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