not corresponding to the reference map . Thus about 40% of the cytosolic proteome was identified . In addition to the 2784 protein-coding genes, the H . salinarum genome contains more than 6000 spurious open reading frames longer than 100 codons . Proteomic information permitted an improvement in genome annotation by validating and correcting gene assignments . The correlation between theoretical pI and gel position is exceedingly good and was used as a tool to improve start codon assignments . The fraction of identified chromosomal proteins was much higher than that of those encoded on the plasmids . In combination with analysis of the GC content this observation permitted an unambiguous identification of an episomal insert of 60 kbp ("AT-rich island") in the chromosome, as well as a 70 kbp region from the chromosome that has integrated into one of the megaplasmids and carries a series of essential genes . About 63% of the chromosomally encoded proteins larger than 25 kDa were identified, proving the efficacy of 2-DE MALDI-TOF MS PMF technology . The analysis of the integral membrane proteome by tandem mass spectrometric techniques added another 141 identified proteins not identified by the 2-DE approach (see following paper).

Mikrobiologiia, 2004 Sep-Oct, 73(5), 598 - 612
{Microbial diversity studies at the Winogradsky Institute of Microbiology}; Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 A resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP; Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho 1-1, Toyonaka, Osaka 560-0043, JapanPhotoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium . Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50 . Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state . The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type . This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type . Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond . Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.

Environ Microbiol, 2004 Dec, 6(12), 1287 - 91
Isolation and cultivation of Walsby's square archaeon; Bolhuis H et al.; In 1980, A . E . Walsby described a square halophilic archaeon . This archaeon is of specific interest because of its unique shape and its abundance in hypersaline ecosystems, which suggests an important ecophysiological role . Ever since its discovery, the isolation and cultivation of 'Walsby's square archaeon' has been a holy grail for many microbiologists working on halophiles . Despite their abundance and easy recognition by microscopy, all cultivation attempts have failed up to now, marking the organism as one of the unculturables . Cultivation of the square archaeon is essential to understand their ecophysiological role and the nature of their unique morphologically features . Here, we report the isolation and cultivation of the enigmatic square archaeon that we propose to name Haloquadratum walsbyi . Pure cultures are easily maintained in simple artificial hypersaline media . Initial growth experiments revealed a tolerance to high concentrations of MgCl(2) (>2 M) in the presence of 3.3 M NaCl . Fresh cultures contained extraordinary large cells (>40 x 40 microm) without any visible division structures, ranking them among the largest prokaryotes known to date . The genome was estimated to contain approximately three million basepairs.

FEMS Microbiol Lett, 2004 Dec 1, 241(1), 21 - 6
Regulation of acetate and acetyl-CoA converting enzymes during growth on acetate and/or glucose in the halophilic archaeon Haloarcula marismortui; Brasen C et al.; Haloarcula marismortui formed acetate during aerobic growth on glucose and utilized acetate as growth substrate . On glucose/acetate mixtures diauxic growth was observed with glucose as the preferred substrate . Regulation of enzyme activities, related to glucose and acetate metabolism was analyzed . It was found that both glucose dehydrogenase (GDH) and ADP-forming acetyl-CoA synthetase (ACD) were upregulated during periods of glucose consumption and acetate formation, whereas both AMP-forming acetyl-CoA synthetase (ACS) and malate synthase (MS) were downregulated . Conversely, upregulation of ACS and MS and downregulation of ACD and GDH were observed during periods of acetate consumption . MS was also upregulated during growth on peptides in the absence of acetate . From the data we conclude that a glucose-inducible ACD catalyzes acetate formation whereas acetate activation is catalyzed by an acetate-inducible ACS; both ACS and MS are apparently induced by acetate and repressed by glucose.

Mol Microbiol, 2004 Dec, 54(5), 1307 - 18
An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea; Levin I et al.; Whereas tetrahydrofolate is an essential cofactor in all bacteria, the gene that encodes the enzyme dihydrofolate reductase (DHFR) could not be identified in many of the bacteria whose genomes have been entirely sequenced . In this communication we show that the halophilic archaea Halobacterium salinarum and Haloarcula marismortui contain genes coding for proteins with an N-terminal domain homologous to dihydrofolate synthase (FolC) and a C-terminal domain homologous to dihydropteroate synthase (FolP) . These genes are able to complement a Haloferax volcanii mutant that lacks DHFR . We also show that the Helicobacter pylori dihydropteroate synthase can complement an Escherichia coli mutant that lacks DHFR . Activity resides in an N-terminal segment that is homologous to the polypeptide linker that connects the dihydrofolate synthase and dihydropteroate synthase domains in the haloarchaeal enzymes . The purified recombinant H . pylori dihydropteroate synthase was found to be a flavoprotein.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2347 - 52
Shewanella marisflavi sp . nov . and Shewanella aquimarina sp . nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea; Yoon JH et al.; Two Gram-negative, motile, non-spore-forming, rod-shaped organisms, strains SW-117(T) and SW-120(T), were isolated from sea water of the Yellow Sea in Korea and subjected to a polyphasic taxonomic study . Strains SW-117(T) and SW-120(T) simultaneously contained both menaquinones (MK) and ubiquinones (Q) as isoprenoid quinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8 . The major fatty acid detected in the two strains was iso-C(15 : 0) . The DNA G+C content of strains SW-117(T) and SW-120(T) was 51 and 54 mol%, respectively . Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-117(T) and SW-120(T) fall within the radiation of the cluster comprising Shewanella species . Strains SW-117(T) and SW-120(T) showed a 16S rRNA gene sequence similarity of 97.4 % and a DNA-DNA relatedness level of 10.1 % . Strains SW-117(T) and SW-120(T) exhibited 16S rRNA gene sequence similarity levels of 93.8-98.5 % and 92.4-97.0 %, respectively, to Shewanella species . Strain SW-117(T) exhibited DNA-DNA relatedness levels of 8.3-20.3 % to the type strains of six phylogenetically related Shewanella species . On the basis of phenotypic, phylogenetic and genetic data, strains SW-117(T) and SW-120(T) were classified in the genus Shewanella as two distinct novel species, for which the names Shewanella marisflavi sp . nov . (type strain, SW-117(T)=KCCM 41822(T)=JCM 12192(T)) and Shewanella aquimarina sp . nov . (type strain, SW-120(T)=KCCM 41821(T)=JCM 12193(T)) are proposed, respectively.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2275 - 9
Haloferax sulfurifontis sp . nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring; Elshahed MS et al.; A pleomorphic, extremely halophilic archaeon (strain M6(T)) was isolated from a sulfide- and sulfur-rich spring in south-western Oklahoma (USA) . It formed small (0.8-1.0 mm), salmon pink, elevated colonies on agar medium . The strain grew in a wide range of NaCl concentrations (6 % to saturation) and required at least 1 mM Mg(2+) for growth . Strain M6(T) was able to reduce sulfur to sulfide anaerobically . 16S rRNA gene sequence analysis indicated that strain M6(T) belongs to the family Halobacteriaceae, genus Haloferax; it showed 96.7-98.0 % similarity to other members of the genus with validly published names and 89 % similarity to Halogeometricum borinquense, its closest relative outside the genus Haloferax . Polar lipid analysis and DNA G+C content further supported placement of strain M6(T) in the genus Haloferax . DNA-DNA hybridization values, as well as biochemical and physiological characterization, allowed strain M6(T) to be differentiated from other members of the genus Haloferax . A novel species, Haloferax sulfurifontis sp . nov., is therefore proposed to accommodate the strain . The type strain is M6(T) (=JCM 12327(T)=CCM 7217(T)=DSM 16227(T)=CIP 108334(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2231 - 5
Porphyrobacter donghaensis sp . nov., isolated from sea water of the East Sea in Korea; Yoon JH et al.; Two Gram-negative, motile, non-spore-forming, bacteriochlorophyll a-containing slightly halophilic strains, SW-132(T) and SW-158, were isolated from sea water of the East Sea in Korea, and subjected to a polyphasic taxonomic study . The two isolates were characterized chemotaxonomically as having Q-10 as the predominant respiratory lipoquinone and major amounts of unsaturated fatty acids C(18 : 1)omega7c and C(17 : 1)omega6c . The DNA G+C contents of the two strains were in the range 66.8-65.9 mol% . The 16S rRNA gene sequences of strains SW-132(T) and SW-158 were 99.9 % (1 nt difference) similar and their mean level of DNA-DNA relatedness was 86 % . The 16S rRNA gene sequence analysis showed that strains SW-132(T) and SW-158 are phylogenetically closely related to Porphyrobacter species and Erythromicrobium ramosum . Similarities between the 16S rRNA gene sequences of strains SW-132(T) and SW-158 and the type strains of Porphyrobacter species and E . ramosum ranged from 97.8 to 99.0 % . DNA-DNA relatedness data indicated that strains SW-132(T) and SW-158 are members of a genomic species that is separate from the four Porphyrobacter species . On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strains SW-132(T) (=KCTC 12229(T)=DSM 16220(T)) and SW-158 (=KCTC 12230) are classified as a novel Porphyrobacter species, for which the name Porphyrobacter donghaensis sp . nov . is proposed.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2163 - 7
Transfer of Bacillus halodenitrificans Denariaz et al . 1989 to the genus Virgibacillus as Virgibacillus halodenitrificans comb . nov; Yoon JH et al.; A Gram-variable, endospore-forming moderately halophilic rod, strain SF-121, was isolated from a marine solar saltern of the Yellow Sea in Korea . The result of 16S rRNA gene sequence analysis showed that strain SF-121 has highest sequence similarity (99.7 %) with the type strain of Bacillus halodenitrificans . Phylogenetic analyses based on 16S rRNA gene sequences revealed that B . halodenitrificans DSM 10037(T) and strain SF-121 are more closely related to the genus Virgibacillus than to the genus Bacillus . Strain SF-121 and B . halodenitrificans DSM 10037(T) exhibited 16S rRNA gene similarity levels of 95.3-97.5 % with the type strains of Virgibacillus species and 94.0 % with the type strain of Bacillus subtilis . DNA-DNA relatedness and phenotypic data indicated that B . halodenitrificans DSM 10037(T) and strain SF-121 are members of the same species . B . halodenitrificans DSM 10037(T) and strain SF-121 exhibited DNA-DNA relatedness values of 9-11 % with the type strains of Virgibacillus carmonensis and Virgibacillus marismortui . On the basis of the phenotypic, chemotaxonomic, phylogenetic and genetic data, B . halodenitrificans should be reclassified in the genus Virgibacillus as Virgibacillus halodenitrificans comb . nov.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2037 - 42
Halomonas koreensis sp . nov., a novel moderately halophilic bacterium isolated from a solar saltern in Korea; Lim JM et al.; A moderately halophilic bacterium, strain SS20(T), capable of growing at salinities of 1-20 % (w/v) NaCl was isolated from a solar saltern of the Dangjin area in Korea and was characterized taxonomically . Strain SS20(T) was a Gram-negative bacterium comprising motile, short rods . Its major cellular fatty acids were C(18 : 1)omega7c, C(19 : 0)omega8c cyclo and C(16 : 0) . The DNA G+C content was 70 mol% and the predominant ubiquinone was Q-9 . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SS20(T) belonged to the genus Halomonas . The levels of 16S rRNA gene sequence similarity to the type strains of Halomonas species were in the range 93.0-97.5 % . The levels of DNA-DNA relatedness between strain SS20(T) and the type strains of phylogenetically closely related Halomonas species were in the range 5.3-12.3 % . On the basis of physiological and molecular properties, strain SS20(T) represents a novel species of the genus Halomonas, for which the name Halomonas koreensis sp . nov . is proposed . The type strain is SS20(T) (=KCTC 12127(T)=JCM 12237(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 1981 - 5
Erythrobacter aquimaris sp . nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea; Yoon JH et al.; Three Gram-negative, non-motile, non-spore-forming, slightly halophilic rods (strains SW-110(T), SW-116 and SW-140) were isolated from sea water of a tidal flat of the Yellow Sea in Korea and subjected to a polyphasic taxonomic study . The three isolates did not produce bacteriochlorophyll a and were characterized chemotaxonomically by having ubiquinone-10 as the predominant respiratory lipoquinone and C(18 : 1)omega7c and C(17 : 1)omega6c as the major fatty acids . The DNA G+C content of the three isolates was between 62.2 and 62.9 mol% . Strains SW-110(T), SW-116 and SW-140 showed no difference in their 16S rRNA gene sequences, and their mean level of DNA-DNA relatedness was 94.8 % . Phylogenetic analyses based on 16S rRNA gene sequences showed that the three strains form a distinct phylogenetic lineage within the cluster comprising Erythrobacter species . Similarities between the 16S rRNA gene sequences of strains SW-110(T), SW-116 and SW-140 and the type strains of Erythrobacter species ranged from 98.4 % (with Erythrobacter longus) to 97.7 % (with Erythrobacter flavus) . Levels of DNA-DNA relatedness between strains SW-110(T), SW-116 and SW-140 and the type strains of all recognized Erythrobacter species were in the range 5.3-12.7 % . On the basis of polyphasic taxonomic data, strains SW-110(T), SW-116 and SW-140 were classified as a novel Erythrobacter species, for which the name Erythrobacter aquimaris sp . nov . is proposed . The type strain is SW-110(T) (=KCCM 41818(T)=JCM 12189(T)).

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 1921 - 6
Chromohalobacter sarecensis sp . nov., a psychrotolerant moderate halophile isolated from the saline Andean region of Bolivia; Quillaguaman J et al.; A moderately halophilic, aerobic, motile, Gram-negative, rod-shaped bacterium (strain LV4(T)) was isolated from saline soil around the lake Laguna Verde in the Bolivian Andes . The organism is a heterotroph, able to utilize various carbohydrates as a carbon source . It showed tryptophan deaminase, oxidase and catalase activity, but was unable to produce indole or H(2)S; nitrate was not reduced . The G+C content of the genomic DNA was 56.1 mol% . The pH range for growth was 5-10, temperature range was 0-45 degrees C and the range of NaCl concentrations was 0-25 % (w/v) . On the basis of 16S rRNA gene sequence analysis, strain LV4(T) was found to be closely related to Chromohalobacter canadensis DSM 6769(T) and Pseudomonas beijerinckii DSM 7218(T); however, its DNA-DNA relatedness with these type strains was low . Strain LV4(T) resembled other Chromohalobacter species with respect to various physiological, biochemical and nutritional characteristics but also exhibited differences . Thus, a novel species, Chromohalobacter sarecensis sp . nov., is proposed, with LV4(T) (=CCUG 47987(T)=ATCC BAA-761(T)) as the type strain.

Appl Environ Microbiol, 2004 Nov, 70(11), 6678 - 85
Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber; Corcelli A et al.; Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria . Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S . ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum . A novel sulfonolipid, giving rise to the molecular ion {M-H}- of m/z 660, has been identified . The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13'-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid . This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W . Godchaux and E . R . Leadbetter, J . Bacteriol . 153:1238-1246, 1983) and of diatoms (R . Anderson, M . Kates, and B . E . Volcani, Biochim . Biophys . Acta 528:89-106, 1978) . Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter . This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.

Appl Environ Microbiol, 2004 Nov, 70(11), 6535 - 41
Glutamine, glutamate, and alpha-glucosylglycerate are the major osmotic solutes accumulated by Erwinia chrysanthemi strain 3937; Goude R et al.; Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli . Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way . Unexpectedly, the pools of endogenous osmolytes show different compositions . Instead of the commonly accumulated glutamate and trehalose, E . chrysanthemi strain 3937 promotes the accumulation of glutamine and alpha-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine . The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium . Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable . In addition, the balance between the osmolytes depended on the osmolality of the medium . Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations . Interestingly, the ammonium content of the medium also influenced the pool of osmolytes . During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes . Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.

J Biotechnol, 2004 Nov 9, 114(3), 225 - 37
Cassette-based presentation of SIV epitopes with recombinant gas vesicles from halophilic archaea; Stuart ES et al.; In earlier studies we demonstrated recombinant gas vesicles from Halobacterium sp . NRC-1, expressing a model six amino acid insert, or native vesicles displaying chemically coupled TNP, each were immunogenic, and antigenic . Long-lived responses displaying immunologic memory were elicited without exogenous adjuvant . Here we report the generation and expression of cassettes containing SIV derived DNA . The results indicate a cassette-based display/delivery system derived from recombinant halobacterial gas vesicle genes is highly feasible . Data specifically support four conclusions: (i) Recombinants carrying up to 705 bp of SIV DNA inserted into the gvpC gene form functional gas vesicles; (ii) SIV peptides contained as part of the expressed recombinant, surface exposed GvpC protein are recognized by antibody elicited in monkeys exposed to native SIV in vivo; (iii) in the absence of adjuvant, mice immunized with the recombinant gas vesicle (r-GV) preparations mount a solid, titratable antibody response to the test SIV insert that is long lived and exhibits immunologic memory; (iv) recombinant organelles, created through the generation of cassettes encoding epitopes inserted into the gvpC DNA, can be used to construct a multiepitope display (MED) library, a potentially cost effective vehicle to express and deliver peptides of SIV, HIV or other pathogens.

Genome Res, 2004 Nov, 14(11), 2221 - 34
Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea; Baliga NS et al.; We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea . The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62% . Comparison of the genome architectures of Halobacterium sp . NRC-1 and H . marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former . Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm . A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms . Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp . NRC-1--suggesting H . marismortui is significantly more physiologically capable of exploiting diverse environments . In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile . Finally, as expected from the larger genome, H . marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp . NRC-1.

J Bacteriol, 2004 Nov, 186(22), 7763 - 72
Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii; Reuter CJ et al.; The halophilic archaeon Haloferax volcanii produces three different proteins (alpha1, alpha2, and beta) that assemble into at least two 20S proteasome isoforms . This work reports the cloning and sequencing of two H . volcanii proteasome-activating nucleotidase (PAN) genes (panA and panB) . The deduced PAN proteins were 60% identical with Walker A and B motifs and a second region of homology typical of AAA ATPases . The most significant region of divergence was the N terminus predicted to adopt a coiled-coil conformation involved in substrate recognition . Of the five proteasomal proteins, the alpha1, beta, and PanA proteins were the most abundant . Differential regulation of all five genes was observed, with a four- to eightfold increase in mRNA levels as cells entered stationary phase . In parallel with this mRNA increase, the protein levels of PanB and alpha2 increased severalfold during the transition from exponential growth to stationary phase, suggesting that these protein levels are regulated at least in part by mechanisms that control transcript levels . In contrast, the beta and PanA protein levels remained relatively constant, while the alpha1 protein levels exhibited only a modest increase . This lack of correlation between the mRNA and protein levels for alpha1, beta, and PanA suggests posttranscriptional mechanisms are involved in regulating the levels of these major proteasomal proteins . Together these results support a model in which the cell regulates the ratio of the different 20S proteasome and PAN proteins to modulate the structure and ultimately the function of this central energy-dependent proteolytic system.

Syst Appl Microbiol, 2004 Sep, 27(5), 535 - 40
Vibrio ponticus sp . nov., a neighbour of V fluvialis-V . furnissii clade, isolated from gilthead sea bream, mussels and seawater; Macian MC et al.; A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain . Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source . Phylogenetic analysis locate these marine bacteria in the vicinity of the V . fluvialis-V . furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively) . DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio . We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.

J Biol Chem, 2004 Dec 17, 279(51), 53160 - 6 Epub 2004 Oct 08.
In the Archaea Haloferax volcanii, membrane protein biogenesis and protein synthesis rates are affected by decreased ribosomal binding to the translocon; Ring G et al.; In the haloarchaea Haloferax volcanii, ribosomes are found in the cytoplasm and membrane-bound at similar levels . Transformation of H . volcanii to express chimeras of the translocon components SecY and SecE fused to a cellulose-binding domain substantially decreased ribosomal membrane binding, relative to non-transformed cells, likely due to steric hindrance by the cellulose-binding domain . Treatment of cells with the polypeptide synthesis terminator puromycin, with or without low salt washes previously shown to prevent in vitro ribosomal membrane binding in halophilic archaea, did not lead to release of translocon-bound ribosomes, indicating that ribosome release is not directly related to the translation status of a given ribosome . Release was, however, achieved during cell starvation or stationary growth, pointing at a regulated manner of ribosomal release in H . volcanii . Decreased ribosomal binding selectively affected membrane protein levels, suggesting that membrane insertion occurs co-translationally in Archaea . In the presence of chimera-incorporating sterically hindered translocons, the reduced ability of ribosomes to bind in the transformed cells modulated protein synthesis rates over time, suggesting that these cells manage to compensate for the reduction in ribosome binding . Possible strategies for this compensation, such as a shift to a post-translational mode of membrane protein insertion or maintained ribosomal membrane-binding, are discussed.

Proteomics, 2004 Nov, 4(11), 3632 - 41
Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum; Park SJ et al.; Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl) . Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations . In this study, the protein expression profile of H . salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE) . As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips . The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry . Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins . Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application . IMPDH gene (1.6 kb fragment) expected to exist in H . salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector . Recombinant IMPDH purified from H . salinarum has a higher activity in the presence of salt than in the absence of salt.

Appl Environ Microbiol, 2004 Oct, 70(10), 6220 - 9
Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments; Kaye JZ et al.; Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts) . The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts . Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures . Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions . The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses . Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure . Profiles of cytosolic and membrane proteins of Halomonas axialensis and H . hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two . The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.

J Microbiol, 2004 Sep, 42(3), 174 - 80
Growth and physiological properties of wild type and mutants of Halomonas subglaciescola DH-1 in saline environment; Ryu HJ et al.; A halophilic bacterium was isolated from fermented seafood . The 16S rDNA sequence identity between the isolate and Halomonas subglaciescola AJ306801 was above 95% . The isolate that did not grow in the condition without NaCl or in the condition with other sodium (Na+) or chloride ions (Cl-) instead of NaCl was named H . subglaciescola DH-1 . Two mutants capable of growing without NaCl were obtained by random mutagenesis, of which their total soluble protein profiles were compared with those of the wild type by two-dimensional electrophoresis . The external compatible solutes (betaine and choline) and cell extract of the wild type did not function as osmoprotectants, and these parameters within the mutants did not enhance their growth in the saline environment . In the proton translocation test, rapid acidification of the reactant was not detected for the wild type, but it was detected for the mutant in the condition without NaCl . From these results, we derived the hypothesis that NaCl may be absolutely required for the energy metabolism of H . subglaciescola DH-1 but not for its osmoregulation, and the mutants may have another modified proton translocation system that is independent of NaCl, except for those mutants with an NaCl-dependent system .

Proteomics, 2004 Nov, 4(11), 3622 - 31
Identification of a novel protein D3UPCA from Halobacterium salinarum and prediction of its function; Lee MS et al.; Halobacterium salinarum is an extremely halophilic archaea, which is able to live in highly saline environments . In a recent study, several halophilic archaea were found to have the ability to biodegrade organic hydrocarbon pollutants, but protein information regarding hydrocarbon degradation and tolerance in halophilic archaea has been relatively rare . In this study, the protein expression profile of H . salinarum cultured under different diesel concentrations (0, 2 and 4%) was investigated by two-dimensional gel electrophoresis . Proteins which increased their expression levels in diesel media were identified using matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry analysis . Among these, a protein spot (named D3UPCA) which was up-regulated about nine-fold and found to have COG3388, an uncharacterized protein conserved in archaea, was selected in order to further characterize its functions . The D3UPCA coding gene (named d3upca) was cloned and expressed in Escherichia coli, and purified by the glutathione-S-transferase-fusion method . The function of the protein was estimated using various bioinformatics tools and was predicted to be related to the regulation of transcription and/or translation of genes needed to tolerate stresses associated within the presence of diesel oil.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1805 - 9
Nocardiopsis salina sp . nov., a novel halophilic actinomycete isolated from saline soil in China; Li WJ et al.; A moderately halophilic actinomycete strain, designated YIM 90010T, was isolated from a soil sample collected from a hypersaline habitat in Xinjiang Province, China, and then investigated using a polyphasic taxonomic approach . The strain produced abundant aerial mycelia and fragmented substrate mycelia on most media tested; the optimum NaCl concentration for growth was 10 % (w/v) and the optimum growth temperature and pH were 28 degrees C and 7.2, respectively . Chemotaxonomically and phylogenetically, the strain was related to members of the genus Nocardiopsis . The isolate contained chemotaxonomic markers that were diagnostic for the genus Nocardiopsis, i.e . meso-diaminopimelic acid, no diagnostic sugars, and MK-10(H6), MK-10(H8) and MK-12 as the predominant menaquinones . The major fatty acids were iso- and anteiso-branched acids combined with tuberculostearic acid (Me C(18 : 0)), straight-chain saturated fatty acids and unsaturated fatty acids . The G + C content was 73.1 mol% . Phylogenetic analysis confirmed that strain YIM 90010T was a member of the genus Nocardiopsis and most closely related to Nocardiopsis kunsanensis (97.6 % similarity) and Nocardiopsis xinjiangensis (98.1 % similarity) . It can be differentiated from these species by using phenotypic characteristics, phylogenetic analysis and DNA-DNA hybridization results . On the basis of the polyphasic evidence, a novel species, Nocardiopsis salina sp . nov., is proposed . The type strain of the species is YIM 90010T (= KCTC 19003T = CCTCC AA 204009T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1799 - 803
Marinobacter flavimaris sp . nov . and Marinobacter daepoensis sp . nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea; Yoon JH et al.; Two Gram-negative, motile, non-spore-forming and slightly halophilic rods (strains SW-145T and SW-156T) were isolated from sea water of the Yellow Sea in Korea . Strains SW-145T and SW-156T grew optimally at 37 and 30-37 degrees C, respectively, and in the presence of 2-6 % (w/v) NaCl . Strains SW-145T and SW-156T were chemotaxonomically characterized as having ubiquinone-9 as the predominant respiratory lipoquinone and C(16 : 0), C(18 : 1)omega9c, C(16 : 1)omega9c and C(12 : 0) 3-OH as the major fatty acids . The DNA G + C contents of strains SW-145T and SW-156T were 58 and 57 mol%, respectively . Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-145T and SW-156T fell within the evolutionary radiation enclosed by the genus Marinobacter . The 16S rRNA gene sequences of strains SW-145T and SW-156T were 94.8 % similar . Strains SW-145T and SW-156T exhibited 16S rRNA gene sequence similarity levels of 94.3-98.1 and 95.4-97.7 %, respectively, with respect to the type strains of all Marinobacter species . Levels of DNA-DNA relatedness, together with 16S rRNA gene sequence similarity values, indicated that strains SW-145T and SW-156T are members of two species that are distinct from seven Marinobacter species with validly published names . On the basis of phenotypic properties and phylogenetic and genotypic distinctiveness, strains SW-145T (= KCTC 12185T = DSM 16070T) and SW-156T (= KCTC 12184T = DSM 16072T) should be placed in the genus Marinobacter as the type strains of two distinct novel species, for which the names Marinobacter flavimaris sp . nov . and Marinobacter daepoensis sp . nov . are proposed.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1793 - 7
Idiomarina fontislapidosi sp . nov . and Idiomarina ramblicola sp . nov., isolated from inland hypersaline habitats in Spain; Martinez-Canovas MJ et al.; Two bacterial strains, F23T and R22T, have been isolated from hypersaline habitats in Malaga (S . Spain) and Murcia (E . Spain) . The novel strains, similar to previously described Idiomarina species, are slightly curved rods, Gram-negative, chemo-organotrophic, strictly aerobic and motile by a single polar flagellum . Both strains produce catalase and oxidase . They hydrolyse aesculin, gelatin, casein, Tween 20, Tween 80 and DNA but not starch or tyrosine . The strains differ from the hitherto described Idiomarina species in their capacity to produce extracellular polysaccharides and their different patterns of carbon sources and antimicrobial susceptibility . They are moderate halophiles capable of growing in NaCl concentrations of 0.5 to 25 % w/v, the optimum being 3-5 % w/v . Cellular fatty acids are predominantly iso-branched . The main fatty acids in strain FP23T are 15 : 0 iso (26.75 %), 16 : 1omega7c (11.33 %) and 16 : 0 (11.73 %) whilst 15 : 0 iso (24.69 %), 17 : 0 iso (12.92 %) and 17 : 1omega9c (11.03 %) are predominant in strain R22T . The DNA G + C composition is 46.0 mol% in strain FP23T and 48.7 mol% in strain R22T . Phylogenetic analyses indicate conclusively that the two strains belong to the genus Idiomarina . DNA-DNA hybridization revealed that they represent novel species . In the light of the polyphasic evidence accumulated in this study, it is proposed that they be classified as novel species of the genus Idiomarina, with the names Idiomarina fontislapidosi sp . nov . (type strain F23T = CECT 5859T = LMG 22169T) and Idiomarina ramblicola sp . nov . (type strain R22T = CECT 5858T = LMG 22170T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1789 - 91
Halorubrum xinjiangense sp . nov., a novel halophile isolated from saline lakes in China; Feng J et al.; A novel halophilic archaeon, strain BD-1T, was isolated from Xiao-Er-Kule Lake in Xinjiang, China . The taxonomy of strain BD-1T was studied by polyphasic methods . According to 16S rRNA gene sequence similarity, strain BD-1T was phylogenetically related to Halorubrum trapanicum (98.4 %), Halorubrum sodomense (98.0 %), Halorubrum distributum (97.8 %) and Halorubrum coriense (97.3 %) . Strain BD-1T is able to grow at 10 degrees C and further differs physiologically from the above species in the assimilation of sugars . The G + C content of DNA is 68.0 % (Tm) . The DNA-DNA relatedness values to Hrr . trapanicum and Hrr . distributum are 47 and 24 %, respectively . It is concluded that strain BD-1T represents a novel species of the genus Halorubrum, for which the name Halorubrum xinjiangense sp . nov . is proposed . The type strain is BD-1T (= AS 1.3527T = JCM 12388T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1765 - 71
Glaciecola polaris sp . nov., a novel budding and prosthecate bacterium from the Arctic Ocean, and emended description of the genus Glaciecola; Van Trappen S et al.; Four strains of cold-adapted, strictly aerobic and facultative oligotrophic bacteria were isolated from polar seas and investigated using a polyphasic taxonomic approach . Two strains (LMG 21857T and LMG 21854) derive from Arctic sea water whereas the other two strains (LMG 21855 and LMG 21858) were isolated from Antarctic sea water . Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the gamma-subclass of the Proteobacteria and are related to the genus Glaciecola, with 98.0-99.7 % sequence similarity to Glaciecola mesophila and 94.2-95.3 % sequence similarity to Glaciecola punicea, their nearest phylogenetic neighbours . Two strains (LMG 21855 and LMG 21858) were identified as G . mesophila, whereas DNA-DNA hybridization results and differences in phenotypic characteristics showed that the other two strains (LMG 21857T and LMG 21854) constitute a novel species within the genus Glaciecola, with a DNA G + C content of 44.0 mol% . The isolates are Gram-negative, chemoheterotrophic, motile, rod-shaped cells that are psychrotolerant and moderately halophilic . Buds can be produced on mother cells and on prosthecae . Branch formation of prosthecae occurs . Whole-cell fatty acid profiles of the isolates are very similar and include C(16 : 0) and C(16 : 1)omega7c as the major fatty acid components . On the basis of genotypic and phenotypic properties, a novel species of the genus Glaciecola is described, for which the name Glaciecola polaris sp . nov . is proposed, with isolate LMG 21857T (= CIP 108324T = ARK 150T) as the type strain . An emended description of the genus Glaciecola is presented.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1757 - 64
Description of Algoriphagus aquimarinus sp . nov., Algoriphagus chordae sp . nov . and Algoriphagus winogradskyi sp . nov., from sea water and algae, transfer of Hongiella halophila Yi and Chun 2004 to the genus Algoriphagus as Algoriphagus halophilus comb . nov . and emended descriptions of the genera Algoriphagus Bowman et al . 2003 and Hongiella Yi and Chun 2004; Nedashkovskaya OI et al.; Four marine heterotrophic, aerobic, pink-pigmented and non-motile bacterial strains were isolated from sea water and algae collected in the Sea of Japan . In a polyphasic taxonomic study, 16S rRNA gene sequence analysis indicated that the strains were phylogenetically highly related to Algoriphagus ratkowskyi LMG 21435T, a member of the phylum Bacteroidetes . Further phenotypic, chemotaxonomic and genomic analyses revealed that the strains represent three novel species of the genus Algoriphagus, for which the following names are proposed: Algoriphagus aquimarinus sp . nov., Algoriphagus chordae sp . nov . and Algoriphagus winogradskyi sp . nov., with type strains KMM 3958T (= LMG 21971T = CCUG 47101T), KMM 3957T (= LMG 21970T = CCUG 47095T) and KMM 3956T (= LMG 21969T = CCUG 47094T), respectively . The species Hongiella halophila Yi and Chun 2004 is transferred to the genus Algoriphagus as Algoriphagus halophilus comb . nov . because of its close phylogenetic relatedness to Algoriphagus species and analogous phenotypic and chemotaxonomic properties . The above-mentioned novel species descriptions and species reclassification justify emended descriptions of the genera Algoriphagus and Hongiella.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1747 - 52
Desulfovibrio alaskensis sp . nov., a sulphate-reducing bacterium from a soured oil reservoir; Feio MJ et al.; A novel sulphate-reducing bacterium (Al1T) was recovered from a soured oil well in Purdu Bay, Alaska . Light and atomic force microscopy observations revealed that cells were Gram-negative, vibrio-shaped and motile by means of a single polar flagellum . The carbon and energy sources used by the isolate and the salinity, temperature and pH ranges facilitating its growth proved to be typical of a partial lactate-oxidizing, moderately halophilic, mesophilic, sulphate-reducing bacterium . Analysis of the fatty acid profile revealed that C(18 : 0), isoC(15 : 0) and isoC(17 : 1)omega7c were the predominant species . Fatty acid profile and complete 16S rRNA gene sequencing demonstrated the similarity between strain Al1T and members of the genus Desulfovibrio . The position of strain Al1T within the phylogenetic tree indicated that it clustered closely with Desulfovibrio vietnamensis DSM 10520T (98.9 % sequence similarity), a strain recovered from a similar habitat . However, whole-cell protein profiles, Fourier-transform infrared studies and DNA-DNA hybridization demonstrated that, in spite of the high level of 16S rRNA gene sequence similarity, there is sufficient dissimilarity at the DNA sequence level between D . vietnamensis DSM 10520T and strain Al1T (10.2 % similarity) to propose that strain Al1T belongs to a separate species within the genus Desulfovibrio . Based on the results obtained, the name Desulfovibrio alaskensis sp . nov . is therefore proposed, with Al1T (= NCIMB 13491T = DSM 16109T) as the type strain.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1735 - 40
Salipiger mucescens gen . nov., sp . nov., a moderately halophilic, exopolysaccharide-producing bacterium isolated from hypersaline soil, belonging to the alpha-Proteobacteria; Martinez-Canovas MJ et al.; Salipiger mucescens gen . nov., sp . nov . is a moderately halophilic, exopolysaccharide-producing, Gram-negative rod isolated from a hypersaline habitat in Murcia in south-eastern Spain . The bacterium is chemoheterotrophic and strictly aerobic (i.e . unable to grow under anaerobic conditions either by fermentation or by nitrate or fumarate respiration) . It does not synthesize bacteriochlorophyll a . Catalase and phosphatase are positive . It does not produce acids from carbohydrates . It cannot grow with carbohydrates or amino acids as sole sources of carbon and energy . It grows best at 9-10 % w/v NaCl and requires the presence of Na+ but not Mg2+ or K+, although they do stimulate its growth somewhat when present . Its major fatty-acid component is 18 : 1omega7c (78.0 %) . The predominant respiratory lipoquinone found in strain A3T is ubiquinone with ten isoprene units . The G + C content is 64.5 mol% . Phylogenetic analyses strongly indicate that this strain forms a distinct line within a clade containing the genus Roseivivax in the subclass alpha-Proteobacteria . The similarity value with Roseivivax halodurans and Roseivivax halotolerans is 94 % . In the light of the polyphasic evidence gathered in this study it is proposed that the isolate be classified as representing a new genus and species, Salipiger mucescens gen . nov., sp . nov . The proposed type strain is strain A3T (= CECT 5855T = LMG 22090T = DSM 16094T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1723 - 8
Halomonas organivorans sp . nov., a moderate halophile able to degrade aromatic compounds; Garcia MT et al.; A group of moderately halophilic bacteria able to degrade aromatic organic compounds contaminating hypersaline habitats in southern Spain have been isolated and characterized . The taxonomic position of these strains was determined using phenotypic, phylogenetic and genotypic methods . The G + C content of their DNA ranged from 61.0 to 62.9 mol% . DNA-DNA hybridization studies showed that they constitute a genospecies, having DNA-DNA hybridization values of 90-100 % . Analysis of the complete 16S rRNA gene sequence revealed a high level of similarity with members of the genus Halomonas, sharing 98 % 16S rRNA gene sequence similarity with the type strains of Halomonas salina and Halomonas halophila . However, phenotypic differences and the low level of DNA-DNA hybridization suggest the placement of these strains as a novel species within the genus Halomonas . The name Halomonas organivorans sp . nov . is proposed, with strain G-16.1T (= CECT 5995T = CCM 7142T) as the type strain . This novel species of Halomonas is characterized by its ability to use a wide range of organic compounds (benzoic acid, p-hydroxybenzoic acid, cinnamic acid, salicylic acid, phenylacetic acid, phenylpropionic acid, phenol, p-coumaric acid, ferulic acid and p-aminosalicylic acid), and it could be useful for the decontamination of polluted saline habitats.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1699 - 702
Paracoccus haeundaensis sp . nov., a Gram-negative, halophilic, astaxanthin-producing bacterium; Lee JH et al.; An aerobic, non-motile, Gram-negative, orange-pigmented, rod-shaped, astaxanthin-producing marine bacterium was isolated from the Haeundae Coast, Korea . This strain, BC74171T, produced carotenoids, mainly astaxanthin . All the type strains of the genus Paracoccus were compared with strain BC74171T using 16S rRNA gene sequence analysis, fatty acid patterns and physiological reaction profiles . Based on the results of these analyses, it is proposed that strain BC74171T represents a novel species, Paracoccus haeundaensis sp . nov . The type strain is BC74171T (= KCCM 10460T = LMG P-21903T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1693 - 7
Desulfovibrio bastinii sp . nov . and Desulfovibrio gracilis sp . nov., moderately halophilic, sulfate-reducing bacteria isolated from deep subsurface oilfield water; Magot M et al.; Two moderately halophilic, mesophilic, sulfate-reducing bacteria were isolated from production-water samples from Emeraude Oilfield, Congo . Motile, vibrioid cells of SRL4225T grew optimally at a concentration of 4 % NaCl, at pH 5.8-6.2, with a minimal pH for growth of 5.2, showing that it is a moderately acidophilic bacterium . Cells of SRL6146T were motile, curved or vibrioid, long and thin rods . Optimal growth was obtained at a concentration of 5-6 % NaCl, at pH 6.8-7.2 . The nutritional requirements showed that many of the characteristics of these strains overlap with those of known Desulfovibrio species . On the basis of 16S rRNA gene sequence analysis and DNA-DNA hybridization studies, both strains are members of the genus Desulfovibrio . However, they are not closely related to any species of the genus that have validly published names . It is therefore proposed that the two strains are members of two novel species of the genus Desulfovibrio with the names Desulfovibrio bastinii sp . nov . (type strain SRL4225T = DSM 16055T = ATCC BAA-903T) and Desulfovibrio gracilis sp . nov . (type strain SRL6146T = DSM 16080T = ATCC BAA-904T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1585 - 9
Serinicoccus marinus gen . nov., sp . nov., a novel actinomycete with L-ornithine and L-serine in the peptidoglycan; Yi H et al.; A Gram-positive bacterial strain containing L-ornithine as the diagnostic diamino acid was isolated from a sea-water-sample from the East Sea, Korea . A phylogenetic analysis based on 16S rRNA gene sequences showed that strain JC1078T represents a phyletic line within the suborder Micrococcineae of the order Actinomycetales, adjacent to the genus Ornithinimicrobium . The highest sequence similarity values to the isolate were observed against Ornithinimicrobium humiphilum (94.3 %) and Kytococcus sedentarius (94.1 %) . The strain was strictly aerobic and moderately halophilic with optimal growth at 2-3 % (w/v) NaCl . Cells were non-motile, non-sporulating and coccoid-shaped . The cell wall contains L-ornithine, glutamic acid, alanine, glycine and serine . The major menaquinone was MK-8(H4) . The predominant cellular fatty acids were of the iso- and anteiso-methyl-branched types . The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unknown glycolipid . The acyl type of the glycan chain of peptidoglycan is acetyl . The DNA G + C content was 72 mol% . The combination of physiological, biochemical and chemotaxonomical data clearly separated the marine isolate from other members of the suborder Micrococcineae . On the basis of polyphasic evidence, it is proposed to classify strain JC1078T in a novel genus and species, for which the name Serinicoccus marinus gen . nov., sp . nov . is proposed . The type strain is JC1078T (= IMSNU 14026T = KCTC 9980T = DSM 15273T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1483 - 7
Novosphingobium pentaromativorans sp . nov., a high-molecular-mass polycyclic aromatic hydrocarbon-degrading bacterium isolated from estuarine sediment; Sohn JH et al.; A Gram-negative, yellow-pigmented, halophilic bacterial strain US6-1T, which degrades high-molecular-mass polycyclic aromatic hydrocarbons of two to five rings, was isolated from muddy sediment of Ulsan Bay, Republic of Korea . The 16S rRNA gene of the isolate showed high sequence similarity to Novosphingobium subarcticum (96.23 %) and Sphingopyxis alaskensis (96.18 %); however, the isolate formed a distinct phyletic line within the genus Novosphingobium . DNA-DNA relatedness between US6-1T and the closest strain N . subarcticum revealed that strain US6-1T was independent from this species . Isolate US6-1T had ubiquinone 10 and a DNA G + C ratio of 61.1 mol% . Major fatty acids were octadecanoic acid (18 : 1omega7), hexadecanoic acid (16 : 1omega7) and 2-hydroxy-myristic acid (14 : 0 2-OH) . On the basis of polyphasic taxonomic evidence, strain US6-1T is proposed to represent a novel species in the genus Novosphingobium for which the name Novosphingobium pentaromativorans sp . nov . is proposed . The type strain is US6-1T (= KCTC 10454T = JCM 12182T).

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1431 - 7
Characterization of Pseudoalteromonas distincta-like sea-water isolates and description of Pseudoalteromonas aliena sp . nov; Ivanova EP et al.; Seven melanogenic Pseudoalteromonas distincta-like strains, KMM 3562T, KMM 3536, KMM 3537, KMM 3538, KMM 3539, KMM 3615 and KMM 3629, which expressed tyrosinases were isolated from sea-water samples collected from different locations in Amursky Bay (Sea of Japan, Pacific Ocean) and characterized to clarify their taxonomic position . By 16S rRNA gene sequence analysis, the bacteria were shown to belong to the genus Pseudoalteromonas . The G + C content of the DNAs of the strains was 41-43 mol% . The level of DNA similarity among these strains was conspecific (92-97 %), indicating that they represented a single genospecies . However, DNA from the strains isolated from sea water showed only 63-65 % genetic relatedness with the DNA of the type strain P . distincta . The novel organisms grew mainly between 4 and 30 degrees C, were neutrophilic and slightly halophilic (four strains had a narrow range of growth between 3 and 6 % NaCl, w/v), were haemolytic and cytotoxic and were able to degrade starch, gelatin and Tween 80 . The predominant fatty acids, including 16 : 0, 16 : 1omega7, 17 : 1omega7 and 18 : 1omega7, were typical of the genus Pseudolateromonas . The phylogenetic, genetic and physiological properties of the seven strains placed them within a novel species, Pseudoalteromonas aliena sp . nov., the type strain of which is SW19T (= KMM 3562T = LMG 22059T).

Extremophiles . 2004 Sep 17; {Epub ahead of print}
Organic solvent tolerance of halophilic alpha-amylase from a Haloarchaeon, Haloarcula sp . strain S-1; Fukushima T et al.; A halophilic archaeon, Haloarcula sp . strain S-1, produced extracellular organic solvent-tolerant alpha-amylase . Molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This amylase exhibited maximal activity at 50 degrees C in buffer containing 4.3 M NaCl, pH 7.0 . Moreover, the enzyme was active and stable in various organic solvents (benzene, toluene, and chloroform, etc.) . Activity was not detected at low ionic strengths, but it was detected in the presence of chloroform at low salt concentrations . On the other hand, no activity was detected in the presence of ethyl alcohol and acetone.

Proc Natl Acad Sci U S A, 2004 Sep 21, 101(38), 13780 - 5 Epub 2004 Sep 13.
Iron-oxo clusters biomineralizing on protein surfaces: structural analysis of Halobacterium salinarum DpsA in its low- and high-iron states; Zeth K et al.; The crystal structure of the Dps-like (Dps, DNA-protecting protein during starvation) ferritin protein DpsA from the halophile Halobacterium salinarum was determined with low endogenous iron content at 1.6-A resolution . The mechanism of iron uptake and storage was analyzed in this noncanonical ferritin by three high-resolution structures at successively increasing iron contents . In the high-iron state of the DpsA protein, up to 110 iron atoms were localized in the dodecameric protein complex . For ultimate iron storage, the archaeal ferritin shell comprises iron-binding sites for iron translocation, oxidation, and nucleation . Initial iron-protein interactions occur through acidic residues exposed along the outer surface in proximity to the iron entry pore . This narrow pore permits translocation of ions toward the ferroxidase centers via two discrete steps . Iron oxidation proceeds by transient formation of tri-iron ferroxidase centers . Iron storage by biomineralization inside the ferritin shell occurs at two iron nucleation centers . Here, a single iron atom provides a structural seed for iron-oxide cluster formation . The clusters with up to five iron atoms adopt a geometry that is different from natural biominerals like magnetite but resembles iron clusters so far known only from bioinorganic model compounds.

J Mol Recognit, 2004 Sep-Oct, 17(5), 382 - 9
Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation; Ebel C et al.; The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell . We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering . This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.

Mol Biol Evol, 2004 Dec, 21(12), 2340 - 2351 Epub 2004 Sep 8.
Evolution of the RNA Polymerase B' Subunit Gene (rpoB') in Halobacteriales: a Complementary Molecular Marker to the SSU rRNA Gene; Walsh DA et al.; Many prokaryotes have multiple ribosomal RNA operons . Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies . For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic . The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales . We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales . To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree . The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages . A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species . Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny . AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree . We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer . This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene . In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.

Microbiology, 2004 Sep, 150(Pt 9), 3051 - 63
Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T; Calderon MI et al.; The synthesis of the compatible solute ectoine, mediated by the ectABC gene products, is the main mechanism used by the halophilic bacterium Chromohalobacter salexigens to cope with osmotic stress . Evidence was found that this process is regulated at the transcriptional level . S1 protection analyses performed with RNA extracted from cells grown in minimal medium at low (0.75 M NaCl) or high (2.5 M NaCl) osmolarity suggested the existence of four promoters upstream of ectA . Two of these (PectA1 and PectA2) might be recognized by the main vegetative sigma factor sigma(70), and one (PectA3) might be dependent on the general stress sigma factor sigma(S) . The S1 protection assays suggest that PectA1 and PectA3 may be osmoregulated promoters . In addition, an internal promoter showing sequences homologous to promoters dependent on the heat-shock sigma factor sigma(32) was found upstream of ectB . Transcription from PectA in C . salexigens followed a pattern typical of sigma(S)-dependent promoters, and was reduced by 50 % in an E . coli rpoS background . These data strongly suggest the involvement of the general stress sigma factor sigma(S) in ectABC transcription in C . salexigens . Expression of PectA-lacZ and PectB-lacZ trancriptional fusions was very high at low salinity, suggesting that ectABC may be a partially constitutive system . Both transcriptional fusions were induced during continuous growth at high temperature and their expression was reduced in cells grown in the presence of osmoprotectants (ectoine or glycine betaine) or the DNA gyrase inhibitor nalidixic acid . Moreover, PectA-lacZ expression was negatively modulated in cells grown with an excess of iron (FeCl(3)) . Measurement of ectoine levels in the presence of glycine betaine at different NaCl concentrations suggests that an additional post-transcriptional control may occur as well.

Biophys J, 2004 Sep, 87(3), 1858 - 72
Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy; Larsen DS et al.; Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira halophila . The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses . This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model . This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I(0) and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state . Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment . This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.

Environ Microbiol, 2004 Oct, 6(10), 1096 - 101
Microbial diversity of extant stromatolites in the hypersaline marine environment of Shark Bay, Australia; Burns BP et al.; Stromatolites have been present on Earth, at various levels of distribution and diversity, for more than 3 billion years . Today, the best examples of stromatolites forming in hypersaline marine environments are in Hamelin Pool at Shark Bay, Western Australia . Despite their evolutionary significance, little is known about their associated microbial communities . Using a polyphasic approach of culture-dependent and culture-independent methods, we report the discovery of a wide range of microorganisms associated with these biosedimentary structures . There are no comparable reports combining these methodologies in the survey of cyanobacteria, bacteria, and archaea in marine stromatolites . The community was characterized by organisms of the cyanobacterial genera Synechococcus, Xenococcus, Microcoleus, Leptolyngbya, Plectonema, Symploca, Cyanothece, Pleurocapsa and Nostoc . We also report the discovery of potentially free-living Prochloron . The other eubacterial isolates and clones clustered into seven phylogenetic groups: OP9, OP10, Marine A group, Proteobacteria, Low G+C Gram-positive, Planctomycetes and Acidobacteria . We also demonstrate the presence of sequences corresponding to members of halophilic archaea of the divisions Euryarchaeota and Crenarchaeota and methanogenic archaea of the order Methanosarcinales . This is the first report of such archaeal diversity from this environment . This study provides a better understanding of the microbial community associated with these living rocks.

J Bacteriol, 2004 Sep, 186(18), 6198 - 207
Novel xylose dehydrogenase in the halophilic archaeon Haloarcula marismortui; Johnsen U et al.; During growth of the halophilic archaeon Haloarcula marismortui on D-xylose, a specific D-xylose dehydrogenase was induced . The enzyme was purified to homogeneity . It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+ . In addition to D-xylose, D-ribose was oxidized at similar kinetic constants, whereas D-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km) . With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)-coding for a 39.9-kDA protein-was identified in the partially sequenced genome of H . marismortui . The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli . The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates . Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases . Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H . marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H . marismortui . Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H . marismortui.

Arch Microbiol, 2004 Oct, 182(4), 277 - 87 Epub 2004 Aug 31.
Unusual ADP-forming acetyl-coenzyme A synthetases from the mesophilic halophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum; Brasen C et al.; ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea Acety - CoA + ADP + Pi<==>acetate + ATP CoA), has been studied only in few hyperthermophilic euryarchaea . Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum . ACD from H . marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa) . The encoding gene was identified in the partially sequenced genome of H . marismortui and functionally expressed in Escherichia coli . The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts . The ACD catalyzed the reversible ADP- and Pi-dependent conversion of acetyl-CoA to acetate . In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate . In the genome of P . aerophilum, the ORFs PAE3250 and PAE 3249, which code for alpha and beta subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs . The two ORFs were separately expressed in E . coli and the recombinant subunits alpha (50 kDa) and beta (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability . The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates . In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes .

Extremophiles . 2004 Aug 25; {Epub ahead of print}
Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress; Gonzalez-Hernandez JC et al.; The comparative analysis of growth, intracellular content of Na(+) and K(+), and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress . The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl . D . hansenii strains grew better and accumulated more Na(+) than S . cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S . cerevisiae strains under similar conditions . By two methods, we found that D . hansenii showed a higher production of trehalose, compared to S . cerevisiae; S . cerevisiae active dry yeast contained more trehalose than a regular commercial strain ( S . cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl . In our experiments, it was found that D . hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts) . However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol . We suggest that the elevated production of trehalose in D . hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.

J Food Prot, 2004 Aug, 67(8), 1736 - 42
Activity of two histidine decarboxylases from Photobacterium phosphoreum at different temperatures, pHs, and NaCl concentrations; Morii H et al.; The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine . The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C . Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible enzymes . This article reports the effect of various growth and reaction conditions, such as temperature, pH, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P . phosphoreum . The histidine decarboxylase activity of the cell-free extracts was highest in 7 degrees C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at pH 6.0 . Moreover, percent activity of the constitutive and inducible enzymes was highest for the inducible enzyme in cultures grown at 7 degrees C and pH 7.5 and in 5% NaCl . The temperature and pH dependences of histidine decarboxylase differed between the constitutive and inducible enzymes; that is, the activity of histidine decarboxylases was optimum at 30 degrees C and pH 6.5 for the inducible enzyme and 40 degrees C and pH 6.0 for the constitutive enzyme . The differences in the temperature and pH dependences between the two enzymes extended the activity range of histidine decarboxylase under reaction conditions . On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two enzymes . Additionally, the effects of reaction temperature, pH, and NaCl concentration on the constitutive enzyme activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive enzyme activity reflected the whole histidine decarboxylase activity.

Extremophiles, 2004 Aug, 8(4), 325 - 34 Epub 2004 May 15.
Thialkalivibrio halophilus sp . nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake; Banciu H et al.; A new chemolithoautotrophic, facultatively alkaliphilic, extremely salt-tolerant, sulfur-oxidizing bacterium was isolated from an alkaline hypersaline lake in the Altai Steppe (Siberia, Russia) . According to 16S rDNA analysis and DNA-DNA hybridization, strain HL 17T was identified as a new species of the genus Thialkalivibrio belonging to the gamma subdivision of the Proteobacteria for which the name Thialkalivibrio halophilus is proposed . Strain HL 17T is an extremely salt-tolerant bacterium growing at sodium concentrations between 0.2 and 5 M, with an optimum of 2 M Na+ . It grew at high concentrations of NaCl and of Na2CO3/NaHCO3 (soda) . Strain HL 17T is a facultative alkaliphile growing at pH range 7.5-9.8, with a broad optimum between pH 8.0 and 9.0 . It used reduced inorganic sulfur compounds (thiosulfate, sulfide, polysulfide, elemental sulfur, and tetrathionate) as energy sources and electron donors . In continuous culture under energy limitation, thiosulfate was stoichiometrically oxidized to sulfate . In sodium carbonate medium under alkaline conditions, the maximum growth rate was similar, while the biomass yield was lower as compared with the NaCl-grown culture . The maximum sulfur-oxidizing capacity measured in washed cells was higher in the soda buffer independent of the growth conditions . The compatible solute content of the biomass was higher in the sodium chloride-grown culture than in the sodium carbonate/bicarbonate-grown culture . The data suggest that the osmotic pressure differences between soda and NaCl solutions might be responsible for the difference observed in compatible solutes production . This may have important implications in overall energetic metabolism of high salt adaptation.

Philos Trans R Soc Lond B Biol Sci, 2004 Aug 29, 359(1448), 1269 - 75; discussion 1275, 1323-8
The effect of water on protein dynamics; Zaccai G; Neutron diffraction and spectroscopy were applied to describe the hydration and dynamics of a soluble protein and a natural membrane from extreme halophilic Archaea . The quantitative dependence of protein motions on water activity was clearly illustrated, and it was established that a minimum hydration shell is required for the systems to access their functional resilience, i.e . a dynamics state that allows biological activity.

Appl Environ Microbiol, 2004 Aug, 70(8), 4424 - 31
Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp; Tokunaga H et al.; It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained . We have addressed this question by identifying the gene responsible for multidrug resistance . A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp . strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane . The mutant produced an elevated level of the 58-kDa outer membrane protein . This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did . An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts . The hrdC gene encoding the 58-kDa outer membrane protein has been cloned . Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr . The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC . Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells . These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.

Genome Biol . 2004;5(8):R52 . Epub 2004 Jul 12.
Comprehensive de novo structure prediction in a systems-biology context for the archaea Halobacterium sp . NRC-1; Bonneau R et al.; BACKGROUND: Large fractions of all fully sequenced genomes code for proteins of unknown function . Annotating these proteins of unknown function remains a critical bottleneck for systems biology and is crucial to understanding the biological relevance of genome-wide changes in mRNA and protein expression, protein-protein and protein-DNA interactions . The work reported here demonstrates that de novo structure prediction is now a viable option for providing general function information for many proteins of unknown function . RESULTS: We have used Rosetta de novo structure prediction to predict three-dimensional structures for 1,185 proteins and protein domains (<150 residues in length) found in Halobacterium NRC-1, a widely studied halophilic archaeon . Predicted structures were searched against the Protein Data Bank to identify fold similarities and extrapolate putative functions . They were analyzed in the context of a predicted association network composed of several sources of functional associations such as: predicted protein interactions, predicted operons, phylogenetic profile similarity and domain fusion . To illustrate this approach, we highlight three cases where our combined procedure has provided novel insights into our understanding of chemotaxis, possible prophage remnants in Halobacterium NRC-1 and archaeal transcriptional regulators . CONCLUSIONS: Simultaneous analysis of the association network, coordinated mRNA level changes in microarray experiments and genome-wide structure prediction has allowed us to glean significant biological insights into the roles of several Halobacterium NRC-1 proteins of previously unknown function, and significantly reduce the number of proteins encoded in the genome of this haloarchaeon for which no annotation is available.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1317 - 21
Marinibacillus campisalis sp . nov., a moderate halophile isolated from a marine solar saltern in Korea, with emended description of the genus Marinibacillus; Yoon JH et al.; A Gram-positive, motile, round to ellipsoidal, endospore-forming, rod-shaped bacterial strain, SF-57T, was isolated from a marine solar saltern in Korea . This organism grew between 4 and 39 degrees C, with optimum growth at 30 degrees C . Strain SF-57T grew in the presence of 0.5-15.0% NaCl, with optimum growth at 2-3% NaCl . The peptidoglycan type of strain SF-57T was A1alpha linked directly through l-Lys . In strain SF-57TT, menaquinone-7 (MK-7) was the predominant isoprenoid quinone and anteiso-C(15 : 0) was the major fatty acid . The DNA G+C content was 41.8 mol% . Phylogenetic analyses based on 16S rRNA gene sequences showed that strain SF-57T formed a coherent cluster with Marinibacillus marinus, with a bootstrap resampling value of 100% . The level of 16S rRNA gene sequence similarity between strain SF-57T and M . marinus DSM 1297T was 98.9% . The mean DNA-DNA relatedness level between strain SF-57T and the type strain of M . marinus was 20.6% . Based on phenotypic properties, phylogenetic analyses and genomic data, strain SF-57T merits placement in the genus Marinibacillus as a representative of a novel species, for which the name Marinibacillus campisalis sp . nov . is proposed . The type strain is SF-57T (=KCCM 41644T=JCM 11810T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1197 - 201
Alteromonas litorea sp . nov., a slightly halophilic bacterium isolated from an intertidal sediment of the Yellow Sea in Korea; Yoon JH et al.; A Gram-negative, motile, non-spore-forming, rod-shaped bacterium, designated strain TF-22T, was isolated from an intertidal sediment in Korea . This organism grew optimally at 30-37 degrees C and in the presence of 2-5% (w/v) NaCl . It did not grow without NaCl or in the presence of more than 14% (w/v) NaCl . Strain TF-22T was characterized chemotaxonomically as having ubiquinone-8 as the predominant respiratory lipoquinone and C(16 : 0), C(16 : 1) omega7c and/or iso-C(15 : 0) 2-OH and C(18 : 1) omega7c as the major fatty acids . The DNA G+C content of strain TF-22T was 46.0 mol% . Phylogenetic analyses based on 16S rDNA sequences showed that strain TF-22T falls within the gamma-subclass of the Proteobacteria and forms a coherent cluster with Alteromonas macleodii and Alteromonas marina . Levels of 16S rDNA similarity between strain TF-22T and the type strains of two Alteromonas species were in the range 98.1-98.6% . The level of DNA-DNA relatedness between strain TF-22T and the type strains of two Alteromonas species was 15.7-18.5% . Therefore, on the basis of phenotypic properties, phylogeny and genomic distinctiveness, strain TF-22T should be placed in the genus Alteromonas as a novel species, for which the name Alteromonas litorea sp . nov . is proposed . The type strain is TF-22TT (=KCCM 41775T=JCM 12188T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1157 - 63
Alteromonas stellipolaris sp . nov., a novel, budding, prosthecate bacterium from Antarctic seas, and emended description of the genus Alteromonas; Van Trappen S et al.; Seven novel, cold-adapted, strictly aerobic, facultatively oligotrophic strains, isolated from Antarctic sea water, were investigated by using a polyphasic taxonomic approach . The isolates were Gram-negative, chemoheterotrophic, motile, rod-shaped cells that were psychrotolerant and moderately halophilic . Buds were produced on mother and daughter cells and on prosthecae . Prostheca formation was peritrichous and prosthecae could be branched . Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the gamma-Proteobacteria and are related to the genus Alteromonas, with 98.3% sequence similarity to Alteromonas macleodii and 98.0% to Alteromonas marina, their nearest phylogenetic neighbours . Whole-cell fatty acid profiles of the isolates were very similar and included C(16 : 0), C(16 : 1)omega7c, C(17 : 1)omega8c and C(18 : 1)omega8c as the major fatty acid components . These results support the affiliation of these isolates to the genus Alteromonas . DNA-DNA hybridization results and differences in phenotypic characteristics show that the strains represent a novel species with a DNA G+C content of 43-45 mol% . The name Alteromonas stellipolaris sp . nov . is proposed for this novel species; the type strain is ANT 69aT (=LMG 21861T=DSM 15691T) . An emended description of the genus Alteromonas is given.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1111 - 6
Microbulbifer maritimus sp . nov., isolated from an intertidal sediment from the Yellow Sea, Korea; Yoon JH et al.; A Gram-negative, rod-shaped, non-spore-forming, slightly halophilic bacterium (strain TF-17T) was isolated from an intertidal sediment from the Yellow Sea, Korea . Pigment of strain TF-17T was similar to that of Microbulbifer elongatus, but different from those of Microbulbifer hydrolyticus and Microbulbifer salipaludis . Strain TF-17T was distinguishable from M . elongatus by some phenotypic properties, including motility, optimal growth temperature and others . Phylogenetic analysis based on 16S rDNA sequences showed that strain TF-17TT clustered with the type strains of the three Microbulbifer species with validly published names . Strain TF-17T exhibited 16S rDNA sequence similarity levels of 95.1-95.7% to the type strains of the three Microbulbifer species . The predominant respiratory lipoquinone found in strain TF-17T was ubiquinone-8 . The major fatty acid was iso-C(15 : 0) and significant amounts of iso-C(11 : 0) 3-OH and iso-C(17 : 1)omega9c were also present . The DNA G+C content of strain TF-17T was 59.9 mol% . Levels of DNA-DNA relatedness between strain TF-17T and the type strains of the three Microbulbifer species were in the range 10.0-13.0% . On the basis of phenotypic and phylogenetic data and genotypic distinctiveness, strain TF-17T (=KCCM 41774T=JCM 12187T) is proposed as the type strain of a novel species of the genus Microbulbifer, Microbulbifer maritimus sp . nov.

Methods Mol Biol, 2004, 267, 209 - 23
Gene transfer and expression of recombinant proteins in moderately halophilic bacteria; Afendra AS et al.; Moderately halophilic bacteria (MHB) of the genera Halomonas and Chromohalobacter have been used as hosts for the expression of heterologous proteins of biotechnological interest, thus expanding their potential to be used as cell factories for various applications . This chapter deals with the methodology for the construction of recombinant plasmids, their transfer to a number of MHB, and the assaying of the corresponding heterologous proteins activity . The transferred genes include (1) inaZ, encoding the ice nucleation protein of the plant pathogen Pseudomonas syringae, (2) gfp, encoding a green fluorescent protein from the marine bioluminescent jellyfish Aequorea victoria, and (3) the alpha-amylase gene from the hyperthermophilic archeon Pyrococcus woesei . Vector pHS15, which was designed for expression of heterologous proteins in both E . coli and MHB, was used for the subcloning and transfer of the above genes . The recombinant constructs were introduced to MHB by assisted conjugal transfer from E . coli donors . The expression and function of the recombinant proteins in the MHB transconjugants is described.

Methods Mol Biol, 2004, 267, 183 - 208
Genetic tools for the manipulation of moderately halophilic bacteria of the family Halomonadaceae; Vargas C et al.; Moderately halophilic bacteria of the family Halomonadaceae (Halomonas, Chromohalobacter, and Zymobacter) have promising applications in biotechnology as a source of compatible solutes (stabilizers of biomolecules and cells), salt-tolerant enzymes, biosurfactants, and extracellular polysaccharides, among other products . In addition, they offer a number of advantages to be used as cell factories, alternative to conventional prokaryotic hosts like E . coli or Bacillus, for the production of recombinant proteins: (1) their high salt tolerance decreases to a minimum the necessity for aseptic conditions, resulting in cost-reducing conditions; (2) they are very easy to grow and maintain in the laboratory, and their nutritional requirements are simple; and (3) the majority can use a large range of compounds as a sole carbon and energy source . In this decade, the efforts of our group and others have made possible the genetic manipulation of this bacterial group . In this review, the most relevant tools are described, with emphasis given to cloning vectors, genetic exchange mechanisms, mutagenesis approaches, and reporter genes . Due to its relevance for genetic studies, complementary sections describing the influence of salinity on the susceptibility of moderately halophilic bacteria to antimicrobials, as well as the growth media most routinely used, culture conditions, and nucleic acid isolation procedures for these microorganisms, are included.

Rev Biol Trop, 2001 Dec, 49 Suppl 2, 273 - 8
Requiem for an eastern Pacific seagrass bed; Cortes J; Few papers concerning seagrasses of the eastern Pacific have been published . This paper presents the first ecological data on the seagrass, Ruppia maritima, from a non-lagoonal setting in the eastern Pacific . A 5000 m2 patch formed by R . maritima, at Playa Iguanita, Bahia Culebra, Pacific coast of Costa Rica was studied . Plant density and leaf length of R . maritima were determined along two transects on different dates . Above and below ground biomass were calculated along one transect . Plant density ranged from 1590 to 8630 individuals m(-2) along the two transects, with means of 5990 +/- 1636 and 6100 +/- 1876 plants m(-2) for transect 1 and 2, respectively . Longest leaf length per plant varied between 0.5 and 23.0 cm . Leaf biomass (LB) ranged from 10 to 97 gm(-2), and root-rhizome biomass (RB) from 31 to 411 gm(-2), resulting in RB:LB ratios of 3.07 to 15.27 . Total biomass at Bahia Culebra was lower than at tropical lagoons on the Pacific coast of Mexico, but higher than in the Gulf of Mexico . The below ground: above ground biomass ratio was much higher at Bahia Culebra than at other sites on the Pacific coast of Mexico and the Gulf of Mexico . Another seagrass present at Bahia Culebra was Halophila baillonii, with low densities on the deepest section of the patch . At least 44 invertebrate species associated with the seagrass bed have also been identified . The patch at Playa Iguanita and other sites within Bahia Culebra, as well as their associated organisms, disappeared after a severe storm in June 1996 . No seagrasses have been found in the area or in any other location on the Pacific coast of Costa Rica since then.

Carbohydr Res, 2004 Aug 2, 339(11), 1985 - 93
The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549(T); Silipo A et al.; Novel lipooligosaccharide components were isolated and identified from the lipooligosaccharide fraction of the halophilic marine bacterium Pseudoalteromonas issachenkonii type strain KMM 3549(T) . The complete structure was achieved by chemical analysis, 2D NMR spectroscopy and MALDI mass spectrometry as the following: All sugars are d-pyranoses . Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate, residues and substituents in italic are not stoichiometrically linked . In addition, by MALDI mass spectrometry of the intact LOS, the lipid A moiety was also identified as a mixture of penta-, tetra- and triacylated species.

FEBS Lett, 2004 Jul 16, 570(1-3), 87 - 92
Facilitated folding and subunit assembly in Escherichia coli and in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag; Ishibashi M et al.; We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation . Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E . coli . This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E . coli . The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK . Folded His-HsNDK isolated from E . coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 degrees C, while the native HsNDK purified from H . salinarum dissociated to dimer in 0.2 M NaCl . The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt . These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.

Plant Physiol, 2004 Jul, 135(3), 1697 - 709 Epub 2004 Jul 09.
Comparative genomics in salt tolerance between Arabidopsis and aRabidopsis-related halophyte salt cress using Arabidopsis microarray; Taji T et al.; Salt cress (Thellungiella halophila), a halophyte, is a genetic model system with a small plant size, short life cycle, copious seed production, small genome size, and an efficient transformation . Its genes have a high sequence identity (90%-95% at cDNA level) to genes of its close relative, Arabidopsis . These qualities are advantageous not only in genetics but also in genomics, such as gene expression profiling using Arabidopsis cDNA microarrays . Although salt cress plants are salt tolerant and can grow in 500 mm NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation . This suggests that the salt tolerance in salt cress results from mechanisms that are similar to those operating in glycophytes . To elucidate the differences in the regulation of salt tolerance between salt cress and Arabidopsis, we analyzed the gene expression profiles in salt cress by using a full-length Arabidopsis cDNA microarray . In salt cress, only a few genes were induced by 250 mm NaCl stress in contrast to Arabidopsis . Notably a large number of known abiotic- and biotic-stress inducible genes, including Fe-SOD, P5CS, PDF1.2, AtNCED, P-protein, beta-glucosidase, and SOS1, were expressed in salt cress at high levels even in the absence of stress . Under normal growing conditions, salt cress accumulated Pro at much higher levels than did Arabidopsis, and this corresponded to a higher expression of AtP5CS in salt cress, a key enzyme of Pro biosynthesis . Furthermore, salt cress was more tolerant to oxidative stress than Arabidopsis . Stress tolerance of salt cress may be due to constitutive overexpression of many genes that function in stress tolerance and that are stress inducible in Arabidopsis.

Plant Physiol, 2004 Jul, 135(3), 1718 - 37 Epub 2004 Jul 09.
Salt cress . A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles; Inan G et al.; Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle . It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure . It is capable of copious seed production . Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C . It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity . Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly . Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress . Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis . Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation . We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci . Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions . Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress . Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress . In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

Environ Technol, 2004 May, 25(5), 543 - 53
Treatment of hypersaline industrial wastewater by a microbial consortium in a sequencing batch reactor; Lefebvre O et al.; Hypersaline effluents are produced by various industrial activities . Such wastewater, rich in both organic matter and salt (> 35 g l(-1)), is difficult to treat by conventional wastewater treatment processes . It is necessary to use halophilic bacteria . In this study, a bench-scale sequencing batch reactor (SBR) was inoculated with halophilic sediments in order to treat an agri-food effluent containing 120 g salt l(-1) . The micro-organisms were able to treat carbon and nitrogen, provided the pH in the reactor was neutralised with phosphoric acid . Soluble COD and Soluble TKN removal attained 83% and 72% respectively . 16S rDNA identification of the halophilic microbial community showed high diversity.

Extremophiles, 2004 Oct, 8(5), 377 - 84 Epub 2004 Jun 18.
Salt-dependent studies of NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii; Madern D et al.; The salt-dependent stability of recombinant dimeric isocitrate dehydrogenase {ICDH; isocitrate: NADP oxidoreductase (decarboxylating), EC 1.1.1.42} from the halophilic archaeon Haloferax volcanii (Hv) was investigated in various conditions . Hv ICDH dissociation/deactivation was measured to probe the respective effect of anions and cations on stability . Surprisingly, enzyme stability was found to be mainly sensitive to cations and very little (or not) sensitive to anions . Divalent cations induced a strong shift of the active/inactive transition towards low salt concentration . A high resistance of Hv ICDH to chemical denaturation was also found . The data were analysed and are discussed in the framework of the solvation stability model for halophilic proteins.

Syst Appl Microbiol, 2004 May, 27(3), 323 - 33
Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences; Okamoto T et al.; Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids . Ten strains were selected for physiological and phylogenetic characterization in detail . All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant . Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments . The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC) . The phylogenetic relationships of H . variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB . The ectC-based tree was inconsistent with the other phylogenetic trees . For that reason, ectC was inferred to derive from horizontal transfer.

J Drug Target, 2003, 11(8-10), 515 - 24
Archaeosomes as self-adjuvanting delivery systems for cancer vaccines; Krishnan L et al.; Archaeal ether glycerolipid vesicles (archaeosomes) efficiently deliver exogenous antigen for induction of humoral and cell-mediated immunity . Because induction of CD8 cytotoxic T cells is critical for protective vaccination against tumors, we compared the ability of various archaeosome lipid compositions to evoke a strong CD8 CTL response to entrapped antigen . Subcutaneous immunization of mice with ovalbumin (OVA) entrapped in all archaeosome lipid compositions evoked a primary (day 10) splenic CTL response indicating processing for MHC class I presentation . Interestingly, several polar lipid compositions from halophilic archaea were very potent to adjuvant this early CTL response . Despite this, the lytic units reduced substantially by weeks 6-7 . More importantly, at >50 weeks, only Methanobrevibacter smithii and Thermoplasma acidophilum both rich in bipolar membrane-spanning caldarchaeols, demonstrated recall memory CTLs . Immunization of mice with OVA entrapped in M . smithii, Halobacterium salinarum or T . acidophilum vesicles provided prophylactic protection against challenge with OVA-expressing solid tumors at 6 weeks . Even a dose of 3 microg OVA in archaeosomes significantly delayed tumor growth . Tumor protection was also noted in a therapeutic design wherein OVA-archaeosomes were injected concurrent with the tumor challenge . Interestingly, antigen-free T . acidophilum but not antigen-free H . salinarum archaeosomes provided innate therapeutic protection . Vaccination with a CTL peptide epitope from the melanoma differentiation antigen, tyrosinase-related protein 2, in archaeosomes induced a protective CD8 response against B16OVA metastasis, indicating potential for targeting self, tumor antigens . Thus, lipid structural properties of archaea may differentially modulate primary, long-term and/or innate immunity, impacting adjuvant choice for vaccine design.

Biochimie, 2004 Apr-May, 86(4-5), 295 - 303
Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein; Madern D et al.; Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs . In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt . Its amino acid composition does not display the strong acidic character specific of halophilic proteins . The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor . Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl . The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.

J Microbiol Methods, 2004 Jul, 58(1), 31 - 8
Glycine betaine as a cryoprotectant for prokaryotes; Cleland D et al.; Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes . These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing . This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant . In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D) . GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae . GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve . For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA . Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants . GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria . These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens .

J Bacteriol, 2004 Jun, 186(12), 3980 - 90
Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes; Boucher Y et al.; More than one copy of rRNA operons, which code for both the small-subunit (SSU) and large-subunit (LSU) rRNA, are often found in prokaryotes . It is generally assumed that all rRNA operons within a single cell are almost identical . A notable exception is the extremely halophilic archaeal genus Haloarcula, most species of which are known to harbor highly divergent rRNA operons that differ at approximately 5% of the nucleotide positions in the SSU gene and at 1 to 2% of the nucleotide positions in the LSU gene . We report that such intragenomic heterogeneity is not unique to Haloarcula, as high levels of intragenomic sequence variation have been observed for the SSU genes of two other genera of extreme halophiles, Halosimplex and Natrinema . To investigate this in detail, the two rRNA operons of Halosimplex carlsbadense and the four operons of Natrinema sp . strain XA3-1 were cloned and completely sequenced . The SSU and LSU genes of H . carlsbadense show the highest levels of intragenomic heterogeneity observed so far in archaea (6.7 and 2.6%) . The operons of Natrinema sp . strain XA3-1 have additional unusual characteristics, such as identical internal transcribed spacers, while one of four SSU genes is 5% divergent and all LSU genes differ from each other by 0.9 to 1.9% . The heterogeneity among the Natrinema sp . strain XA3-1 LSU genes is localized in hot spots, and one of these regions is shown to be the result of a recombination event with a distantly related halophile . This is the first example of interspecies recombination between rRNA genes in archaea, and the recombination occurred over one of the largest phylogenetic distances ever reported for such an event . We suggest that intragenomic heterogeneity of rRNA operons is an ancient and stable trait in several lineages of the Halobacteriales . The impact of this phenomenon on the taxonomy of extremely halophilic archaea is discussed.

Photochem Photobiol Sci, 2004 Jun, 3(6), 537 - 42 Epub 2004 Feb 20.
Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II); Yoshida H et al.; Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile . This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G)via a protonated Schiff base . Various mutants were engineered to have a single cysteine in the F-helix . In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate . The fluorescence of the isolated protein increased with increasing illumination . These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule . This reaction occurred only at the M-state and not at the O-state . The implications are discussed.

Photochem Photobiol Sci, 2004 Jun, 3(6), 531 - 6 Epub 2004 May 06.
Tryptophan fluorescence monitors structural changes accompanying signalling state formation in the photocycle of photoactive yellow protein; Gensch T et al.; Photoactive yellow protein, a small, water-soluble blue-light absorbing photoreceptor protein from Ectothiorhodospira(Halorhodospira){space}halophila has a structure with two hydrophobic cores, of which the main one houses its light-sensitive chromophore (p-coumaric acid), separated by a central {small beta}-sheet . This photoreceptor protein contains a single tryptophan residue (W119) that is situated at the interface between the central beta-sheet and its N-terminal cap . The fluorescence properties of W119 in the dark state pG (lambda(max)= 328 nm; Phi(fl)= 0.01; nearly pH-independent) are typical for a buried tryptophan in a hydrophobic environment with significant quenching by nearby amino acid residues . Signalling state formation leads to pH-dependent fluorescence changes: At pH values <6.5 the fluorescence emission increases, with a minor blue shift of the emission maximum . Above this pH, the emission maximum of the tryptophan shifts considerably to the red, whereas its total intensity decreases . These results further support the contention that signalling state formation in PYP leads to significant changes in the structure of this protein, even at sites that are at a considerable distance from the chromophore . The nature of these changes in pB, however, depend upon the pH imposed upon the protein: At slightly alkaline pH, which presumably is closest to the pH to which this protein is exposed in vivo, these changes lead to an exposure of the part of the central beta-sheet harbouring W119 . At slightly acidic pH the polarity of the environment of W119 is hardly affected by the formation of the signalling state but the quenching of its fluorescence emission, possibly by nearby amino acids, is reduced . On the other hand, its accessibility for quenching by small molecules in the solution is enhanced at acidic and alkaline pH in the signalling state (pB) compared to the dark state (pG) . This latter observation points towards a more flexible structure of the N-terminal cap, having a looser interaction with the central beta-sheet in pB.

Photochem Photobiol Sci, 2004 Jun, 3(6), 519 - 30 Epub 2004 Apr 20.
Photoactive yellow protein, bacteriophytochrome, and sensory rhodopsin in purple phototrophic bacteria; Kyndt JA et al.; The purple photosynthetic bacteria contain a large variety of sensory and regulatory proteins, and those responding to light are among the most interesting . These currently include bacteriophytochrome (Bph), sensory rhodopsin (SR), and photoactive yellow protein (PYP), which all appear to function as light sensors . We herein interpret new findings within the context of current knowledge . For greater detail, the reader is referred to comprehensive reviews on these topics . Of the three proteins, only PYP has been well-characterized in terms of structure and physical-chemical properties in the purple bacteria, although none have well-defined functions . New findings include a cluster of six genes in the Thermochromatium tepidum genome that encodes presumed sensory rhodopsin and phototaxis proteins . T . tepidum also has a gene for PYP fused to bacteriophytochrome and diguanylate cyclase domains . The genes for PYP and its biosynthetic enzymes are associated with those for gas vesicle formation in Rhodobacter species, suggesting that one function of PYP is to regulate cell buoyancy . The association of bacteriophytochrome genes with those for reaction centers and light-harvesting proteins in Rhodopseudomonas palustris suggests that the photosynthetic antenna as well as the reaction center are regulated by Bphs . Furthermore, Rc . centenum PPR is reversibly photobleached at 702 nm rather than red-shifted as in other phytochromes, suggesting that PPR senses the intensity of white light rather than light quality . PYP from Halorhodospira(aka Ectothiorhodospira)halophila is of special interest because it has become the structural prototype for the PAS domain, a motif that is found throughout the phylogenetic tree and which plays important roles in many signaling pathways . Thus, the structural and photochemical characterization of PYP, utilizing site-directed mutagenesis, provides insights into the mechanism of signal transduction.

J Bioenerg Biomembr, 2004 Feb, 36(1), 17 - 24
Transport of compatible solutes in extremophiles; Pfluger K et al.; Salt-tolerant as well as moderately halophilic and halophilic organisms have to maintain their turgor . One strategy is to accumulate small organic compounds, compatible solutes, by de novo synthesis or uptake . From a bioenergetic point of view, uptake is preferred over biosynthesis . The transport systems catalyzing uptake of compatible solutes are of primary or secondary nature and coupled to ATP hydrolysis or ion (H+, Na+) symport . Expression of the transporter genes as well as the activity of the transporters is regulated by salinity/osmolarity and one of the key questions is how salinity or osmolarity is sensed and the signal transmitted as far as to gene expression and transporter activation . Recent studies shed light on the nature and the activation mechanisms of solute transporters in extremophiles, and this review summarizes current knowledge on the structure, function and osmo- or salt-regulation of transporters for compatible solutes in extremophiles.

J Basic Microbiol, 2004, 44(3), 232 - 40
Major outer membrane proteins in moderately halophilic eubacteria of genera Chromohalobacter and Halomonas; Tokunaga H et al.; Outer and inner membrane fractions of Chromohalobacter marismortui and Halomonas elongata were isolated by differential detergent solubilization, and profiles of membrane proteins, especially major outer membrane proteins, were analyzed . These type strains possessed one extremely abundant outer membrane protein which showed similarity in amino-terminal amino acid sequence with the outer membrane porin proteins in other Gram-negative bacteria . Three halophilic eubacterial strains isolated from saline environments were also characterized . Strains 160 and 43 were found to be Chromohalobacter spp . and strain 40 to be a Halomonas sp . by sequence analysis of their 16 S ribosomal RNA genes . Extremely abundant porin proteins with an apparent molecular mass of 49 kDa were found in Chromohalobacter sp.160 and Halomonas sp . 40, but no major outer membrane protein was detected in Chromohalobacter sp . 43, suggesting strain 43 was most likely a naturally defective porin mutant . Porin proteins from Chromohalobacter spp . and Halomonas spp . showed the same migration on SDS-polyacrylamide gel electrophoresis with or without heat-treatment, indicating that these porin proteins did not form a SDS-resistant trimeric structure, which was detected in most of the Gram-negative bacterial porin proteins .

Proteins, 2004 Jun 1, 55(4), 1070 - 7
Direct measure of functional importance visualized atom-by-atom for photoactive yellow protein: application to photoisomerization reaction; Yamada A et al.; Photoreceptor proteins serve as efficient nano-machines for the photoenergy conversion and the photosignal transduction of living organisms . For instance, the photoactive yellow protein derived from a halophilic bacterium has the p-coumaric acid chromophore, which undergoes an ultrafast photoisomerization reaction after light illumination . To understand the structure-function relationship at the atomic level, we used a computational method to find functionally important atoms for the photoisomerization reaction of the photoactive yellow protein . In the present study, a "direct" measure of the functional significance was quantitatively evaluated for each atom by calculating the partial atomic driving force for the photoisomerization reaction . As a result, we revealed the reaction mechanism in which the specific role of each functionally important atom has been well characterized in a systematic manner . In addition, we observed that this mechanism is strongly conserved during the thermal fluctuation of the photoactive yellow protein . We compared the experimental data of fluorescence decay constant of several different mutants and the present analysis . As a result, we found that the reaction rate constant is decreased when a large positive driving force is missing .

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 935 - 40
Psychroflexus tropicus sp . nov., an obligately halophilic Cytophaga-Flavobacterium-Bacteroides group bacterium from an Hawaiian hypersaline lake; Donachie SP et al.; A Gram-negative bacterium designated LA1(T) was isolated from water collected in hypersaline Lake Laysan on Laysan Island in the Northwestern Hawaiian Islands . Cells occurred singly as fine rods to short filaments . Growth in 50 % strength marine broth occurred optimally when the medium contained 7.5-10 % (w/v) NaCl . The major fatty acids in LA1(T) grown at 15 and 30 degrees C were 12-methyl tetradecanoic acid and 13-methyl tetradecanoic acid, respectively . The nucleotide sequence of the 16S rRNA gene showed that LA1(T) belonged in the Cytophaga-Flavobacterium-Bacteroides (CFB) group in the domain Bacteria . The closest described neighbour in terms of 16S rRNA gene sequence identity was Psychroflexus torquis ACAM 623(T) (94.4 % over 1423 bases), an obligate psychrophile from Antarctic sea-ice . The G+C content of 35.0 mol% was consistent with this affiliation . Phenotypic and genotypic analyses, including DNA hybridization, indicated that LA1(T) could be assigned to the genus Psychroflexus but, based on significant differences, including growth at 43 degrees C, it constitutes a novel species, Psychroflexus tropicus sp . nov., for which LA1(T) (=ATCC BAA-734(T)=DSM 15496(T)) is the type strain.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 803 - 8
Bacillus hwajinpoensis sp . nov . and an unnamed Bacillus genomospecies, novel members of Bacillus rRNA group 6 isolated from sea water of the East Sea and the Yellow Sea in Korea; Yoon JH et al.; Two Gram-positive or -variable, endospore-forming, slightly halophilic strains (SW-72(T) and SW-93) were isolated from sea water of the East Sea and the Yellow Sea in Korea, respectively, and subjected to polyphasic taxonomic study . Both strains had cell-wall peptidoglycan that was based on meso-diaminopimelic acid and MK-7 as the predominant menaquinone . The two strains contained large amounts of saturated and branched fatty acids, with anteiso-C(15 : 0) as the major fatty acid . The DNA G+C contents of strains SW-72(T) and SW-93 were 40.9 and 41.0 mol%, respectively . Phylogenetic analysis based on 16S rDNA sequences showed that strains SW-72(T) and SW-93 fall within the radiation of the cluster that comprises members of the genus Bacillus, particularly Bacillus rRNA group 6 . There were five nucleotide differences between the 16S rDNA sequences of strains SW-72(T) and SW-93 . The mean level of DNA-DNA relatedness between strains SW-72(T) and SW-93 was 21.5 % . Strains SW-72(T) and SW-93 showed 93.1-95.2 % 16S rDNA sequence similarity to the type strains of Bacillus species that are assigned to rRNA group 6 . Strains SW-72(T) and SW-93 could not be differentiated clearly by using their phenotypic properties . On the basis of phenotypic properties, phylogeny and genomic data, it is proposed that strain SW-72(T) (=KCCM 41641(T)=JCM 11807(T)) should be placed in the genus Bacillus as the type strain of a novel species, Bacillus hwajinpoensis sp . nov., and that strain SW-93 (=KCCM 41640=JCM 11806) should be placed in the genus Bacillus as an unnamed Bacillus genomospecies.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 759 - 63
Methanobacterium aarhusense sp . nov., a novel methanogen isolated from a marine sediment (Aarhus Bay, Denmark); Shlimon AG et al.; Strain H2-LR(T), a 5-18 micro m long and 0.7 micro m wide filamentous, mesophilic, moderately halophilic, non-motile hydrogenotrophic methanogen, was isolated from marine sediment of Aarhus Bay, Denmark, 1.7 m below the sediment surface . On the basis of 16S rRNA gene comparison with sequences of known methanogens, strain H2-LR(T) could be affiliated to the genus Methanobacterium . The strain forms a distinct line of descent within this genus, with Methanobacterium oryzae (95.9 % sequence identity) and Methanobacterium bryantii (95.7 % sequence identity) as its closest relatives . The 16S rRNA-based affiliation was supported by comparison of the mcrA gene, which encodes the alpha-subunit of methyl-coenzyme M reductase . Strain H2-LR(T) grew only on H(2)/CO(2) . The DNA G+C content is 34.9 mol% . Optimum growth temperature was 45 degrees C . The strain grew equally well at pH 7.5 and 8 . No growth or methane production was observed below pH 5 or above pH 9 . Strain H2-LR(T) grew well within an NaCl concentration range of 100 and 900 mM . No growth or methane production was observed at 1 M NaCl . At 50 mM NaCl, growth and methane production were reduced . Based on 16S rRNA gene sequence analysis, the isolate is proposed to represent a novel taxon within the genus Methanobacterium, namely Methanobacterium aarhusense sp . nov . The type strain is H2-LR(T) (=DSM 15219(T)=ATCC BAA-828(T)).

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 721 - 5
Halomonas boliviensis sp . nov., an alkalitolerant, moderate halophile isolated from soil around a Bolivian hypersaline lake; Quillaguaman J et al.; Halomonas boliviensis sp . nov . is proposed for two moderately halophilic, psychrophilic, alkalitolerant bacteria, LC1(T) (=DSM 15516(T)=ATCC BAA-759(T)) and LC2 (=DSM 15517=ATCC BAA-760), both of which were isolated from a soil sample around the lake Laguna Colorada, located at 4300 m above sea level in the south-west region of Bolivia . The bacteria are aerobic, motile, Gram-negative rods that produce colonies with a cream pigment . Moreover, they are heterotrophs that are able to utilize various carbohydrates as carbon sources . The organisms reduce nitrate and show tryptophan deaminase activity . The genomic DNA G+C contents were 51.4 mol% for isolate LC1(T) and 52.6 mol% for isolate LC2 . Based on 16S rDNA sequence analysis, isolates LC1(T) and LC2 were identified as members of the genus Halomonas and clustered closely with Halomonas variabilis DSM 3051(T) and Halomonas meridiana DSM 5425(T) . However, DNA-DNA relatedness between the new isolates and the closest related Halomonas species was low.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 675 - 80
Kordia algicida gen . nov., sp . nov., an algicidal bacterium isolated from red tide; Sohn JH et al.; A bacterium (named OT-1(T)) that showed algicidal activity was isolated from sea water of Masan Bay, Korea, during an outbreak of red tide . Phylogenetic analysis based on 16S rDNA sequences showed that the isolate formed a distinct phyletic lineage within the family Flavobacteriaceae of the Cytophaga-Flavobacterium-Bacteroides group . No species with a validly published name showed >/=93 % 16S rRNA gene sequence similarity to strain OT-1(T) . The isolate had major amounts of iso-branched and 3-hydroxy iso-branched fatty acids and menaquinone 6 and a DNA G+C content of 34 mol%; these chemotaxonomic characters also supported the placement of the organism in the family Flavobacteriaceae . The strain was Gram-negative, yellow-pigmented, non-motile, non-gliding, flexirubin-negative, strictly aerobic, catalase-negative, oxidase-positive and halophilic . Na(+), Ca(2+) and Mg(2+) ions were obligately required for growth . The strain utilized various sugars as sole carbon sources and degraded gelatin, skimmed milk and starch . Several phenotypic characters can be used to differentiate the test strain from phylogenetically related marine bacterial genera . On the basis of polyphasic evidence, it is proposed that strain OT-1(T) should be assigned to the family Flavobacteriaceae as Kordia algicida gen . nov., sp . nov . The type strain is OT-1(T) (=KCTC 8814P(T)=NBRC 1000336(T)).

Environ Microbiol, 2004 Jun, 6(6), 591 - 5
Isolation of haloarchaea that grow at low salinities; Purdy KJ et al.; Summary Archaea, the third domain of life, were long thought to be limited to environmental extremes . However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles . Until now, none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles . We have cultivated three novel halophilic Archaea (haloarchaea) genotypes from sediments in which the pore-water salinity was close to that of sea water . All previously reported haloarchaeal isolates are obligate extreme halophiles requiring at least 9% (w/v) NaCl for growth and are typically the dominant heterotrophic organisms in salt and soda lakes, salt deposits and salterns . Two of these three newly isolated genotypes have lower requirements for salt than previously cultured haloarchaea and are capable of slow growth at sea-water salinity (2.5% w/v NaCl) . Our data reveal the existence of Archaea that can grow in non-extreme conditions and of a diverse community of haloarchaea existing in coastal salt marsh sediments . Our findings suggest that the ecological range of these physiologically versatile prokaryotes is much wider than previously supposed.

Genome Res, 2004 Jun, 14(6), 1025 - 35 Epub 2004 May 12.
Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1; Baliga NS et al.; We report a remarkably high UV-radiation resistance in the extremely halophilic archaeon Halobacterium NRC-1 withstanding up to 110 J/m2 with no loss of viability . Gene knockout analysis in two putative photolyase-like genes (phr1 and phr2) implicated only phr2 in photoreactivation . The UV-response was further characterized by analyzing simultaneously, along with gene function and protein interactions inferred through comparative genomics approaches, mRNA changes for all 2400 genes during light and dark repair . In addition to photoreactivation, three other putative repair mechanisms were identified including d(CTAG) methylation-directed mismatch repair, four oxidative damage repair enzymes, and two proteases for eliminating damaged proteins . Moreover, a UV-induced down-regulation of many important metabolic functions was observed during light repair and seems to be a phenomenon shared by all three domains of life . The systems analysis has facilitated the assignment of putative functions to 26 of 33 key proteins in the UV response through sequence-based methods and/or similarities of their predicted three-dimensional structures to known structures in the PDB . Finally, the systems analysis has raised, through the integration of experimentally determined and computationally inferred data, many experimentally testable hypotheses that describe the metabolic and regulatory networks of Halobacterium NRC-1 .

J Appl Microbiol, 2004, 96(6), 1215 - 21
Effect of culture conditions on lactic acid production of Tetragenococcus species; Kobayashi T et al.; AIMS: To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T . halophilus . METHOD AND RESULTS: The growth, lactic acid production and pH reduction ability of five strains of T . muriaticus and T . halophilus in MRS broth medium under various culture conditions such as salt concentration (3, 7, 15 and 23% NaCl), temperature (20, 30 and 40 degrees C), and initial medium pH (5.8, 6.5 and 7.5) were investigated . Those of T . halophilus were seriously affected by a high salinity (23% NaCl); in contrast, those of T . muriaticus were affected by a low initial pH (5.8) . CONCLUSIONS: The results indicate that high saline concentrations and low pH values have significant impact on the growth, lactic acid production and pH reduction ability of T . halophilus and T . muriaticus, respectively . SIGNIFICANCE AND IMPACT OF THE STUDY: This study appears to be important in biopreservation during the manufacture of fermented food products . Both T . muriaticus and T . halophilus may support each other in reducing pH in hypersaline or low pH environment . To our knowledge, this is the first report on the fermentation ability of T . muriaticus.

J Bacteriol, 2004 May, 186(10), 3187 - 94
Arsenic resistance in Halobacterium sp . strain NRC-1 examined by using an improved gene knockout system; Wang G et al.; The genome sequence of Halobacterium sp . strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic . These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB) . We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts . Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 Deltaura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate . In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate . We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase . These results indicate that Halobacterium sp . strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts . Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid . The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal . Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite . We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.

Prikl Biokhim Mikrobiol, 2004 Mar-Apr, 40(2), 153 - 8
{Transglycosylation of stevioside by cyclodextrin glucanotransferases of various group of microorganisms}; Abelian VA et al.; Cyclodextrin glucanotransferases (CGTases, EC 2.4.1.19), produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli, were used for the transglycosylation of stevioside to remove bitterness and aftertaste, with cyclodextrins (CDs) being used as donors . It was shown that CGTases produced by extremophiic microorganisms are effective biocatalysts . Optimal temperature and pH of these enzymes were at pH 6.5-7.5 and 45 degrees C, respectively . The optimal stevioside-to-CD ratio and total concentration of dry matter for the synthesis of best-taste product were 1:1 (w/w) and 11.6%, respectively.

Fish Shellfish Immunol, 2004 Apr, 16(4), 487 - 99
Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea; Bulgakov AA et al.; The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study . A method was developed for the isolation of the Manila clam lectin (MCL) . Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation . SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits . The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography . The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM . MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation . The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc . In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins . MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria . It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans . Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.

Yeast, 2004 Apr 15, 21(5), 403 - 12
Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii; Gonzalez-Hernandez JC et al.; Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na(+) or K(+) . After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium . However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations . This yeast shows a cation uptake activity of both (86)Rb(+) and (22)Na(+) with saturation kinetics, and much higher affinity for (86)Rb(+) than for (22)Na(+) . The pH dependence of the kinetics constants was similar for both cations, and although K(m) values were higher at pH 8.0, there was also an increase in the V(max) values . The accumulation of (22)Na(+) was found to be increased in cells grown in the presence of 0.6 M NaCl . (86)Rb(+) was also accumulated more in these cells, but to a slightly greater extent . The inhibition kinetics of the uptake of (22)Na(+) by K(+), and that of (86)Rb(+) by Na(+) was found to be non-competitive . It can be concluded that Na(+) in D . hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations .

Biochem J, 2004 Aug 1, 381(Pt 3), 795 - 802
Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp . NRC-1: archaeal RNase HI can cleave an RNA-DNA junction; Ohtani N et al.; All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene . We found that the genome of a halophilic archaeon, Halobacterium sp . NRC-1, contains an open reading frame with similarity to Type 1 RNase H . The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue . The B . subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E . coli RNase HI, and the Vng0255c has the similar deletion . As this deletion apparently conferred a complete loss of RNase H activity on the B . subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity . However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E . coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain . These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H . This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3'-side of ribonucleotide and 5'-side of deoxyribonucleotide . It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.

J Mol Biol, 2004 Feb 27, 336(4), 829 - 42
Heat capacity effects of water molecules and ions at a protein-DNA interface; Bergqvist S et al.; The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics . In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (deltaCp) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations . Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change . We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP . We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity . In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations . The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change . Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone . Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.

Environ Pollut, 1994, 85(1), 77 - 85
Seasonal variation and local distribution of metals in the seagrass Halophila stipulacea (Forsk.) Aschers . In the Antikyra Gulf, Greece; Malea P; Accumulation of Fe, Pb, Zn, Cu, Cd, Na, K, Ca and Mg concentrations by the seagrass Halophila stipulacea (Forsk.) Aschers . was studied at eight stations of the Antikyra Gulf (Viotia, Greece) . This area was of interest because the contribution of bauxite to the mineral substrate and the discharge of an aluminium factory's wastes in it . Fe, Zn and K concentrations showed a significant seasonal variation with the same pattern (maximum mean value in summer and autumn), unlike Cu, Na and Mg concentrations which showed the opposite pattern . The observed patterns were mainly attributed to the dependence of metal concentrations in the plant on the tissue-age, the growth dynamics of the seagrass and the environment . All metal concentrations in the plant present uniform distribution in the inner part of the Gulf . The leaves of H . stipulacea accumulated higher Zn, Na, K and Mg concentrations than the stems, roots and rhizoids . There was a positive correlation between Cu, Zn, Cd and Na concentrations in the above-ground and below-ground plant parts.

J Food Prot, 2004 Apr, 67(4), 809 - 12
Bacteriology of unshelled frozen blue swimming crab (Portunus pelagicus); Giuffrida A et al.; In this study, 45 (10 whole specimens and 35 frozen claws) frozen samples of Portunus pelagicus imported into Sicily (Italy) from the west coast of Africa were examined to assess their bacteriological characteristics and suitability for consumption . Bacteriological examination was performed on two subsamples for each whole crab . The first was the body and claw muscle; the second was a pool of viscera and gills . In the case of frozen claws, each muscle claw was a sample . An aerobic plate count at 30 degrees C (mesophilic aerobic plate count {MAPC}) and 18 degrees C (psychrotrophic aerobic plate count {PAPC}) for 3 days, sulfite-reducing anaerobes, total coliforms, Escherichia coli, enterococci, and Aeromonas spp . were enumerated . Detection of halophilic Vibrio spp . was also performed using salt polymixin broth as an enrichment medium and thiosulfate citrate bile salts sucrose agar as a selective medium; a further morphological and biochemical identification of suspected colonies was performed . The bacterial load of muscle and viscera and gills was low . The MAPC ranged from 0.78 to 3.26 log CFU/g, and the PAPC ranged from 0.48 to 2.41 log CFU/g . Vibrio spp., Aeromonas spp., sulfite-reducing anaerobes, and E . coli were never isolated from muscles or viscera and gills . In contrast to the findings of others, this study showed good bacteriological quality of crabs imported into Sicily from the west coast of Africa . This study also demonstrated the positive influence of the characteristics of environment of origin and postharvest handling hygiene; these parameters could be useful in the context of the application of the hazard analysis critical control point system to this production.

Protein Pept Lett, 2004 Apr, 11(2), 125 - 32
Electrostatic and hydrophobic interactions play a major role in the stability and refolding of halophilic proteins; Arakawa T et al.; In general, halophilic proteins are stable only in the presence of salts at high concentrations . Not only is high salt concentration important for structural stability of halophilic proteins, but also refolding of a denatured halophilic protein requires high salt concentration . This review summarizes the importance of electrostatic charge shielding and hydrophobic interactions in the stability and refolding of halophilic proteins.

J Clin Microbiol, 2004 Apr, 42(4), 1840 - 2
Problems in identification of Francisella philomiragia associated with fatal bacteremia in a patient with chronic granulomatous disease; Friis-Moller A et al.; Francisella philomiragia is a rare gram-negative, halophilic coccobacillus with bizarre spherical forms on primary isolation . A case of F . philomiragia bacteremia in a 24-year-old patient with chronic granulomatous disease is reported . Identification of F . philomiragia was problematic with conventional tests but was done correctly and rapidly by kit 16S ribosomal DNA sequencing.

Appl Environ Microbiol, 2004 Apr, 70(4), 2230 - 9
Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring; Elshahed MS et al.; The archaeal community in a sulfide- and sulfur-rich spring with a stream water salinity of 0.7 to 1.0% in southwestern Oklahoma was studied by cloning and sequencing of 16S rRNA genes . Two clone libraries were constructed from sediments obtained at the hydrocarbon-exposed source of the spring and the microbial mats underlying the water flowing from the spring source . Analysis of 113 clones from the source library and 65 clones from the mat library revealed that the majority of clones belonged to the kingdom Euryarchaeota, while Crenarchaeota represented less than 10% of clones . Euryarchaeotal clones belonged to the orders Methanomicrobiales, Methanosarcinales, and Halobacteriales, as well as several previously described lineages with no pure-culture representatives . Those within the Halobacteriales represented 36% of the mat library and 4% of the source library . All cultivated members of this order are obligately aerobic halophiles . The majority of halobacterial clones encountered were not affiliated with any of the currently described genera of the family Halobacteriaceae . Measurement of the salinity at various locations at the spring, as well as along vertical gradients, revealed that soils adjacent to spring mats have a much higher salinity (NaCl concentrations as high as 32%) and a lower moisture content than the spring water, presumably due to evaporation . By use of a high-salt-plus-antibiotic medium, several halobacterial isolates were obtained from the microbial mats . Analysis of 16S rRNA genes indicated that all the isolates were members of the genus Haloferax . All isolates obtained grew at a wide range of salt concentrations, ranging from 6% to saturation, and all were able to reduce elemental sulfur to sulfide . We reason that the unexpected abundance of halophilic Archaea in such a low-salt, highly reduced environment could be explained by their relatively low salt requirement, which could be satisfied in specific locations of the shallow spring via evaporation, and their ability to grow under the prevalent anaerobic conditions in the spring, utilizing zero-valent sulfur compounds as electron acceptors . This study demonstrates that members of the Halobacteriales are not restricted to their typical high-salt habitats, and we propose a role for the Halobacteriales in sulfur reduction in natural ecosystems.

Extremophiles, 2004 Feb, 8(1), 53 - 61 Epub 2003 Nov 13.
Salt-induced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi; Turk M et al.; The halophilic melanized yeast-like fungi Hortaea werneckii, Phaeotheca triangularis, and the halotolerant Aureobasidium pullulans, isolated from salterns as their natural environment, were grown at different NaCl concentrations and their membrane lipid composition and fluidity were examined . Among sterols, besides ergosterol, which was the predominant one, 23 additional sterols were identified . Their total content did not change consistently or significantly in response to raised NaCl concentrations in studied melanized fungi . The major phospholipid classes were phosphatidylcholine and phosphatidylethanolamine, followed by anionic phospholipids . The most abundant fatty acids in phospholipids contained C16 and C18 chain lengths with a high percentage of C18:2Delta9,12 . Salt stress caused an increase in the fatty acid unsaturation in the halophilic H . werneckii and halotolerant A . pullulans but a slight decrease in halophilic P . triangularis . All the halophilic fungi maintained their sterol-to-phospholipid ratio at a significantly lower level than did the salt-sensitive Saccharomyces cerevisiae and halotolerant A . pullulans . Electron paramagnetic resonance (EPR) spectroscopy measurements showed that the membranes of all halophilic fungi were more fluid than those of the halotolerant A . pullulans and salt-sensitive S . cerevisiae, which is in good agreement with the lipid composition observed in this study.

Extremophiles, 2004 Feb, 8(1), 23 - 8 Epub 2003 Sep 26.
Halobacillus locisalis sp . nov., a halophilic bacterium isolated from a marine solar saltern of the Yellow Sea in Korea; Yoon JH et al.; A Gram-positive, motile, endospore-forming and rod-shaped halophilic bacterial strain MSS-155 (KCTC 3788 and KCCM 41687) was isolated from a marine solar saltern of the Yellow Sea in Korea and was subjected to a polyphasic taxonomic study . This organism grew at temperature of 10.0-42.0 degrees C with an optimum of 35 degrees C . Strain MSS-155 grew optimally in the presence of 10% NaCl and did not grow in the absence of NaCl . The cell wall peptidoglycan type of strain MSS-155 was A4beta based on l-Orn- d-Asp . Strain MSS-155 was also characterized chemotaxonomically by having menaquinone-7 (MK-7) as the predominant isoprenoid quinone and anteiso-C15:0 as the major fatty acid . The DNA G + C content was 44.0 mol% . Phylogenetic analysis based on 16S rDNA sequences showed that strain MSS-155 falls within the radiation of the cluster comprising Halobacillus species . Levels of 16S rDNA sequence similarity between strain MSS-155 and the type strains of four Halobacillus species were in the range 97.6-98.8% . Strain MSS-155 exhibited levels of DNA-DNA relatedness of 6.2-11.2% to the type strains of Halobacillus species described previously . On the basis of phenotypic properties, phylogeny, and genomic data, strain MSS-155 should be placed in the genus Halobacillus as a member of a novel species, for which we propose the name Halobacillus locisalis sp . nov.

Curr Microbiol, 2004 May, 48(5), 348 - 53
A taxonomic study to establish the relationship between exopolysaccharide-producing bacterial strains living in diverse hypersaline habitats; Martinez-Canovas MJ et al.; This study was undertaken to identify exopolysaccharide-producing bacteria gathered from 18 hypersaline habitats . Phenotypic studies performed with 134 isolates revealed the majority of them to be Gram-negative rods with respiratory metabolism, belonging to the genus Halomonas . A numerical analysis of the 114 phenotypic data showed that at an 80% similarity level most of the strains (121) could be grouped into six phenotypic groups . Phenon A included 25 new isolates and the reference strain of Halomonas eurihalina, and phenon B was formed by 77 new isolates and Halomonas maura . Phenon C was also related to H . maura although to a lesser extent than strains in group B . Three phena (D, E, and F) could not be grouped with any of the reference strains and may represent new taxa; their G + C contents and DNA-DNA hybridization data corroborated this hypothesis . Results of this work proved that the most abundant halophilic species EPS producer in hypersaline habitats was H . maura, followed by H . eurihalina.

Water Environ Res, 2004 Jan-Feb, 76(1), 56 - 66
Reuse of textile reactive azo dyebaths following biological decolorization; Lee YH et al.; The objective of this work was to evaluate the feasibility of biological decolorization and reuse of textile reactive azo dyebaths with the goal of developing an in-plant, closed-loop decolorization system leading to water conservation and wastewater minimization . Decolorization of three spent dyebaths was investigated using a suspended-growth, halophilic mixed culture under anoxic conditions . A high rate and extent of color removal ranging from 77 to 90% was achieved . The three azo dyebaths and their decolorization products did not inhibit the halophilic culture . Although single dyeings using the biologically renovated dyebaths as process water resulted in highly reproducible and consistent fabric color shades, pH adjustment of the renovated dyebaths during the dyeing cycle was necessary . The extent of color removal using a biofilm reactor ranged from 86 to 95% during five repetitive dyebath decolorization and reuse cycles without any inhibition and residual color buildup . Repetitive dyeing with the same biologically decolorized dyebath for up to five cycles using a biofilm reactor and dyebath pH adjustment during the redyeing process resulted in almost identical fabric color shades as standard dyeing using fresh water . Excellent reproducibility in fabric color shades was also obtained by using biologically decolorized dyebaths in cross dyeings with different dyeing recipes . To our knowledge, this is the first report on the feasibility of biological decolorization of spent reactive dyebaths using a halophilic mixed culture and the direct reuse of the renovated dyebath as process water in the dyeing process . The results of this study demonstrate that in-plant application of closed-loop dyeing and decolorization is feasible and has the potential to not only decrease wastewater volume and treatment cost, but also minimize water use as well as the discharge of textile pollutants such as salt and dyes.

Protein Eng Des Sel, 2004 Feb, 17(2), 191 - 200 Epub 2004 Feb 13.
Natural protein engineering: a uniquely salt-tolerant, but not halophilic, alpha-type carbonic anhydrase from algae proliferating in low- to hyper-saline environments; Bageshwar UK et al.; Dunaliella salina is a unicellular green alga thriving in environments ranging from fresh water to hyper-saline lakes, such as the Dead Sea . An unusual, internally duplicated, 60 kDa alpha-type carbonic anhydrase (dCA I), located on the surface of this alga, is expected to function over a broad range of salinities . It would therefore differ from other carbonic anhydrases that already lose activity at low salinities and also from halophilic proteins that require high salinities for conformational stability . Enzymatic analyses indeed indicated that dCA I retained activity at salt concentrations ranging from low salt to at least 1.5 M NaCl or KCl for CO(2) hydration, 2.0 M NaCl for esterase activity and 0.5 M for bicarbonate dehydration . Although measurements at higher salinities were constrained by the interference of salt in the respective assayed reactions, activity was noticeable even at 4.0 M NaCl . Comparisons of the internally duplicated dCA I to single-domain derivatives indicated that inter-domain interactions played a decisive role in the stability, activity, salt tolerance and pH responses of dCA I . Hence dCA I is a uniquely salt- tolerant protein, retaining an active conformation over a large range of salinities and, as a Zn metalloenzyme, largely immune to the specific inhibitory effects of anions . Its unique features make dCA I a useful model to understand the physico-chemical basis of halotolerance and protein-salt interactions in general.

Biochem Soc Trans, 2004 Apr, 32(Pt 2), 269 - 72
Hyperthermophilic and salt-dependent formyltransferase from Methanopyrus kandleri; Shima S et al.; Methanopyrus kandleri is a hyperthermophilic methanogenic archaeon, which grows on H(2) and CO(2) as its sole energy source . Its growth temperature optimum is 98 degrees C . One of the interesting characteristics of this archaeon is its high intracellular salt content . The organism has been reported to contain the trianionic cDPG (cyclic 2,3-diphosphoglycerate) and K+ at concentrations of 1.1 and 3 M, respectively . Reflecting the high cellular salt concentration, the enzymes in this organism are adapted not only to high temperature but also to high salt concentrations . The formyltransferase from M . kandleri was characterized extensively with respect to thermo- and halophilicity . The crystal structure of the formyltransferase at 1.73 A shows the enzyme to be composed of four identical subunits of molecular mass 32 kDa . The formyltransferase is thermostable and active only at relatively high concentrations of potassium phosphate (1 M) or other salts with strongly hydrated anions (strong salting-out salts) . Potassium phosphate and potassium cDPG were found to be equivalent in activating and stabilizing the enzyme . At low concentrations of these salts, the enzyme is inactive and thermolabile . It was shown by equilibrium sedimentation analysis that the enzyme is in a monomer/dimer/tetramer equilibrium, the equilibrium constant being dependent on the concentration of salts: the higher oligomeric species increase with increasing salt concentrations . Evidence was provided that the monomer is both inactive and thermolabile . Experiments using a mutation which is directed to break surface ion pairs between two dimers indicated that dimerization is required for activity and tetramerization leads to thermostability.

J Mol Evol, 2004 Mar, 58(3), 348 - 58
New insights into the evolutionary history of type 1 rhodopsins; Ruiz-Gonzalez MX et al.; Type 1 (archaeal) rhodopsins and related rhodopsin-like proteins had been described in a few halophile archaea, gamma-proteobacteria, a single cyanobacteria, some fungi, and a green alga . In exhaustive database searches, we detected rhodopsin-related sequences derived not only from additional fungal species but also from organisms belonging to three groups in which opsins had hitherto not been described: the alpha-proteobacterium Magnetospirillum magnetotacticum, the cryptomonad alga Guillardia theta, and the dinoflagellate Pyrocystis lunula . Putative plant and human type 1 rhodopsin sequences found in the databases are demonstrated to be contaminants of fungal origin . However, a highly diverged sequence supposedly from the plant Oryza sativa was found that is, together with the Pyrocystis sequence, quite similar to gamma-proteobacterial rhodopsins . These close relationships suggest that at least one horizontal gene transfer event involving rhodopsin genes occurred between prokaryotes and eukaryotes . Alternative hypotheses to explain the current phylogenetic range of type 1 rhodopsins are suggested . The broader phylogenetic range found is compatible with an ancient origin of type 1 rhodopsins, their patchy distribution being caused by losses in multiple lineages . However, the possibility of ancient horizontal transfer events between distant relatives cannot be dismissed.

Extremophiles, 2004 Aug, 8(4), 259 - 68 Epub 2004 Mar 20.
Identification of archaea and some extremophilic bacteria using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; Krader P et al.; Archaea and a number of groups of environmentally important bacteria, e.g., sulfate-reducing bacteria, anoxygenic phototrophs, and some thermophiles, are difficult to characterize using current methods developed for phenotypically differentiating heterotrophic bacteria . We have evaluated matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) as a rapid method for identifying different groups of extremophilic prokaryotes using a linear mass spectrometer (Micromass, UK) . The instrument is designed to acquire mass-spectral patterns from prokaryotic cell-wall components between masses of 500 and 10,000 Da in a statistically robust manner and create a database that can be used for identification . We have tested 28 archaea (10 genera, 20 spp.) and 42 bacteria (25 genera, 37 spp.) and found that all species yield reproducible, unique mass-spectral profiles . As a whole, the profiles for the archaea had fewer peaks and showed less differentiation compared to the bacteria, perhaps reflecting fundamental differences in cell-wall structure . The halophilic archaea all had consistent patterns that showed little differentiation; however, the software was able to consistently distinguish Halobacterium salinarium, Halococcus dombrowski, and Haloarcula marismortui from one another, although it could not always correctly distinguish four strains of Hb . salinarium from one another . The method was able to reliably identify 10(5) cells of either Albidovulum inexpectatum or Thermococcus litoralis and could detect as low as 10(3) cells . We found that the matrix, alpha-cyano-4-hydroxy-cinnamic acid yielded better spectra for archaea than 5-chloro-2-mercapto-benzothiazole . Overall, the method was rapid, required a minimum of sample processing, and was capable of distinguishing and identifying a very diverse group of prokaryotes.

J Mol Biol, 2004 Mar 5, 336(5), 997 - 1010
Membrane binding of ribosomes occurs at SecYE-based sites in the Archaea Haloferax volcanii; Ring G et al.; Whereas ribosomes bind to membranes at eukaryal Sec61alphabetagamma and bacterial SecYEG sites, ribosomal membrane binding has yet to be studied in Archaea . Accordingly, functional ribosomes and inverted membrane vesicles were prepared from the halophilic archaea Haloferax volcanii . The ability of the ribosomes to bind to the membranes was determined using a flotation approach . Proteolytic pretreatment of the vesicles, as well as quantitative analyses, revealed the existence of a proteinaceous ribosome receptor, with the affinity of binding being comparable to that found in Eukarya and Bacteria . Inverted membrane vesicles prepared from cells expressing chimeras of SecE or SecY fused to a cytoplasmically oriented cellulose-binding domain displayed reduced ribosome binding due to steric hindrance . Pretreatment with cellulose drastically reduced ribosome binding to chimera-containing but not wild-type vesicles . Thus, as in Eukarya and Bacteria, ribosome binding in Archaea occurs at Sec-based sites . However, unlike the situation in the other domains of Life, ribosome binding in haloarchaea requires molar concentrations of salt . Structural information on ribosome-Sec complexes may provide insight into this high salt-dependent binding.

Appl Spectrosc, 2004 Mar, 58(3), 317 - 22
Structural changes in the cells of some bacteria during population growth: a Fourier transform infrared-attenuated total reflectance study; Ede SM et al.; Structural changes occurring in the cells of several bacteria during their growth curves have been investigated by Fourier transform infrared (FT-IR) spectroscopy using the sampling technique of attenuated total reflectance (ATR) . Spectra reflect all of the components of the cells, including the cell walls, cell membranes, internal structures, and the cytoplasm . The bacteria studied were Bacillus stearothermophilus, Halobacterium salinarum, Halococcus morrhuae, and Acetobacter aceti . All species showed significant spectral changes during their growth curves, indicating structural changes in the cells during increases in cell numbers . The major change for B . stearothermophilus was in the lipid content, which was at a maximum during the exponential phase of the growth curve . For the halophiles H . salinarum and H . morrhuae, the major change was that the concentration of sulfate ion in the cells varied during the growth curve and was at a maximum during the mid-part of the exponential phase of the growth curve . A . aceti cells showed increasing polysaccharide content during the growth curve as well as maximum lipid content during the exponential phase of growth.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 211 - 5
Glycerol metabolism in the extremely halophilic bacterium Salinibacter ruber; Sher J et al.; Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by glycerol . In contrast to glucose consumption, which starts only after more easily degradable substrates present in yeast extract have been depleted, glycerol is consumed during the earliest growth phases . When U-(14)C-labeled glycerol was added to the culture, up to 25% of the radioactivity was incorporated by the cells . Glycerol kinase activity was detected only in cells grown in the presence of glycerol (up to 90 nmol mg protein(-1) min(-1)) . This enzyme functioned over salt concentrations from 0.6 to 2.8 M KCl . No significant activity of NAD-dependent glycerol dehydrogenase was found . It is suggested that Salinibacter may use glycerol as one of its principal substrates in its habitat, the saltern crystallizer ponds.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 203 - 9
Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions; Kogej T et al.; The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns . They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls . The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions . We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol . Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography . This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.

Eur J Biochem, 2004 Apr, 271(7), 1382 - 90
Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcanii; Lichi T et al.; Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites . Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea . In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii . Purified H . volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H . volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein . Such studies revealed that both FtsY and the FtsY NG domain bound to H . volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids . Indeed, membrane association of FtsY was also detected in intact H . volcanii cells . The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY-membrane association in Bacteria) . Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H . volcanii SRP54, a protein that did not otherwise interact with the membrane.

J Bacteriol, 2004 Apr, 186(7), 2179 - 94
Reconstruction of the central carbohydrate metabolism of Thermoproteus tenax by use of genomic and biochemical data; Siebers B et al.; The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach . A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T . tenax . Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism . T . tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism . For the EMP pathway some new, unexpected enzymes were identified . The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T . tenax . No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T . tenax . Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO2 fixation under autotrophic growth conditions . Almost all genes necessary for glycogen and trehalose metabolism were identified in the T . tenax genome.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 571 - 6
Aestuariibacter salexigens gen . nov., sp . nov . and Aestuariibacter halophilus sp . nov., isolated from tidal flat sediment, and emended description of Alteromonas macleodii; Yi H et al.; Two strictly aerobic, halophilic strains of the gamma-Proteobacteria, designated JC2042(T) and JC2043(T), were obtained from a sediment sample of getbol, the Korean tidal flat . Comparative 16S rDNA sequence studies revealed that the test strains were related most closely to the type strains of the genera Alteromonas (93.5-95.5 %) and Glaciecola (91.1-93.3 %) . Phylogenetic analyses demonstrated that strains JC2042(T) and JC2043(T) formed a distinct monophyletic clade within the family Alteromonadaceae and clustered distantly with the genera Alteromonas and Glaciecola . Physiological, biochemical and chemotaxonomic data also indicated that the two getbol isolates were significantly different from members of these two genera and others with validly published names . Cells were rod-shaped and motile with a polar flagellum . The major isoprenoid quinone was Q8 . The predominant cellular fatty acids were C(16 : 0), C(18 : 1)omega7c and a mixture of C(16 : 1)omega7c and iso-C(15 : 0) 2-OH . DNA G+C contents were 48-54 mol% . On the basis of this polyphasic study, Aestuariibacter gen . nov . is proposed with two novel species, Aestuariibacter salexigens sp . nov . (type strain, JC2042(T)=IMSNU 14006(T)=KCTC 12042(T)=DSM 15300(T)) and Aestuariibacter halophilus sp . nov . (type strain, JC2043(T)=IMSNU 14007(T)=KCTC 12043(T)=DSM 15266(T)) . Aestuariibacter salexigens is the type species of the genus . In addition, an emended description of Alteromonas macleodii is proposed.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 499 - 511
Halomonas neptunia sp . nov., Halomonas sulfidaeris sp . nov., Halomonas axialensis sp . nov . and Halomonas hydrothermalis sp . nov.: halophilic bacteria isolated from deep-sea hydrothermal-vent environments; Kaye JZ et al.; To assess the physiological and phylogenetic diversity of culturable halophilic bacteria in deep-sea hydrothermal-vent environments, six isolates obtained from low-temperature hydrothermal fluids, sulfide rock and hydrothermal plumes in North and South Pacific Ocean vent fields located at 1530-2580 m depth were fully characterized . Three strains were isolated on media that contained oligotrophic concentrations of organic carbon (0.002 % yeast extract) . Sequencing of the 16S rRNA gene indicated that all strains belonged to the genus Halomonas in the gamma-subclass of the Proteobacteria . Consistent with previously described species, the novel strains were slightly to moderately halophilic and grew in media containing up to 22-27 % total salts . The isolates grew at temperatures as low as -1 to 2 degrees C and had temperature optima of 30 or 20-35 degrees C . Both the minimum and optimum temperatures for growth were similar to those of Antarctic and sea-ice Halomonas species and lower than typically observed for the genus as a whole . Phenotypic tests revealed that the isolates were physiologically versatile and tended to have more traits in common with each other than with closely related Halomonas species, presumably a reflection of their common deep-sea, hydrothermal-vent habitat of origin . The G+C content of the DNA for all strains was 56.0-57.6 mol%, and DNA-DNA hybridization experiments revealed that four strains (Eplume1(T), Esulfide1(T), Althf1(T) and Slthf2(T)) represented novel species and that two strains (Eplume2 and Slthf1) were related to Halomonas meridiana . The proposed new species names are Halomonas neptunia (type strain Eplume1(T)=ATCC BAA-805(T)=CECT 5815(T)=DSM 15720(T)), Halomonas sulfidaeris (type strain Esulfide1(T)=ATCC BAA-803(T)=CECT 5817(T)=DSM 15722(T)), Halomonas axialensis (type strain Althf1(T)=ATCC BAA-802(T)=CECT 5812(T)=DSM 15723(T)) and Halomonas hydrothermalis (type strain Slthf2(T)=ATCC BAA-800(T)=CECT 5814(T)=DSM 15725(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 487 - 91
Shewanella gaetbuli sp . nov., a slight halophile isolated from a tidal flat in Korea; Yoon JH et al.; A Gram-negative, motile, non-spore-forming, rod-shaped strain, TF-27(T) (=KCCM 41648(T)=JCM 11814(T)), was isolated from a tidal flat in Korea . This organism grew well at 25-35 degrees C, with optimum growth at 30 degrees C . Strain TF-27(T) grew optimally in the presence of 2 % NaCl; it did not grow without NaCl or in the presence of >8 % NaCl . Strain TF-27(T) simultaneously contained both menaquinones and ubiquinones as isoprenoid quinones . The predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8 . The major fatty acids in strain TF-27(T) were iso-C(15 : 0) (20.6 %) and iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (21.1 %) . The DNA G+C content of strain TF-27(T) was 42 mol% . Phylogenetic analyses based on 16S rDNA sequences showed that strain TF-27(T) falls within the radiation of the cluster that is encompassed by the genus Shewanella.Levels of 16S rDNA sequence similarity between strain TF-27(T) and the type strains of Shewanella species were 93.2-96.8 % . On the basis of phenotypic properties and phylogenetic data, strain TF-27(T) should be placed in the genus Shewanella as a novel species, for which the name Shewanella gaetbuli sp . nov . is proposed.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 389 - 92
Taxonomic study of Halorubrum distributum and proposal of Halorubrum terrestre sp . nov; Ventosa A et al.; Halorubrum distributum (basonym, Halobacterium distributum) is an extremely halophilic, aerobic archaeon isolated from saline soils, which was described on the basis of phenotypic features of several strains . The designated type strain of the species (1m(T)=VKM B-1733(T)=JCM 9100(T)) was shown recently to differ from the other strains . In this study, Halorubrum distributum isolates have been characterized with regard to phenotypic features, polar lipid content, comparison of 16S rRNA gene sequences and DNA-DNA hybridization . On the basis of these data, a novel species that includes the other isolates is proposed, with the name Halorubrum terrestre sp . nov . The type strain of this novel species is 4p(T) (=VKM B-1739(T)=JCM 10247(T)) . The DNA G+C content of this novel species is 64.2-64.9 mol% (64.4 mol% for the type strain).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 377 - 80
Thalassomonas ganghwensis sp . nov., isolated from tidal flat sediment; Yi H et al.; A Gram-negative, aerobic, halophilic bacterium, designated strain JC2041(T), was isolated from a sediment sample of getbol, the Korean tidal flat . Results of 16S rDNA sequence analyses indicated a moderate relationship to Thalassomonas viridans within the gamma-Proteobacteria (94.9 % similarity) . Depending on the tree-making algorithm used, the isolate either formed a monophyletic clade with T . viridans or was recovered as a sister group of a class containing the genera Thalassomonas and Colwellia . Phenotypic features of the getbol isolate were similar to those of T . viridans, but several physiological and chemotaxonomic properties, including nitrate reduction, amylase, lecithinase, Tweenase and utilization of 13 carbon sources, distinguished strain JC2041(T) from T . viridans . The polyphasic data presented in this study indicate that the isolate should be classified as a novel species in the genus Thalassomonas . The name Thalassomonas ganghwensis sp . nov . is therefore proposed for the getbol isolate; the type strain is JC2041(T) (=IMSNU 14005(T)=KCTC 12041(T)=DSM 15355(T)).

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3591 - 6 Epub 2004 Feb 27.
CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea; Woodson JD et al.; The existence of a pathway for salvaging the coenzyme B(12) precursor dicyanocobinamide (Cbi) from the environment was established by genetic and biochemical means . The pathway requires the function of a previously unidentified amidohydrolase enzyme that converts adenosylcobinamide to adenosylcobyric acid, a bona fide intermediate of the de novo coenzyme B(12) biosynthetic route . The cbiZ gene of the methanogenic archaeon Methanosarcina mazei strain Gol was cloned, was overproduced in Escherichia coli, and the recombinant protein was isolated to homogeneity . HPLC, UV-visible spectroscopy, MS, and bioassay data established adenosylcobyric as the corrinoid product of the CbiZ-catalyzed reaction . Inactivation of the cbiZ gene in the extremely halophilic archaeon Halobacterium sp . strain NRC-1 blocked the ability of this archaeon to salvage Cbi . cbiZ function restored Cbi salvaging in a strain of the bacterium Salmonella enterica, whose Cbi-salvaging pathway was blocked . The salvaging of Cbi through the CbiZ enzyme appears to be an archaeal strategy because all of the genomes of B(12)-producing archaea have a cbiZ ortholog . Reasons for the evolution of two distinct pathways for Cbi salvaging in prokaryotes are discussed.

Astrobiology, 2003 Winter, 3(4), 785 - 811
The search for life on Europa: limiting environmental factors, potential habitats, and Earth analogues; Marion GM et al.; The putative ocean of Europa has focused considerable attention on the potential habitats for life on Europa . By generally clement Earth standards, these Europan habitats are likely to be extreme environments . The objectives of this paper were to examine: (1) the limits for biological activity on Earth with respect to temperature, salinity, acidity, desiccation, radiation, pressure, and time; (2) potential habitats for life on Europa; and (3) Earth analogues and their limitations for Europa . Based on empirical evidence, the limits for biological activity on Earth are: (1) the temperature range is from 253 to 394 K; (2) the salinity range is a(H2O) = 0.6-1.0; (3) the desiccation range is from 60% to 100% relative humidity; (4) the acidity range is from pH 0 to 13; (5) microbes such as Deinococcus are roughly 4,000 times more resistant to ionizing radiation than humans; (6) the range for hydrostatic pressure is from 0 to 1,100 bars; and (7) the maximum time for organisms to survive in the dormant state may be as long as 250 million years . The potential habitats for life on Europa are the ice layer, the brine ocean, and the seafloor environment . The dual stresses of lethal radiation and low temperatures on or near the icy surface of Europa preclude the possibility of biological activity anywhere near the surface . Only at the base of the ice layer could one expect to find the suitable temperatures and liquid water that are necessary for life . An ice layer turnover time of 10 million years is probably rapid enough for preserving in the surface ice layers dormant life forms originating from the ocean . Model simulations demonstrate that hypothetical oceans could exist on Europa that are too cold for biological activity (T < 253 K) . These simulations also demonstrate that salinities are high, which would restrict life to extreme halophiles . An acidic ocean (if present) could also potentially limit life . Pressure, per se, is unlikely to directly limit life on Europa . But indirectly, pressure plays an important role in controlling the chemical environments for life . Deep ocean basins such as the Mariana Trench are good analogues for the cold, high-pressure ocean of Europa . Many of the best terrestrial analogues for potential Europan habitats are in the Arctic and Antarctica . The six factors likely to be most important in defining the environments for life on Europa and the focus for future work are liquid water, energy, nutrients, low temperatures, salinity, and high pressures.

Astrobiology, 2003 Winter, 3(4), 761 - 70
Physical simulation for low-energy astrobiology environmental scenarios; Gormly S et al.; Speculations about the extent of life of independent origin and the potential for sustaining Earth-based life in subsurface environments on both Europa and Mars are of current and relevant interest . Theoretical modeling based on chemical energetics has demonstrated potential options for viable biochemical metabolism (metabolic pathways) in these types of environments . Also, similar environments on Earth show microbial activity . However, actual physical simulation testing of specific environments is required to confidently determine the interplay of various physical and chemical parameters on the viability of relevant metabolic pathways . This testing is required to determine the potential to sustain life in these environments on a specific scenario by scenario basis . This study examines the justification, design, and fabrication of, as well as the culture selection and screening for, a psychrophilic/halophilic/anaerobic digester . This digester is specifically designed to conform to physical testing needs of research relating to potential extent physical environments on Europa and other planetary bodies in the Solar System . The study is a long-term effort and is currently in an early phase, with only screening-level data at this time . Full study results will likely take an additional 2 years . However, researchers in electromagnetic biosignature and in situ instrument development should be aware of the study at this time, as they are invited to participate in planning for future applications of the digester facility.

Extremophiles, 2004 Jun, 8(3), 193 - 200 Epub 2004 Feb 21.
Alkalimonas amylolytica gen . nov., sp . nov., and Alkalimonas delamerensis gen . nov., sp . nov., novel alkaliphilic bacteria from soda lakes in China and East Africa; Ma Y et al.; Two related novel alkaliphilic and slightly halophilic bacteria are described . They are strain N10 from Lake Chahannor in China and strain 1E1 from Lake Elmenteita in East Africa . Both strains are strictly aerobic, heterotrophic, alkaliphilic, mesophilic, and require NaCl for growth . The optimal conditions for growth were at pH 10-10.5 and 2-3% (w/v) NaCl . Cells of both strains were Gram-negative, rod-shaped, non-spore-forming, and motile with a single polar flagellum . Cellular fatty acids in both strains were predominantly saturated and mono-unsaturated straight-chain fatty acids (16:0, 16:1omega7c and 18:1omega7c) . The major isoprenoid quinone of both strains was Q8 . The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and phosphatidylethanolamine . The guanine plus cytosine (G + C) content of the DNA was 52.5 mol% and 55.4 mol%, respectively . Phylogenetic analysis revealed that the two strains formed a distinct lineage within the gamma-3 subclass of the Proteobacteria . The strains shared a 16S rDNA sequence similarity of 96.1% and showed less than 93.7% of sequence similarity to any other known species . Based on polyphasic data, the two strains were differentiated from currently recognized genera and represent a new genus, Alkalimonas gen . nov., with two species, Alkalimonas amylolytica sp . nov . (type strain is N10T = AS 1.3430) and Alkalimonas delamerensis sp . nov . ( type strain is 1E1(P, T) = CBS 391.94) . The GenBank accession numbers for the 16S rRNA gene sequence of strains N10 and 1E1 are AF250323 and X92130, respectively .

South Med J, 2004 Feb, 97(2), 163 - 8
Varied clinical presentations of Vibrio vulnificus infections: a report of four unusual cases and review of the literature; Ulusarac O et al.; Vibrio vulnificus is a Gram-negative, motile, curved bacillus of the family Vibrionaceae that is a rare cause of gastroenteritis, septicemia, and wound infections in humans . V . vulnificus is halophilic, flourishes in warm temperatures, and is part of the bacterial flora of the marine environment . The location of our health care setting, on the Gulf of Mexico, has given us the opportunity to observe a wide variety of clinical presentations of infections caused by this organism . In the first case, a 27-year-old man struck by lightning while windsurfing was found pulseless in the water and was resuscitated . The patient subsequently developed cardiac arrhythmias, respiratory failure, and necrotizing fasciitis, blood cultures yielded V . vulnificus . After antibiotic therapy and several fasciotomies, the patient recovered . The second case was that of a 43-year-old Asian man employed as an oyster shucker who presented with complaints of redness, tearing, and photophobia of the right eye . The diagnosis of corneal ulcer secondary to V . vulnificus was made after culture of the right eye revealed the organism . The third case involved a 46-year-old man who presented with complaints of abdominal pain, nausea, chills, and bullous lesions on the lower extremities . He developed disseminated intravascular coagulation, and cultures of the lesions on his lower extremities showed V . vulnificus . Initially, the patient denied any exposure to raw seafood or seawater, but he eventually remembered eating raw oysters 3 days before his illness . The fourth case is that of a 32-year-old, human immunodeficiency virus-positive, hepatitis C-positive woman with cirrhosis who presented with productive cough, chills, fever, and red spots on her extremities and buttocks . Blood cultures revealed V . vulnificus and the patient was treated with antibiotics and improved clinically . These four cases illustrate the wide range of clinical presentations associated with this organism.

Biosci Biotechnol Biochem, 2004 Feb, 68(2), 397 - 406
Ureases of extreme halophiles of the genus Haloarcula with a unique structure of gene cluster; Mizuki T et al.; We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method . Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts . Only 4 strains of the genus Haloarcula, Har . aidinensis, Har . hispanica, Har . japonica, and Har . marismortui were finally shown as the urease producers . A partially purified urease from Har . hispanica was a typical halophilic enzyme in that it showed maximum activity at 18-23% NaCl and lost the activity irreversibly in the absence of NaCl . Partial genes (1596 bp) of the urease encoding from upstream of the beta subunit down to the N-terminal 139 amino acids of the alpha subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains . Strains of other genera, which were urease-negative, did not yield PCR products . The deduced amino acid sequences of the beta subunit and partial alpha subunit were similar to each other (92-100% similarities) and to those from other organisms . Analysis of the draft genome sequence of Har . marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was beta-alpha-gamma-UreG-UreD-UreE-UreF . This order is quite unique, since in other microorganisms the order is gamma-beta-alpha-UreE-UreF-UreG-UreD in most cases . No open reading frames were detected in the PCR-amplified upstream of the beta subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.

Orig Life Evol Biosph, 2004 Feb, 34(1-2), 159 - 69
Polysaccharides from extremophilic microorganisms; Nicolaus B et al.; Several marine thermophilic strains were analyzed for exopolysaccharide production . The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions . We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains . The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character . The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter(-1) was obtained with mannose as carbon and energy source . NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different alpha-D-mannoses and three different beta-D-glucoses . It seems to be close to some xantan polymers . EPS 2 was a mannan . Four different alpha-D-mannoses were found as the repeating unit . Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.

Biochemistry, 2004 Feb 24, 43(7), 1809 - 20
Rhodobacter capsulatus photoactive yellow protein: genetic context, spectral and kinetics characterization, and mutagenesis; Kyndt JA et al.; A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence . However, the PYP had not been characterized as a protein . We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag . The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 nm, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery . RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline . This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state . The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP . Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP . However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics . Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr . The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized . These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.

Extremophiles, 2004 Jun, 8(3), 175 - 84 Epub 2004 Feb 11.
Marinococcus halophilus DSM 20408T encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM; Vermeulen V et al.; In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium . To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M . halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport . The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM . The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs) . The transporter responsible for the uptake of glycine betaine in M . halophilus is encoded by an open reading frame of 1,482 bp called betM . The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family . The affinity of whole cells of E . coli MKH13 for ectoine (Ks =1.6 microM) and betaine (Ks = 21.8 microM) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates . An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E . coli MKH13 cells, demonstrating that both systems are osmoregulated .

Bioinformatics, 2004 May 22, 20(8), 1248 - 53 Epub 2004 Feb 10.
Poorly conserved ORFs in the genome of the archaea Halobacterium sp . NRC-1 correspond to expressed proteins; Shmuely H et al.; MOTIVATION: A large fraction of open reading frames (ORFs) identified as 'hypothetical' proteins correspond to either 'conserved hypothetical' proteins, representing sequences homologous to ORFs of unknown function from other organisms, or to hypothetical proteins lacking any significant sequence similarity to other ORFs in the databases . Elucidating the functions and three-dimensional structures of such orphan ORFs, termed ORFans or poorly conserved ORFs (PCOs), is essential for understanding biodiversity . However, it has been claimed that many ORFans may not encode for expressed proteins . RESULTS: A genome-wide experimental study of 'paralogous PCOs' in the halophilic archaea Halobacterium sp . NRC-1 was conducted . Paralogous PCOs are ORFs with at least one homolog in the same organism, but with no clear homologs in other organisms . The results reveal that mRNA is synthesized for a majority of the Halobacterium sp . NRC-1 paralogous PCO families, including those comprising relatively short proteins, strongly suggesting that these Halobacterium sp . NRC-1 paralogous PCOs correspond to true, expressed proteins . Hence, further computational and experimental studies aimed at characterizing PCOs in this and other organisms are merited . Such efforts could shed light on PCOs' functions and origins, thereby serving to elucidate the vast diversity observed in the genetic material.

Appl Environ Microbiol, 2004 Feb, 70(2), 1222 - 5
Biodegradation of benzene by halophilic and halotolerant bacteria under aerobic conditions; Nicholson CA et al.; A highly enriched halophilic culture was established with benzene as the sole carbon source by using a brine soil obtained from an oil production facility in Oklahoma . The enrichment completely degraded benzene, toluene, ethylbenzene, and xylenes within 1 to 2 weeks . Also, {14C}benzene was converted to 14CO2, suggesting the culture's ability to mineralize benzene . Community structure analysis revealed that Marinobacter spp . were the dominant members of the enrichment.

Appl Environ Microbiol, 2004 Feb, 70(2), 943 - 53
Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes; Allers T et al.; Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry . Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism . We describe here isolation of H . volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system . DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively . We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter . The use of these selectable markers in deletion of the lhr gene of H . volcanii is described.

Mol Microbiol, 2004 Jan, 51(2), 579 - 88
In vivo studies on putative Shine-Dalgarno sequences of the halophilic archaeon Halobacterium salinarum; Sartorius-Neef S et al.; The involvement of Shine-Dalgarno sequences in the translation of mRNA in halophilic archaea was investigated for two gvp genes involved in gas vesicle formation in Halobacterium salinarum PHH1 . With the exception of gvpA and gvpO, all reading frames of the p-gvpDEFGHIJKLM and p-gvpACNO mRNAs contained upstream of the AUG start codon a putative Shine-Dalgarno (SD) sequence that is complementary to the 3'-end of the small ribosomal subunit RNA . The importance of the SD sequences of gvpG and gvpH was investigated in Haloferax volcanii transformants, and an alteration of the SD sequence resulted in a reduction of the amount of the GvpG or GvpH protein . For a more quantitative analysis the region upstream of gvpH was fused to the bgaH reading frame encoding an enzyme with beta-galactosidase activity as reporter . Scanning mutagenesis within the mRNA leader demonstrated that mutations adjacent to the putative SD sequence GGAGGUCA did not influence the efficiency of translation, whereas constructs harbouring an altered SD sequence yielded only 5-50% of the beta-galactosidase activities obtained with the wild-type SD element . A complete mutation of the SD sequence still yielded 20% of the wild-type activity . Alterations in the spacing of the SD sequence and the translation initiation codon of gvpH indicated that a distance of 4 or 10 nucleotides yielded a similar beta-galactosidase activity as found with the 7 nt spacing of the SD element in wild type, whereas a distance of 1 nt resulted in the loss of translation . A complete deletion of the 5'-UTR resulting in a leaderless mRNA yielded an enhanced beta-galactosidase activity in transformants implying that the initiation of translation involved a mechanism other than a specific mRNA-rRNA interaction.

FEBS Lett, 2004 Jan 30, 558(1-3), 7 - 12
Highly efficient renaturation of beta-lactamase isolated from moderately halophilic bacteria; Tokunaga H et al.; Most, if not all, beta-lactamases reported to date are irreversibly denatured at 60-70 degrees C . Here, we found that a halophilic beta-lactamase from the moderately halophilic bacterium Chromohalobacter sp . 560 was highly stable against heat inactivation: it retained approximately 75% of its activity after boiling for 5 min in the presence of 0.2 M NaCl, suggesting that the protein either incompletely denatures during the boiling process or readily renatures upon cooling to the assay temperature . Circular dichroism showed a complete unfolding at 60 degrees C and a full reversibility, indicating that the observed activity after boiling is due to efficient refolding following heat denaturation . The enzyme showed optimal activity at 50-60 degrees C, indicating that an increase in activity with temperature offsets the thermal denaturation . The gene bla was cloned, and the primary structure of the enzyme was deduced to be highly abundant in acidic amino acid residues, one of the characteristics of halophilic proteins . Despite its halophilic nature, the enzyme refolds in low salt media after heat denaturation.

Yeast, 2004 Jan 30, 21(2), 119 - 26
Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+); Thome PE; A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant . DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids . Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes . The sequence has been submitted to the GenBank database under Accession No . AY333427 .

J Mol Evol, 2004 Jan, 58(1), 115 - 21
The rDNA ITS region in the lessepsian marine angiosperm Halophila stipulacea (Forssk.) Aschers . (Hydrocharitaceae): intragenomic variability and putative pseudogenic sequences; Ruggiero MV et al.; Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea . This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869) . Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin . In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis . A high degree of intraindividual variability of ITS sequences was found . Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals . Features of ITS functional sequences and pseudogenes are described . Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 163 - 7
Marinospirillum insulare sp . nov., a novel halophilic helical bacterium isolated from kusaya gravy; Satomi M et al.; A novel species that belongs to the genus Marinospirillum is described on the basis of phenotypic characteristics, phylogenetic analysis of 16S rRNA and gyrB gene sequences and DNA-DNA hybridization . Four strains of helical, halophilic, Gram-negative, heterotrophic bacteria were isolated from kusaya gravy, which is fermented brine that is used for the production of traditional dried fish in the Izu Islands of Japan . All of the new isolates were motile by means of bipolar tuft flagella, of small cell size, coccoid-body-forming and aerophilic; it was concluded that they belong to the same bacterial species, based on DNA-DNA hybridization values (>70% DNA relatedness) . DNA G+C contents of the new strains were 42-43 mol% and they had isoprenoid quinone Q-8 as the major component . Phylogenetic analysis of 16S rRNA gene sequences indicated that the new isolates were members of the genus Marinospirillum; sequence similarity of the new isolates to Marinospirillum minutulum, Marinospirillum megaterium and Marinospirillum alkaliphilum was 98.5, 98.2 and 95.2%, respectively . Phylogenetic analysis based on the gyrB gene indicated that the new isolates had enough phylogenetic distance from M . minutulum and M . megaterium to be regarded as different species, with 84.7 and 78.7% sequence similarity, respectively . DNA-DNA hybridization showed that the new isolates had <36% DNA relatedness to M . minutulum and M . megaterium, supporting the phylogenetic conclusion . Thus, a novel species is proposed: Marinospirillum insulare sp . nov . (type strain, KT=LMG 21802T=NBRC 100033T).

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 157 - 62
Hongiella mannitolivorans gen . nov., sp . nov., Hongiella halophila sp . nov . and Hongiella ornithinivorans sp . nov., isolated from tidal flat sediment; Yi H et al.; Three marine strains of the Cytophaga-Flavobacterium-Bacteroides group, designated JC2050T, JC2051T and JC2052T, were obtained from a single sediment sample of getbol, the Korean tidal flat . Comparative 16S rDNA sequence studies revealed that the test strains were not closely related to any validly published genera and that these strains were only distantly related to the genus Cyclobacterium (88.7-91.2%) . Phylogenetic analyses demonstrated that the three getbol isolates formed a distinct monophyletic clade within the family Cytophagaceae . Physiological, biochemical and chemotaxonomic data also indicated that these three getbol isolates differed significantly from members of other genera and were sufficiently different from each other to be recognized as separate species . On the basis of polyphasic evidence, a new genus, Hongiella gen . nov., is proposed, with three novel species, Hongiella mannitolivorans sp . nov . (type strain JC2050T=IMSNU 14012T=DSM 15301T), Hongiella halophila sp . nov . (type strain JC2051T=IMSNU 14013T=DSM 15292T) and Hongiella ornithinivorans sp . nov . (type strain JC2052T=IMSNU 14014T=DSM 15282T) . Hongiella mannitolivorans is the type species of the genus.

Biol Chem, 2003 Dec, 384(12), 1565 - 73
Effects of iron limitation on the respiratory chain and the membrane cytochrome pattern of the Euryarchaeon Halobacterium salinarum; Hubmacher D et al.; The effects of iron limitation on the electron transport chain of the extremely halophilic Euryarchaeon Halobacterium salinarum were analyzed . When iron was growth-limiting, the respiratory rates as well as the inhibition pattern of the membranes were significantly different from membranes of iron replete cells . Changes in the availability of iron cause the formation of different respiratory pathways including different entry sites for electrons, different terminal oxidases of the respiratory chain, and drastic changes of the cytochrome composition and of the relative amounts of cytochromes . Under iron-limiting conditions, mainly low-potential cytochromes were measured . EPR spectroscopic studies revealed that the amount of proteins containing iron-sulfur clusters is reduced in membranes under iron-limiting growth conditions . Taken together, our results strongly suggest for the first time an important role of iron supply for the bioenergetics of an Archaeon.

BMC Genomics . 2004 Jan 12;5(1):3.
Low-pass sequencing for microbial comparative genomics; Goo YA et al.; BACKGROUND: We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense . Approximately one thousand single pass genomic sequences per genome were obtained . The data were analyzed by comparative genomic analyses using the completed Halobacterium sp . NRC-1 genome as a reference . Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe . RESULTS: As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits . All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins . Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H . lacusprofundi and H . marismortui . The core biological functions that govern cellular and genetic mechanisms of H . sp . NRC-1 appear to be conserved in these four other halophiles . Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles . The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H . sp . NRC-1 and H . lacusprofundi . CONCLUSION: Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively . Identification of multiple IS elements in the genome of H . lacusprofundi and H . marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H . sp . NRC-1 . Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions . Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics.

J Gen Appl Microbiol, 2003 Oct, 49(5), 279 - 86
Isolation and characterization of halophilic lactic acid bacteria isolated from "terasi" shrimp paste: a traditional fermented seafood product in Indonesia; Kobayashi T et al.; Lactic acid bacteria from "terasi" shrimp paste, a highly popular fermented seafood in Indonesia were isolated and characterized . Viable cell counts were 10(4) to 10(6) cfu/g on MRS medium . All the isolates were catalase-negative, gram-positive cocci and were able to grow at 15% NaCl . Numerical phenotypic analysis showed that the isolates clustered into one group . However, they could be classified into two types: the Tetragenococcus halophilus group and the T . muriaticus group as revealed by a restriction fragment length polymorphism (RFLP) analysis and sequencing of the 16S rRNA gene . This study is the first to show that both species of Tetragenococcus are distributed in Indonesian fermented foods.

Curr Microbiol, 2003 Nov, 47(5), 388 - 94
The 492-bp RM07 DNA fragment from the halophilic Archaea confers promoter activity in all three domains of life; Yang Y et al.; A 492-bp DNA fragment, designated RM07, was isolated from the chromosomal DNA of the halophilic Archaea, Halobacterium halobium, and was shown to confer promoter activity in Escherichia coli . Sequence analysis revealed that RM07 contained three consensus sequences of the archaeal distal promoter element as well as the typical -35 and -10 box sequences of bacterial promoters . Promoter probe analysis confirmed that RM07 conferred promoter activity in all three domains of life: Archaea (Haloferax volcanii), Eukarya (Saccharomyces cerevisiae) and Bacteria (Escherichia coli) . Deletion analysis and site-directed mutagenesis further identified the functional regions within RM07 required for promoter activity . This is the first report of a DNA fragment from Archaea that confers promoter activity in all three domains of life, suggesting that the promoter structure and activity may be viewed as a bridge narrowing the gaps among the different domains of life.

J Mol Biol, 2004 Jan 2, 335(1), 343 - 56
The 2.9A resolution crystal structure of malate dehydrogenase from Archaeoglobus fulgidus: mechanisms of oligomerisation and thermal stabilisation; Irimia A et al.; The crystal structure of malate dehydrogenase from the hyperthermophilic archaeon Archeoglobus fulgidus, in complex with its cofactor NAD, was solved at 2.9A resolution . The crystal structure shows a compact homodimer with one coenzyme bound per subunit . The substrate binding site is occupied by a sulphate ion . In order to gain insight into adaptation mechanisms, which allow the protein to be stable and active at high temperatures, the 3D structure was compared to those of several thermostable and hyperthermostable homologues, and to halophilic malate dehydrogenase . The hyperthermostable A . fulgidus MalDH protein displays a reduction of the solvent-exposed surface, an optimised compact hydrophobic core, a high number of hydrogen bonds, and includes a large number of ion pairs at the protein surface . These features occur concomitantly with a reduced number of residues in the protein subunit, due to several deletions in loop regions . The loops are further stiffened by ion pair links with secondary structure elements . A . fulgidus malate dehydrogenase is the only dimeric protein known to date that belongs to the {LDH-like} MalDH family . All the other known members of this family are homo-tetramers . The crystal structures revealed that the association of the dimers to form tetramers is prevented by several deletions, taking place at the level of two loops that are known to be essential for the tetramerisation process within the LDH and {LDH-like} MalDH enzymes.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2073 - 8
Marinobacter excellens sp . nov., isolated from sediments of the Sea of Japan; Gorshkova NM et al.; Five strains of halophilic, Gram-negative marine bacteria (KMM 3809(T), KMM 3814, KMM 3815, KMM 3817 and KMM 3818) were isolated from sediments collected from Chazhma Bay, Sea of Japan . Phylogenetic 16S rRNA gene sequence-based analysis placed these bacteria in a clade within the genus Marinobacter in the gamma-Proteobacteria . KMM 3809(T) showed highest 16S rRNA gene sequence similarity of 97.3 % to Marinobacter litoralis and 96.9 % to Marinobacter hydrocarbonoclasticus and Marinobacter aquaeolei . DNA-DNA hybridization between the five isolates was at the conspecific level (94-96 %) and that among the closest phylogenetic neighbours ranged from 45.0 to 62.5 % . The new organisms were susceptible to polymyxin . Predominant fatty acids were C(16 : 0), C(16 : 1)omega9c, C(16 : 1)omega7c and C(18 : 1)omega9c . Phylogenetic evidence, along with phenotypic and genotypic characteristics, showed that the bacteria constituted a novel species of the genus Marinobacter . The name Marinobacter excellens sp . nov . is proposed for this species, with the type strain KMM 3809(T) (=CIP 107686(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1967 - 71
Mesonia algae gen . nov., sp . nov., a novel marine bacterium of the family Flavobacteriaceae isolated from the green alga Acrosiphonia sonderi (Kütz) Kornm; Nedashkovskaya OI et al.; The taxonomic position of four heterotrophic, aerobic, Gram-negative, non-motile and moderately halophilic marine bacteria, isolated from the green alga Acrosiphonia sonderi (Kutz) Kornm, was established . 16S rDNA sequence analysis indicated that the strains studied are members of the family Flavobacteriaceae, in which they form a distinct lineage . On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, the novel bacteria were classified as Mesonia algae gen . nov., sp . nov . The type strain is KMM 3909(T) (=KCTC 12089(T)=CCUG 47092(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1885 - 8
Oceanisphaera litoralis gen . nov., sp . nov., a novel halophilic bacterium from marine bottom sediments; Romanenko LA et al.; A polyphasic taxonomic study was performed to characterize a new bacterial isolate, designated KMM 3654(T), from a marine bottom sand sample . The strain was Gram-negative, encapsulated, aerobic, moderately halophilic and grew between 0.5 and 10 % NaCl and at 4-42 degrees C . Its DNA G+C content was 56.4 mol% . Isolate KMM 3654(T) was phylogenetically closely related to members of the genus Oceanimonas, showing 96.7 and 95.6 % 16S rRNA gene sequence similarity to Oceanimonas doudoroffii DSM 7028(T) and Oceanimonas baumannii ATCC 700832(T), respectively . Strain KMM 3654(T) shared some physiological and chemotaxonomic properties with these two Oceanimonas species, but differed from them in morphology, growth at 4 degrees C, urease activity, weak phenol degradation and utilization of phenylacetate . On the basis of phenotypic and phylogenetic evidence, Oceanisphaera litoralis gen . nov., sp . nov . is proposed, with the type strain KMM 3654(T) (=DSM 15406(T)).

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1873 - 9
Idiomarina loihiensis sp . nov., a halophilic gamma-Proteobacterium from the Lō'ihi submarine volcano, Hawai'i; Donachie SP et al.; During an investigation of bacterial diversity at hydrothermal vents on the Lo'ihi Seamount, Hawai'i, a novel bacterium (designated L2-TR(T)) was cultivated, which shares 99.9 % 16S rRNA gene sequence similarity over 1415 nt with an uncultured eubacterium from sediment at a depth of 11 000 m in the Mariana Trench . The nearest cultivated neighbour of L2-TR(T), however, is Idiomarina abyssalis KMM 227(T), with which it shares 98.9 % 16S rRNA sequence similarity . L2-TR(T) differed from I . abyssalis KMM 227(T) in several phenotypic respects, including growth at 46 degrees C and in medium that contained 20 % (w/v) NaCl . DNA-DNA hybridization data showed that L2-TR(T) did not belong to the species I . abyssalis (43.4 % DNA-DNA reassociation) . Cells of L2-TR(T) were Gram-negative rods, 0.35 microm wide and 0.7-1.0 microm long, which were occasionally up to 1.8 microm in length . Cells were motile by a single polar or subpolar flagellum . The major fatty acid in L2-TR(T) was iso-C(15 : 0) (32.6 %) . The DNA G+C content was 47.4 mol% . Phenotypic and genotypic analyses indicated that L2-TR(T) could be assigned to the genus Idiomarina but, based on significant phenotypic and genotypic differences, constituted a novel species within this genus, Idiomarina loihiensis sp . nov., of which L2-TR(T) (=ATCC BAA-735(T)=DSM 15497(T)) is the type strain.

Genome Res, 2003 Dec, 13(12), 2577 - 87
Comparative genome analysis of Vibrio vulnificus, a marine pathogen; Chen CY et al.; The halophile Vibrio vulnificus is an etiologic agent of human mortality from seafood-borne infections . We applied whole-genome sequencing and comparative analysis to investigate the evolution of this pathogen . The genome of biotype 1 strain, V . vulnificus YJ016, was sequenced and includes two chromosomes of estimated 3377 kbp and 1857 kbp in size, and a plasmid of 48,508 bp . A super-integron (SI) was identified, and the SI region spans 139 kbp and contains 188 gene cassettes . In contrast to non-SI sequences, the captured gene cassettes are unique for any given Vibrio species and are highly variable among V . vulnificus strains . Multiple rearrangements were found when comparing the 5.3-Mbp V . vulnificus YJ016 genome and the 4.0-Mbp V . cholerae El Tor N16961 genome . The organization of gene clusters of capsular polysaccharide, iron metabolism, and RTX toxin showed distinct genetic features of V . vulnificus and V . cholerae . The content of the V . vulnificus genome contained gene duplications and evidence of horizontal transfer, allowing for genetic diversity and function in the marine environment . The genomic information obtained in this study can be applied to monitoring vibrio infections and identifying virulence genes in V . vulnificus.

Arch Microbiol, 2004 Jan, 181(1), 17 - 25 Epub 2003 Dec 04.
Phylogenetic and physiological characterization of a filamentous anoxygenic photoautotrophic bacterium ' Candidatus Chlorothrix halophila' gen . nov., sp . nov., recovered from hypersaline microbial mats; Klappenbach JA et al.; We report the phylogenetic and physiological characterization of a mesophilic and halophilic member of the filamentous anoxygenic phototrophic (FAP) bacteria, provisionally named ' Candidatus Chorothrix halophila' gen . nov . sp . nov., that has been maintained in a highly enriched culture in our laboratory for over a decade . Phylogenetic analysis of small-subunit RNA-encoding sequences places ' Candidatus Chlorothrix halophila' in a clade that includes cultivated members of the genera Chloroflexus and Oscillochloris . Physiological studies demonstrated sulfide-dependent photosynthetic uptake of (14)C-labeled bicarbonate . Enzymatic assays for the activity of propionyl-coenzyme A synthase indicated that ' Candidatus Chlorothrix halophila' does not use the 3-hydroxypropionate cycle of Chloroflexus aurantiacus OK-70-fl for autotrophic carbon assimilation . New concepts regarding the taxonomy and phylogeny of FAP bacteria have emerged from this work.

Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2257 - 8 Epub 2003 Nov 27.
Crystallization of a novel alpha-amylase, AmyB, from the thermophilic halophile Halothermothrix orenii; Tan TC et al.; This is a report on the structure determination of AmyB, the second alpha-amylase from Halothermothrix orenii, by X-ray crystallography . This bacterium was isolated from saltpans where conditions consisted of both high temperatures and high NaCl content . AmyB is a 599-residue protein which is stable and significantly active at 358 K in starch solution containing up to 10%(w/v) NaCl . The purified recombinant AmyB protein crystallizes in the monoclinic space group C2, with unit-cell parameters a = 225.85, b = 77.16, c = 50.13 A, beta = 99.32 degrees, using the hanging-drop vapour-diffusion method . The crystal diffracts X-rays to a resolution limit of 1.97 A.

Environ Microbiol, 2003 Nov, 5(11), 1094 - 102
Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions; Mormile MR et al.; Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years . Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls) . U-Th disequilibrium dating indicates that these halite layers were deposited at 9,600 and 97,000 years before present (ybp) respectively . Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-specific 16S rDNA primers or placed into haloarchaea culture medium . Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloarchaea-containing prepared crystals (positive controls), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (negative controls) resulted in no growth . Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobacterium salinarum . DNA-DNA hybridization experiments confirmed that BBH 001 was closely related to H . salinarum (81-75% hybridization) and its ascription to this haloarchaea species . The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites.

Curr Microbiol, 2003 Oct, 47(4), 347 - 51
Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila; Baliarda A et al.; Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium . DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes . When expressed in the heterologous host E . coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T . halophila (i.e . carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine) . In E . coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction . This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.

Annu Rev Genet, 2003, 37, 283 - 328
Lateral gene transfer and the origins of prokaryotic groups; Boucher Y et al.; Lateral gene transfer (LGT) is now known to be a major force in the evolution of prokaryotic genomes . To date, most analyses have focused on either (a) verifying phylogenies of individual genes thought to have been transferred, or (b) estimating the fraction of individual genomes likely to have been introduced by transfer . Neither approach does justice to the ability of LGT to effect massive and complex transformations in basic biology . In some cases, such transformation will be manifested as the patchy distribution of a seemingly fundamental property (such as aerobiosis or nitrogen fixation) among the members of a group classically defined by the sharing of other properties (metabolic, morphological, or molecular, such as small subunit ribosomal RNA sequence) . In other cases, the lineage of recipients so transformed may be seen to comprise a new group of high taxonomic rank ("class" or even "phylum") . Here we review evidence for an important role of LGT in the evolution of photosynthesis, aerobic respiration, nitrogen fixation, sulfate reduction, methylotrophy, isoprenoid biosynthesis, quorum sensing, flotation (gas vesicles), thermophily, and halophily . Sometimes transfer of complex gene clusters may have been involved, whereas other times separate exchanges of many genes must be invoked.

Int J Food Microbiol, 2003 Dec 15, 89(1), 31 - 9
Retention of virulence in viable but non-culturable halophilic Vibrio spp; Baffone W et al.; The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V . parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking . Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC . When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model . For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture . Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures . Isolation was obtained in 25% of the animals challenged with the VBNC V . alginolyticus strain, in 37.5% of those challenged with the VBNC V . parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V . parahaemolyticus ATCC 43996 . The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity . The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model . The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 81 - 6
Megaplasmids in Gram-negative, moderately halophilic bacteria; Argandona M et al.; We have discovered that many Halomonas species harbour large extrachromosomal DNA elements . Using currently available protocols it is technically very difficult to identify large plasmids in bacteria, and even more so when they are coated in mucous polysaccharide . We used culture conditions suitable for both halophilic and halophilic exopolysaccharide-producing bacteria and applied a modified gel electrophoresis method to locate and visualise the megaplasmids . Almost all the species of Halomonas studied harbour two plasmids of about 70 kb and 600 kb and some species carry other smaller extrachromosomal DNA elements . The common presence of these megaplasmids may well be related to the survival strategies of the bacteria in their special surroundings.

Arch Microbiol . 2000 Dec 9;:52-61 {Epub ahead of print}
Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus; Johnsen U et al.; The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by (13)C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments . H . saccharolyticus grown on complex media containing glucose or fructose specifically (13)C-labeled at C1 and C3, formed acetate and small amounts of lactate . The (13)C-labeling patterns, analyzed by (1)H- and (13)C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway . Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea . Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase . During growth of H . saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed . After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased . Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose . Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses . The data indicate that, in the archaeon H . saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.

Protein Pept Lett, 2003 Oct, 10(5), 449 - 57
A new dehydrogenase specific towards aromatic aldehydes from a halophilic bacterium; La Cara F et al.; A new enzyme showing a dehydrogenase activity towards aromatic aldehydes was isolated, purified and characterized from a halophilic strain isolated from saline environment . The enzyme is a monomer of 54 kDa; it is rather thermostable (optimal temperature: 50 degrees C) showing a broad spectrum of activity in a large pH range with the maximum at pH 9.5 . The substrate specificity and the effect of ions were evaluated and compared with analogous described proteins.

Adv Exp Med Biol, 2001, 491, 109 - 26
Binding properties and applications of Aplysia gonad lectin; Gilboa-Garber N et al.; Adult Aplysia gonad contains high levels of a galactophilic lectin (MW around 65 kDa; composed of 2 subunits of apparent single species) . It binds galactose and various alpha/beta-galactosides (but not N-acetylgalactosamine), in addition to an outstanding high affinity for galacturonic acid . This lectin is relatively resistant to heating up to 70 degrees C and to alkaline pH, but sensitive to proteolysis and low pH . It resembles galectins in binding to poly LacNAc (preferentially branched) complexes at low temperatures (0 degrees-4 degrees C) more avidly than at room temperature or at 37 degrees C, but differs from them in being Ca(2+)-dependent . It agglutinates papain/sialidase-treated erythrocytes more strongly than untreated cells and stimulates mitosis in peripheral human lymphocytes (inducing IL-2 formation) . This lectin also enhances neurite outgrowth and increases their viability, while suppressing cell tumorigenicity . It is useful for histochemical/ cytochemical studies of galacturonic acid in plant tissues and fungi and for the study of cell surface composition of various prokaryotic (including halophilic Archaea) and eukaryotic cells and for their typing . It is useful as a reagent for I-antigen detection in adult human erythrocytes (anti-I), exhibiting strongest agglutination of O(h) Bombay-type erythrocytes and also exhibits sensitivity to the T antigen . It binds galactosylated molecules in human body fluids (shown by hemagglutination--inhibition tests), including saliva, seminal fluid and milk (detecting individual divergence) and in fowl egg albumens (exhibiting highest affinity for that of pigeon) . Therefore, it might be valuable as a probe and fishhook for fishing compounds exhibiting anti-bacterial/neoplastic cell adhesion activities.

Appl Environ Microbiol, 2003 Oct, 69(10), 6334 - 6
The high salt requirement of the moderate halophile Chromohalobacter salexigens DSM3043 can be met not only by NaCl but by other ions; O'Connor K et al.; The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na+, K+, Rb+, or NH4+, Cl-, Br-, NO3-, or SO4(2-) ions . To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously.

Planta Med, 2003 Aug, 69(8), 779 - 81
Halophilols A and B, two new stilbenes from Iris halophila; Wang YQ et al.; A new monomeric stilbene, halophilol A (1), and a new tetrastilbene, halophilol B (2), along with three known oligostilbenes were isolated from the seeds of Iris halophila . Their structures were established on the basis of the spectral data . The oligostilbene skeleton is encountered for the first time in the Iridaceae family . Bioactivity tests showed that 1 had moderate cytotoxicity against KB and HMEC cell lines (IC50 = 17.28 microM, 22.47 microM respectively), while 2 was inactive.

Syst Appl Microbiol, 2003 Sep, 26(3), 382 - 9
Methylophaga natronica sp . nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region; Doronina N et al.; A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described . The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission . It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium . Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids . The major ubiquinone is Q-8 . The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol . Diphosphatidylglycerol is also present . Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl . Cells accumulate ectoine and glutamate as the main osmoprotectants . The G + C content of the DNA is 45.0 mol% . Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).

Diabetes Metab, 2003 Sep, 29(4 Pt 1), 363 - 74
Low rate of glucose 6-phosphate hydrolysis in liver cells is a physiological feature of non-diabetic wild sand rats (Psammomys obesus); Koceir EA et al.; OBJECTIVE: In this study we have compared glucose metabolism and liver gluconeogenesis in wild adult desert gerbil Psammomys obesus fed with their natural halophilic plants and Wistar rats fed on a laboratory chow . Psammomys obesus is a natural model of insulin resistance when fed a rodent laboratory chow . METHODS: Basal glucose and insulin were determined in plasma of fasting animals . Hepatocyte gluconeogenesis from lactate-plus-pyruvate was investigated in perifused hepatocytes by assessing simultaneously glucose synthesis rate and intracellular oxaloacetate, phosphoenolpyruvate, 3-phosphoglycerate, fructose 6-phosphate and glucose 6-phosphate (G6P) under true steady state conditions . RESULTS: Fasting blood glucose (2.8 +/- 0.1 vs 4.8 +/- 0.4 mmol.L(- 1)) and plasma insulin concentration (129 +/- 14 vs 150 +/- 21 pmol.L(-1)) were significantly lower in Psammomys as compared to albino rats . Maximal gluconeogenic rate was also lower in Psammomys (2.3 +/- 0.3 vs 5.1 +/- 0.3 micromol x min(-1) x g dry cells(-1)) . This effect was related to a slower hydrolysis of G6P . CONCLUSION: A lower G6P hydrolysis in Psammomys as compared to wistar was the main difference between the two groups of liver cells . Such feature may represent the major metabolic adaptation permitting Psammomys to survive despite its severe restrictive natural conditions . Indeed, a low G6P hydrolysis allows an insulin resistance state, with a high lipogenic activity, but associated with low blood glucose . The rise in blood glucose occurring when Psammomys are fed with exogenous carbohydrates perturbs such delicate metabolic equilibrium, resulting thus in a diabetic state because of the deleterious effect of hyperglycemia.

J Bacteriol, 2003 Oct, 185(20), 5959 - 66
An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp . strain NRC-1; Berquist BR et al.; We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1 . The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid . Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation . Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication . Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp . The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted . Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved . Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication . We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.

Nucleic Acids Res Suppl, 2003, (3), 313 - 4
Gene cloning of ftsZ1 homolog from extremely halophilic archaeon Haloarcula japonica; Ozawa K et al.; The gene encoding FtsZ1 was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1 . Nucleotide sequencing analysis of the possible ftsZ1 gene revealed that the structural gene consisted of an open reading frame of 1,038 nucleotides encoding 346 amino acids . Transcription of the ftsZ1 gene in Ha . japonica was confirmed by RT-PCR.

Res Microbiol, 2003 Sep, 154(7), 483 - 90
Development and use of fluorescent 16S rRNA-targeted probes for the specific detection of Methylophaga species by in situ hybridization in marine sediments; Janvier M et al.; Methylotrophic bacteria are widespread in nature . They may play an important role in the cycling of carbon and in the metabolism of dimethylsulfide in a marine environment . Bacteria belonging to the genus Methylophaga are a unique group of aerobic, halophilic, non-methane-utilizing methylotrophs . Two 16S rRNA-targeted oligonucleotide probes were developed for the specific detection of Methylophaga species, marine methylobacteria, by fluorescence in situ hybridization . Probe MPH-730 was highly specific for all members of the genus Methylophaga while probe MPHm-994 targeted exclusively M . marina . The application of these probes were demonstrated by the detection of Methylophaga species in enrichment cultures from various marine sediments . All isolates recovered were visualized by using the genus specific probe MPH-730 . The results were confirmed by 16S rDNA sequencing which demonstrated that all selected isolates belong to Methylophaga . Five isolates could be detected by the M . marina-specific probe MPHm-994 and were confirmed by rRNA gene restriction pattern (ribotyping) . With the development of these specific probes, fluorescence in situ hybridization shows that the genus Methylophaga is widespread in marine samples.

Biochim Biophys Acta, 2003 Sep 22, 1633(3), 179 - 88
Novel polar lipids of halophilic eubacterium Planococcus H8 and archaeon Haloferax volcanii; Sprott GD et al.; As part of a study to identify novel lipids with immune adjuvant activity, a structural comparison was made between the polar lipids from two halophiles, an archaeon Haloferax volcanii and a eubacterium Planococcus H8 . H . volcanii polar lipid extracts consisted of 44% archaetidylglycerol methylphosphate, 35% archaetidylglycerol, 4.7% of archaeal cardiolipin, 2.5% archaetidic acid, and 14% sulfated glycolipids 1 and 2 . Nuclear magnetic resonance (NMR) and Fast atom bombardment mass spectrometry (FAB MS) data determined the glycolipids to be 6-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-{sn-2,3-di-O-phytanylglycerol} and a novel glycocardiolipin 6'-HSO(3)-D-Man(p)-alpha1-2-D-Glc(p)-alpha1,1-{sn-2,3-di-O-phytanylglycerol}-6-{phospho-sn-2,3-di-O-phytanylglycerol} . The polar lipids of Planococcus H8 consisted of 49% saturated phosphatidylglycerol and cardiolipin (9:1, w/w), and surprisingly 51% of the photosynthetic membrane lipid sulfoquinovosyldiacylglycerol (SQDG) . This study documents archaeal cardiolipin and a novel glycocardiolipin in H . volcanii (lacking purple membrane), and is the first report of SQDG in a non-photosynthetic, halophilic bacterium.

J Protein Chem, 2003 May, 22(4), 345 - 51
Activation of halophilic nucleoside diphosphate kinase by a non-ionic osmolyte, trimethylamine N-oxide; Ishibashi M et al.; The folding and activity of halophilic enzymes are believed to require the presence of salts at high concentrations . When the inactivated nucleoside diphosphate kinase (NDK) from extremely halophilic archaea was incubated with low salt media, no activity was regained over the course of 8 days . When it was incubated with approximately 2 M NaCl or 3 M KCl, however, it gradually regained activity . To our surprise, trimethylamine N-oxide (TMAO) also was able to induce activation at 4.0 M . The enzyme activity and secondary structure of refolded NDK in 4 M TMAO were comparable with those of the native NDK or the refolded NDK in 3.8 M NaCl . TMAO is not an electrolyte, meaning that the presence of concentrated salts is not an absolute requirement, and that charge shielding or ion binding is not a sole factor for the folding and activation of NDK . Although both NaCl and TMAO are effective in refolding NDK, the mechanism of their actions appears to be different: the effect of protein concentration and pH on refolding is qualitatively different between these two, and at pH 8.0 NDK could be refolded in the presence of 4 M TMAO only when low concentrations of NaCl are included.

J Lipid Res, 2004 Jan, 45(1), 194 - 201 Epub 2003 Sep 16.
Osmotic shock stimulates de novo synthesis of two cardiolipins in an extreme halophilic archaeon; Lopalco P et al.; The present report illustrates the response to osmotic stress of an extreme halophilic archaeon, Halorubrum sp., isolated from the saltern ponds of Margherita di Savoia in southern Italy . The hypotonic stress induces relevant changes in the membrane lipid composition: archaeal cardiolipin content markedly increases, whereas phosphatidylglycerol (PG) decreases . Membranes isolated from this archaeon after cell disruption by osmotic shock are highly enriched in archaeal cardiolipin and reveal the presence of a novel phospholipid . Electrospray ionization mass spectrometry and NMR analyses revealed that this novel lipid has the structure of a sulfo-diglyco-diether-phosphatidic acid, i.e., a phospholipid dimer or a novel cardiolipin analogue . As NMR analyses showed that the sugars in the novel phospholipid dimer are the same and in the same order of a sulfated diglycosyl diphytanylglycerol diether (S-DGD-5) present as a major lipid component in the archaeon membranes, the novel phospholipid dimer was named S-DGD-5-PA . We conclude that osmotic shock induces a specific increase in the membrane content of the two cardiolipins and suggest that PG and S-DGD-5 are intermediates for the de novo synthesis of archaeal cardiolipin and S-DGD-5-PA, respectively.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1625 - 30
Alteromonas marina sp . nov., isolated from sea water of the East Sea in Korea; Yoon JH et al.; Two Gram-negative, motile, non-spore-forming and moderately halophilic rods (strains SW-47(T) and SW-49) were isolated from sea water of the East Sea in Korea and subjected to a polyphasic taxonomic study . The two strains grew optimally between 30 and 37 degrees C, and grew at 4 and 44 degrees C but not at temperatures above 45 degrees C . They grew optimally in the presence of 2-5 % (w/v) NaCl, but did not grow in the absence of NaCl . Strains SW-47(T) and SW-49 had ubiquinone-8 (Q-8) as the predominant respiratory lipoquinone and C(16 : 1) omega7c and/or iso-C(15 : 0) 2OH, C(16 : 0) and C(18 : 1) omega7c as the major fatty acids, which are consistent with the corresponding data for Alteromonas macleodii . The DNA G+C contents of strains SW-47(T) and SW-49 were 45 and 44 mol%, respectively . Strains SW-47(T) and SW-49 showed a high level of 16S rDNA sequence similarity (99.9 %) and a mean level of DNA-DNA relatedness of 96.5 % . Phylogenetic analyses based on 16S rDNA sequences showed that the two strains form a coherent cluster with A . macleodii . Strains SW-47(T) and SW-49 exhibited levels of 16S rDNA sequence similarity of 99.3 and 99.1 %, respectively, with A . macleodii DSM 6062(T) and of less than 89.4 % with other species used in the phylogenetic analyses . Alteromonas fuliginea CIP 105339(T) was found to be more closely related to the genus Pseudoalteromonas than to the genus ALTEROMONAS: On the basis of phenotypic properties and phylogenetic and genomic data, strains SW-47(T) and SW-49 represent a new species of the genus Alteromonas, for which the name Alteromonas marina (type strain SW-47(T)=KCCM 41638(T)=JCM 11804(T)) is proposed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1591 - 4
Saccharomonospora paurometabolica sp . nov., a moderately halophilic actinomycete isolated from soil in China; Li WJ et al.; A novel, moderately halophilic actinomycete, strain YIM 90007(T), was isolated from a soil sample collected from the Xinjiang Province, China, and characterized . The optimum growth temperature of the strain was between 35 and 37 degrees C and growth occurred optimally in 10 % (w/v) NaCl . The cell wall of strain YIM 90007(T) contained meso-diaminopimelic acid . Whole-cell sugars were galactose, arabinose and ribose . The principal menaquinone was MK-9(H(4)), while MK-9(H(2)) was found in smaller amounts . The phospholipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and hydroxyphosphatidylethanolamine . The predominant cellular fatty acids were of the iso- and anteiso-branched and unbranched types; significant amounts of 2-hydroxy fatty acids were also found but 10-methyl-branched fatty acids were missing . The DNA G+C content of strain YIM 900007(T) was 71 mol% . Phylogenetic analysis based on 16S rRNA gene sequences showed strain YIM 90007(T) to be closely related to Saccharomonospora halophila (98.7 % similarity) . DNA-DNA hybridization revealed a relatedness of 53.8 % between strain YIM 90007(T) and S . halophila DSM 44411(T) . Based on physiological and biochemical characteristics, phylogenetic analysis (based on 16S rRNA gene sequences) and DNA-DNA relatedness, it is concluded that strain YIM 90007(T) represents a novel species of the genus Saccharomonospora, for which the name Saccharomonospora paurometabolica (type strain YIM 90007(T)=CCTCC AA001018(T)=CCRC 16315(T)=DSM 44619(T)) is proposed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1545 - 9
Prauserella halophila sp . nov . and Prauserella alba sp . nov., moderately halophilic actinomycetes from saline soil; Li WJ et al.; The actinomycete strains YIM 90001(T) and YIM 90005(T) were isolated on starch-casein agar {20 % (w/v) NaCl, pH 7.0} from a soil sample collected from Xinjiang Province in the west of China . The two isolates were aerobic and Gram-positive . The substrate mycelium was fragmented and an aerial mycelium was well developed on Czapek medium . The aerial mycelium formed long spore chains with branched short or long spore chains at maturity, which were straight to flexuous, and spores were non-motile . Based on the results of metabolic, chemotaxonomic and molecular analyses, the two isolates were identified as two new species of the genus Prauserella, for which the names Prauserella halophila for strain YIM 90001(T) (=DSM 44617(T)=CCTCC AA001015(T)) and Prauserella alba for strain YIM 90005(T) (=DSM 44590(T)=CCTCC AA001016(T)) are proposed.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1421 - 5
Streptomonospora alba sp . nov., a novel halophilic actinomycete, and emended description of the genus Streptomonospora Cui et al . 2001; Li WJ et al.; A halophilic actinomycete, strain YIM 90003(T), was isolated from a soil sample collected from Xinjiang Province, China, by using starch-casein agar with a salt concentration of 20 % (w/v), pH 7.0 . The strain grew well on most media tested . No diffusible pigment was produced . Aerial mycelium and substrate mycelium were well developed on most media . The aerial mycelium formed short spore chains, bearing non-motile, straight to flexuous spores with wrinkled surfaces . The cell walls of strain YIM 90003(T) contained meso-diaminopimelic acid as the diagnostic diamino acid . Cell-wall hydrolysates contained galactose and arabinose . Menaquinone composition varied with the medium used for cell cultivation; on glucose-yeast extract medium supplemented with 10 % NaCl, the major menaquinone was MK-9(H(4)), while, on vitamin-enriched ISP 2 medium, the major menaquinones were MK-10(H(2)), MK-9(H(8)) and MK-10(H(4)) . Phospholipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, diphosphatidyl glycerol, methylphosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and an unidentified phospholipid . 16S rRNA gene sequence analysis showed Streptomonospora salina as the closest phylogenetic neighbour . On the basis of these analyses, strain YIM 90003(T) is a member of the genus Streptomonospora, though its properties do not match the generic description fully with respect to the menaquinone composition and peptidoglycan amino acid . Analyses of mechanically disrupted cell walls of the type species, Streptomonospora salina DSM 44593(T), and strain YIM 90003(T), purified by tryptic digestion and subsequent SDS treatment, revealed the exclusive presence of meso-diaminopimelic acid as the diagnostic diamino acid of peptidoglycan . Thus, the genus description of Streptomonospora, indicating the presence of several amino acids usually not found in the peptidoglycan moiety, is therefore emended . DNA-DNA hybridization and comparison of physiological and chemotaxonomic characteristics demonstrated strain YIM 90003(T) to be different from Streptomonospora salina . The name Streptomonospora alba sp . nov . is proposed, with strain YIM 90003(T) (=CCTCC AA001013(T)=DSM 44588(T)) as the type strain.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1383 - 7
Marinobacter lipolyticus sp . nov., a novel moderate halophile with lipolytic activity; Martin S et al.; In the course of a screening programme in hypersaline habitats of southern Spain to isolate halophilic bacteria that are able to produce different extracellular enzymes, a novel, moderately halophilic bacterium (strain SM19(T)) that displays lipolytic activity has been isolated and characterized . Strain SM19(T) is a Gram-negative rod that grows optimally in culture media that contain 7.5 % NaCl . The DNA G+C content was 57.0 mol% . According to phenotypic and genotypic data, this strain was assigned to the genus MARINOBACTER: However, 16S rDNA sequence similarity between strain SM19(T) and species of the genus Marinobacter was <96.7 %; this value is sufficiently low to propose its designation as a novel species . In addition, DNA-DNA hybridization with reference strains of close phylogenetic relatives was between 11 and 19 % . On the basis of these data, the inclusion of strain SM19(T) in the genus Marinobacter as a novel species is proposed, with the name Marinobacter lipolyticus sp . nov . The type strain of the novel species is SM19(T) (=DSM 15157(T)=NCIMB 13907(T)=CIP 107627(T)=CCM 7048(T)).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1297 - 303
Bacillus marisflavi sp . nov . and Bacillus aquimaris sp . nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea; Yoon JH et al.; Two Gram-positive or Gram-variable, endospore-forming, moderately halophilic rods (strains TF-11(T) and TF-12(T)) were isolated from a tidal flat of the Yellow Sea in Korea and were subjected to a polyphasic taxonomic study . Strains TF-11(T) and TF-12(T) had cell-wall peptidoglycan based on meso-diaminopimelic acid . The predominant menaquinone found in the two strains was MK-7 . The cellular fatty acid profiles of both strains contained large amounts of branched and saturated fatty acids . The major fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0) . The DNA G+C contents of strains TF-11(T) and TF-12(T) were respectively 49 and 38 mol% . Phylogenetic analysis based on 16S rDNA sequences showed that strains TF-11(T) and TF-12(T) fell within the radiation of the cluster comprising Bacillus species . The level of 16S rDNA sequence similarity between strains TF-11(T) and TF-12(T) was 98.3 % . Strains TF-11(T) and TF-12(T) exhibited levels of 16S rDNA sequence similarity of less than 96.0 and 96.3 %, respectively, to Bacillus species . The mean level of DNA-DNA relatedness between the two strains was approximately 7 % . On the basis of phenotypic and phylogenetic data and genomic distinctiveness, strains TF-11(T) and TF-12(T) should be placed in the genus Bacillus as two distinct novel species, for which the names Bacillus marisflavi sp . nov . (type strain TF-11(T)=KCCM 41588(T)=JCM 11544(T)) and Bacillus aquimaris sp . nov . (type strain TF-12(T)=KCCM 41589(T)=JCM 11545(T)) are proposed.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 181 - 6
Occurrence of two different glutamate dehydrogenase activities in the halophilic bacterium Salinibacter ruber; Bonete MJ et al.; Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII . GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts . GDHII depended on high salt concentrations for both activity and stability . It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8 . No activating effect was found when NaCl was replaced by KCl . Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5 . Both enzymes were activated by certain amino acids (L-leucine, L-histidine, L-phenylalanine) and by nucleotides such as ADP or ATP . A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.

Biochim Biophys Acta, 2003 Sep 8, 1623(1), 47 - 51
NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway; Martinez-Espinosa RM et al.; Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source . The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin . This ferredoxin has been purified and characterised . Air-oxidized H . mediterranei ferredoxin has a UV-visible absorption spectra typical of 2Fe-type ferredoxins with an A420/A280 of 0.21 . The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a {2Fe-2S} cluster . The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1) . The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Biosci Biotechnol Biochem, 2003 Aug, 67(8), 1809 - 12
Organic solvent tolerance of halophilic archaea; Usami R et al.; Organic solvent tolerance was tested in type strains of type species of the sixteen genera of Halobacteriaceae, the halophilic archaea . Most of the strains were observed to grow in the presence of hexylether (log Pow=5.1), but none grew in the presence of n-octane (log Pow=4.9) except Halogeometricum borinquense JCM 10706T and Halorubrum saccharovorum JCM 8865T . On the other hand, two strains, Haloarcula spp . OHF-1 and 2 isolated from a French solar salt were found to show stronger tolerance even to isooctane (log Pow=4.8) . Growth of some strains was retarded by the presence of n-decane but reached to the same cell densities at late stationary phase . Final cell densities of some strains were greatly repressed by the presence of the solvent.

Biochemistry, 2003 Sep 2, 42(34), 10054 - 9
Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy; Groot ML et al.; Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light . Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates . Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy . Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur . The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process . We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps . Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore . Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.

Extremophiles, 2003 Aug, 7(4), 319 - 26 Epub 2003 May 27.
Mauran, an exopolysaccharide produced by the halophilic bacterium Halomonas maura, with a novel composition and interesting properties for biotechnology; Arias S et al.; Mauran is an anionic, sulfated heteropolysaccharide with a high uronic-acid content, synthesized by strain S-30 of the halophilic bacterium Halomonas maura . Under optimum environmental and nutritional conditions, it is capable of producing up to 3.8 g of mauran per liter of medium . Aqueous solutions of mauran are highly viscous and display pseudoplastic, viscoelastic and thixotropic behavior . Its viscosity is stable over a wide pH range (3-11), after freezing-thawing processes, and in the presence of sucrose, salts, surfactants and alpha-hydroxyl acids . It has a high capacity for binding lead and other cations . Its molecular mass when collected from an MY medium supplemented with 2.5% w/v salt during the stationary growth phase is 4.7x10(6) Da.

Extremophiles, 2003 Aug, 7(4), 299 - 306 Epub 2003 Apr 24.
Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei; Perez-Pomares F et al.; The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source . When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration . Here we report methods to concentrate and purify the enzyme . The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa . These results indicated that the halophilic alpha-amylase is a monomeric enzyme . The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl . The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C . The results also implicated several prototropic groups in the catalytic reaction.

Microb Ecol, 2003 Oct, 46(3), 291 - 301 Epub 2003 Aug 14.
Characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (T-RFLP); Ovreas L et al.; The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA . These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches . Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied . The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments . The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity . At 37% salinity the diversity was reduced again to nearly half that at 22% salinity . Our results revealed that the community "genome" had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome . The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds . They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity . The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT . The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques . In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP . One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.

Nucleic Acids Symp Ser, 2000, (44), 155 - 6
Cloning and sequencing of ftsZ homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1; Kazumichi O et al.; The gene encoding FtZ was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1 . Nucleotide sequencing analysis of the possible ftsZ gene revealed that the structural gene consisted of an open reading frame of 1,182 nucleotides encoding 394 amino acids . The deduced amino acid sequence of the Ha . japonica FtsZ showed high identities with those Halobacterium salinarom, Haloferax volcanii and Haloferax mediterranei FtsZs.

Nucleic Acids Symp Ser, 2000, (44), 83 - 4
Threonyl-tRNA synthetase of archaea: importance of the discriminator base in the aminoacylation of threonine tRNA; Ishikura H et al.; To investigate the contribution of the discriminator base of archaeal tRNA(Thr) in aminoacylation by threonyl-tRNA synthetase (ThrRS), cross-species aminoacylation between Escherichia coli and Haloferax volcanii, halophilic archaea, was studied . It was found that E . coli ThrRS threonylated the H . volcanii tRNA(Thr) but that E . coli threonine tRNA was not aminoacylated by H . volcanii ThrRS . Results of a threonylation experiment using in vitro mutants of E . coli threonine tRNA showed that only the mutant tRNA(Thr) having U73 was threonylated by H . volcanii ThrRS . These findings indicate that the discriminator base U73 of H . volcanii tRNA(Thr) is a strong determinant for the recognition by ThrRS.

Nucleic Acids Symp Ser, 2000, (44), 1 - 2
The gene encoding a novel halorhodopsin-like protein of extremely halophilic archaeon Haloarcula japonica strain TR-1; Yatsunami R et al.; The gene encoding the halorhodopsin (hR)-like protein was cloned and sequenced from Haloarcula japonica strain TR-1 . The structural gene consisted of an open reading frame of 828 nucleotides encoding 276 amino acids . The deduced amino acid sequence of the Ha . japonica hR-like protein showed the highest homology to those of cruxhalorhodopsins . Furthermore, we have demonstrated that the Ha . japonica chR gene is transcriptionally induced by light.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1169 - 74
Erythrobacter flavus sp . nov., a slight halophile from the East Sea in Korea; Yoon JH et al.; Two gram-negative, motile, non-spore-forming, yellow-pigmented and slightly halophilic strains (SW-46T and SW-52) were isolated from sea water of the East Sea, Korea, and subjected to a polyphasic taxonomic study . Strains SW-46T and SW-52 were characterized chemotaxonomically by having ubiquinone-10 (Q-10) as the predominant respiratory lipoquinone and C18:1 omega7c as the major fatty acid . Their DNA G + C content was 64.0-64.1 mol% . Strains SW-46T and SW-52 showed 1 bp difference in their 16S rDNA sequences and a mean DNA-DNA relatedness level of 94.4% . Phylogenetic analysis based on 16S rDNA sequences showed that strains SW-46T and SW-52 fall within the alpha-subclass of the Proteobacteria and form a coherent cluster with Erythrobacter longus, Erythrobacter litoralis and Erythrobacter citreus . Levels of 16S rDNA similarity between strains SW-46T and SW-52 and the type strains of these three Erythrobacter species were 96.5-97.9% . Levels of DNA-DNA relatedness between strains SW-46T and SW-52 and the type strains of E . longus, E . litoralis and E . citreus were 3.6-14.7% . Therefore, on the basis of phenotypic properties, phylogeny and genomic data, strains SW-46T and SW-52 should be placed in the genus Erythrobacter as a novel species, for which the name Erythrobacter flavus sp . nov . is proposed . The type strain is SW-46T (= KCCM 41642T = JCM 11808T).

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1115 - 22
Aurantimonas coralicida gen . nov., sp . nov., the causative agent of white plague type II on Caribbean scleractinian corals; Denner EB et al.; A bacterium previously isolated from a diseased colony of the scleractinian coral Dichocoenia stokesi (common name elliptical star coral) was subjected to a detailed polyphasic taxonomic characterization . The isolate, designated WP1T, was halophilic and strictly aerobic and formed golden-orange-pigmented colonies after prolonged incubation . Cells of WP1T were gram-negative, rod-shaped and showed a characteristic branching rod morphology . Chemotaxonomically, WP1T was characterized by having Q-10 as the major respiratory lipoquinone and sym-homospermidine as the main component of the cellular polyamine content . The predominant constituent in the cellular fatty acid profile was C18:1 omega7c, along with C19:0 cyclo omega8c and C16:0 . Other fatty acids present in smaller amounts were C17:0, C18:0, C16:1 omega7c, C20:1 omega7c and C18:1 2-OH . The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine . Minor amounts of diphosphatidylglycerol, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine were present . The G + C content of the genomic DNA was 66.3 mol% . Phylogenetic analysis of the 16S rRNA gene sequence showed that WP1T represents a separate subline of descent within the order 'Rhizobiales' of the 'Alphaproteobacteria' . The new line of descent falls within the group of families that includes the Rhizobiaceae, Bartonellaceae, Brucellaceae and 'Phyllobacteriaceae', with no particular relative within this group . The 16S rRNA gene sequence similarity to all established taxa within this group was not higher than 92.0% (to Mesorhizobium mediterraneum) . To accommodate this emerging coral pathogen, the creation of a new genus and species is proposed, Aurantimonas coralicida gen . nov., sp . nov . (type strain WP1T = CIP 107386T = DSM 14790T).

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1059 - 63
Halobacillus karajensis sp . nov., a novel moderate halophile; Amoozegar MA et al.; A moderately halophilic, gram-positive, spore-forming bacterium was isolated from surface saline soil of the Karaj region, Iran . The strain, designated MA-2T, was strictly aerobic with rod-shaped cells that occurred singly, in pairs or short chains . It contained L-Om-D-Asp-type peptidoglycan and the major respiratory lipoquinone was MK-7 . It was non-motile and had an ellipsoidal endospore located centrally or subterminally . Growth occurred at 10-49 degrees C and in the pH range 6.0-9.6 . Strain MA-2T grew at salinities of 1-24% (w/v) NaCl, showing optimal growth at 10% (w/v) . The DNA G + C content was 41.3 mol% . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MA-2T was associated with Bacillus rRNA group 1 . The micro-organisms showing the closest phylogenetic relationship to strain MA-2T were Halobacillus litoralis and Halobacillus trueperi . On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA-DNA similarity data, it is proposed that strain MA-2T (= DSM 14948T = LMG 21515T) should be placed in the genus Halobacillus as the type strain of a novel species, Halobacillus karajensis sp . nov.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1043 - 9
Clostridium caminithermale sp . nov., a slightly halophilic and moderately thermophilic bacterium isolated from an Atlantic deep-sea hydrothermal chimney; Brisbarre N et al.; A strictly anaerobic, slightly halophilic and moderately thermophilic, sporulating rod designated strain DVird3T was isolated from deep-sea hydrothermal vent samples collected at a depth of approximately 800 m on the Atlantic Ocean Ridge . Strain DVird3T possessed a few laterally inserted flagella, had a DNA G + C content of 33.1 mol% and grew optimally at pH 6.6 and at 45 degrees C . Growth was observed at temperatures between 20 and 58 degrees C and at pH values between 5.8 and 8.2 . The optimum NaCl concentration for growth was 3% sea salt (30 g l(-1)); no growth was observed in the presence of 15 or 60 g sea salt l(-1) . Strain DVird3T is heterotrophic and utilizes some sugars and various single amino acids . Acetate was the main fatty acid detected from carbohydrate fermentation, together with H2 and CO2 . Gelatin was used as an energy source . It performed the Stickland reaction . Phylogenetically, strain DVird3T branched with members of cluster XI of the order Clostridiales, with Clostridium halophilum as its closest relative (similarity of 94.6%) . On the basis of its phenotypic, genotypic and phylogenetic characteristics, strain DVird3T (= DSM 15212T = CIP 107654T) is proposed as the type strain of a novel species of the genus Clostridium, Clostridium caminithermale sp . nov.

Extremophiles, 2003 Oct, 7(5), 423 - 31 Epub 2003 Jul 18.
Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium; Wejse PL et al.; The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8 . Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography . The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively . Stimulation of activity by Ca(2+), Mn(2+), Mg(2+), Ba(2+), Li(2+), NaN(3) and isopropanol was observed . The K(m) and V(max) values determined for Xyl 1 with 4- O-methyl- d-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively . The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein . Xylobiose and xylotriose were the major end products for both endoxylanases . The xylanases were stable at pH 4-11 showing pH optima around pH 6 . Xyl 1 shows maximal activity at 60 degrees C, Xyl 2 at 65 degrees C (at 4 M NaCl) . The xylanases showed high temperature stability with half-lives at 60 degrees C of 97 min and 192 min respectively . Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.

Mol Cell Proteomics, 2003 Aug, 2(8), 506 - 24 Epub 2003 Jul 18.
Proteomic Analysis of an Extreme Halophilic Archaeon, Halobacterium sp . NRC-1; Goo YA et al.; Halobacterium sp . NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate . Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified . This represents approximately one-fifth of the theoretical proteome of Halobacterium . Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions . There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively . Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified . Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1 . A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected . This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations.

Syst Appl Microbiol, 2003 Jun, 26(2), 189 - 96
Enhygromyxa salina gen . nov., sp . nov., a slightly halophilic myxobacterium isolated from the coastal areas of Japan; Iizuka T et al.; Six isolates of novel marine myxobacteria, designated strains SHK-1T, SMK-1-1, SMK-1-3, SMK-10, SKK-2, and SMP-6, were obtained from various coastal samples (mud, sands and algae) collected around Japan . All of the isolates had Gram-negative rod-shaped cells, motile by gliding and grew aerobically . They showed bacteriolytic action, fruiting body formation, and NaCl requirement for growth with an optimum concentration of 1.0-2.0% (w/v) . In addition, divalent cationic components of seawater, such as Mg2+ or Ca2+, were also needed for growth . The major respiratory quinone was MK-7 . The G+C content of genomic DNA ranged from 65.6 to 67.4 mol% (by HPLC) . The isolates shared almost identical 16S rDNA sequences, and clustered with a recently described marine myxobacterium, Plesiocystis pacifica, as their closest relative on a phylogenetic tree (95.9-96.0% similarity) . Physiological and chemotaxonomic differences between the new strains and strains of the genus Plesiocystis justify the proposal of a new genus . Therefore, we propose to classify the six isolates into a new taxon of marine myxobacteria with the name, Enhygromyxa salina gen . nov., sp . nov . The type strain is SHK-1(T) (JCM 11769(T) = DSM 15217(T) = AJ 110011(T)).

Syst Appl Microbiol, 2003 Jun, 26(2), 172 - 6
Bacillus aeolius sp . nov . a novel thermophilic, halophilic marine Bacillus species from Eolian Islands (Italy); Gugliandolo C et al.; Phylogenetic relationships of a thermophilic, halophilic, aerobic spore-forming strain 4-1(T), isolated from the water of a shallow sea hot spring at Vulcano Island (Italy), revealed its relatedness to members of the genus Bacillus . Chemotaxonomic and phenotypic properties of strain 4-1(T) are sufficiently different from related moderately thermophilic species, e.g., B . smithii, B . fumarioli, B . oleronius, B . sporothermodurans and B . infernus to describe strain 4-1(T) as a new Bacillus species, for which the name Bacillus aeolius sp . nov . is proposed . Strain 4-1(T) is characterised by the potential biotechnological important properties such as exopolysaccharide production, surfactant activity, and utilisation of hydrocarbons.

J Gen Appl Microbiol, 2003 Apr, 49(2), 95 - 100
Taxonomic homogeneity of a salt-tolerant lactic acid bacteria isolated from shoyu mash; Hanagata H et al.; Forty-seven salt-tolerant lactic acid bacteria, which had been isolated from different places and grown in 15% NaCl, were examined to assess their taxonomic heterogeneity . Among the isolates, 42 were isolated from shoyu mash during the acid fermentation phase, 2 were from miso and 3 were from anchovy pickles . All isolates were identified as Tetragenococcus halophilus on the basis of DNA relatedness values . We further examined 102 phenotypic characteristics of them . The isolates exhibited differences in only 16, supporting the conclusion obtained from the DNA relatedness analysis.

J Biol Chem, 2003 Sep 19, 278(38), 36556 - 62 Epub 2003 Jun 23.
Conformational changes detected in a sensory rhodopsin II-transducer complex; Bergo V et al.; Sensory rhodopsins (SRs) are light receptors that belong to the growing family of microbial rhodopsins . SRs have now been found in all three major domains of life including archaea, bacteria, and eukaryotes . One of the most extensively studied sensory rhodopsins is SRII, which controls a blue light avoidance motility response in the halophilic archaeon Natronobacterium pharaonis . This seven-helix integral membrane protein forms a tight intermolecular complex with its cognate transducer protein, HtrII . In this work, the structural changes occurring in a fusion complex consisting of SRII and the two transmembrane helices (TM1 and TM2) of HtrII were investigated by time-resolved Fourier transform infrared difference spectroscopy . Although most of the structural changes observed in SRII are conserved in the fusion complex, several distinct changes are found . A reduction in the intensity of a prominent amide I band observed for SRII indicates that its structural changes are altered in the fusion complex, possibly because of the close interaction of TM2 with the F helix, which interferes with the F helix outward tilt . Deprotonation of at least one Asp/Glu residue is detected in the transducer-free receptor with a pKa near 7 that is abolished or altered in the fusion complex . Changes are also detected in spectral regions characteristic of Asn and Tyr vibrations . At high hydration levels, transducer-fusion interactions lead to a stabilization of an M-like intermediate that most likely corresponds to an active signaling form of the transducer . These findings are discussed in the context of a recently elucidated x-ray structure of the fusion complex.

Extremophiles, 2003 Oct, 7(5), 409 - 13 Epub 2003 Jun 19.
Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization; Rossello-Mora R et al.; The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp . and the conspicuous square Archaea had been detected . Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines . The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate . However, Salinibacter spp . only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated . No glycerol incorporation was observed for any component of the microbial community.

Cell Mol Biol Lett, 2003, 8(2), 285 - 96
Visual and archaeal rhodopsins: similarities, differences and controversy; Bryl K; Rhodopsins are currently known to belong to two distinct protein families . The visual rhodopsins, found in eyes throughout the animal kingdom, are photosensory pigments . Archaeal rhodopsins, found in extreme halophiles, function as light-driven proton pumps (bacteriorhodopsins), chloride ion pumps (halorhodopsins), or photosensory receptors (sensory rhodopsins) . Light absorption by rhodopsins triggers their characteristic photoconversion extending into the (milli)second time range . There are three main paradigms of rhodopsins photoconversion . (1) Initiation of the trans-cis isomerization is the very primary consequence of light absorption . (2) Rhodopsins store light energy via the charge-separation mechanism (the charge of Schiff base is separated from its counterion) . (3) Full trans-cis isomerization of the chromophore is a prerequisite for the full biological activity of rhodopsins . These paradigms will be questioned.

RNA, 2003 Jul, 9(7), 794 - 801
Aminoacylation of an unusual tRNA(Cys) from an extreme halophile; Evilia C et al.; The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium . This raises the fundamental question of how halophiles can maintain the specificity of protein-nucleic acid interactions that are particularly sensitive to high salts in mesophiles . Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase . Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts . This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution . Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core . This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 829 - 32
Marinomonas primoryensis sp . nov., a novel psychrophile isolated from coastal sea-ice in the Sea of Japan; Romanenko LA et al.; Two bacterial strains (KMM 3633(T) and KMM 3634) were isolated from marine coastal sea-ice and characterized by using phenotypic and molecular methods . The isolates were aerobic, Gram-negative, psychrophilic, halophilic and motile by means of a single polar flagellum . The DNA G + C content was 45.3-45.6 mol% . The major cellular fatty acids were C(16 :0), C(16 : 1)omega9c and C(18 : 1)omega7c . Comparison of almost-complete 16S rDNA sequences demonstrated that the strains were phylogenetically closely related to each other (99.5% sequence identity), and related to Marinomonas species (94.4-96.4% identity) . DNA-DNA reassociation between KMM 3633(T) and KMM 3634 occurred at a level of 92% . Based on phenotypic, chemotaxonomic and phylogenetic properties, the name Marinomonas primoryensis sp . nov . is proposed for strains KMM 3633(T) and KMM 3634; the type strain is KMM 3633(T) (= JCM 11775(T) = NRIC 523(T)).

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 711 - 20
Marinilactibacillus psychrotolerans gen . nov., sp . nov., a halophilic and alkaliphilic marine lactic acid bacterium isolated from marine organisms in temperate and subtropical areas of Japan; Ishikawa M et al.; A novel marine lactic acid rod bacterium has been described for eight strains isolated from living and decomposing marine organisms collected from temperate and subtropical areas of Japan . The isolates were Gram-positive, catalase-negative, non-sporulating and motile with peritrichous flagella . They were slightly halophilic, highly halotolerant and alkaliphilic; the optimum NaCl concentration for growth was 2.0-3.75% (w/v) with a range from 0 to 17.0-20.5% (depending on the strain); the optimum pH was between 8.0 and 9.5 with a range from 6.0 to 10.0 . They were psychrotolerant, growing well at -1.8 degrees C with a maximum at 40-45 degrees C and the optimum at 37-40 degrees C . Lactate yields were 87-100% per consumed glucose; the residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 . The product composition was markedly affected by the pH of fermentation medium; at higher pH, the yield of lactate decreased (60-65% at pH 9.0) and that of other products increased conversely . The cell-wall peptidoglycan type was type A4beta, Orn-D-Glu, whereas that of the genus Alkalibacterium, the phylogenetically closest lactic acid bacterium, was type A4beta, Orn-D-Asp . The major cellular fatty acids were C16 : 0, C16 : 1delta9, C18 : 0 and C18 : 1delta9 (oleic acid) . The G + C content of the DNA was 34.6-36.2 mol% . The eight isolates were phenotypically homogeneous and formed a single genomic species . The 16S rRNA gene sequence analysis indicated that the isolates constituted an independent phylogenetic lineage within the radiation of lactic acid bacteria with 96.2% similarity to the genus Alkalibacterium . The secondary structure and the nucleotide sequence of the V6 region of the 16S rRNA were characteristic of the organism among other related lactic acid genera . On the bases of phenotypic and phylogenetic distinctness, the organism was proposed to belong to a new genus and species, Marinilactibacillus psychrotolerans gen . nov., sp . nov . The type strain, M13-2(T) (G + C = 36.2 mol%), has been deposited in the IAM, NBRC, NCIMB and NRIC culture collections as IAM 14980(T), NBRC 100002(T), NCIMB 13873(T) and NRIC 0510(T), respectively.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 687 - 93
Halobacillus salinus sp . nov., isolated from a salt lake on the coast of the East Sea in Korea; Yoon JH et al.; A Gram-positive, rod-shaped, endospore-forming, halophilic bacterium (strain HSL-3(T)) was isolated from a salt lake near Hwajinpo beach on the East Sea in Korea and was subjected to a polyphasic taxonomic study . Strain HSL-3(T) grew optimally in the presence of 2-10% (w/v) NaCl . Strain HSL-3(T) showed poor growth in the absence of NaCl and grew in the presence of less than 23% NaCl . The cell wall peptidoglycan type of strain HSL-3(T) was A4beta based on L-Orn-D-Asp . The predominant menaquinone found in strain HSL-3(T) was menaquinone-7 (MK-7) . Strain HSL-3(T) had a cellular fatty acid profile containing large amounts of branched fatty acids; the major fatty acids were anteiso-C(15 : 0), iso-C(15 : 0) and iso-C(16 : 0) . The DNA G + C content of strain HSL-3(T) was 45 mol% . Phylogenetic analysis based on 16S rDNA sequences showed that strain HSL-3(T) falls within the radiation of the cluster comprising Halobacillus species . Strain HSL-3(T) exhibited levels of 16S rDNA similarity of 97.4-98.4% to the type strains of Halobacillus species . Levels of DNA-DNA relatedness between strain HSL-3(T) and the type strains of all validly named Halobacillus species were in the range 7.3-9.2% . On the basis of phenotypic and phylogenetic data and the genomic distinctiveness, strain HSL-3(T) (= KCCM 41590(T) = JCM 11546(T)) should be placed in the genus Halobacillus as the type strain of a novel species, for which the name Halobacillus salinus sp . nov . is proposed.

Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 647 - 51
Glaciecola mesophila sp . nov., a novel marine agar-digesting bacterium; Romanenko LA et al.; Alteromonas-like strains KMM 241(T) and KMM 642, isolated from marine invertebrate specimens, were investigated to clarify their taxonomic position . The novel isolates were aerobic, Gram-negative, motile, slightly halophilic and heterotrophic and hydrolysed polysaccharides . They did not hydrolyse urea, gelatin or casein and produced acid weakly from carbohydrates . The DNA G + C content ranged between 44.6 and 44.8 mol% . DNA-DNA similarity between the two strains was 71% . Comparison of the 16S rRNA gene sequence of strain KMM 241(T) revealed 94.5-94.8% similarity to Glaciecola species . The novel strains shared several phenotypic and physiological properties with members of Glaciecola, but they differed in their lack of pigment production, their minimal and maximal growth temperatures and their ability to hydrolyse agar and carrageenan and in the utilization of organic compounds . On the basis of phenotypic and physiological characteristics as well as phylogenetic analysis, the isolates should be assigned to a novel species, Glaciecola mesophila sp . nov . The type strain is strain KMM 241(T) (=DSM 15026(T)).

Folia Microbiol (Praha), 2003, 48(2), 211 - 7
Physiological characterization of osmotolerant yeast Pichia sorbitophila and comparison with a putative synonym Pichia farinosa; Maresova L et al.; The osmotolerant yeast Pichia sorbitophila was found to differ from other yeast species, not only from the conventional ones (Saccharomyces cerevisiae, Schizosaccharomyces pombe), but also from those widely known as osmotolerant (Debaryomyces hansenii, Zygosaccharomyces rouxii) . P . sorbitophila was able to survive extremely high extracellular concentrations of salts (e.g., saturated solution of KCl) and other osmolytes (70% glucitol), although it is not classified as halophilic (or osmophilic) . P . sorbitophila assimilated a broad range of carbon and nitrogen sources with extreme effectiveness . On solid media, P . sorbitophila created colonies of variable shapes and sizes in relation to media composition, number of colonies on the plate and cultivation conditions . Colonies were able to produce long-distance signals between each other that resulted in growth inhibition of the facing parts of both colonies, but were not inhibited by colonies of other yeast species growing on the same plate . Though sometimes P . sorbitophila has been indicated as a synonym of P . farinosa, comparative physiological studies together with PCR amplification of P . farinosa DNA fragments homologous to known P . sorbitophila genes provided a strong indication that this strain should be classified as a separate species.

FEMS Microbiol Lett, 2003 Jun 6, 223(1), 83 - 7
Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber; Oren A et al.; Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by a small number of sugars (glucose, maltose, starch at 1 g l(-1)) . Glucose consumption starts after other substrates have been depleted . Glucose metabolism proceeds via a constitutive, salt-inhibited hexokinase and a constitutive salt-dependent nicotinamide adenine dinucleotide phosphate (NADP)-linked glucose-6-phosphate dehydrogenase . Glucose dehydrogenase and fructose-1,6-bisphosphate aldolase activity could not be detected . It is therefore suggested that Salinibacter metabolizes glucose by the classic Entner-Doudoroff pathway and not by the Embden-Meyerhof glycolytic pathway or by the modified Entner-Doudoroff pathway present in halophilic Archaea of the family Halobacteriaceae, in which the phosphorylation step is postponed . However, activity of 2-keto-3-deoxy-6-phosphogluconate aldolase could not be detected in extracts of Salinibacter cells, whether or not grown in the presence of glucose.

Mol Microbiol, 2003 Jun, 48(6), 1647 - 64
Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system; Kim SY et al.; Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections . We tested whether V . vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum-sensing system 1 and/or 2 . Although there was no evidence for signalling system 1, we found that V . vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V . harveyi through signalling system 2 . Maximal autoinducer 2 (AI-2) activity was observed during mid-exponential to early stationary phase and disappeared in the late stationary phase when V . vulnificus was grown in heart infusion broth containing 2.5% NaCl . V . vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0) . From a cosmid library of V . vulnificus type strain ATCC 29307, we have identified the AI-2 synthase gene (luxSVv) showing 80% identity with that of V . harveyi (luxSVh) at the amino acid level . To investigate the pathogenic role of luxSVv, a deletion mutant of the clinical isolate V . vulnificus MO6-24/O was constructed . The luxSVv mutant showed a significant delay in protease production and an increase in haemolysin production . The decreased protease and increased haemolysin activities were restored to the isogenic wild-type level by complementation with the wild-type luxSVv allele . The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild-type bacteria . Transcriptional activities of the haemolysin gene (vvhA) and protease gene (vvpE) were also observed in the mutant using chromosomal PvvhA::lacZ and PvvpE::lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation . The mutation resulted in an attenuation of lethality to mice . Intraperitoneal LD50 of the luxSVv mutant increased by 10- and 750-fold in ferric ammonium citrate-non-overloaded and ferric ammonium citrate-overloaded mice respectively . The time required for the death of mice was also significantly delayed in the luxSVv mutant . Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation . Taken together, the V . vulnificus LuxS quorum-sensing system seems to play an important role in co-ordinating the expression of virulence factors.

Int J Food Microbiol, 2003 Jul 15, 84(1), 13 - 20
Potential osmoprotectants for the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila; Baliarda A et al.; The physiological responses of the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila (formely known as P . halophila), subjected to osmotic stress in the presence of molecules known to act as osmoprotectants for other bacteria were studied . In a defined medium, glycine betaine, dimethylsulfonioacetate, choline, proline and L-carnitine were able to relieve inhibition of growth at 0.8 M NaCl . The five compounds were shown to efficiently compete with glycine betaine transport, suggesting the existence of common transporter(s) for these molecules . T . halophila, the most tolerant strain, exhibited a larger spectrum of compatible solutes including dimethylsulfonioacetate, dimethylsulfoniopropionate and ectoine . Preliminary data suggest that restoration of growth by ectoine under osmotic constraint seems specific to the genus Tetragenococcus.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 686 - 9
The archaeal twin-arginine translocation pathway; Hutcheon GW et al.; The twin-arginine translocation (Tat) pathway is a system with the unique ability to export proteins in a fully folded conformation . Its main components are TatA, TatB and TatC, all of which are required for Tat-dependent export . The Tat pathway is found in several Archaea, and in most of them a moderate number of predicted Tat-dependent substrates are present . Putative substrates include those binding cofactors such as iron-sulphur clusters and molybdopterin . In these Archaea, the role of the Tat pathway seems to be similar to that of bacteria: the export of a small subset of proteins that fold before translocation across the cytoplasmic membrane . The exception to this is the Tat system of the halophilic archaeon Halobacterium sp . NRC-1 . In this organism, the majority of extra-cytoplasmic proteins are predicted to use the Tat pathway, which is, most likely, a specific adaptation to its particular lifestyle in highly saline conditions.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 681 - 5
The chaperonins: perspectives from the Archaea; Lund PA et al.; Heat-shock protein (Hsp) 60 chaperones are almost ubiquitous and almost always essential . They can be divided on the basis of sequence homology into two broad types: group I (found in bacteria, mitochondria and chloroplasts) and group II (found in Archaea and the eukaryotic cytosol) . Of the two, the group I chaperones are the better understood . Data on their structure, mechanism of action and cellular role will be briefly presented . The group II chaperones are less well studied . In eukaryotes they form large complexes with 8-fold symmetry containing eight different subunits, all of which are essential . They appear to have a major role in the folding of actin and tubulin, although they may also act on other substrates . No crystal structures are available for these complexes . The situation in the Archaea is simpler, with organisms containing between one and three genes for these chaperones . A 2.6 A structure exists for one archaeal group II chaperone complex . Some progress has been made in defining the reaction cycle of the archaeal group II chaperones and this has shown that they have some properties distinct from the group I chaperones . To date, the in vivo role and importance of the archaeal group II Hsp60 chaperones has not been determined . We have now shown that in the halophilic archaeon Haloferax volcanii not all the genes for these proteins are essential . Further analysis of these proteins in the Archaea should be very productive in yielding more information about these important chaperones and their cellular functions.

Biochem Soc Trans, 2003 Jun, 31(Pt 3), 677 - 80
Halophilic adaptation of protein-DNA interactions; Bergqvist S et al.; Pyrococcus woesei ( Pw ) is an archaeal organism adapted to living in conditions of elevated salt and temperature . Thermodynamic data reveal that the interaction between the TATA-box-binding protein (TBP) from this organism and DNA has an entirely different character to the same interaction in mesophilic counterparts . In the case of the Pw TBP, the affinity of its interaction with DNA increases with increasing salt concentration . The opposite effect is observed in all known mesophilic protein-DNA interactions . The halophilic behaviour can be attributed to sequestration of cations into the protein-DNA complex . By mutating residues in the Pw TBP DNA-binding site, potential sites of cation interaction can be removed . These mutations have a significant effect on the binding characteristics, and the halophilic nature of the Pw TBP-DNA interaction can be reversed, and made to resemble that of a mesophile, in just three mutations . The genes of functionally homologous proteins in organisms existing in different environments show that adaptation is most often accompanied by mutation of an existing protein . However, the importance of any individual residue to a phenotypic characteristic is usually difficult to assess amongst the multitude of changes that occur over evolutionary time . Since the halophilic nature of this protein can be attributed to only three mutations, this reveals that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.

Extremophiles, 2003 Jun, 7(3), 221 - 8 Epub 2003 Apr 02.
Screening and characterization of the protease CP1 produced by the moderately halophilic bacterium Pseudoalteromonas sp . strain CP76; Sanchez-Porro C et al.; A total of 26 proteolytic moderate halophiles were isolated and characterized . Most isolates were members of the genus Salinivibrio (16 strains), while others were identified as Bacillus (4 strains), Salinicoccus (2 strains), or members of the gamma-Proteobacteria (4 strains) . Strain CP76 was selected as the best producer of an extracellular protease, designated CP1, and was used for further studies . Sequence analysis of the 16S rRNA gene in addition to phenotypic tests led to the placement of this organism in the genus Pseudoalteromonas . Maximal protease production was detected at the end of the exponential growth phase . This CP1 protease was purified and biochemically characterized, showing optimal activity at 55 degrees C, pH 8.5, and high tolerance to a wide range of NaCl concentrations (0 to 4 M NaCl) . The most interesting features of this enzyme are its moderate thermoactivity, its activity at a range of pH values (6-10), and, especially, its salt tolerance (optimal activity at 7.5% total salts) . The purified protease has a molecular mass of 38 kDa, and the N-terminal amino acid sequence determined showed similarity to metalloproteases previously described . The protease activity was strongly inhibited by EDTA, PMSF, and Pefabloc . No significant inhibition was detected with E-64, bestatin, chymostatin, or leupeptin . These results suggest that Pseudoalteromonas sp . strain CP76 produces an extracellular metalloprotease moderately thermotolerant and stable at high salt concentrations.

Extremophiles, 2003 Jun, 7(3), 205 - 11 Epub 2003 Mar 14.
Identification and characterization of the carAB genes responsible for encoding carbamoylphosphate synthetase in Halomonas eurihalina; Llamas I et al.; Halomonas eurihalina is a moderately halophilic bacterium which produces exopolysaccharides potentially of great use in many fields of industry and ecology . Strain F2-7 of H . eurihalina synthesizes an anionic exopolysaccharide known as polymer V2-7, which not only has emulsifying activity but also becomes viscous under acidic conditions, and therefore we consider it worthwhile making a detailed study of the genetics of this strain . By insertional mutagenesis using the mini-Tn 5 Km2 transposon we isolated and characterized a mutant strain, S36 K, which requires both arginine and uracil for growth and does not excrete EPS . S36 K carries a mutation within the carB gene that encodes the synthesis of the large subunit of the carbamoylphosphate synthetase enzyme, which in turn catalyzes the synthesis of carbamoylphosphate, an important precursor of arginine and pyrimidines . We describe here the cloning and characterization of the carAB genes, which encode carbamoylphosphate synthetase in Halomonas eurihalina, and discuss this enzyme's possible role in the pathways for the synthesis of exopolysaccharides in strain F2-7.

Extremophiles, 2003 Jun, 7(3), 195 - 203 Epub 2003 Feb 08.
Growth physiology and competitive interaction of obligately chemolithoautotrophic, haloalkaliphilic, sulfur-oxidizing bacteria from soda lakes; Sorokin DY et al.; Two different groups of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria belonging to the genera Thioalkalimicrobium and Thioalkalivibrio have recently been discovered in highly alkaline and saline soda lakes . To understand response to their extreme environment and different occurrence in soda lakes, the growth kinetics and competitive behavior of several representatives have been characterized in detail using batch and pH-controlled continuous cultivation . The bacteria belong to the true alkaliphiles, growing within the pH range 7.5-10.6 with maximum growth rate and maximum growth yield at pH 9.5-10 . On the basis of their response to salt content, three groups can be identified . All the Thioalkalimicrobium strains and some of the Thioalkalivibrio strains belonged to the moderate halophiles . Some of the Thioalkalivibrio strains from hypersaline soda lakes were extremely salt-tolerant and capable of growth in saturated soda brines . The Thioalkalimicrobium strains demonstrated relatively high specific growth rates, low growth yield, high maintenance, and extremely high rates of thiosulfate and sulfide oxidation . In contrast, the Thioalkalivibrio strains, in general, were slow-growing, high-yield organisms with lower maintenance and much lower rates of oxidation of sulfide and thiosulfate . Moreover, the latter survived starvation much better than Thioalkalimicrobium . Different growth characteristics and salt resistance appear to determine the outcome of the enrichment cultures from different soda lakes: Thioalkalimicrobium dominated in the enrichments with freshly obtained samples from diluted soda lakes at low-medium salinity, while Thioalkalivibrio was the predominant organism in enrichments from aged samples and at hypersaline conditions . In mixed thiosulfate-limited chemostat cultures at low salinity, Thioalkalimicrobium strains (mu(max)=0.33 h(-1)) out-competed Thioalkalivibrio strains (mu(max)=0.15 h(-1)) at D>0.02 h(-1) . The overall results suggest that Thioalkalimicrobium and Thioalkalivibrio represent two different ecological strategies.

Infect Immun, 2003 Jun, 71(6), 3213 - 20
Purification and characterization of enterotoxigenic El Tor-like hemolysin produced by Vibrio fluvialis; Kothary MH et al.; The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin . This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200 . It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions . The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice . Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step . Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus . This homology was further confirmed by PCR analysis using a 5' primer derived from the amino-terminal sequence of the hemolysin and a 3' primer derived from the El Tor hemolysin structural gene . The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V . cholerae.

Mol Microbiol, 2003 May, 48(4), 1089 - 105
Construction and usage of a onefold-coverage shotgun DNA microarray to characterize the metabolism of the archaeon Haloferax volcanii; Zaigler A et al.; Haloferax volcanii is a moderately halophilic archaeon that can grow aerobically and anaerobically with a variety of substrates . We undertook a novel approach for the characterization of metabolic adaptations, i.e . transcriptome analysis with a onefold-coverage shotgun DNA microarray . A genomic library was constructed and converted into a polymerase chain reaction (PCR) product library, which was used to print two DNA microarrays, a 960-spot test array used for optimization of microarray analysis and a 2880-spot onefold-coverage array . H . volcanii cultures were shifted from casamino acid-based metabolism to glucose-based metabolism, and the transcriptome changes were analysed with the onefold-coverage array at five time points covering the transition phase and the onset of exponential growth with the new carbon source . About 10% of all genes were found to be more than 2.5-fold regulated at at least one time point . The genes fall into five clusters of kinetically co-regulated genes . For members of all five clusters, the results were verified by Northern blot analyses . The identity of the regulated genes was determined by sequencing . Many co-regulated genes encode proteins of common functions . Expected as well as a variety of unexpected findings allowed predictions about the central metabolism, the transport capacity and the cellular composition of H . volcanii growing on casamino acids and on glucose . The microarray analyses are in accordance with the growth rates and ribosome contents of H . volcanii growing on the two carbon sources . Analysis of the results revealed that onefold-coverage shotgun DNA microarrays are well suited to characterize the regulation of metabolic pathways as well as protein complexes in response to changes in environmental conditions.

Radiat Res, 2003 Jun, 159(6), 722 - 9
Radiation-dependent limit for the viability of bacterial spores in halite fluid inclusions and on Mars; Kminek G et al.; When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed . We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B . subtilis and S . marismortui spores in solution as well as for dry spores of B . subtilis and B . thuringiensis . S . marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897-900, 2000) . B . thuringiensis is a soil bacterium that is genetically similar to the human pathogens B . anthracis and B . cereus (Helgason et al., Appl . Environ . Microbiol . 66, 2627-2630, 2000) . To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time . Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods . In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.

FEMS Yeast Res, 2003 Jun, 3(4), 347 - 62
Outlines for the definition of halotolerance/halophily in yeasts: Candida versatilis (halophila) CBS4019 as the archetype?
Silva-Graca M, Neves L, Lucas C.
Candida versatilis (halophila) CBS4019 was chosen to study the physiological reactions of long-term exposure to extremely high salt concentrations . In general, our results show a significant increase in enzyme expression during growth under stress conditions . Although glycerol and mannitol pathways are not under glucose repression, they were found to be metabolically regulated . Glycerol-3P-dehydrogenase used either of its cofactors NADPH or NADH, being in favor of NADPH during growth with high salt concentrations . This ability of interchanging cofactors, an increased fermentation rate, and the observed mannitol pathway activity are suggested to contribute to the yeasts' redox stability . Enzymes per se were not salt-tolerant in vitro . Consistently, intracellular sodium was low and intracellular potassium, a requirement for growth, was high . The concept of halophily and its applicability to yeasts is discussed.

Dis Aquat Organ, 2003 Mar 31, 54(2), 119 - 26
Carriage of potentially fish-pathogenic bacteria in Sparus aurata cultured in Mediterranean fish farms; Pujalte MJ et al.; A bacteriological survey of gilthead sea bream Sparus aurata from different fish farms and culture systems on the Spanish Mediterranean coast was conducted . Three different studies were performed . Study A included hatchery-reared larvae; Study B, periodic examination of randomly sampled growing fish; and Study C, growing fish sampled only during mortality/morbidity events . In Studies B and C, sea cages, earth ponds and indoor tanks were surveyed, and in both cases diseased (showing clinical signs) and non-diseased fish were included . In Study A, a shift from Vibrio spp . (30 d after hatching) to oxidative species (60 d after hatching) was detected, and no mortality events were registered . The percentage of fish yielding bacterial growth were similar in Studies B and C, reaching 57.4 and 61.3%, respectively . A statistically significant relationship between the bacterial carriage and the type of facility was only found in Study B, showing that fish from sea cages had a higher bacterial occurrence than fish from other facilities . A statistically significant relationship between bacterial carriage and signs of disease was found, although the pattern differed in each study . Thus, in Study B only 36.2% of fish yielding abundant bacterial growth were diseased, versus 68.0% in Study C . In total, 25.0% of the fish examined were diseased . Bacterial species composition was similar in asymptomatic and diseased fish, except for a group of V . ichthyoenteri-like isolates that occurred almost exclusively in asymptomatic fish . Dominant bacterial species were V . harveyi and V . splendidus, followed by V . ichthyoenteri-like isolates, Photobacterium damselae ssp . damselae and V . fisheri . Non-fermenters were less frequent but, among them, unidentified halophilic Cytophaga-Flavobacterium isolates and Pseudoalteromonas haloplanktis were the most abundant . An association of individual species with disease was not clear, which suggests the involvement of mixed infections.

J Bacteriol, 2003 May, 185(10), 3049 - 59
Synthesis of catalytically active form III ribulose 1,5-bisphosphate carboxylase/oxygenase in archaea; Finn MW et al.; Ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the biological reduction and assimilation of carbon dioxide gas to organic carbon; it is the key enzyme responsible for the bulk of organic matter found on earth . Until recently it was believed that there are only two forms of RubisCO, form I and form II . However, the recent completion of several genome-sequencing projects uncovered open reading frames resembling RubisCO in the third domain of life, the archaea . Previous work and homology comparisons suggest that these enzymes represent a third form of RubisCO, form III . While earlier work indicated that two structurally distinct recombinant archaeal RubisCO proteins catalyzed bona fide RubisCO reactions, it was not established that the rbcL genes of anaerobic archaea can be transcribed and translated to an active enzyme in the native organisms . In this report, it is shown not only that Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina acetivorans, and Methanosarcina barkeri possess open reading frames with the residues required for catalysis but also that the RubisCO protein from these archaea accumulates in an active form under normal growth conditions . In addition, the form III RubisCO gene (rbcL) from M . acetivorans was shown to complement RubisCO deletion strains of Rhodobacter capsulatus and Rhodobacter sphaeroides under both photoheterotrophic and photoautotrophic growth conditions . These studies thus indicate for the first time that archaeal form III RubisCO functions in a physiologically significant fashion to fix CO(2) . Furthermore, recombinant M . jannaschii, M . acetivorans, and A . fulgidus RubisCO possess unique properties with respect to quaternary structure, temperature optima, and activity in the presence of molecular oxygen compared to the previously described Thermococcus kodakaraensis and halophile proteins.

Extremophiles, 2003 Aug, 7(4), 261 - 6 Epub 2003 May 01.
Metabolism of chloride in halophilic prokaryotes; Muller V et al.; While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions . Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin . Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance . However, some species show a specific requirement for chloride . In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent . In this organism the expression of a large number of proteins is chloride regulated . Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride . A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber . It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms.

Extremophiles, 2003 Aug, 7(4), 327 - 34 Epub 2003 May 01.
Tindallia californiensis sp . nov., a new anaerobic, haloalkaliphilic, spore-forming acetogen isolated from Mono Lake in California; Pikuta EV et al.; A novel extremely haloalkaliphilic, strictly anaerobic, acetogenic bacterium strain APO was isolated from sediments of the athalassic, meromictic, alkaline Mono Lake in California . The Gram-positive, spore-forming, slightly curved rods with sizes 0.55-0.7x1.7-3.0 microm were motile by a single laterally attached flagellum . Strain APO was mesophilic (range 10-48 degrees C, optimum of 37 degrees C); halophilic (NaCl range 1-20% (w/v) with optimum of 3-5% (w/v), and alkaliphilic (pH range 8.0-10.5, optimum 9.5) . The novel isolate required sodium ions in the medium . Strain APO was an organotroph with a fermentative type of metabolism and used the substrates peptone, bacto-tryptone, casamino acid, yeast extract, l-serine, l-lysine, l-histidine, l-arginine, and pyruvate . The new isolate performed the Stickland reaction with the following amino acid pairs: proline + alanine, glycine + alanine, and tryptophan + valine . The main end product of growth was acetate . High activity of CO dehydrogenase and hydrogenase indicated the presence of a homoacetogenic, non-cycling acetyl-CoA pathway . Strain APO was resistant to kanamycin but sensitive to chloramphenicol, tetracycline, and gentamycin . The G+C content of the genomic DNA was 44.4 mol% (by HPLC method) . The sequence of the 16S rRNA gene of strain APO possessed 98.2% similarity with the sequence from Tindallia magadiensis Z-7934, but the DNA-DNA hybridization value between these organisms was only 55% . On the basis of these physiological and molecular properties, strain APO is proposed to be a novel species of the genus Tindallia with the name Tindallia californiensis sp . nov., (type strain APO = ATCC BAA-393 = DSM 14871).

Biophys J, 2003 May, 84(5), 3226 - 39
Stark spectroscopy on photoactive yellow protein, E46Q, and a nonisomerizing derivative, probes photo-induced charge motion; Premvardhan LL et al.; The change in the electrostatic properties on excitation of the cofactor of wild-type photoactive yellow protein (WT-PYP) have been directly determined using Stark-effect spectroscopy . We find that, instantaneously on photon absorption, there is a large change in the permanent dipole moment, /Delta(-->)mu/, (26 Debye) and in the polarizability, (-)Deltaalpha, (1000 A(3)) . We expect such a large degree of charge motion to have a significant impact on the photocycle that is associated with the important blue-light negative phototactic response of Halorhodospira halophila . Furthermore, changing E46 to Q in WT-PYP does not significantly alter its electrostatic properties, whereas, altering the chromophore to prevent it from undergoing trans-cis isomerization results in a significant diminution of /Delta(-->)mu/ and (-)Deltaalpha . We propose that the enormous charge motion that occurs on excitation of 4-hydroxycinnamyl thioester, the chromophore in WT-PYP, plays a crucial role in initiating the photocycle by translocation of the negative charge, localized on the phenolate oxygen in the ground state, across the chromophore . We hypothesize that this charge motion would consequently increase the flexibility of the thioester tail thereby decreasing the activation barrier for the rotation of this moiety in the excited state.

J Struct Biol, 2003 May, 142(2), 247 - 55
Crystal structure of a nucleoside diphosphate kinase from Bacillus halodenitrificans: coexpression of its activity with a Mn-superoxide dismutase; Chen CJ et al.; We found that when grown under anaerobic conditions the moderate halophile, gram-positive bacterium Bacillus halodenitrificans (ATCC 49067) synthesizes large amounts of a polypeptide complex that contains a heme center capable of reversibly bind nitric oxide . This complex, when exposed to air, dissociates and reassociates into two active components, a Mn-containing superoxide dismutase (SOD) and a nucleoside diphosphate kinase (BhNDK) . The crystal structure of this latter enzyme has been determined at 2.2A resolution using molecular replacement method, based on the crystal structure of Drosophila melanogaster NDK . The model contains 149 residues of a total 150 residues and 34 water molecules . BhNDK consists of a four-stranded antiparallel beta-sheet, whose surfaces are partially covered by six alpha-helices, and its overall and active site structures are similar to those of homologous enzymes . However, the hexameric packing of BhNDK shows that this enzyme is different from both eukaryotic and gram-negative bacteria . The need for the bacterium to presynthesize both SOD and NDK precursors which are activated during the anaerobic-aerobic transition is discussed.

Biochem Biophys Res Commun, 2003 May 2, 304(2), 405 - 10
Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus; Ran Kim Y et al.; Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells . To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes . The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells . We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein . The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice . This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V . vulnificus.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 595 - 602
Jeotgalicoccus halotolerans gen . nov., sp . nov . and Jeotgalicoccus psychrophilus sp . nov., isolated from the traditional Korean fermented seafood jeotgal; Yoon JH et al.; Two Gram-positive, non-motile, non-spore-forming, halotolerant and moderately halophilic cocci (strains YKJ-101T and YKJ-115T) were isolated from the traditional Korean fermented seafood jeotgal, and were investigated using a polyphasic taxonomic approach . Phylogenetic analysis of 16S rDNA sequences showed that strains YKJ-101T and YKJ-115T are most closely related to the cluster comprising two Salinicoccus species . The peptidoglycan type of the strains is A3alpha, based on L-Lys-Gly(3-4)-L-Ala(Gly), and the predominant menaquinone is MK-7 . Strains YKJ-101T and YKJ-115T have cellular fatty acid profiles containing major amounts of saturated, unsaturated and branched fatty acids; the major fatty acids are anteiso-C15 : 0 and iso-C15 : 0 . The cellular polar lipids are phosphatidylglycerol, diphosphatidylglycerol and unidentified phospholipids . Strains YKJ-101T and YKJ-115T have identical DNA G + C contents of 42 mol% . The 16S rDNA similarity between strains YKJ-101T and YKJ-115T is 98% and the mean level of DNA-DNA relatedness between the two strains is 13.4% . On the basis of phenotypic and phylogenetic data and genomic distinctiveness, it is proposed that strains YKJ-101T and YKJ-115T should be placed in a new genus, Jeotgalicoccus gen . nov., as two distinct new species, for which the names Jeotgalicoccus halotolerans sp . nov . and Jeotgalicoccus psychrophilus sp . nov . are proposed . The type strains are YKJ-101T (=KCCM 41448T =JCM 11198T) and YKJ-115T (=KCCM 41449T =JCM 11199T), respectively.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 563 - 8
Marinobacter litoralis sp . nov., a moderately halophilic bacterium isolated from sea water from the East Sea in Korea; Yoon JH et al.; A Gram-negative, motile, non-spore-forming and moderately halophilic rod-shaped strain, SW-45T, was isolated from sea water of the East Sea in Korea . The organism grew optimally at 30-37 degrees C and grew at 4 and 46 degrees C . It grew in the presence of 0.5-18% (v/w) NaCl, with an optimum of 2-7% NaCl . Strain SW-45T was chemotaxonomically characterized by having ubiquinone-9 (Q-9) as the major respiratory lipoquinone and C16 : 0, C18 : 1omega9c and C16 : 1omega9c as the predominant fatty acids . The DNA G + C content was found to be 55 mol% . Phylogenetic analysis based on 16S rDNA sequences showed that strain SW-45T forms a coherent cluster with the clade comprising the two Marinobacter species . 16S rDNA sequence similarities between strain SW-45T and the Marinobacter species was 94.9% to Marinobacter hydrocarbonoclasticus DSM 8798T and 95.3% to Marinobacter aquaeolei DSM 11845T . Levels of DNA-DNA relatedness between strain SW-45T and the type strains of M . hydrocarbonoclasticus and M . aquaeolei were respectively 4.3 and 5.5% . On the basis of phenotypic properties, phylogeny and genomic data, strain SW-45T (=KCCM 41591T =JCM 11547T) should be placed in the genus Marinobacter as a member of a novel species, for which the name Marinobacter litoralis sp . nov . is proposed . As part of this study, the major respiratory lipoquinone of M . hydrocarbonoclasticus and M . aquaeolei was also found to be Q-9.

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 491 - 9
Shewanella marinintestina sp . nov., Shewanella schlegeliana sp . nov . and Shewanella sairae sp . nov., novel eicosapentaenoic-acid-producing marine bacteria isolated from sea-animal intestines; Satomi M et al.; Three novel Shewanella species are described on the basis of phenotypic, chemotaxonomic and phylogenetic studies . A total of six novel halophilic, aerobic organisms with the ability to produce eicosapentaenoic acid (EPA) were isolated from various sea animals in Japan . Cells of all six isolates were Gram-negative, rod-shaped and motile by means of polar flagella . They were able to produce large amounts of EPA (about 20% of the total fatty acids) and had isoprenoid quinones Q-7 and Q-8 as major components . Analysis of the nearly complete 16S rRNA gene sequences of the novel isolates showed that they are very close phylogenetically (sequence similarity > 99%) and the closest species was Shewanella pealeana, with 97% sequence similarity . However, analysis of gyrB sequences indicated that the novel isolates were divided into three groups at sufficient phylogenetic distance to indicate that they are different species (< 90% sequence similarity) . DNA-DNA hybridization experiments supported this conclusion . The first group (three strains) had positive reactions for lipase, DNase, ONPG and trimethylamine oxide (TMAO) reduction and had G + C contents of 43 mol% (determined by HPLC) . The second group (two strains) was positive for urease, DNase, ONPG and TMAO reduction but not lipase . Their G + C content was 45 mol% . The third group (one strain) was negative for ONPG, DNase and TMAO reduction and had a G + C content of 43 mol% . Strains of the second group, but not those of the first or third groups, grew at 32 degrees C . On the basis of the polyphasic taxonomic data, the novel strains isolated from intestines of sea animals are placed in three novel species of the genus Shewanella: Shewanella marinintestina sp . nov . (type strain: JCM 11558T =LMG 21403T), Shewanella schlegeliana sp . nov . (type strain: JCM 11561T =LMG 21406T) and Shewanella sairae sp . nov . (type strain: JCM 11563T =LMG 21408T).

Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 449 - 54
Psychrobacter jeotgali sp . nov., isolated from jeotgal, a traditional Korean fermented seafood; Yoon JH et al.; Two Gram-negative, non-motile, non-spore-forming and moderately halophilic cocci (strains YKJ-103T and YKJ-105) were isolated from the traditional Korean fermented seafood, jeotgal . The two strains grew optimally at 25-30 degrees C and grew at 4 and 36 degrees C, but not above 37 degrees C . They grew in the presence of 0-10% (w/v) NaCl with an optimum of 2-3% (w/v) NaCl . Strains YKJ-103T and YKJ-105 were chemotaxonomically characterized by having ubiquinone-8 (0-8) as the predominant isoprenoid quinone and C18:1omega9c as the major fatty acid . The polar lipid analysis indicated the presence of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine . The DNA G + C contents of strains YKJ-103T and YKJ-105 were 44 and 43 mol%, respectively . Strains YKJ-103T and YKJ-105 showed no difference in their 16S rDNA sequences, and their mean level of DNA-DNA relatedness was 92.3% . Phylogenetic analysis based on 16S rDNA sequences showed that the two strains form a distinct phylogenetic lineage within the cluster comprising Psychrobacter species . Strains YKJ-103T and YKJ-105 exhibited 16S rDNA similarities of 96.6% with the type strain of Psychrobacter proteolyticus, the closest Psychrobacter species, and of 94.5-95.9% with type strains of other Psychrobacter species . On the basis of phenotypic properties, phylogenetic and genomic data, strains YKJ-103T and YKJ-105 should be placed in the genus Psychrobacter as members of a new species, for which the name Psychrobacter jeotgali sp . nov . is proposed . The type strain of the new species is strain YKJ-103T (=KCCM 41559T = JCM 11463T).

Environ Technol, 2003 Mar, 24(3), 383 - 90
Using ammonium-tolerant yeast isolates: Candida halophila and Rhodotorula glutinis to treat high strength fermentative wastewater; Yang Q et al.; Two ammonium-tolerant yeast strains were isolated from sludge samples contaminated with monosodium glutamate manufacturing wastewater and were identified as Candida haplophila and Rhodotorula glutinis . The tolerance of the two yeast isolates to ammonia and their chemical oxygen demand (COD) removal perfomances were evaluated under batch and bench-scale conditions . The mixture of the two isolates was found to grow well in an artificial medium containing 25% (NH4)2SO4 and could effectively remove COD from monosodium glutamate wastewater even when the concentrations of NH4+-N and free NH3-N reached as high as 18,977 and 879 mg l(-1) respectively . A fixed-bed yeast reactor, which was initially inoculated with the yeast mixture, permitted a constant COD removal rate of over 80% during a period of near 2-month continuous running even when the influent COD was increased from 8,000 to 25,000 mg l(-1) . The effluent was accompanied with suspended solids (SS) of over 4,500 mg l(-1), which was mainly composed of yeast cells and could be considered as a source of animal forage additive . The residual COD of effluents from the yeast reactor could be further reduced to under 500 mg l(-1) by a combination process of activated sludge treatment and coagulation technologies.

Mikrobiologiia, 2003 Jan-Feb, 72(1), 84 - 92
{Mats of Microcoleus from alkaliphilic and halophilic communities}; Gerasimenko LM et al.; A detailed description of the macrostructure, the ultrastructure, and the species diversity of alkaliphilic mat from Lake Khilganta (Buryatiya) is presented . The structure of this mat was found to be similar to that of halophilic mats from hypersaline lagoons of Lake Sivash (Crimea) that we studied earlier . Microcoleus chthonoplastes was the dominant form of cyanobacteria in both mats (in the alkaliphilic mat, Phormidium molle was another dominant form) . Both mats had a pronounced laminated structure . However, unlike halophilic mats with calcium carbonate and gypsum laminas, the alkaliphilic mat contained calcium phosphate laminas instead of gypsum ones . The species diversity of microorganisms in the alkaliphilic mat was at least as rich as that in the halophilic mat; however, in the halophilic mat, the distribution of organisms between layers was more clear-cut . In the alkaliphilic mat, the highest species diversity was observed in the upper mat layers at the boundary between zones of oxygenic and anoxygenic photosynthesis . This fact can be explained by the ephemeral nature of soda lakes.

FEMS Microbiol Lett, 2003 Apr 11, 221(1), 53 - 7
Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis; Ye X et al.; We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7 . The plasmid has a G+C content of 61.1% . A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found . Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins . A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain . Sequence analysis using the GC skew procedure predicted a possible origin of replication . A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H . salinarum R1 . Several methods failed to completely cure H . salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host . Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.

FEMS Yeast Res, 2003 May, 3(3), 247 - 60
Physiological studies on long-term adaptation to salt stress in the extremely halotolerant yeast Candida versatilis CBS 4019 (syn . C . halophila); Silva-Graca M et al.; Candida halophila CBS 4019 (syn . C . versatilis) is an extremely salt-tolerant yeast . It was chosen to study the physiology of long-term resistance to salt stress in cells cultivated at increasing NaCl concentrations up to 4 or 5 M . Growth under stress was slow, severely affected not by salt, but rather by initial external pH . Growing on glucose, glycerol and mannitol were produced . Glycerol is the osmolyte and is transported by H(+)/symport . Transport-driven accumulation was though not affected by salt . The role of mannitol is unknown . Internal pH and intracellular volume were constant during growth at all initial pH/salt combinations . H(+)-ATPase activity was not affected by salt.

Structure (Camb), 2003 Apr, 11(4), 375 - 85
Crystal structure of halophilic dodecin: a novel, dodecameric flavin binding protein from Halobacterium salinarum; Bieger B et al.; A novel, 68 amino acid long flavoprotein called dodecin has been discovered in the proteome of Halobacterium salinarum by inverse structural genomics . The 1.7 A crystal structure of this protein shows a dodecameric, hollow sphere-like arrangement of the protein subunits . Unlike other known flavoproteins, which bind only monomeric flavin cofactors, the structure of the dodecin oligomer comprises six riboflavin dimers . The dimerization of these riboflavins along the re-faces is mediated by aromatic, antiparallel pi staggering of their isoalloxazine moieties . A unique aromatic tetrade is formed by further sandwiching of the riboflavin dimers between the indole groups of two symmetry-related Trp36s . So far, the dodecins represent the smallest known flavoproteins . Based on the structure and the wide spread occurrences in pathogenic and soil eubacteria, a function in flavin storage or protection against radical or oxygenic stress is suggested for the dodecins.

Appl Environ Microbiol, 2003 Apr, 69(4), 2126 - 32
Cloning, expression, and purification of choline dehydrogenase from the moderate halophile Halomonas elongata; Gadda G et al.; Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate . Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures . To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro . In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase . The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose . The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 micro mol of O(2) min(-1) mg(-1) mM(-1) at pH 7 and 25 degrees C, respectively . With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V(max) values for choline and betaine-aldehyde were 10.9 and 5.7 micro mol of O(2) min(-1) mg(-1), respectively . These V(max) values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor . Altogether, the kinetic data are consistent with the conclusion that H . elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.

Extremophiles, 2003 Apr, 7(2), 95 - 9 Epub 2002 Oct 26.
Marine acidophilic sulfur-oxidizing bacterium requiring salts for the oxidation of reduced inorganic sulfur compounds; Kamimura K et al.; An acidophilic sulfur-oxidizing bacterium was isolated from seawater, and designated as strain SH . Strain SH was a Gram-negative, rod-shaped and motile bacterium, which had an optimum temperature and pH value for growth of 30 degrees C and 4.0, respectively . The mol% guanine plus cytosine of the DNA was 46.0 . Chemolithotrophic growth was observed with elemental sulfur and tetrathionate at pH 4.0, and was not observed with ferrous ion . The isolate was able to utilize carbon dioxide as a carbon source, and was unable to grow heterotrophically with yeast extract or glucose . The growth of strain SH was activated in medium supplemented with NaCl . However, LiCl and KCl did not sustain the growth of strain SH . The results indicate that strain SH was an acidophilic, halophilic, and obligately chemolithotrophic sulfur-oxidizing bacterium . Phylogenetic analysis based on 16S rDNA sequences indicated that strain SH had a close relationship to Acidithiobacillus thiooxidans . The oxidizing activities of sulfur and sulfite with resting cells were stimulated not only by the addition of NaCl, but also by KCl and LiCl . The oxidation of sulfite was inhibited by ionophores, carbonyl cyanide- m-chlorophenylhydrazone (CCCP), and monensin, and respiratory inhibitors, KCN and 2-heptyl-4-hydroxy-quinoline-N-oxode (HQNO).

Extremophiles, 2003 Apr, 7(2), 87 - 93 Epub 2002 Nov 14.
Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis; Waino M et al.; The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities . Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth . A broad NaCl optimum was observed for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl . Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities . When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta-xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl . The enzymes were also shown to be slightly thermophilic; beta-xylanase activity exhibiting two optima at 55 degrees and 70 degrees C, while beta-xylosidase activity was optimal at 65 degrees C . SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants . To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 331 - 8
Description of Alcanivorax venustensis sp . nov . and reclassification of Fundibacter jadensis DSM 1 21 78T (Bruns and Berthe-Corti 1999) as Alcanivorax jadensis comb . nov., members of the emended genus Alcanivorax; Fernandez-Martinez J et al.; Two strains of a novel bacterium were isolated independently of each other, from different depths in the Mediterranean Sea, within a time period of 7 months, using two different isolation approaches that were focused on different objectives . Both strains, designated ISO1 and ISO4T, were halophilic, Gram-negative, strictly aerobic, straight rods that were oxidase- and catalase-positive . Both strains produced mucoid colonies in some defined minimal media and were able to grow with organic acids and some alkanes; they were also able to accumulate intracellular poly-beta-hydroxybutyrate granules . The G + C content of the DNA of strain ISO4T was 66 mol% . Comparative analysis of 16S rRNA gene sequences showed that the closest described species to the novel strains were Alcanivorax borkumensis and Fundibacter jadensis, both of the gamma-Proteobacteria . Both of these recognized species were originally isolated from North Sea waters and are able to degrade aliphatic compounds, a property shared with strains ISO1 and ISO4T . However, strains ISO1 and ISO4T were different from A . borkumensis and F . jadensis, not only in their 16S rDNA sequences but also in the motility of their cells (by polar flagella) and by the presence of C19:Ocyclo in their cellular fatty acids, among other differential features . On the basis of biochemical and molecular data, it is suggested that strains ISO1 and ISO4T be recognized as a novel species of the genus Alcanivorax, for which the name Alcanivorax venustensis (ISO4T =DSM 13974T =CECT 5388T) is proposed . On the basis of its high phenotypic similarity and close phylogenetic relatedness to A . borkumensis, it is also proposed that F . jadensis (DSM 12178T) be reclassified as Alcanivorax jadensis in the genus Alcanivorax, and that the description of the genus Alcanivorax be emended.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 317 - 21
Nocardiopsis xinjiangensis sp . nov., a halophilic actinomycete isolated from a saline soil sample in China; Li MG et al.; An actinomycete strain (YIM 90004T) was isolated from a saline soil sample from the Xinjiang Province, People's Republic of China . The strain displayed abundant aerial and substrate mycelia, and short spore chains were borne on the aerial mycelium . The spore chains were composed of non-motile, smooth-surfaced, rod-shaped spores . The cell wall of strain YIM 90004T contained mainly meso-diaminopimelic acid, alanine and glutamic acid (cell wall type III) . Glucose, ribose, galactose, xylose and arabinose were the whole-cell sugars of the strain . The predominant phospholipids were phosphatidylinositol and phosphatidylglycerol; MK-10(H2) and MK-10(H4) were the predominant menaquinones . The DNA G + C content of strain YIM 90004T was 74.3 mol% . Chemotaxonomic properties and 16S rDNA analysis placed strain YIM 90004T in the genus Nocardiopsis . On the basis of phenotypic and phylogenetic analyses and DNA-DNA hybridization results, strain YIM 90004T (= CCRC 16285T = CCTCC AA99004T = DSM 44589T) is considered to represent a novel species of the genus Nocardiopsis, for which the name Nocardiopsis xinjiangensis is proposed.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 223 - 9
Methylophaga alcalica sp . nov., a novel alkaliphilic and moderately halophilic, obligately methylotrophic bacterium from an East Mongolian saline soda lake; Doronina NV et al.; A moderately haloalkaliphilic and obligately methylotrophic bacterium (strain M39T) with the ribulose monophosphate pathway of carbon assimilation is described . Cells of this methanol and methylamine utilizer are aerobic, Gram-negative, asporogenous, motile short rods, multiplying by binary fission . It is auxotrophic for vitamin B12 and requires NaHCO3 or NaCl for growth in alkaline medium . Its cellular fatty acid profile consists primarily of straight-chain saturated C16:0 and unsaturated C16:1 and C18:1 acids . The major ubiquinone is Q-8 . The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol . Diphosphatidylglycerol is also present . Optimal growth conditions are 25-29 degrees C, pH 9.0-9.5 and 3-4 % (w/v) NaCl . Cells accumulate the cyclic amino acid ectoine as the main compatible solute . The DNA G + C content is 48.3 mol% . Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-30 %) with the type strains of marine methylotrophs belonging to the genus Methylophaga, the novel isolate M39T (=VKM B-2251T =ATCC BAA-297T) was classified as the type strain of Methylophaga alcalica sp . nov.

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 153 - 63
Isolation and characterization of spirilloid purple phototrophic bacteria forming red layers in microbial mats of Mediterranean salterns: description of Halorhodospira neutriphila sp . nov . and emendation of the genus Halorhodospira; Hirschler-Rea A et al.; Microbial mats developing in the hypersaline lagoons of a commercial saltern in the Salin-de-Giraud (Rhone delta) were found to contain a red layer fully dominated by spirilloid phototrophic purple bacteria underlying a cyanobacterial layer . From this layer four strains of spirilloid purple bacteria were isolated, all of which were extremely halophilic . All strains were isolated by using the same medium under halophilic photolithoheterotrophic conditions . One of them, strain SG 3105 was a purple non-sulfur bacterial strain closely related to Rhodovibrio sodomensis with a 16S rDNA sequence similarity of 98.8% . The three other isolated strains, SG 3301T, SG 3302 and SG 3304, were purple sulfur bacteria and were found to be very similar . The cells were motile by a polar tuft of flagella . Photosynthetic intracytoplasmic membranes of the lamellar stack type contained BChl a and spirilloxanthin as the major carotenoid . Phototrophic growth with sulfide as electron donor was poor; globules of elemental sulfur were present outside the cells . In the presence of sulfide and CO2 good growth occurred with organic substrates . Optimum growth occurred in the presence of 9-12% (w/v) NaCl at neutral pH (optimal pH 6.8-7) and at 30-35 degrees C . The DNA base composition of strains SG 3301T and SG 3304 were 74.5 and 74.1 mol% G + C, respectively . According to the 16S rDNA sequences, strains SG 3301T and SG 3304 belonged to the genus Halorhodospira, but they were sufficiently separated morphologically, physiologically and genetically from other recognized Halorhodospira species to be described as a new species of the genus . They are, therefore, described as Halorhodospira neutriphila sp . nov . with strain SG 3301T as the type strain (=DSM 15116T).

Int J Syst Evol Microbiol, 2003 Jan, 53(Pt 1), 53 - 7
Microbulbifer salipaludis sp . nov., a moderate halophile isolated from a Korean salt marsh; Yoon JH et al.; A Gram-negative, non-motile, non-spore-forming, moderately halophilic rod (strain SM-1T) was isolated from salt marsh around the junction of the Youngsan River and the Yellow Sea in Korea and subjected to a polyphasic taxonomic study . This organism grew optimally at 37 degrees C and was able to grow at 10 and 45 degrees C . It grew optimally in the presence of 2-3% (w/v) NaCl . The major fatty acids in strain SM-1T were iso-C15:0 and C16:0 . Strain SM-1T and Microbulbifer hydrolyticus DSM 11525T were characterized by having ubiquinone-8 as the predominant respiratory lipoquinone . The DNA G+C content of strain SM-1T was 59 mol% . Phylogenetic analysis based on 16S rDNA sequences showed that strain SM-1T formed a coherent cluster with M . hydrolyticus; this relationship was supported by a bootstrap resampling value of 100% . The level of 16S rDNA identity between strain SM-1T and the type strain of M . hydrolyticus was 98.6% . The mean level of DNA-DNA relatedness between strain SM-1T and the type strain of M . hydrolyticus was 20.6% . Therefore, on the basis of phenotypic properties, phylogeny and genomic data, strain SM-1T should be placed in the genus Microbulbifer as a member of a novel species, for which the name Microbulbifer salipaludis sp . nov . is proposed . The type strain of the novel species is strain SM-1T (=KCCM 41586T =JCM 11542T).

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