Microorganisms

J Microsc, 2005 Jan, 217(Pt 1), 109 - 16
Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology; Kassies R et al.; Summary We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region . Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods . The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects . The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection . We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution . Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 91 - 4
Planococcus stackebrandtii sp . nov., isolated from a cold desert of the Himalayas, India; Mayilraj S et al.; The taxonomic position of a bacterium isolated from a cold desert of the Himalayas, India, was analysed by using a polyphasic approach . The isolated strain, designated K22-03(T), had phenotypic characteristics that matched those of the genus Planococcus and it represents a novel species . The almost-complete 16S rRNA gene sequence (1464 bases) of the novel strain was compared with those of previously studied Planococcus type strains and confirmed that the strain belongs to the genus Planococcus . 16S rRNA gene sequence analysis indicated that strain K22-03(T) differs from all other species of Planococcus by at least 2.5 % . DNA-DNA hybridization showed that it had low genomic relatedness with Planomicrobium mcmeekinii (MTCC 3704(T), 23 %), Planococcus psychrophilus (MTCC 3812(T), 61 %), Planococcus antarcticus (MTCC 3854(T), 45 %) and Planomicrobium okeanokoites (MTCC 3703(T), 51 %), the four species with which it was most closely related based on 16S rRNA gene sequence analysis (97-97.5 % similarity) . Therefore, strain K22-03(T) should be recognized as a novel species, for which the name Planococcus stackebrandtii sp . nov . is proposed . The type strain is K22-03(T) (=MTCC 6226(T)=DSM 16419(T)=JCM 12481(T)).

Science, 2005 Jan 14, 307(5707), 214 - 5
Microbiology . TB--a new target, a new drug; Cole ST et al.; Genetique Moleculaire Bacterienne and Biochimie Structurale Units, Institut Pasteur, Paris 75724 Cedex 15, France . stcole@pasteur.fr

Chembiochem . 2005 Jan 13; {Epub ahead of print}
A New Type of Metalloprotein: The Mo Storage Protein from Azotobacter vinelandii Contains a Polynuclear Molybdenum-Oxide Cluster; Fenske D et al.; Azotobacter vinelandii is a diazotrophic bacterium characterized by the outstanding capability of storing Mo in a special storage protein, which guarantees Mo-dependent nitrogen fixation even under growth conditions of extreme Mo starvation . The Mo storage protein is constitutively synthesized with respect to the nitrogen source and is regulated by molybdenum at an extremely low concentration level (0-50 nM) . This protein was isolated as an alpha(4)beta(4) octamer with a total molecular mass of about 240 kg mol(-1) and its shape was determined by small-angle X-ray scattering . The genes of the alpha and beta subunits were unequivocally identified; the amino acid sequences thereby determined reveal that the Mo storage protein is not related to any other known molybdoprotein . Each protein molecule can store at least 90 Mo atoms . Extended X-ray absorption fine-structure spectroscopy identified a metal-oxygen cluster bound to the Mo storage protein . The binding of Mo (biosynthesis and incorporation of the cluster) is dependent on adenosine triphosphate (ATP); Mo release is ATP-independent but pH-regulated, occurring only above pH 7.1 . This Mo storage protein is the only known noniron metal storage system in the biosphere containing a metal-oxygen cluster.

J Biol Inorg Chem . 2005 Jan 14; {Epub ahead of print}
Isoprenoid biosynthesis in chloroplasts via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE) from Arabidopsis thaliana is a {4Fe-4S} protein; Seemann M et al.; The mevalonate-independent methylerythritol phosphate pathway is widespread in bacteria . It is also present in the chloroplasts of all phototrophic organisms . Whereas the first steps, are rather well known, GcpE and LytB, the enzymes catalyzing the last two steps have been much less investigated . 2-C-Methyl-D: -erythritol 2,4-cyclodiphosphate is transformed by GcpE into 4-hydroxy-3-methylbut-2-enyl diphosphate, which is converted by LytB into isopentenyl diphosphate or dimethylallyl diphosphate . Only the bacterial GcpE and LytB enzymes have been investigated to some extent, but nothing is known about the corresponding plant enzymes . In this contribution, the prosthetic group of GcpE from the plant Arabidopsis thaliana and the bacterium Escherichia coli has been fully characterized by Mossbauer spectroscopy after reconstitution with (57)FeCl(3), Na(2)S and dithiothreitol . It corresponds to a {4Fe-4S} cluster, suggesting that both plant and bacterial enzymes catalyze the reduction of 2-C-methyl-D: -erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate via two consecutive one-electron transfers . In contrast to the bacterial enzyme, which utilizes NADPH/flavodoxin/flavodoxin reductase as a reducing shuttle system, the plant enzyme could not use this reduction system . Enzymatic activity was only detected in the presence of the 5-deazaflavin semiquinone radical.

J Antimicrob Chemother . 2005 Jan 13; {Epub ahead of print}
Molecular evaluation of antibiotic susceptibility of Tropheryma whipplei in axenic medium; Boulos A et al.; Whipple's disease is a rare multisystem chronic infection, involving the intestinal tract as well as various other organs . Tropheryma whipplei is a slow-growing facultative intracellular bacterium that remains poorly understood . In vitro antibiotic susceptibility testing has previously been assessed in cells using a real-time quantitative PCR assay . In this study, we have evaluated the antibiotic susceptibility of three strains of T . whipplei grown in axenic medium using the same assay . The active compounds in axenic medium were doxycycline, macrolide compounds, penicillin G, streptomycin, rifampicin, chloramphenicol, thiamphenicol, teicoplanin, vancomycin, amoxicillin, gentamicin, aztreonam, levofloxacin and ceftriaxone, with MICs in the range 0.06-1 mg/L . Cefalothin was less active, with MICs in the range 2-4 mg/L . We found that co-trimoxazole was active with MICs in the range 0.5-1 mg/L, and sulfamethoxazole alone was active with MICs in the range 0.5-1 mg/L . MICs of trimethoprim varied from 64-128 mg/L . Co-trimoxazole was effective in vitro, but this activity was due to sulfamethoxazole alone . These results were in accordance with the fact that T . whipplei does not contain the encoding gene for dihydrofolate reductase, the target for trimethoprim.

Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 668 - 74
Successful recombinant production of Allochromatium vinosum cytochrome c' requires coexpression of cmm genes in heme-rich Escherichia coli JCB712; Evers TH et al.; Cytochrome c' from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN(-) . This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available . Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography . Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c' . Characterization using circular dichroism, UV-vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization.

Environ Microbiol, 2005 Jan, 7(1), 22 - 33
Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A . felis; Moosvi SA et al.; Summary Three novel strains of methylotrophic Afipia felis were isolated from several locations on Signy Island, Antarctica, and a fourth from estuary sediment from the River Douro, Portugal . They were identified as strains of the alpha-2 proteobacterium A . felis by 16S rRNA gene sequence analysis . Two strains tested were shown to contain the fdxA gene, diagnostic for A . felis . All strains grew with methanesulfonate (and two strains with dimethylsulfone) as sole carbon substrate . Growth on methanesulfonate required methanesulfonate monooxygenase (MSAMO), using NADH as the reductant and stimulated by reduced flavin nucleotides and Fe(II) . Polymerase chain reaction amplification of DNA from an Antarctic strain showed a typical msmA gene for the alpha-hydroxylase of MSAMO, and both Antarctic and Portuguese strains contained mxaF, the methanol dehydrogenase large subunit gene . This is the first report of methanesulfonate-degrading bacteria from the Antarctic and of methylotrophy in Afipia, and the first description of any bacterium able to use both methanesulfonate and dimethylsulfone . In contrast, the type strain of A . felis DSM 7326(T) was not methylotrophic, but grew in defined mineral medium with a wide range of single simple organic substrates . Free-living Afipia strains occurring widely in the natural environment may be significant as methylotrophs, degrading C(1)-sulfur compounds, including the recalcitrant organosulfur compound methanesulfonate.

Appl Environ Microbiol, 2005 Jan, 71(1), 417 - 22
Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces; Shinohara R et al.; The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium . A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants . Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease . In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease . Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent . Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation . Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.

Appl Environ Microbiol, 2005 Jan, 71(1), 123 - 30
Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR; Segawa T et al.; The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR . Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum . Bacterial colonies that developed in an in situ meltwater medium at 4 degrees C were revealed to be V . paradoxus . The biomasses of C . psychrophilum, J . lividum, and V . paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 x 10(5)-fold, 1.5 x 10(5)-fold, and 1.0 x 10(4)-fold increases, respectively), suggesting their rapid growth in the surface snow . The biomasses of C . psychrophilum and J . lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season . In contrast, the biomass of V . paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October . The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow . Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

Science, 2005 Jan 7, 307(5706), 105 - 8
Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes; Seshadri R et al.; Dehalococcoides ethenogenes is the only bacterium known to reductively dechlorinate the groundwater pollutants, tetrachloroethene (PCE) and trichloroethene, to ethene . Its 1,469,720-base pair chromosome contains large dynamic duplicated regions and integrated elements . Genes encoding 17 putative reductive dehalogenases, nearly all of which were adjacent to genes for transcription regulators, and five hydrogenase complexes were identified . These findings, plus a limited repertoire of other metabolic modes, indicate that D . ethenogenes is highly evolved to utilize halogenated organic compounds and H2 . Diversification of reductive dehalogenase functions appears to have been mediated by recent genetic exchange and amplification . Genome analysis provides insights into the organism's complex nutrient requirements and suggests that an ancestor was a nitrogen-fixing autotroph.

J Clin Microbiol, 2005 Jan, 43(1), 393 - 401
Latency-related gene encoded by bovine herpesvirus 1 promotes virus growth and reactivation from latency in tonsils of infected calves; Perez S et al.; Infection of calves with bovine herpesvirus 1 (BHV-1) results in transient immunosuppression that may lead to bacterium-induced pneumonia and, occasionally, death . Although sensory neurons in the trigeminal ganglia (TG) are the primary site of BHV-1 latency, viral genomes are detected in the tonsils of latently infected calves . Dexamethasone (DEX) consistently induces reactivation from latency, and viral gene expression is detected in TG and tonsils . In sensory neurons of latently infected calves, the latency-related (LR) gene is abundantly expressed and is required for reactivation from latency . In the present study, we compared the abilities of wild-type (wt) BHV-1 and a strain with a mutation in the LR gene (the LR mutant strain) to grow in the tonsils of infected calves and reactivate from latency . Lower levels of the LR mutant virus were detected in the tonsils of acutely infected calves . LR mutant viral DNA was consistently detected by PCR in the tonsils of latently infected calves, suggesting that the establishment of a latent or persistent infection occurred . Although the LR mutant did not reactivate from latency in vivo after DEX treatment, explantation of tonsil tissue from calves latently infected with the LR mutant yielded infectious virus . Relative to wt BHV-1, the LR mutant did not induce explant-induced reactivation as efficiently . These studies indicate that the LR gene promotes virus shedding from tonsil tissue during acute infection and reactivation from latency in tonsil tissue in vivo . We suggest that incorporation of the LR gene mutation into existing modified live vaccines would prevent reactivation from latency in neural and nonneural sites and would thus prevent transmission to other animals.

J Clin Microbiol, 2005 Jan, 43(1), 41 - 8
Multispacer typing technique for sequence-based typing of Bartonella quintana; Foucault C et al.; Bartonella quintana is a worldwide fastidious bacterium of the Alphaproteobacteria responsible for bacillary angiomatosis, trench fever, chronic lymphadenopathy, and culture-negative endocarditis . The recent genome sequencing of a B . quintana isolate allowed us to propose a genome-wide sequence-based typing method . To ensure sequence discrimination based on highly polymorphic areas, we amplified and sequenced 34 spacers in a large collection of B . quintana isolates . Six of these exhibited polymorphisms and allowed the characterization of 4 genotypes . However, the strain variants suggested by the noncoding sequences did not correlate with the results of pulsed-field gel electrophoresis (PFGE), which suggested a higher degree of variability . Modification of the PFGE profile of one isolate after nine subcultures confirmed that rearrangement frequencies are high in this species, making PFGE unreliable for epidemiological purposes . The low extent of sequence heterogeneity in the species suggests a recent emergence of this bacterium as a human pathogen . Direct typing of natural samples allowed the identification of a fifth genotype in the DNA extracted from a human body louse collected in Burundi . We have named the typing technique herein described multispacer typing.

Proc Natl Acad Sci U S A . 2005 Jan 4; {Epub ahead of print}
Brucella coopts the small GTPase Sar1 for intracellular replication; Celli J et al.; The pathogen Brucella abortus resides inside macrophages within a unique, replication-permissive organelle that is derived from the endoplasmic reticulum (ER) . Although dependent on the Brucella type IV secretion system VirB, the mechanisms governing the biogenesis of this compartment remain elusive . Here, we investigated a putative role of the early secretory pathway in ER membrane accretion by the Brucella-containing vacuoles (BCVs) . We show that BCVs interact with ER exit sites (ERES), and blockade of Sar1 activity, which disrupts ERES, prevents intracellular replication of Brucella . In cells expressing the dominant interfering form Sar1{T39N}, BCVs do not acquire ER membranes, suggesting that they are unable to mature into replicative organelles . By contrast, treatments that block subsequent secretory events do not affect bacterial replication . We propose that Sar1-dependent ERES functions, but not subsequent secretory events, are essential for the biogenesis of the Brucella replicative compartment and, thus, bacterial replication . These results assign an essential role for Sar1 in pathogenesis of an intracellular bacterium.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
Functional consequences of the organization of the photosynthetic apparatus in R . sphaeroides: 1 . Quinone domains and excitation transfer in chromatophores and reaction center-antenna complexes; Comayras F et al.; The purpose of this study was to gain information on functional consequences of the supramolecular organization of the photosynthetic apparatus in the bacterium Rhodobacter sphaeroides . Isolated complexes of the reaction center (RC) with its core antenna ring (LH1) were studied, in their dimeric (native) form or as monomers, with respect to excitation transfer and distribution of the quinone pool . Similar issues were examined in chromatophore membranes . The relationship between the fluorescence yield and the amount of closed centers is indicative of a very efficient excitation transfer between the two monomers in isolated dimeric complexes . A similar dependence was observed in chromatophores, suggesting that excitation transfer in vivo from a closed RC-LH1 unit is also essentially directed to its partner in the dimer . The isolated complexes were found to retain 25-30 % of the endogenous quinone acceptor pool and the distribution of this pool among the complexes suggests a cooperative character for the association of quinones to the protein complexes . In chromatophores, the decrease in the amount of photoreducible quinones when inhibiting a fraction of the centers implies a confinement of the quinone pool over small domains, including 1-6 reaction centers . We suggest that the crowding of membrane proteins may not be the sole reason for quinone confinement and that a quinone-rich region is formed around the RC-LH1 complexes.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
Functional consequences of the organization of the photosynthetic apparatus in R . sphaeroides: 2 . A study of PufX- membranes; Comayras F et al.; In the bacterium R . sphaeroides, polypeptide PufX is indispensable for photosynthetic growth . Its deletion is known to have important consequences on the organization of the photosynthetic apparatus . In the WT, the RC-LH1 complexes (reaction center and antenna) are associated in dimers and the LH1 does not fully encircle the RC . In the absence of PufX, the complexes become monomeric and the LH1 ring closes around the RC . We analyze functional consequences of PufX deletion . Some effects can be ascribed to the monomerization of the RC-LH1 complexes: the number of RCs that share a common antenna for excitation transfer, or a common quinone pool, both become smaller . We examined the kinetic effects of the closed LH1 ring on quinone turnover: the diffusion across the LH1 entails a delay of about 1 ms and the barrier appears located directly against the quinone binding (QB) pocket . The diffusion of ubiquinol from the RC to the cytochrome bc1 complex is about two-fold slower in the mutant, suggesting an increased distance between the two complexes . The properties of the QB pocket (binding of inhibitors, stabilization of QB- and rate of QBH2 formation) appeared modified in the mutant . Another specificity of PufX- is the accumulation of closed centers in the QA- state as the secondary acceptor pool becomes reduced, which is probably the origin of the photosynthetic incompetence . We suggest that this is related to the QB pocket alterations . The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
How an enzyme binds the C1-carrier tetrahydromethanopterin: Structure of the tetrahydromethanopterin dependent formaldehyde-activating enzyme (Fae) from methylobacterium extorquens AM1; Acharya P et al.; Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate (H4F) analogue involved as C1-carrier in the metabolism of various groups of microorgansims . How H4MPT is bound to the respective C1-unit converting enzymes remained elusive . We describe here the structure of the homopentameric formaldehyde-activating enzyme (Fae) from Methylobacterium extorquens AM1 established at 2.0 A without and at 1.9 A with methylene-H4MPT bound . Methylene-H4MPT is bound in a 'S'-shaped conformation into the cleft formed between two adjacent subunits . Coenzyme binding is accompanied by side chain rearrangements up to 5 A and leads to a rigidification of the C-terminal arm, a formation of a new hydrophobic cluster and an inversion of the amide side chain of Gln88 . Methylene-H4MPT in Fae shows a characteristic kink between the tetrahydropyrazine and the imidazolidine rings of 70 degrees that is more pronounced than that reported for free methylene-H4MPT in solution (50 degrees ) . Fae is an essential enzyme for energy metabolism and formaldehyde detoxification of this bacterium and catalyses the formation of methylene-H4MPT from H4MPT and formaldehyde . The molecular mechanism of this reaction involving His22 as acid catalyst is discussed.

Plasmid, 2005 Jan, 53(1), 1 - 13 Epub 2004 Dec 16.
The plasmids of Borrelia burgdorferi: essential genetic elements of a pathogen; Stewart PE et al.; The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, has an unusual genome comprised of a linear chromosome and the largest plasmid complement of any characterized bacterium . Certain plasmid-encoded elements are required for virulence and viability, both in vitro and in vivo . The genetic tools to manipulate B . burgdorferi are sufficiently developed for precise molecular genetic investigations . B . burgdorferi now represents a prime system with which to address basic questions of plasmid biology and plasmid contributions to bacterial virulence and disease pathogenesis.

J Bacteriol, 2005 Jan, 187(2), 534 - 43
Analysis of the Precursor rRNA Fractions of Rapidly Growing Mycobacteria: Quantification by Methods That Include the Use of a Promoter (rrnA P1) as a Novel Standard; Menendez Mdel C et al.; Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their rrn operons . Mycobacterium chelonae and M . fortuitum are members of the rapidly growing mycobacterial group . They carry a total of five promoters each, encoded, respectively, by one and two rrn operons per genome . Quantification of precursor rrn transcriptional products (pre-rrn) has allowed detection of different promoter usage during cell growth . Bacteria growing in several culture media with different nutrient contents were compared . Balanced to stationary phases were analyzed . Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium . Some biological implications are discussed . Sequences of the several promoters showed motifs that could be correlated to their particular level of usage . A product corresponding to the first rrnA promoter in both species, namely, rrnA P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed . This product was used as a standard to quantitate rRNA gene expression by real-time PCR when M . fortuitum infected macrophages . It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its rrn operons is preferentially used under such conditions.

Cell Motil Cytoskeleton, 2005 Feb, 60(2), 104 - 20
Actin and alpha-actinin dynamics in the adhesion and motility of EPEC and EHEC on host cells; Shaner NC et al.; Two pathogenic Escherichia coli, Enteropathogenic E . coli (EPEC) and Enterohemorrhagic E . coli (EHEC), adhere to the outside of host cells and induce cytoskeletal rearrangements leading to the formation of membrane-encased pedestals comprised of actin filaments and other associated proteins beneath the bacteria . The structure of the pedestals induced by the two pathogens appears similar, although those induced by EHEC are shorter in length . Fluorescence Recovery After Photobleaching (FRAP) was used to determine potential differences of actin polymerization in EPEC and EHEC induced pedestals in cultured PtK2 cells expressing either Green or Yellow Fluorescent Protein (GFP or YFP) fused to actin or alpha-actinin . When all the fluorescent actin in a pedestal on EPEC-infected cells was photobleached, fluorescence recovery first occurred directly beneath the bacterium in a band that grew wider at a rate of one micron/minute . Consistently observed in all EPEC-induced pedestals, whether they were stationary, lengthening, or translocating, the rate of actin polymerization that occurred at the pedestal tip was approximately 1 mum/min . Overall, a much slower rate of actin polymerization was measured in long EHEC-induced pedestals . In contrast to the dynamics of GFP-actin, recovery of GFP-alpha-actinin fluorescence was not polarized, with the actin cross-linking protein exchanging all the length of the EPEC/EHEC induced pedestals . Surprisingly, the depolymerization and retrograde flow of pedestal actin, as well as pedestal translocations, were inhibited reversibly by either 2,3-butanedione monoxime (BDM) or by a combination of sodium azide and 2-deoxy D-glucose, leading to an increase in the lengths of the pedestals . A simple physical model was developed to describe elongation and translocation of EPEC/EHEC pedestals in terms of actin polymerization and depolymerization dynamics . Cell Motil . Cytoskeleton 60:104-120, 2005 . (c) 2004 Wiley-Liss, Inc.

Rev Esp Cardiol, 2002 Jul, 55(Supl.1), 2 - 9
Mycoplasma Pneumoniae and Chlamydia Pneumoniae are associated to inflammation and rupture of the atherosclerotic coronary plaques; F Ramires JA et al.; In this review we report recent findings of our lab showing that Mycoplasma pneumoniae and Chlamydia pneumoniae are present in higher amount, associated with adventitial inflammation and positive vessel remodeling, in thrombosed coronary artery segments (CAS) of patients who died due to acute myocardial infarction . CD8T cell was the predominant lymphocytes in the plaque and CD24(B) cell in the adventitia . The mean numbers of lymphocytes were significantly higher in adventitia than in the plaque . Vulnerable plaques were usually associated with focal positive vessel remodeling and large lipidic atheromas . Mycoplasma is the only bacterium that needs cholesterol for proliferation . We hypothesized that the association of Mycoplasma pneumoniae and Chlamydia pneumoniae increases virulence of both bacteria, inducing inflammation and rupture of the plaque . The search of CMV and Helicobacter pylori resulted negative.

Hum Fertil (Camb), 2004 Dec, 7(4), 271 - 6
Chlamydia trachomatis and male fertility; Pacey A et al.; There is increasing evidence that the function of human spermatozoa can be significantly affected by direct exposure to the bacterium Chlamydia trachomatis . This may contribute to sub-fertility in infected individuals by a route that is independent of any damage to the reproductive epithelium . In addition, if a C . trachomatis infection is undiagnosed it could contribute to poor outcomes in assisted conception techniques such as in vitro fertilization . The antibiotics routinely used in IVF culture systems are largely ineffective against chlamydia, emphasizing the importance of screening patients prior to treatment . Moreover, given the many thousands of semen samples provided for analysis by men in primary care (many of which will never undergo assisted conception treatment), it is suggested that this may represent a wasted opportunity to provide screening (and treatment) for the infection using an appropriate test specimen and without the need for additional hospital visits.

J Inorg Biochem, 2005 Feb, 99(2), 415 - 23
Single crystal EPR study of electronic structure and exchange interactions for copper(II)(l-arginine)(2)(SO(4)).(H(2)O)(6): a model system to study exchange interactions between unpaired spins in proteins; Santana RC et al.; We report EPR measurements at 9.77 and 34.1 GHz in powder and single crystal samples of the ternary copper amino acid complex Cu(l-arginine)(2)(SO(4)).(H(2)O)(6) . The single crystal Electron Paramagnetic Resonance spectra display a single resonance for all magnetic field orientations in the ca and cb crystal planes . In the ab plane they display two resonances for most orientations of the magnetic field, and only one resonance for orientations close to the crystal axes . This behavior is a result of the selective collapse of the resonances corresponding to the four copper sites in the unit cell produced by the exchange interactions between copper ions . From the characteristics of the collapse and the angular dependences of the position and width of the resonances we evaluate the g-tensors of the copper molecules and estimate exchange interactions |J(1)/k(B)|=0.9 K and |J(2)/k(B)|=0.009 K between copper neighbors at 5.908 A and at 15.684 A, respectively . J(1) is assigned to a syn-anti equatorial-apical carboxylate bridge with a total bond length of 7.133 A . J(2) is assigned to a long bridge of 12 atoms with a total bond length of 19.789 A, that includes two hydrogen bonds . The results are discussed in terms of the crystal and electronic structure of Cu(l-arginine)(2)(SO(4)).(H(2)O)(6) . We show that J(2) is in excellent agreement with the observed magnetic interaction between the reduced quinone acceptors in the photosynthetic reaction center protein of the bacterium Rb . sphaeroides, which is transmitted along a similar chemical path containing two hydrogen bonds . Our findings indicate that it is valid to estimate values for the exchange interactions between redox centers in proteins transmitted along long chemical paths containing sigma and H-bonds, from data obtained in model systems, and emphasize the importance of measuring exchange interactions in biologically relevant model systems.

Carbohydr Res, 2005 Jan 17, 340(1), 69 - 74
Structure of an acidic polysaccharide from the agar-decomposing marine bacterium Pseudoalteromonas atlantica strain IAM 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid; Perepelov AV et al.; The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated . The polysaccharide was studied by (1)H and (13)C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses . After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated . It was found that the polysaccharide has pentasaccharide repeating units with following structure:

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2519 - 28
Involvement of Sulfide:Quinone Oxidoreductase in Sulfur Oxidation of an Acidophilic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans NASF-1; Wakai S et al.; The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined . The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO . Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO . Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells . Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol . The transcription of three genes, encoding an aa(3)-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction . The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells . A model is proposed for the oxidation of reduced inorganic sulfur compounds in A . ferrooxidans NASF-1 cells.

Infect Immun, 2005 Jan, 73(1), 155 - 65
NF-kappaB activation during Rickettsia rickettsii infection of endothelial cells involves the activation of catalytic IkappaB kinases IKKalpha and IKKbeta and phosphorylation-proteolysis of the inhibitor protein IkappaBalpha; Clifton DR et al.; Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium . R . rickettsii infection induces a biphasic pattern of the nuclear factor-kappaB (NF-kappaB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h . To elucidate the underlying mechanisms, we investigated the expression of NF-kappaB subunits, p65 and p50, and IkappaB proteins, IkappaBalpha and IkappaBbeta . The transcript and protein levels of p50, p65, and IkappaBbeta remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IkappaBalpha at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IkappaBalpha mRNA . The level of IkappaBalpha mRNA gradually returned toward baseline, whereas that of total IkappaBalpha protein remained lower than the corresponding controls . The activities of IKKalpha and IKKbeta, the catalytic subunits of IkappaB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R . rickettsii-infected ECs, revealed significant increases at 2 h after infection . The activation of IKK and early phase of NF-kappaB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae . The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKalpha and IKKbeta, leading to attenuation of nuclear translocation of NF-kappaB . Also, increased activity of IKKalpha was evident later during the infection, coinciding with the late phase of NF-kappaB activation . Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R . rickettsii-induced NF-kappaB activation . Since NF-kappaB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

Cell Microbiol, 2005 Jan, 7(1), 29 - 38
Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation; Ge Y et al.; Summary The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis . Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated . bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils . The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A . phagocytophilum . Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable . By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A . phagocytophilum-infected neutrophils maintained high membrane potential . Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A . phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils . Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation . These results suggest that A . phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2361 - 3
Crystallization and preliminary X-ray characterization of the atypical glutaminyl-tRNA synthetase from Deinococcus radiodurans; Deniziak MA et al.; The glutaminyl-tRNA synthetase (GlnRS) from the radiation-resistant bacterium Deinococcus radiodurans differs from known GlnRSs and other tRNA synthetases by the presence of an additional C-terminal domain resembling the C-terminal region of the GatB subunit of tRNA-dependent amidotransferase (AdT) . This atypical synthetase was overexpressed in Escherichia coli, purified and crystallized in the presence of PEG 3350 . Orthorhombic crystals were obtained that belong to space group P2(1)2(1)2(1) and diffract to 2.3 A resolution . The crystal structure was solved by molecular replacement using the structure of E . coli GlnRS as a search model.

J Infect Chemother, 2004 Dec, 10(6), 316 - 25
Animal models for the study of Helicobacter-induced gastric carcinoma; Kodama M et al.; Helicobacter pylori is considered to have a close association with gastric cancer . Many epidemiological studies have shown a strong association between chronic H . pylori infection and subsequent development of gastric carcinoma in humans . To clarify this link more clearly, it is necessary to use this bacterium in experimental studies to develop gastric carcinoma in suitable experimental animals . Persistent H . pylori infection was seen in the Japanese monkey model, and has recently been achieved in the Mongolian gerbil model . In these models, the sequential histopathological changes in the gastric mucosa are very similar to those in humans . The Japanese monkey model showed advances in atrophic change and p53 point mutations in the gastric mucosa during long-term observation . The Mongolian gerbil model demonstrated that H . pylori infection enhances gastric carcinogenesis in combination with known carcinogens such as N-methyl-N-nitrosourea (MNU) and N-methyl-N-nitro-N'-nitrosoguanidine (MNNG), and also showed that H . pylori infection alone can result in the development of gastric carcinoma . These important results provide a starting point for further studies to clarify the mechanism of gastric carcinogenesis as a result of H . pylori infection and assist in the planning of eradication therapy to prevent gastric carcinoma.

Biophys J . 2004 Dec 21; {Epub ahead of print}
Multiple scattering X-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers; Giachini L et al.; Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, QB . On the basis of X-ray diffraction (XRD) at 2.5 A resolution a site, formed by AspH124, HisH126 and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+) . Using Zn K-edge X-ray absorption fine structure (XAFS) spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films . XAFS data were analysed by combining ab-initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included . Results for complexes characterized by a Zn to RC stoichiometry close to 1 indicate that Zn(2+) binds two O and two N atoms in the first coordination shell . Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue and a water molecule . Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue . The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of XRD data, but detects a significant contraction of the first shell . Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.

Syst Appl Microbiol, 2004 Nov, 27(6), 653 - 60
Mycobacterium fluoranthenivorans sp . nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant; Hormisch D et al.; Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons . The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species . Based on comparative 16S rRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M . diernhoferi as its closest neighbour . The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates . Strain FA4T is able to degrade aflatoxin B1 . This biological attribute might be useful in biological detoxification processes of foods and feeds . From the investigated characteristics it is concluded that strain FA4T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp . nov . The type strain of Mycobacterium fluoranthenivorans is FA4T (DSM 44556T = CIP 108203T).

Biofizika, 2004 Nov-Dec, 49(6), 1069 - 74
{A study of the content of pigments in the light-harvesting antenna of the green bacterium from the new family Oscillochloridaceae}
{Physicochemical properties of two-component polyhydroxyalkanoates {poly(3HB/3HV)}}
{No authors listed}

A series of two-component polyhydroxyalkanoates consisting of hydroxybutyrate and hydroxyvalerate monomer at different ratios were synthesized using the bacterium Ralstonia eutropha B5786 . The properties of polyhydroxyalkanoates were compared with those of the homopolymer of hydroxybutyric acid by X-ray structure analysis, IR spectroscopy, differential scanning calorimetry, and viscosimetry . With an increase in the molar fraction of hydroxyvalerate, an equalization of the ratio of the crystalline and amorphous phases in the copolymer was observed . The degree of crystallinity of the polymer decreased from 70-80 to 45-50%; in the range of an increase in the hydroxyvalerate molar fraction from several to 25-30 mol%, the dependence was linear . The temperature characteristics, the melting temperature (T(m)), and the degradation temperature (T(d)) were lower in polyhydroxyalkanoates than in polyhydroxybutyrate, for which T(m) and T(d) were 168-170 and 260-265 degrees C, respectively . In the copolymer, as the molar fraction of hydroxyvalerate grew, both parameters decreased . In the range of variation of monomer ratio studied, they decreased to 150-160 and 200-220 degrees C, respectively . No distinct correlation between the composition of the polymer and its molecular mass was found.

Dis Aquat Organ, 2004 Nov 4, 61(3), 227 - 33
Experimental infection of Pacific white shrimp Litopenaeus vannamei with Necrotizing Heptopancreatitis (NHP) bacterium by per os exposure; Vincent AG et al.; Necrotizing Hepatopancreatitis Bacterium (NHPB), which causes Necrotizing Hepatopancreatitis, was successfully transmitted in individually isolated Kona stock Litopenaeus vannamei through per os exposure . Animals (140) were individually exposed orally to a 0.05 g piece of an NHPB-infected hepatopancreas and 120 control animals were each exposed to a 0.05 g piece of NHPB-negative hepatopancreas . Shrimp were maintained in Sterilite containers with approximately 41 of artificial seawater at 30 per thousand salinity and 30 degrees C for 60 d . Mortality of infected shrimp was observed from Day 16 to Day 51 post-exposure . Infected animals sustained reduced feeding activity and displayed empty guts . Some infected animals developed a pale hepatopancreas noticeable through the carapace . Survival probabilities fit a Weibull distribution and parametric survival analysis revealed lowered survival due to NHPB infection . Median survival time of NHPB-infected animals was 34.5 d . After correcting for background daily mortality in the controls, mean acute daily mortality of NHPB was estimated at 0.09, a value much lower than that estimated for other diseases in Kona stock L . vannamei such as White Spot Syndrome Virus (0.40) and Taura Syndrome Virus (0.30) . A chronic, or carrier, state was not demonstrated in NHPB epizootics because all NHPB-positive animals experienced mortality and no animals surviving to 60 d post-exposure were diagnosed NHPB-positive through PCR or histology.

Eur Biophys J . 2004 Dec 18; {Epub ahead of print}
Sizing-up finite fluorescent particles with nanometer-scale precision by convolution and correlation image analysis; Gennerich A et al.; Determining the positions, shapes and sizes of finite living particles such as bacteria, mitochondria or vesicles is of interest in many biological processes . In fluorescence microscopy, algorithms that can simultaneously localize such particles as a function of time and determine the parameters of their shapes and sizes at the nanometer scale are not yet available . Here we develop two such algorithms based on convolution and correlation image analysis that take into account the position, orientation, shape and size of the object being tracked, and we compare the precision of the two algorithms using computer simulations . We show that the precision of both algorithms strongly depends on the object's size . In cases where the diameter of the object is larger than about four to five times the beam waist radius, the convolution algorithm gives a better precision than the correlation algorithm (it leads to more precise parameters), while for smaller object diameters, the correlation algorithm gives superior precision . We apply the convolution algorithm to sequences of confocal laser scanning micrographs of immobile Escherichia coli bacteria, and show that the centroid, the front end, the rear end, the left border and the right border of a bacterium can be determined with a signal-to-noise-dependent precision down to ~5 nm.

J Physiol Pharmacol, 2004 Jul, 55 Suppl 2, 5 - 17
Duodenal mucosal protection by bicarbonate secretion and its mechanisms; Konturek SJ et al.; Proximal portion of duodenum is exposed to intermittent pulses of gastric H(+) discharged by the stomach . This review summarizes the mechanisms of duodenal mucosal integrity, mainly the role of mucus-alkaline secretion and the mucous barrier protecting surface epithelium against gastric H(+) . The mucous barrier protects the leaky duodenal epithelium against each pulse of gastric H(+), which penetrates this barrier and diffuses into duodenocytes, but fails to damage them due to; a) an enhanced expression of cyclooxygenase-1 (COX-1), with release of protective prostaglandins (PG) and of nitric oxide (NO) synthase (NOS) with, however, production of NO, stimulating duodenal HCO(3)(-) secretion and b) the release of several neurotransmitters also stimulating HCO(3)(-) secretion such as vasoactive intestimal peptide (VIP), pituitary adenylate-cyclase activating polypeptide (PACAP), acetylcholine, melatonin, leptin and ghrelin released by enteric nerves and mucosal cells . At the apical duodenocyte membrane at least two HCO(3)(-)/Cl(-) anion exchangers operate in response to luminal H(+) to provide adequate extrusion of HCO(3)(-) into duodenal lumen . In the basolateral portion of duodenocyte membrane, both non-electrogenic (NBC) and electrogenic (NBC(n)) Na(+) HCO(3)(-) cotransporters are activated by the exposure to duodenal acidification, causing inward movement of HCO(3)(-) from extracellular fluid to duodenocytes . There are also at least three Na(+)/H(+) (NHE1-3) amiloride-sensitive exchangers, eliminating H(+)which diffused into these cells . The Helicobacter pylori (Hp) infection and gastric metaplasia in the duodenum with bacterium inoculating metaplastic mucosa and inhibiting HCO(3)(-) secretion by its endogenous inhibitor, asymetric dimethyl arginine (ADMA), may result in duodenal ulcerogenesis.

Biochem Biophys Res Commun, 2005 Jan 28, 326(4), 777 - 81
Cytokine induction by a bacterial DNA-specific modified base; Tsuchiya H et al.; Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals . Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production . An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice . Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone . Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12 . These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.

Biotechnol Lett, 2004 Oct, 26(19), 1511 - 5
Expression and characterization of a thermostable beta-xylosidase from the hyperthermophile, Thermotoga maritima; Xue Y et al.; A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system . The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE . The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1 . The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C . Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively . The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1) . When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.

Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 17994 - 9 Epub 2004 Dec 15.
The long-range organization of a native photosynthetic membrane; Frese RN et al.; Photosynthesis relies on the delicate interplay between a specific set of membrane-bound pigment-protein complexes that harvest and transport solar energy, execute charge separation, and conserve the energy . We have investigated the organization of the light-harvesting (LH) and reaction-center (RC) complexes in native bacterial photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides by using polarized light spectroscopy, linear dichroism (LD) on oriented membranes . These LD measurements show that in native membranes, which contain LH2 as the major energy absorber, the RC-LH1-PufX complexes are highly organized in a way similar to that which we found previously for a mutant lacking LH2 . The relative contribution of LH1 and LH2 light-harvesting complexes to the LD spectrum shows that LH2 preferentially resides in highly curved parts of the membrane . Combining the spectroscopic data with our recent atomic force microscopy (AFM) results, we propose an organization for this photosynthetic membrane that features domains containing linear arrays of RC-LH1-PufX complexes interspersed with LH2 complexes and some LH2 found in separate domains . The study described here allows the simultaneous assessment of both global and local structural information on the organization of intact, untreated membranes.

J Bacteriol, 2005 Jan, 187(1), 257 - 65
The elastic properties of the caulobacter crescentus adhesive holdfast are dependent on oligomers of N-acetylglucosamine; Li G et al.; The aquatic bacterium Caulobacter crescentus attaches to solid surfaces through an adhesive holdfast located at the tip of its polar stalk, a thin cylindrical extension of the cell membrane . In this paper, the elastic properties of the C . crescentus stalk and holdfast assembly were studied by using video light microscopy . In particular, the contribution of oligomers of N-acetylglucosamine (GlcNAc) to the elasticity of holdfast was examined by lysozyme digestion . C . crescentus cells attached to a surface undergo Brownian motion while confined effectively in a harmonic potential . Mathematical analysis of such motion enabled us to determine the force constant of the stalk-holdfast assembly, which quantifies its elastic properties . The measured force constant exhibits no dependence on stalk length, consistent with the theoretical estimate showing that the stalk can be treated as a rigid rod with respect to fluctuations of the attached cells . Therefore, the force constant of the stalk-holdfast assembly can be attributed to the elasticity of the holdfast . Motions of cells in a rosette were found to be correlated, consistent with the elastic characteristics of the holdfast . Atomic force microscopy analysis indicates that the height of a dried (in air) holdfast is approximately one-third of that of a wet (in water) holdfast, consistent with the gel-like nature of the holdfast . Lysozyme, which cleaves oligomers of GlcNAc, reduced the force constant to less than 10% of its original value, consistent with the polysaccharide gel-like nature of the holdfast . These results also indicate that GlcNAc polymers play an important role in the strength of the holdfast.

J Bacteriol, 2005 Jan, 187(1), 92 - 8
Identification of two new genes involved in diazotrophic growth via the alternative Fe-only nitrogenase in the phototrophic purple bacterium Rhodobacter capsulatus; Sicking C et al.; Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon . In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity . To obtain further information on the role of Anf1, we screened an R . capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait . Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified . A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain . In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum . A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

Extremophiles . 2004 Dec 15; {Epub ahead of print}
Cold-active DnaK of an Antarctic psychrotroph Shewanella sp . Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures; Yoshimune K et al.; Shewanella sp . Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0 degrees C . Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24 degrees C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli . We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E . coli (EcoDnaK): a characteristic of a cold-active enzyme . The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E . coli at 43 degrees C nor lambda phage propagation at an even lower temperature, 30 degrees C . However, the recombinant SheDnaK gene enabled the E . coli mutant to grow at 15 degrees C . This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.

J Biochem (Tokyo), 2004 Sep, 136(3), 363 - 9
Reconstitution and replacement of bacteriochlorophyll a molecules in photosynthetic reaction centers; Kobayashi M et al.; Reaction centers (RCs) of the photosynthetic bacterium Rhodobacter sphaeroides R-26 were reconstituted in liposomes after release of pigments (bacteriochlorophyll a (BChla) and bacteriopheophytin a (BPhea)) by treatment with acetone . As shown by absorption and circular dichroism spectroscopies, the reconstituted RCs had the same arrangement of pigments as the native RC and exhibited photoactivity of the special pair . The recovery yield of RCs of up to 30% was achieved by addition of 7.8-fold excess of BChla in the acetone treatment . Furthermore BChla was partially replaced with Zn-BChla by addition of the pigments during the acetone treatment . About 30% and 50% of the special pair and accessory pigments can be replaced with Zn-BChla, respectively . From this rate, an oxidation-reduction potential of 520 mV (vs . the normal hydrogen electrode NHE) was derived by the simulation of the experimental data, which is 35 mV higher than that of the native RC (484 mV vs . NHE).

FEMS Microbiol Lett, 2004 Dec 15, 241(2), 151 - 6
Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates; Tamburro A et al.; Peptide methionine sulphoxide reductase (MsrA) and glutathione S-transferases (GSTs) are considered as detoxification enzymes . In the xenobiotics-degrading bacterium Ochrobactrum anthropi the two enzymes are co-induced by toxic concentrations of aromatic substrates such as phenol and 4-chlorophenol . In aerobic organisms, degradation of aromatic substrates by mono- and dioxygenases leads to a generation of oxidative stress that causes the occurrence of reactive oxygen species (ROS) . A capillary electrophoretic method, using the intracellular conversion of dihydrorhodamine-123 into rhodamine-123, was developed to measure the content of ROS in the bacteria . The presence of toxic concentrations of the aromatic substrate 4-chlorophenol, an inducer of GST and MsrA, leads to a significant increase in the production of ROS . These results strongly suggest that GST and MsrA enzymes are part of the bacterial defence mechanism against particular oxidative stress conditions . As oxidative stress is known to be present predominantly close to the cytoplasmic membrane, we investigated the subcellular distribution of both MsrA and GST enzymes in this bacterium grown in the presence of 4-chlorophenol . By Western blotting, MsrA and GST was assayed in the cytoplasm as well as in the periplasm . Moreover, immunolocalisation by colloidal gold immunoelectron microscopy identified the two proteins associated with the cell envelope.

J Inorg Biochem, 2005 Jan, 99(1), 280 - 92
CooA, a paradigm for gas sensing regulatory proteins; Roberts GP et al.; The heme-containing transcriptional factor CooA regulates the expression of genes involved in the oxidation of carbon monoxide (CO) in the bacterium Rhodospirillum rubrum . CooA is both a redox sensor and a specific CO sensor, a combination of properties that is unique among heme proteins . Extensive biochemical and genetic analyses, interpreted in the context of a crystal structure of one form of the protein, have allowed the creation of hypotheses concerning the mechanism of CooA activation by CO as well as the basis for its CO specificity . The article details the data in support of these hypotheses and indicates future lines of research.

Clin Exp Med, 2004 Sep, 4(1), 39 - 43
Progress, problems, and perspectives in diagnosis and treatment of Whipple's disease; Mahnel R et al.; Whipple's disease is a rare chronic infectious disorder first described in 1907 by G.H . Whipple . The disorder is caused by the newly identified bacterium Tropheryma whipplei and there is evidence that altered immune functions play a role in the manifestation of the disease . The organ systems mostly affected are the joints and the gut, and in the further course often also the heart, lung, brain, and eyes . The intestinal involvement occurs with abdominal pain and diarrhea, which leads to weight loss, malnutrition, and anemia . In some cases the infection spreads to the central nervous system, which may lead to loss of memory, confusion, or disturbances in gait . In the last few years, several steps towards an improved diagnosis of the disease and characterization of the causative bacterium have been made . While untreated disease may be lethal, treatment is often able to eradicate the organism . At present, therapy is based on observations in small patient groups and personal experience . There are different antibiotic therapy regimens often starting with intravenous application for 2 weeks followed by oral medication for 1 year . The first clinical therapy study is ongoing.

Proc Natl Acad Sci U S A, 2004 Dec 21, 101(51), 17737 - 40 Epub 2004 Dec 10.
Mitochondrial cytochrome c1 is a collapsed di-heme cytochrome; Baymann F et al.; Cytochrome c(1) from mitochondrial complex III and the di-heme cytochromes c in the corresponding enzyme from epsilon-proteobacteria have so far been considered to represent unrelated cytochromes . A missing link protein discovered in the genome of the hyperthermophilic bacterium Aquifex aeolicus, however, provides evidence for a close evolutionary relationship between these two cytochromes . The mono-heme cytochrome c(1) from A . aeolicus contains stretches of strong sequence homology toward the epsilon-proteobacterial di-heme cytochromes . These di-heme cytochromes are shown to belong to the cytochrome c(4) family . Mapping cytochrome c(1) onto the di-heme sequences and structures demonstrates that cytochrome c(1) results from a mutation-induced collapse of the di-heme cytochrome structure and provides an explanation for its uncommon structural features . The appearance of cytochrome c(1) thus represents an extension of the biological protein repertoire quite different from the widespread innovation by gene duplication and subsequent diversification.

Microbiology, 2004 Dec, 150(Pt 12), 4085 - 93
Characterization of a Myxococcus xanthus mutant that is defective for adventurous motility and social motility; Lancero H et al.; Myxococcus xanthus is a gliding bacterium that possesses two motility systems, the adventurous (A-motility) and social (S-motility) systems . A-motility is used for individual cell gliding, while S-motility is used for gliding in multicellular groups . Video microscopy studies showed that nla24 cells are non-motile on agar surfaces, suggesting that the nla24 gene product is absolutely required for both A-motility and S-motility under these assay conditions . S-motility requires functional type IV pili, wild-type LPS O-antigen, and an extracellular matrix of exopolysaccharide (EPS) and protein called fibrils . The results of expression studies and tethering assays indicate that the nla24 mutant has functional type IV pili . The nla24 mutant also produces wild-type LPS . However, several lines of evidence suggest that the nla24 mutant is defective for production of the EPS portion of the fibril matrix . The nla24 mutant is also defective for transcription of two genes (aglU and cglB) known to be required for A-motility, which is consistent with the idea that nla24 cells are defective for A-motility . Based on these findings, it is proposed that the putative transcriptional activator Nla24 regulates a subset of genes that are important for A-motility and S-motility in M . xanthus.

J Invertebr Pathol, 2004 Oct-Nov, 87(2-3), 114 - 22
Insect cellular and chemical limitations to pathogen development: the Colorado potato beetle, the nematode Heterorhabditis marelatus, and its symbiotic bacteria; Armer CA et al.; This research examines possible factors limiting pathogen development and reproduction in a novel host insect . The nematode Heterorhabditis marelatus and its symbiotic bacterium, Photorhabdus luminescens, kill 98% of nematode-treated Colorado potato beetle (CPB) prepupae, but the nematode reproduces in only 1-6% of beetles . We examined nematode/bacterial inhibition at each step of the normal developmental pathway to determine host feature(s) limiting nematode reproduction . We found that in vivo encapsulation of nematodes occurred in only 1.6% of CPB, and in 5% of in vitro hanging drops of hemolymph . Thus, the cellular defense system did not strongly limit nematode reproduction in the CPB . The symbiotic bacterium was negatively affected by a heat-labile factor found in the CPB's hemolymph which often caused the bacterium to switch from the primary form that produces antibiotics and nutrients necessary for the nematodes' development, to a secondary form that provides only limited nutrients . A 58 kDa protein was isolated and bioassayed for activity against P . luminescens, but caused a delay in bacterial growth rather than the primary-secondary form switch . Thus, the identity of the heat-labile factor could not be confirmed as being the 58 kDa protein . The heat-labile factor did not directly affect the nematode . The addition of lipids in the form of olive oil to heated CPB hemolymph allowed nematodes to reproduce in 17% of hanging drops, in contrast to zero reproduction in hemolymph without oil . Reproductive nematodes were smaller when grown in CPB hemolymph than in hemolymph of the highly susceptible Galleria mellonella . These data suggest that both the toxic heat-labile factor and a lack of appropriate nutrients alter the CPB-bacterium-nematode interaction . These factors preclude the use of this otherwise highly effective nematode-bacterial complex in the longterm control of the CPB.

J Bacteriol, 2004 Dec, 186(24), 8385 - 400
Transcriptomic and proteomic characterization of the Fur modulon in the metal-reducing bacterium Shewanella oneidensis; Wan XF et al.; The availability of the complete genome sequence for Shewanella oneidensis MR-1 has permitted a comprehensive characterization of the ferric uptake regulator (Fur) modulon in this dissimilatory metal-reducing bacterium . We have employed targeted gene mutagenesis, DNA microarrays, proteomic analysis using liquid chromatography-mass spectrometry, and computational motif discovery tools to define the S . oneidensis Fur regulon . Using this integrated approach, we identified nine probable operons (containing 24 genes) and 15 individual open reading frames (ORFs), either with unknown functions or encoding products annotated as transport or binding proteins, that are predicted to be direct targets of Fur-mediated repression . This study suggested, for the first time, possible roles for four operons and eight ORFs with unknown functions in iron metabolism or iron transport-related functions . Proteomic analysis clearly identified a number of transporters, binding proteins, and receptors related to iron uptake that were up-regulated in response to a fur deletion and verified the expression of nine genes originally annotated as pseudogenes . Comparison of the transcriptome and proteome data revealed strong correlation for genes shown to be undergoing large changes at the transcript level . A number of genes encoding components of the electron transport system were also differentially expressed in a fur deletion mutant . The gene omcA (SO1779), which encodes a decaheme cytochrome c, exhibited significant decreases in both mRNA and protein abundance in the fur mutant and possessed a strong candidate Fur-binding site in its upstream region, thus suggesting that omcA may be a direct target of Fur activation.

J Bacteriol, 2004 Dec, 186(24), 8221 - 8
HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae; Willby MJ et al.; The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae . HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2 . Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1 . Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle . The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2 . We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function . The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2 . The significance of these findings in the context of attachment organelle assembly is considered.

Ying Yong Sheng Tai Xue Bao, 2004 Aug, 15(8), 1459 - 62
{Effect of organophosphorous insecticides on Chinese chive insect pests and their degradation by pesticide-degrading bacterium}; Jiang J et al.; 3.00 kg(a . i) x hm(-2) phoxin and 2.63 kg(a . i) x hm(-2) methyl parathion were respectively applied to control the Taeniothrips alliorum on Chinese chive . Compared to no pesticide treatment, the decline rate of the insect density was 98.28% and 98.39% at the 3rd day after spraying pesticides, and 89.94% and 94.04% at the 20th day after spraying pesticides, respectively . At the 3rd day after spraying 15.00, 18.00 and 21.00 kg(a . i) x hm(-2) phoxin, the insect density of Bradysia odoriphaga decreased 80.77%, 93.10% and 96.98%, and at the 35th day after spraying, it decreased 92.44%, 95.05% and 96.81%, respectively . The application of pesticide-degrading bacterium had not any effect on controlling insect pests, but could markedly degrade pesticide . At the 3rd day after spraying 45.00 L x hm(-2) pesticide-degrading bacterium to control Taeniothrips alliorum, the degradion rate of phoxin and methyl parathion was 99.52% and 98.83%, and at the 3rd after spraying 75.00 L x hm(-2) pesticide-degrading bacterium to control Bradysia odoriphaga, the degradation rate of three concentrations of phoxin was 100%, 100% and 99.69%, respectively.

J Nat Prod, 2004 Nov, 67(11), 1897 - 9
Petrobactin sulfonate, a new siderophore produced by the marine bacterium Marinobacter hydrocarbonoclasticus; Hickford SJ et al.; Culture of the oil-degrading marine bacterium Marinobacter hydrocarbonoclasticus gave the known siderophore petrobactin (1) and the new metabolite petrobactin sulfonate (2), the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring . The structure of petrobactin sulfonate was elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.

Nat Prod Rep, 2004 Dec, 21(6), 773 - 84 Epub 2004 Dec.
Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleya; Deng H et al.; This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosyntheses in the bacterium Streptomyces cattleya . A particular emphasis is placed on the purification and characterisation of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis . Nature has hardly developed a biochemistry around fluorine, yet fluorinated organics are important commercial entities, therefore a biotransformation from inorganic to organic fluorine is novel and of contemporary interest.

Analyst, 2004 Dec, 129(12), 1234 - 7 Epub 2004 Dec.
Monitoring viral DNA release with capillary electrophoresis; Krylova SM et al.; Viral DNA injection into host cells is one of the primary mechanisms of viral propagation . Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro . Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles . As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA . DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor . After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis . YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles . The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems . This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.

Vet Microbiol, 2004 Dec 9, 104(3-4), 219 - 25
The high prevalence of Helicobacter sp . in porcine pyloric mucosa and its histopathological and molecular characteristics; Park JH et al.; This study examined the prevalence of Helicobacter infection in the pyloric mucosa of pigs and its histopathological and molecular characteristics . Forty porcine pyloric samples were examined for Helicobacter infection by silver staining and PCR assay . The PCR product (376 bp) was digested with NdeII to differentiate between Helicobacter heilmannii and Helicobacter pylori . Another PCR assay run to produce an 1157 bp fragment was performed using a primer set designed from the 16S rRNA gene of Candidatus H . suis, and its product was cloned and sequenced . Infection rates were 62.5% (25/40) and 95.0% (38/40) as determined by silver staining and the PCR assay, respectively . On histopathological examination, lymphoid follicle aggregation in the pyloric mucosa and granulocytic migration into the lumen of pyloric glands were observed in 24 (60.0%) and 33 (82.5%) gastric samples, respectively . All PCR products, except that of H . pylori, were cut into two fragments of 147 and 229 bp by enzymatic digestion with NdeII . Sequencing of the 16S rRNA gene showed that the bacterium had 99.57% (1152 bp/1157 bp) homology to the 16S rRNA gene of Candidatus H . suis.

Heredity . 2004 Nov 24; {Epub ahead of print}
Multiple infections and diversity of cytoplasmic incompatibility in a haplodiploid species; Mouton L et al.; Cytoplasmic incompatibility (CI) is a sperm-egg incompatibility commonly induced by the intracellular endosymbiont bacterium Wolbachia that, in diploid species, results in embryo mortality . In haplodiploid species, two types of CI exist depending on whether the incompatible fertilized eggs develop into males (male development (MD)) or abort (female mortality (FM)) . CI allows multiple infections to be maintained in host populations, and thus allows interactions to occur between co-infecting strains . In Leptopilina heterotoma, three Wolbachia strains coexist naturally (wLhet1, wLhet2, wLhet3) . When these three strains are all present, they induce a CI of FM type, whereas wLhet1 alone expresses a CI phenotype intermediate between MD and FM . Here, we compare CI effects in crosses involving insect lines sharing the same nuclear background, but harboring different mixtures of strains . Mating experiments showed that: (i) wLhet2 and wLhet3 also induce an intermediate CI when acting alone, and show a bidirectional incompatibility; (ii) there is no interaction between the co-infecting strains in CI expression; (iii) the diversity of Wolbachia present within a male host influences the expression of CI: an increase in the number of strains is correlated with a decrease in the proportion of the MD type, which is also correlated with an increase in bacterial density . All these data suggest that the CI of FM type results from a stronger effect than the MD type, which conflicts with the conventional hypotheses used to explain CI diversity in haplodiploids, and could provide some new information about CI mechanisms in insects.Heredity advance online publication, 24 November 2004; doi:10.1038/sj.hdy.6800596.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4703 - 12
Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis: novel features of an unusual ketolide produced through expression of the chm polyketide synthase in Streptomyces fradiae; Ward SL et al.; Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium Streptomyces bikiniensis, contains a 2,3-trans double bond and the neutral sugar D-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides . Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from S . bikiniensis with very high identity to a unique ketosynthase domain of the tylosin PKS . The resulting amplimers were used to identify two overlapping cosmids encompassing the chm PKS . Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it . The chm PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin . Expression of PKS in the heterologous host Streptomyces fradiae, from which the tyl genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-trans enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the chm PKS . The production of a 3-keto macrolide from the chm PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-trans double bond in chalcomycin . From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-D-chalcose was proposed.

Mycorrhiza . 2004 Nov 19; {Epub ahead of print}
Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants; Morandi D et al.; From a pool of Medicago truncatula mutants-obtained by gamma-irradiation or ethyl methanesulfonate mutagenesis-impaired in symbiosis with the N-fixing bacterium Sinorhizobium meliloti, new mutants are described and genetically analysed, and for already reported mutants, complementary data are given on their phenotypic and genetic analysis . Phenotypic data relate to nodulation and mycorrhizal phenotypes . Among the five new mutants, three were classified as {Nod(+) Fix(-) Myc(+)} and the mutations were ascribed to two loci, Mtsym20 (TRV43, TRV54) and Mtsym21 (TRV49) . For the two other new mutants, one was classified as {Nod(-/+) Myc(+)} with a mutation ascribed to gene Mtsym15 (TRV48), and the other as {Nod(-) Myc(-/+)} with a mutation ascribed to gene Mtsym16 (TRV58) . Genetic analysis of three previously described mutants has shown that {Nod(-/+) Myc(+)} TR74 mutant can be ascribed to gene Mtsym14, and that {Nod(-/+) Myc(-/+)} TR89 and TRV9 mutants are ascribed to gene Mtsym2 ( dmi2) . Using a detailed analysis of mycorrhizal phenotype, we have observed a delayed typical arbuscular mycorrhizal formation on some mutants that present thick lens-shaped appressoria . This phenotype was called {Myc(-/+)} and mutants TR25, TR26, TR89, TRV9, P1 and Y6 were reclassified as {Myc(-/+)} . Mutant P1 was reclassified as {Nod(-/+)} because of a late nodulation observed on roots of this mutant.

Infect Immun, 2004 Dec, 72(12), 6852 - 9
Rapid sequential changeover of expressed p44 genes during the acute phase of Anaplasma phagocytophilum infection in horses; Wang X et al.; Anaplasma phagocytophilum immunodominant polymorphic major surface protein P44s have been hypothesized to go through antigenic variation, but the within-host dynamics of p44 expression has not been demonstrated . In the present study we investigated the composition and changes of p44 transcripts in the blood during the acute phase of well-defined laboratory A . phagocytophilum infections in naive equine hosts . Three traveling waves of sequential population changeovers of the p44 transcript species were observed within a single peak of rickettsemia of less than 1 month . During the logarithmic increase, the rapid switch-off of the initial dominant transcript p44-18 occurred regardless of whether the bacterium was transmitted by ticks or by intravenous inoculation . Each of the subsequently dominant p44 transcript species was phylogenetically dissimilar from p44-18 . Development of antibody to the hypervariable region of P44-18 during the rickettsemia suggests the suppression of dominance of immuno-cross-reactive p44 populations . When A . phagocytophilum was preincubated with plasma from the infected horse and then coincubated with HL-60 cells, the dominance of the p44-18 transcript was rapidly suppressed in vitro and most of the newly emerged p44 transcript species were previously undetected in this horse . This work provides experimental evidence of within-host p44 antigenic variation . Results suggest that the rapid and synchronized switch of expression is an intrinsic property of p44s reinitiated after transmission to naive mammalian hosts and shaped upon exposure to immune plasma.

FEMS Microbiol Lett, 2004 Dec 1, 241(1), 33 - 40
Nitrogen regulation in Sinorhizobium meliloti probed with whole genome arrays; Davalos M et al.; Using whole genome arrays, we systematically investigated nitrogen regulation in the plant symbiotic bacterium Sinorhizobium meliloti . The use of glutamate instead of ammonium as a nitrogen source induced nitrogen catabolic genes independently of the carbon source, including two glutamine synthetase genes, various aminoacid transporters and the glnKamtB operon . These responses depended on both the ntrC and glnB nitrogen regulators . Glutamate repressible genes included glutamate synthase and a H+-translocating pyrophosphate synthase . The smc01041-ntrBC operon was negatively autoregulated in a glnB-dependent fashion, indicating an involvement of phosphorylated NtrC . In addition to the nitrogen response, glutamate remodelled expression of carbon metabolism by inhibiting expression of the Entner-Doudoroff and pentose phosphate pathways, and by stimulating gluconeogenetic genes independently of ntrC.

FEBS Lett, 2004 Nov 19, 577(3), 403 - 8
The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain; Melino S et al.; Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms . Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure . We have evaluated the susceptibility to proteolytic degradation of the two domains of the enzyme . The two domains show different resistance to the endoproteinases and, in particular, the N-terminal domain shows to be more stable to digestion during time than the C-terminal one . Cloning and overexpression of the N-terminal domain of the protein was performed to better understand its functional and structural role . The recombinant N-terminal domain of rhodanese A . vinelandii is soluble in water solution and the spectroscopic studies by circular dichroism and heteronuclear NMR spectroscopy indicate a stable fold of the protein with the expected alpha/beta topology . The results indicate that this N-terminal domain has already got all the elements necessary for an C-terminal domain independent folding . Its solution structure by NMR, actually under course, will be a valid contribution to understand the role of this domain in the folding process of the sulfurtransferase.

FEBS Lett, 2004 Nov 19, 577(3), 371 - 5
The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase; Angel Rubio M et al.; The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis . Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond . Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs . Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively . Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.

BMC Biochem . 2004 Nov 22;5(1):17.
Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer; Bollivar DW et al.; BACKGROUND: The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage . This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus . RESULTS: The gene for R . capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database . R . capsulatus PBGS was heterologously expressed in E . coli and purified to homogeneity . Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R . capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites . Studies with these ions and chelators confirm the predictions . A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6 . Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R . capsulatus PBGS . The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric . A protein concentration dependence to the specific activity suggests that the hexameric R . capsulatus PBGS is very active and can dissociate to smaller, less active, species . A homology model of hexameric R . capsulatus PBGS is presented and discussed . CONCLUSION: The evidence presented in this paper supports the unusual position of the R . capsulatus PBGS as not requiring any metal ions for function . Unlike other wild-type PBGS, the R . capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.

Cell, 2004 Nov 24, 119(5), 615 - 27
Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel; Clayton GM et al.; Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP . The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain . Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel . We propose a model for gating in which ligand binding alters the structural relationship within a dimer, directly affecting the position of the adjacent transmembrane helices.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2313 - 7
Mycobacterium pyrenivorans sp . nov., a novel polycyclic-aromatic-hydrocarbon-degrading species; Derz K et al.; The taxonomic position of a polycyclic-aromatic-hydrocarbon-degrading bacterium, strain 17A3(T), isolated from contaminated soil was determined using a combination of phenotypic and genotypic properties . The isolate showed phenotypic properties that were diagnostic for species of the genus Mycobacterium . Comparative 16S rRNA gene sequence analysis assigned 17A3(T) to the 16S rRNA gene subgroup that contains Mycobacterium aurum, Mycobacterium austroafricanum, Mycobacterium vaccae and Mycobacterium vanbaalenii, but it could clearly be distinguished from these species using a combination of physiological, chemotaxonomic markers and internal rRNA gene spacer analyses . The data showed that strain 17A3(T) (=DSM 44605(T)=NRRL B-24244(T)) merits recognition as the type strain of a novel species of the genus Mycobacterium . The name Mycobacterium pyrenivorans sp . nov . is proposed for the species because of its ability to use pyrene as a sole source of carbon and energy.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2203 - 11
Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes; O'Rourke JL et al.; While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis' . Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis . This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes . Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism . By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis . However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species . The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 1943 - 9
Shewanella profunda sp . nov., isolated from deep marine sediment of the Nankai Trough; Toffin L et al.; A novel piezotolerant, mesophilic, facultatively anaerobic, organotrophic, polarly flagellated bacterium (strain LT13a(T)) was isolated from a deep sediment layer in the Nankai Trough (Leg 190, Ocean Drilling Program) off the coast of Japan . This organism used a wide range of organic substrates as sole carbon and energy sources: pyruvate, glutamate, succinate, fumarate, lactate, citrate, peptone and tryptone . Oxygen, nitrate, fumarate, ferric iron and cystine were used as electron acceptors . Maximal growth rates were observed at a hydrostatic pressure of 10 MPa . Hydrostatic pressure for growth was in the range 0.1-50 MPa . Predominant cellular fatty acids were 16 : 1omega7c, 15 : 0 iso, 16 : 0 and 13 : 0 iso . The G+C content of the DNA was 44.9 mol% . On the basis of 16S rRNA gene sequences, strain LT13a(T) was shown to belong to the gamma-Proteobacteria, being closely related to Shewanella putrefaciens (98 %), Shewanella oneidensis (97 %) and Shewanella baltica (96 %) . Levels of DNA homology between strain LT13a(T) and S . putrefaciens, S . oneidensis and S . baltica were <20 %, indicating that strain LT13a(T) represents a novel species . Genetic evidence and phenotypic characteristics showed that isolate LT13a(T) constitutes a novel species of the genus Shewanella . Because of the deep origin of the strain, the name Shewanella profunda sp . nov . is proposed, with LT13a(T) (=DSM 15900(T)=JCM 12080(T)) as the type strain.

Chem Commun (Camb), 2004 Nov 21, (22), 2590 - 1 Epub 2004 Sep 30.
A mixed ladderane/n-alkyl glycerol diether membrane lipid in an anaerobic ammonium-oxidizing bacterium; Sinninghe Damste JS et al.; A novel glycerol diether containing ladderane and tetradecyl moieties has been identified in an anaerobic ammonium-oxidizing bacterium by GC/MS and high-field NMR spectroscopy.

Am Nat, 2004 Nov, 164 Suppl 5, S19 - 32
The evolution of virulence when parasites cause host castration and gigantism; Ebert D; It has been suggested that the harm parasites cause to their hosts is an unavoidable consequence of parasite reproduction with costs not only for the host but also for the parasite . Castrating parasites are thought to minimize their costs by reducing host fecundity, which may minimize the chances of killing both host and parasite prematurely . We conducted a series of experiments to understand the evolution of virulence of a castrating bacterium in the planktonic crustacean Daphnia magna . By manipulating food levels during the infection of D . magna with the bacterium Pasteuria ramosa, we showed that both antagonists are resource-limited and that a negative correlation between host and parasite reproduction exists, indicating resource competition among the antagonists . Pasteuria ramosa also induces enhanced growth of its hosts (gigantism), which we found to be negatively correlated with host fecundity but positively correlated with parasite reproduction . Because infected hosts never recovered from infections, we concluded that gigantism is beneficial only for the parasite . Hosts, however, have evolved counteradaptations . We showed that infected hosts have enhanced reproduction before castration . This shift to earlier reproduction increases overall host fecundity and compromises parasite reproduction . Finally, we showed that this resource conflict is subject to genetic variation among host and parasite genotypes within a population and is therefore likely to be an important force in the coevolution of virulence in this system . A verbal model is presented and suggests that the adaptive value of gigantism is to store host resources, which are liberated after parasitic castration for later use by the growing parasite . This hypothesis assumes that infections are long lasting, that is, that they have a high life expectancy.

Bioinformatics . 2004 Nov 11; {Epub ahead of print}
A parallel graph decomposition algorithm for DNA sequencing with nanopores; Bokhari SH et al.; MOTIVATION: With the potential availability of nanopore devices that can sense the bases of translocating single-stranded DNA (ssDNA), it is likely that 'reads' of length approximately 10(5) will be available in large numbers and at high speed . We address the problem of complete DNA sequencing using such reads . We assume that approximately 10(2) copies of a DNA sequence are split into single strands that break into randomly sized pieces as they translocate the nanopore in arbitrary orientations . The nanopore senses and reports each individual base that passes through, but all information about orientation and complementarity of the ssDNA subsequences is lost . Random errors (both biological and transduction) in the reads create further complications . RESULTS: We have developed an algorithm that addresses these issues . It can be considered an extreme variation of the well-known Eulerian path approach . It searches over a space of de Bruijn graphs until it finds one in which (a) the impact of errors is eliminated and (b) both possible orientations of the two ssDNA sequences can be identified separately and unambiguously . Our algorithm is able to correctly reconstruct real DNA sequences of the order of 10(6) bases (e.g . the bacterium Mycoplasma pneumoniae) from simulated erroneous reads on a modest workstation in about one hour . We describe, and give measured timings of, a parallel implementation of this algorithm on the Cray Multithreaded Architecture (MTA-2) supercomputer, whose architecture is ideally suited to this 'unstructured' problem . Our parallel implementation is crucial to the problem of rapidly sequencing long DNA sequences and also to the situation where multiple nanopores are used to obtain a high-bandwidth stream of reads.

Appl Microbiol Biotechnol . 2004 Nov 6; {Epub ahead of print}
Purification, cloning, and properties of alpha-galactosidase from Saccharopolyspora erythraea and its use as a reporter system; Post DA et al.; An alpha-galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity . The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE . The pH optimum, K(m) for p-nitrophenyl-alpha- d-glucopyranoside (pNPalphaG), K(m) for melibiose and the V(max) are similar to those of other studied alpha-galactosidase enzymes . The N-terminal amino-acid sequence of this protein was determined . PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related alpha-galactosidase enzymes . This fragment was used as a probe to clone the alpha-galactosidase gene, designated melA, from a S . erythraea lambda phage chromosomal library . S . erythraea appears to possess an unique alpha-galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources . Furthermore, the ability to use the product of melA as a reporter enzyme in S . erythraea has been demonstrated . The alpha-galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl-alpha- d-galactosidase (X-alpha-gal) on agar media and pNPalphaG in liquid media.

J Med Entomol, 2000 May, 37(3), 349 - 56
Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) infection in Amblyomma americanum (Acari: Ixodidae) at Aberdeen Proving Ground, Maryland; Stromdahl EY et al.; Human monocytic ehrlichiosis (HME) is a sometimes fatal, emerging tick-borne disease caused by the bacterium Ehrlichia chaffeensis . It is frequently misdiagnosed because its symptoms mimic those of the flu . Current evidence indicates that Amblyomma americanum (L.), the lone star tick, is the major vector of HME . To determine if E . chaffeensis is present in ticks at Aberdeen Proving Ground, MD, questing A . americanum ticks were collected from 33 sites . Nucleic acid was extracted from 34 adult and 81 nymphal pools . Sequences diagnostic for E . chaffeensis from three different loci (16S rRNA, 120-kDa protein, and a variable-length polymerase chain reaction {PCR} target, or VLPT) were targeted for amplification by the PCR . Fifty-two percent of the collection sites yielded pools infected with E . chaffeensis, confirming the presence and widespread distribution of E . chaffeensis at Aberdeen Proving Ground . Analysis with the both the 120-kDa protein primers and the VLPT primers showed that genetic variance exists . A novel combination of variance for the two loci was detected in two tick pools . The pathogenic implications of genetic variation in E . chaffeensis are as yet unknown.

Biochemistry, 2004 Nov 16, 43(45), 14379 - 84
Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides; Narvaez AJ et al.; The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues . One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well . These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV . The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation . These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe . Radicals were produced in the mutants by using light to initiate electron transfer . The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra . The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164 . The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.

Folia Microbiol (Praha), 2004, 49(4), 479 - 83
Effect of short-chain acids on the carboxymethylcellulase activity of the ruminal bacterium Ruminococcus albus; Paggi RA et al.; The addition of 100-300 mmol/L of acetic, propionic, butyric or lactic acids (short-chain acids), or of acetic, propionic, and butyric acids (volatile fatty acids, VFA) mixtures increased the degradation of carboxymethyl cellulose (CMC) by R . albus (7.5 to 46 and 6 to 39 %, respectively) . Differences among individual acids were observed at 300 mmol/L whereas VFA mixtures differed at 100 mmol/L . When assayed at the same concentration, CMCase activity was increased less by NaCl than by the short-chain acids, whereas ethylene glycol decreased the activity . Since osmolarity and/or ionic strength changes in the medium cannot completely account for the observed increases of carboxymethylcellulase (CMCase) activity, it is suggested that the anions of short-chain acids produce changes in the reaction media polarity that contribute to the effects observed . Alterations in the media could also bring about conformational changes in CMCase leading to increased rates of reaction and subsequent increases in CMC degradation . Finally, explanations for the observed phenomena based on the direct effect of the compounds tested on the cellulosome complex, its domains, and/or its component enzymes are proposed.

Cell Mol Biol (Noisy-le-grand), 2004 Jun, 50(4), 311 - 6
High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda; Hata K et al.; We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C . The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra . Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol) . Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.

Nat Biotechnol, 2004 Nov, 22(11), 1399 - 408
Recombinant protein folding and misfolding in Escherichia coli; Baneyx F et al.; The past 20 years have seen enormous progress in the understanding of the mechanisms used by the enteric bacterium Escherichia coli to promote protein folding, support protein translocation and handle protein misfolding . Insights from these studies have been exploited to tackle the problems of inclusion body formation, proteolytic degradation and disulfide bond generation that have long impeded the production of complex heterologous proteins in a properly folded and biologically active form . The application of this information to industrial processes, together with emerging strategies for creating designer folding modulators and performing glycosylation all but guarantee that E . coli will remain an important host for the production of both commodity and high value added proteins.

Appl Environ Microbiol, 2004 Nov, 70(11), 6897 - 900
Degradation of a nonylphenol single isomer by Sphingomonas sp . strain TTNP3 leads to a hydroxylation-induced migration product; Corvini PF et al.; Sphingomonas sp . strain TTNP3 degrades 4(3',5'-dimethyl-3'-heptyl)-phenol and unidentified metabolites that were described previously . The chromatographic analyses of the synthesized reference compound and the metabolites led to their identification as 2(3',5'-dimethyl-3'-heptyl)-1,4-benzenediol . This finding indicates that the nonylphenol metabolism of this bacterium involves unconventional degradation pathways where an NIH shift mechanism occurs.

Appl Environ Microbiol, 2004 Nov, 70(11), 6595 - 602
Effects of the metalloid oxyanion tellurite (TeO32-) on growth characteristics of the phototrophic bacterium Rhodobacter capsulatus; Borghese R et al.; This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus . There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 microg of tellurite (TeO3(2-)) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 microg/ml . The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium . Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (< or =50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (> or =90% viable cells), which was supported by the development of a significant membrane potential (Deltapsi = 120 mV) . These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R . capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.

FEBS Lett, 2004 Nov 5, 577(1-2), 255 - 8
Biochemical characterization of the major adenylyl cyclase, Cya1, in the cyanobacterium Synechocystis sp . PCC 6803; Masuda S et al.; We report herein the biochemical properties of an adenylyl cyclase, Cya1, from the cyanobacterium Synechocystis sp . PCC 6803 . Heterologously expressed Cya1 catalyzed cyclic AMP formation with a Km for ATP of approximately 2.2 microM at pH 7.5 . Although cellular Cya1 activity is increased by blue light illumination {Terauchi and Ohmori, Mol . Microbiol . 52 (2004) 303}, purified Cya1 did not contain any chromophores, and the activity was light-insensitive . This suggests that an unknown blue light-responsive factor interacts with the N-terminal regulatory domain of Cya1 to control its adenylyl cyclase activity . Finally, our results show that the sensor of blue light using FAD (BLUF) protein, Slr1694, does not appear to be involved in the regulation of Cya1-mediated cAMP signal transduction in this bacterium.

Cell Microbiol, 2004 Dec, 6(12), 1167 - 83
TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton; Garmendia J et al.; Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections . Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton . While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker . Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells . A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion . When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line . TccP has a similar biological activity on infected human intestinal explants ex vivo . Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro . These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Biofizika, 2004 Sep-Oct, 49(5), 822 - 8
{A network of hydrogen bonds in the reaction centers of Rhodobacter sphaeroides serves as a regulatory factor of the temperature dependence of the recombination rate constant of photooxidized bacteriochlorophyll and primary quinone acceptors}; Archaea recruited D-Tyr-tRNATyr deacylase for editing in Thr-tRNA synthetase; School of Biological Sciences, University of Liverpool, Crown St., Liverpool L69 7ZB, United Kingdom . drigden@liv.ac.uk

Aminoacyl-tRNA synthetases (AARSs) are key players in the maintenance of the genetic code through correct pairing of amino acids with their cognate tRNA molecules . To this end, some AARSs, as well as seeking to recognize the correct amino acid during synthesis of aminoacyl-tRNA, enhance specificity through recognition of mischarged aminoacyl-tRNA molecules in a separate editing reaction . Recently, an editing domain, of uncertain provenance, idiosyncratic to some archaeal ThrRSs has been characterized . Here, sequence analyses and molecular modeling are reported that clearly show a relationship of the archaea-specific ThrRS editing domains with d-Tyr-tRNATyr deacylases (DTDs) . The model enables the identification of the catalytic site and other substrate binding residues, as well as the proposal of a likely catalytic mechanism . Interestingly, typical DTD sequences, common in bacteria and eukaryotes, are entirely absent in archaea, consistent with an evolutionary scheme in which DTD was co-opted to serve as a ThrRS editing domain in archaea soon after their divergence from eukaryotes . A group of present-day archaebacteria contain a ThrRS obtained from a bacterium by horizontal gene transfer . In some of these cases a vestigial version of the original archaeal ThrRS, of potentially novel function, is maintained.

J Biol Chem, 2005 Jan 14, 280(2), 1086 - 94 Epub 2004 Nov 03.
Identification and Characterization of a Novel Vitamin B12 (Cobalamin) Biosynthetic Enzyme (CobZ) from Rhodobacter capsulatus, Containing Flavin, Heme, and Fe-S Cofactors; McGoldrick HM et al.; One of the most intriguing steps during cobalamin (vitamin B(12)) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework . The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ . However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663 . To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B(12) pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA . Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction . HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG . Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B(12) itself is a modified tetrapyrrole . A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.

Epidemiol Mikrobiol Imunol, 2004 Aug, 53(3), 95 - 9
{The role of iron in bacterial virulence}; Hostacka A et al.; Data on the role of iron in host-bacterium interaction in relation to virulence are summarized . Attention is focused on host iron acquisition pathways in bacteria . Host iron can be acquired by several mechanisms, e.g . from hemoglobin degradation products such as heme and hemin, directly from ferrated transferrin and lactoferrin, indirectly from iron binding proteins by the production of siderophores and from intracellular iron stores (ferritin) . Regulation of iron uptake is discussed.

Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 15921 - 6 Epub 2004 Nov 02.
Visualization of the movement of single histidine kinase molecules in live Caulobacter cells; Deich J et al.; The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle . This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle . Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds . In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane . By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole . This D value is maintained across all cell cycle stages . We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium . We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.

Transpl Infect Dis, 2004 Jun, 6(2), 77 - 80
Cavitary Legionella pneumonia in a liver transplant recipient; Fraser TG et al.; This report describes the clinical course of a liver transplant recipient in whom cavitary pneumonia developed due to Legionella pneumophila . We review the experience with cavitary pulmonary processes caused by Legionella species in liver allograft recipients and describe the diagnostic microbiology of this organism . The clinical course of this patient demonstrates the importance of considering legionellosis in the differential diagnosis of lung abscesses after liver transplantation and the diagnostic difficulties encountered with this bacterium.

Evolution Int J Org Evolution, 2004 Sep, 58(9), 1901 - 8
Incipient evolution of Wolbachia compatibility types; Charlat S et al.; Cytoplasmic incompatibility (CI) is induced in arthropods by the maternally inherited bacterium Wolbachia . When infected males mate with uninfected females or with females bearing a different Wolbachia variant, paternal chromosomes behave abnormally and embryos die . This pattern can be interpreted as resulting from two bacterial effects: One (usually termed mod, for modification) would affect sperm and induce embryo death, unless Wolbachia is also present in the egg, which implies the existence of a second effect, usually termed resc, for rescue . The fact that CI can occur in crosses between males and females infected by different Wolbachia shows that mod and resc interact in a specific manner . In other words, different compatibility types, or mod/resc pairs seem to have diverged from one (or a few) common ancestor(s) . We are interested in the process allowing the evolution of mod/resc pairs . Here this question is addressed experimentally after cytoplasmic injection into a single host species (Drosophila simulans) by investigating compatibility relationships between closely related Wolbachia variants naturally evolving in different dipteran hosts: D . simulans, Drosophila melanogaster, and Rhagoletis cerasi . Our results suggest that closely related bacteria can be totally or partially incompatible . The compatibility relationships observed can be explained using a formal description of the mod and resc functions, implying both qualitative and quantitative variations.

Mikrobiologiia, 2004 Jul-Aug, 73(4), 437 - 42
{The role of malate dehydrogenase isoforms in the regulation of anabolic and catabolic processes in the colorless sulfur bacterium Beggiatoa leptomitiformis D-402}; An unusual tryptophanyl tRNA synthetase interacts with nitric oxide synthase in Deinococcus radiodurans; Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USAIn mammals, nitric oxide synthases (NOSs) produce nitric oxide for signaling and defense functions; in Streptomyces, NOS proteins nitrate a tryptophanyl moiety in synthesis of a phytotoxin . We have discovered that the NOS protein from the radiation-resistant bacterium Deinococcus radiodurans (deiNOS) associates with an unusual tryptophanyl tRNA synthetase (TrpRS) . D . radiodurans contains genes for two TrpRSs: the first has approximately 40% sequence identity to typical TrpRSs, whereas the second, identified as the NOS-interacting protein (TrpRS II), has only approximately 29% identity . TrpRS II is induced after radiation damage and contains an N-terminal extension similar to those of proteins involved in stress responses . Recombinantly expressed TrpRS II binds tryptophan (Trp), ATP, and D . radiodurans tRNA(Trp) and catalyzes the formation of 5' adenyl-Trp and tRNA(Trp), with approximately five times less activity than TrpRS I . Upon coexpression in Escherichia coli, TrpRS II binds to, copurifies with, and dramatically enhances the solubility of deiNOS . Dimeric TrpRS II binds dimeric deiNOS with a stoichiometry of 1:1 and a dissociation constant of 6-30 muM . Upon forming a complex, deiNOS quenches the fluorescence of an ATP analog bound to TrpRS II, and increases its affinity for substrate l-arginine . Remarkably, TrpRS II also activates the NOS activity of deiNOS . These findings reveal a link between bacterial NOS and Trp metabolism in a second organism and may indicate yet another novel biological function for bacterial NOS.

Microb Pathog, 2004 Nov, 37(5), 225 - 30
Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis; Lai XH et al.; Francisella tularensis is a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages . In a previous study, an iglC null mutant of F . tularensis live vaccine strain LVS was generated by allelic replacement and in the current study this iglC mutant was successfully complemented in trans . We characterized the capacity of this iglC mutant and the complemented strain to induce macrophage apoptosis . The iglC mutant did not induce apoptosis in the infected cells . In contrast, the complemented iglC strain was able to multiply in the murine macrophage-like cell line J774A.1 and induced apoptosis similar to that of the wild-type strain . It is the first successful example of complementation in trans of a F . tularensis mutant strain and more importantly this work provides direct evidence that the intracellular growth ability is essential for F . tularensis to induce macrophage apoptosis.

Biochemistry, 2004 Nov 9, 43(44), 14199 - 210
Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides; Francia F et al.; The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides . In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)) . A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain . Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)) . Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes . Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10 . These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation . Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex . The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.

Proteins, 2005 Jan 1, 58(1), 80 - 7
Binding of glutamine to glutamine-binding protein from Escherichia coli induces changes in protein structure and increases protein stability; D'Auria S et al.; Glutamine-binding protein (GlnBP) from Escherichia coli is a monomeric protein localized in the periplasmic space of the bacterium . It is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane . The protein consists of two similar globular domains linked by two peptide hinges, and X-ray crystallographic data indicate that the two domains undergo large movements upon ligand binding . Fourier transform infrared spectroscopy (FTIR) was used to analyze the structure and thermal stability of the protein in detail . The data indicate that glutamine binding induces small changes in the secondary structure of the protein and that it renders the structure more thermostable and less flexible . Detailed analyses of IR spectra show a lower thermal sensitivity of alpha-helices than beta-sheets in the protein both in the absence and in the presence of glutamine . Generalized two-dimensional (2D) analyses of IR spectra reveal the same sequence of unfolding events in the protein in the absence and in the presence of glutamine, indicating that the amino acid does not affect the unfolding pathway of the protein . The data give new insight into the structural characteristics of GlnBP that are useful for both basic knowledge and biotechnological applications . (c) 2004 Wiley-Liss, Inc.

Eur Respir J, 2004 Nov, 24(5), 811 - 3
No serological evidence of Rickettsia helvetica infection in Scandinavian sarcoidosis patients; Planck A et al.; Many agents have been suggested to elicit sarcoidosis, and, recently, an association was presented between this disease and the bacterium Rickettsia helvetica . The aim of this study was to investigate if serological support for such an association could be detected . Sera from 20 well-characterised sarcoidosis patients were investigated for anti-rickettsial immunoglobulin G antibodies with a micro-immunofluorescence technique . R . helvetica, R . conorii and R . typhi served as antigens . In conclusion, none of the investigated sera displayed detectable titres of anti-rickettsial immunoglobulin G antibodies . Thus, the current study does not support an association between rickettsia and sarcoidosis.

J Bacteriol, 2004 Nov, 186(22), 7670 - 9
The operator and early promoter region of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin expression; Tyler JS et al.; The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages . Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium . Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx . We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W . Like lambda, 933W has three operator repeats in the right operator region (OR), but unlike lambda and all other studied lambdoid phages, which have three operator repeats in the left operator region (OL), 933W only has two operator repeats in OL . As was observed with lambda, the 933W OR and OL regions regulate transcription from the early PR and PL promoters, respectively . A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor . This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.

MMWR Morb Mortal Wkly Rep, 2004 Oct 29, 53(42), 985 - 8
Lymphogranuloma venereum among men who have sex with men--Netherlands, 2003-2004; Centers for Disease Control and Prevention (CDC); Lymphogranuloma venereum (LGV) is a systemic, sexually transmitted disease (STD) caused by a variety of the bacterium Chlamydia trachomatis that rarely occurs in the United States and other industrialized countries; the prevalence of LGV is greatest in Africa, Southeast Asia, Central and South America, and Caribbean countries . However, in the Netherlands, which typically has fewer than five cases a year, as of September 2004, a total of 92 cases of LGV had been confirmed during the preceding 17 months among men who have sex with men (MSM) . The first 13 cases, diagnosed during April-November 2003, were reported by local health authorities in Rotterdam in December 2003 . An alert was sent to the Early Warning and Reporting System of the European Union and to the European Surveillance of Sexually Transmitted Infections Network (ESSTI) . In April 2004, a report was made to CDC, and state and local health departments were alerted . Of the 92 cases confirmed in the Netherlands, 30 occurred during 2003 and 62 during 2004 . This report describes the ongoing investigation of the LGV outbreak . Health-care providers should be vigilant for LGV, especially among MSM exposed to persons from Europe, and prepared to diagnose the disease and provide appropriate treatment to patients and their exposed sex partners.

Genetics, 2004 Oct, 168(2), 1009 - 18
Fixation probability favors increased fecundity over reduced generation time; Wahl LM et al.; The cornerstone of population genetics is a probabilistic understanding of the ultimate fate--survival or extinction--of rare mutations . If a mutation is beneficial, it enables its carrier to reproduce faster than native wild-type individuals . In classic derivations and in the considerable body of research that has followed, "faster" has been defined mathematically to mean "able to produce more surviving offspring per generation." Many organisms, however, may increase their reproductive rate by producing the same number of offspring in a shorter generation time: a mutant bacterium, for example, may complete the cell cycle and produce two offspring more quickly than the wild type . We find that the ultimate fixation probability of a mutation conferring a shorter generation time differs from that of a mutation conferring more offspring by a factor of 2 ln(2)-nearly 40% . This predicts a reduction in the overall substitution rate for any mutation that decreases the generation time: fixation probability is biased toward increased offspring number.

Genetics, 2004 Oct, 168(2), 713 - 22
ospC diversity in Borrelia burgdorferi: different hosts are different niches; Brisson D et al.; The outer surface protein C (ospC) locus of the Lyme disease bacterium, Borrelia burgdorferi, is at least an order of magnitude more variable than other genes in the species . This variation is classified into 22 ospC major groups, 15 of which are found in the northeastern United States . The frequency distributions of ospC within populations suggest that this locus is under balancing selection . In multiple-niche polymorphism, a type of balancing selection, diversity within a population can be maintained when the environment is heterogeneous and no one genotype has the highest fitness in all environments . Genetically different individuals within vertebrate species and different vertebrate species constitute diverse environments for B . burgdorferi . We examined four important host species of B . burgdorferi and found that the strains that infected each species had different sets of ospC major groups . We found no variation among conspecific hosts in the ospC major groups of their infecting strains . These results suggest multiple niches create balancing selection at the ospC locus.

Curr Neurol Neurosci Rep, 2004 Nov, 4(6), 435 - 40
Neurosyphilis; Marra CM; Treponema pallidum, the bacterium that causes syphilis, invades the central nervous system early in the course of disease but causes persistent infection in only a subset of infected persons . Individuals with persistent infection or asymptomatic meningitis are at risk for developing symptomatic neurosyphilis if they are not treated with a drug regimen that achieves sufficient drug levels in cerebrospinal fluid to kill the organism . In this article, recent studies that address the risk, diagnosis, and management of neurosyphilis are discussed within the context of a brief review . Particular attention is given to current controversies . In the developed world, these issues are particularly relevant to persons who are infected with HIV.

Naturwissenschaften, 2004 Nov, 91(11), 552 - 5 Epub 2004 Nov.
Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp; Rodriguez H et al.; In vitro gluconic acid formation and phosphate solubilization from sparingly soluble phosphorus sources by two strains of the plant growth-promoting bacteria A . brasilense (Cd and 8-I) and one strain of A . lipoferum JA4 were studied . Strains of A . brasilense were capable of producing gluconic acid when grown in sparingly soluble calcium phosphate medium when their usual fructose carbon source is amended with glucose . At the same time, there is a reduction in pH of the medium and release of soluble phosphate . To a greater extent, gluconic acid production and pH reduction were observed for A . lipoferum JA4 . For the three strains, clearing halos were detected on solid medium plates with calcium phosphate . This is the first report of in vitro gluconic acid production and direct phosphate solubilization by A . brasilense and the first report of P solubilization by A . lipoferum . This adds to the very broad spectrum of plant growth-promoting abilities of this genus.

Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2009 - 12 Epub 2004 Oct 20.
Cloning, expression, purification, crystallization and preliminary X-ray characterization of the full-length single-stranded DNA-binding protein from the hyperthermophilic bacterium Aquifex aeolicus; Clarke DJ et al.; Single-stranded DNA-binding (SSB) proteins stabilize single-stranded DNA, which is exposed by separation of the duplex during DNA replication, recombination and repair . The SSB protein from the hyperthermophile Aquifex aeolicus has been overexpressed in Escherichia coli, purified and characterized and crystals of the full-length protein (147 amino acids; M(r) 17 131.20) have been grown by vapour diffusion from ammonium sulfate pH 7.5 in both the absence and presence of ssDNA {dT(pT)(68)} . All crystals diffract to around 2.9 A resolution and those without bound DNA (native) belong to space group P2(1), with two tetramers in the asymmetric unit and unit-cell parameters a = 80.97, b = 73.40, c = 109.76 A, beta = 95.11 degrees . Crystals containing DNA have unit-cell parameters a = 108.65, b = 108.51, c = 113.24 A and could belong to three closely related space groups (I222, I2(1)2(1)2(1) or I4(1)) with one tetramer in the asymmetric unit . Electrospray mass spectrometry of the crystals confirmed that the protein was intact . Molecular replacement with a truncated E . coli SSB structure has revealed the position of the molecules in the unit cell and refinement of both native and DNA-bound forms is under way.

Biophys J, 2005 Jan, 88(1), 422 - 35 Epub 2004 Oct 22.
Fluorescence Spectroscopy of Conformational Changes of Single LH2 Complexes; Rutkauskas D et al.; We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope . The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm . The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity . These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum . The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same . Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder . Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra.

Infect Immun, 2004 Nov, 72(11), 6330 - 40
The infecting dose of Chlamydia muridarum modulates the innate immune response and ascending infection; Maxion HK et al.; Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract . Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity . However, many factors modify this response, including the bacterial load . To investigate the manner in which the inoculating dose of C . muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C . muridarum ranging from 10(4) to 10(7) inclusion-forming units . We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses . However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses . Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection . Taken together, these data suggest that innate immune cells contribute to control of ascending infection.

Infect Immun, 2004 Nov, 72(11), 6221 - 9
Macrophages from mice with the restrictive Lgn1 allele exhibit multifactorial resistance to Legionella pneumophila; Derre I et al.; Although Legionella pneumophila can multiply in diverse cell types from a variety of species, macrophages from most inbred mouse strains are nonpermissive for intracellular replication and allow little or no growth of the bacteria . This phenomenon is likely genetically controlled by the mouse naip5 (birc1e) gene located within the Lgn1 locus . In this study, we have investigated the resistance of C57BL/6J macrophages to L . pneumophila infection by examining the fate of both the bacterium and the infected cells compared to that in macrophages from the permissive A/J strain . Our results indicate that although the trafficking of the L . pneumophila-containing vacuole is partially disrupted in C57BL/6J macrophages, this cannot account for the severity of the defect in intracellular growth observed in this strain . Infected macrophages are lost shortly after infection, and at later times a larger fraction of the C57BL/6J macrophages in which L . pneumophila undergoes replication are apoptotic compared to those derived from A/J mice . Finally, a loss of bacterial counts occurs after the first round of growth . Therefore, the resistance mechanism of C57BL/6J macrophages to L . pneumophila infection appears to be multifactorial, and we discuss how early and late responses result in clearing the infection.

J Environ Sci (China), 2004, 16(4), 589 - 93
Characterization and phylogenetic analysis of a phenanthrene-degrading strain isolated from oil-contaminated soil; Xia Y et al.; Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp . based on analysis of 16S rDNA sequence, cellular fatty acid composition and physiological-chemical tests . The salicylate hydroxylase and catechol 2, 3-dioxygenase (C230) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol . Alignment showed that both of the C230 and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas . The phylogenetic analysis based on 16S rDNA and C230 gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other . The results of coamplification and sequence determination indicated that GST gene should be located upstream of the C230 gene.

Exp Parasitol, 2004 Sep-Oct, 108(1-2), 18 - 23
Wolbachia infection in the newly described Ecuadorian sand flea, Tunga trimamillata; Luchetti A et al.; Wolbachia pipientis is an intracellular endosymbiont producing reproductive alterations in its hosts . This bacterium have been reported in many arthropods and nematodes . By PCR amplification and sequencing of the 16S rDNA and ftsZ genes we have identified a Wolbachia strain in the newly described sand-flea, Tunga trimamillata . Prevalence of this endosymbiont in the 26 individuals screened is equal to 35% . Sympatric and allopatric specimens of the related species Tunga penetrans were also analysed, but in contrast to literature data, Wolbachia appears absent in the presently analysed 24 specimens . Field studies evidence a female-biased sex-ratio in T . trimamillata, suggesting that Wolbachia may cause sex-ratio distortion in this species . By means of BLAST search and phylogenetic analysis we found that the Wolbachia strain from T . trimamillata pertains to the arthropod-infecting Wolbachia; this strain is highly differentiated from the Wolbachia strain of T . penetrans described in literature.

Electrophoresis, 2004 Oct, 25(20), 3468 - 74
Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum; Aivaliotis M et al.; An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels . In this study, different methods were employed for the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE) . Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins . The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117 . The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE . However, the additional proteins detected by the use of SDS are in the majority more difficult to solubilize than less hydrophobic proteins . Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane.

Shock, 2004 Nov, 22(5), 395 - 402
The role of the mitochondrion in trauma and shock; Hubbard WJ et al.; The mitochondrion of the eukaryotic cell is well known as a "power plant" whose energy is made available via the high-energy phosphate bonds of ATP . This indispensable and superbly adapted organelle appears to have originated as an endosymbiotic bacterium rather than as a eukaryotic creation per se . However, under the dangerous conditions of trauma and shock, the mitochondrion can become destabilized and harm its host cell in a variety of ways . These contrary traits may be, in part, vestiges from the bacterial origins of mitochondria . The mitochondrion can respond to the stress of trauma and shock by opening pores that leak contents into the host cell's cytoplasm, an event that can trigger programmed cell death or necrosis . In addition, the enormous oxygen consumption by mitochondria presents a two-edged sword in that a deranged mitochondrion can produce reactive oxygen species that damage genes and gene products, inflicting considerable harm to the mitochondrion and its host cell . However, although trauma and shock can cause the mitochondrion to wreak havoc in many ways, an adjuvant intervention with exogenous ATP-MgCl2 after trauma and shock appears useful for reducing cell and organ damage under those conditions.

Clin Microbiol Rev, 2004 Oct, 17(4), 697 - 728, table of contents
Mycoplasma pneumoniae and its role as a human pathogen; Waites KB et al.; Mycoplasma pneumoniae is a unique bacterium that does not always receive the attention it merits considering the number of illnesses it causes and the degree of morbidity associated with it in both children and adults . Serious infections requiring hospitalization, while rare, occur in both adults and children and may involve multiple organ systems . The severity of disease appears to be related to the degree to which the host immune response reacts to the infection . Extrapulmonary complications involving all of the major organ systems can occur in association with M . pneumoniae infection as a result of direct invasion and/or autoimmune response . The extrapulmonary manifestations are sometimes of greater severity and clinical importance than the primary respiratory infection . Evidence for this organism's contributory role in chronic lung conditions such as asthma is accumulating . Effective management of M . pneumoniae infections can usually be achieved with macrolides, tetracyclines, or fluoroquinolones . As more is learned about the pathogenesis and immune response elicited by M . pneumoniae, improvement in methods for diagnosis and prevention of disease due to this organism may occur.

J Biol Chem, 2004 Dec 31, 279(53), 55644 - 50 Epub 2004 Oct 15.
Structural evidence that the 32-kilodalton lipoprotein (Tp32) of Treponema pallidum is an L-methionine-binding protein; Deka RK et al.; A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium . The crystal structure of rTp32 (determined at a resolution of 1.85 A) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two alpha/beta domains, linked by two crossovers, with a binding pocket between them . In the pocket, a molecule of L-methionine was detected in the electron density map . Residues from both domains interact with the ligand . One of the crossover regions is comprised of a 3(10)-helix, a feature not typical in other ligand-binding proteins . Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins . Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T . pallidum.

Annu Rev Microbiol, 2004, 58, 611 - 47
Bacterial iron sources: from siderophores to hemophores; Wandersman C et al.; Iron is an essential element for most organisms, including bacteria . The oxidized form is insoluble, and the reduced form is highly toxic for most macromolecules and, in biological systems, is generally sequestrated by iron- and heme-carrier proteins . Thus, despite its abundance on earth, there is practically no free iron available for bacteria whatever biotope they colonize . To fulfill their iron needs, bacteria have multiple iron acquisition systems, reflecting the diversity of their potential biotopes . The iron/heme acquisition systems in bacteria have one of two general mechanisms . The first involves direct contact between the bacterium and the exogenous iron/heme sources . The second mechanism relies on molecules (siderophores and hemophores) synthesized and released by bacteria into the extracellular medium; these molecules scavenge iron or heme from various sources . Recent genetic, biochemical, and crystallographic studies have allowed substantial progress in describing molecular mechanisms of siderophore and hemophore interactions with the outer membrane receptors, transport through the inner membrane, iron storage, and regulation of genes encoding biosynthesis and uptake proteins.

J Gen Appl Microbiol, 2004 Jun, 50(3), 159 - 67
Gluconobacter thailandicus sp . nov., an acetic acid bacterium in the alpha-Proteobacteria; Tanasupawat S et al.; Four strains of acetic acid bacteria were isolated from flowers collected in Thailand . In phylogenetic trees based on 16S rRNA gene sequences and 16S-23S rDNA internal transcribed spacer (ITS) region sequences, the four isolates were located in the lineage of the genus Gluconobacter and constituted a separate cluster from the known Gluconobacter species, Gluconobacter oxydans, Gluconobacter cerinus, and Gluconobacter frateurii . In addition, the isolates were distinguished from the known species by restriction analysis of 16S-23S rDNA ITS region PCR products using three restriction endonucleases Bsp1286I, MboII, and AvaII . The DNA base composition of the isolates ranged from 55.3-56.3 mol% G+C . The four isolates constituted a taxon separate from G . oxydans, G . cerinus, and G . frateurii on the basis of DNA-DNA similarities . Morphologically, physiologically, and biochemically, the four isolates were very similar to the type strains of G . oxydans, G . cerinus, and G . frateurii; however, the isolates were discriminated in their growth at 37 degrees C from the type strains of G . cerinus and G . frateurii, and in their growth on L-arabitol and meso-ribitol from the type strain of G . oxydans . The isolates showed no acid production from myo-inositol or melibiose, which differed from the type strains of the three known species . The major ubiquinone homologue was Q-10 . On the basis of the results obtained, Gluconobacter thailandicus sp . nov . was proposed for the four isolates . The type strain is isolate F149-1(T) (=BCC 14116(T)=NBRC 100600(T)=JCM 12310(T)=TISTR 1533(T)=PCU 225(T)), which had 55.8 mol% G+C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in Bangkok, Thailand.

Curr Microbiol, 2004 Nov, 49(5), 372 - 5
Indirect immunofluorescence microscopy for direct detection of Xylella fastidiosa in xylem sap; Carbajal D et al.; The plant pathogen Xylella fastidiosa is the causative agent of a number of diseases of economically important crops, including Pierce's disease that affects grapevines . Using a commercially available antibody specific for X . fastidiosa, we have established a protocol for microscopic identification of the bacterium by indirect immunofluorescence . This antibody clearly labels an uncharacterized antigen concentrated at a single pole of X . fastidiosa cells, but does not react with a non- Xylella control . This technique was also performed successfully on xylem exudates from several different plant genera and correlated well with standard enzyme-linked immunosorbent assay tests . These results establish a novel method for in situ assessment of X . fastidiosa infection from host plants.

Expert Rev Vaccines, 2004 Oct, 3(5), 577 - 84
Vaccines against Coxiella infection; Zhang G et al.; Coxiella burnetii is an obligate intracellular bacterium that causes a worldwide zoonotic disease, Q fever . Since C . burnetii infection is an occupational hazard and could develop into severe chronic disease in humans, vaccination should be considered to protect individuals at-risk of contact with naturally infected animals or exposure to the agents . Although several vaccines produced from Phase I whole-cell C . burnetii are effective in protecting against the infection in humans, vaccination of previously sensitized people can induce severe local and occasional systemic reactions . Safe use of these vaccines requires screening of potential vaccinees by skin tests, serological tests, or in vitro lymphocyte proliferation assay . Since these procedures are time-consuming and costly, they limit the use of whole-cell vaccines in a mass vaccination program . Efforts have been underway to develop a safer, more effective new-generation vaccine that will not cause adverse reactions when given to someone with pre-existing immunity . This article describes new information relating to the characterization of acquired immunity to C . burnetii infection that will provide a fundamental understanding of the development of protective immunity against Q fever . Recent works focused on development of recombinant vaccines against this pathogen offers promise in the pursuit of a new Q fever vaccine.

Vet Parasitol, 2004 Nov 10, 125(3-4), 313 - 21
Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections; Morchon R et al.; Sera from three groups of cats under different experimental conditions were studied by ELISA to assess the host's immune response against synthetic peptides derived from Dirofilaria immitis (Dipp) and against the surface protein of its endosymbiont, Wolbachia (WSPr) . In experimentally infected cats (Group 1), an increase of IgG antibody against both Dipp and WSPr was observed from 2 months post-infection until the end of the study, 6 months post-infection . In experimentally infected cats, treated against infective larvae (Group 2), anti-Dipp IgG decreased dramatically from 4 months post-infection (3 months post treatment), showing very low values till the end of the study (6.5 months from infection, 5.5 months from treatment), while anti-WSP IgG increased constantly till the end of the study . Of 49 outdoor, asymptomatic cats exposed to a high risk of natural infection (Group 3), 9 were positive for anti-Dipp IgG and for a validated, in-clinic commercial antibody diagnostic kit for cats . Two cats were also found positive for circulating antigens of adult female worm . Anti-WSPr IgG were found in five of nine anti-Dipp IgG-positive sera and from eight ELISADipp-negative sera . Our results confirm the strong IgG response in heartworm infected cats and demonstrate the involvement of the Wolbachia endosymbiont in the immune reaction to the parasite both in experimentally infected cats and in cats exposed to a high risk of natural infection.

Lett Appl Microbiol, 2004, 39(5), 466 - 70
The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins; Myers CR et al.; AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination . METHODS AND RESULTS: In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled . The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes . Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA . The identity of this band was confirmed by its absence in an OmcA-minus mutant . A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method . CONCLUSIONS: Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed . SIGNIFICANCE: The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM . The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g . manganese oxides) and is consistent with its in vivo role.

Expert Rev Anti Infect Ther, 2004 Jun, 2(3), 447 - 54
Whooping cough due to Bordetella parapertussis: an unresolved problem; Watanabe M et al.; Bordetella parapertussis is one of the bacteria that causes whooping cough . However, little attention has been paid to this bacterium because it causes a milder illness than Bordetella pertussis and the rate of detection is low, even though research suggests that pertussis vaccines have limited efficacy against B . parapertussis infection . However, recent studies have revealed high rates of detection in patients with whooping cough in some field studies . In this review, the relevant studies of B . parapertussis are summarized and it is demonstrated that it is now necessary to pay greater attention to infections by this bacterium.

J Ind Microbiol Biotechnol, 2004 Nov, 31(10), 475 - 81 Epub 2004 Nov.
Optimization of Streptomyces peucetius var . caesius N47 cultivation and epsilon-rhodomycinone production using experimental designs and response surface methods; Kiviharju K et al.; Streptomyces peucetius var . caesius is an aerobic bacterium that produces doxorubicin as a secondary metabolite . A mixture design was applied for the screening of suitable complex medium components in the cultivation of S . peucetius var . caesius N47, which is an epsilon-rhodomycinone-accumulating mutant strain . epsilon-Rhodomycinone is a non-glycosylated precursor of doxorubicin . Best growth results were obtained with soy peptone and beef extract . A central composite face-centered (CCF) experimental design was constructed for the investigation of pH, temperature and dissolved oxygen (DO) effects on the cultivation growth phase . Another CCF was applied to the production phase to investigate the effects of aeration, pH, temperature and stirring rate on epsilon-rhodomycinone production . An increase in cultivation temperature increased both cell growth and glucose consumption rate . Best epsilon-rhodomycinone productivities were obtained in temperatures around 30 degrees C . DO control increased all growth phase responses, but aeration in the production phase coupled with pH decrease resulted in rapid epsilon-rhodomycinone decay in the medium . In non-aerated production phases a pH change resulted in better productivity than in experiments without pH change . A pH increase with a temperature decrease seemed most beneficial for productivity . This implies that dynamic control strategies in batch production of epsilon-rhodomycinone could increase the overall process productivity.

FEBS Lett, 2004 Oct 8, 576(1-2), 226 - 30
Production of maltodextrin 1-phosphate by Fibrobacter succinogenes S85; Nouaille R et al.; We show for the first time the occurrence of maltodextrin-1-Phosphate (MD-1P) (DP2) in F . succinogenes S85, a rumen bacterium specialized in cellulolysis which is not able to use maltose and starch . MD-1P were found in intra and extracellular medium of resting cells incubated with glucose . We used 2D 1H NMR technique and TLC to identify their structure and quantify their production with time . It was also shown that these phosphorylated oligosaccharides originated both from exogenous glucose and endogenous glycogen .

J Clin Microbiol, 2004 Oct, 42(10), 4487 - 93
Tropheryma whipplei Infection of an acellular porcine heart valve bioprosthesis in a patient who did not have intestinal Whipple's disease; Dreier J et al.; Rare cases of culture-negative infective endocarditis are caused by Tropheryma whipplei, the uncommon bacterium of Whipple's disease . We evaluated an 80-year-old woman with valvular heart disease but without intestinal Whipple's disease . The diagnosis of aortic valve xenograft culture-negative infection with T . whipplei was established by multiple molecular assays and by electron microscopy . First, a PCR with broad-range primers identified the complete 16S ribosomal DNA of T . whipplei in bioprosthesis tissue . Novel real-time reverse transcription-PCR assays were developed to detect mRNAs encoding recently identified proteins determined from the T . whipplei genome, specifically Whipplei surface protein (TW113) and a DNA polymerase III subunit (TW727) . The positive detection of mRNAs indicated the presence of metabolically active bacteria and suggested the viability of T . whipplei . The quantification of T . whipplei genome equivalents by real-time PCR indicated a high-density bacterial colonization of the valve tissue . Additionally, an ultrastructural examination revealed numerous rod-shaped bacteria consistent in size with T . whipplei in the extracellular collagen matrix of the bioprosthesis . We conclude that extracellular growth of T . whipplei can occur in the microenvironment of biological prosthetic valve tissue and that T . whipplei endocarditis can occur in the absence of intestinal Whipple's disease.

Cell Cycle, 2004 Sep, 3(9), 1182 - 7 Epub 2004 Sep 01.
Rusticyanin, a bacterial electron transfer protein, causes G1 arrest in J774 and apoptosis in human cancer cells; Yamada T et al.; During acid mine drainage, Acidithiobacillus ferrooxidans, a nonpathogenic, acidophilic, lithotrophic bacterium, utilizes rusticyanin to transfer electrons for the oxidation of Fe(2+) to Fe(3+) for deriving its energy . No other function of rusticyanin is known . We demonstrate that purified rusticyanin enters mammalian cells inducing either inhibition of cell cycle progression or caspase-8 mediated apoptosis . Treatment of human melanoma cells with rusticyanin allowed significant generation of reactive oxygen species and active caspase-8, leading to cell death . The ability of rusticyanin to modulate mammalian cell death might be relevant to a role of this cupredoxin in protecting At . ferrooxidans from eukaryotic predators in the environment.

J Biol Chem, 2004 Dec 10, 279(50), 52024 - 32 Epub 2004 Dec 10.
DNA helicase activity of the RecD protein from Deinococcus radiodurans; Wang J et al.; The bacterium Deinococcus radiodurans is extremely resistant to high levels of DNA-damaging agents, including gamma rays and ultraviolet light that can lead to double-stranded DNA breaks . Surprisingly, the organism does not appear to have a RecBCD enzyme, an enzyme that is critical for double-strand break repair in many other bacteria . The D . radiodurans genome does encode a protein whose closest characterized homologues are RecD subunits of RecBCD enzymes in other bacteria . We have purified this novel D . radiodurans RecD protein and characterized its biochemical activities . The D . radiodurans RecD protein is a DNA helicase that unwinds short (20 base pairs) DNA duplexes with either a 5'-single-stranded tail or a forked end, but not blunt-ended or 3'-tailed duplexes . Duplexes with 10-12 nucleotide (nt) 5'-tails are good unwinding substrates and are bound tightly, while DNA with shorter tails (4-8 nt) are poor unwinding substrates and are bound much less tightly . The RecD protein is much less efficient at unwinding slightly longer substrates (52 or 76 base pairs, with 12 nt 5'-tails) . Unwinding of the longer substrates is stimulated somewhat (4-5-fold) by the single-stranded DNA-binding protein from D . radiodurans . These results show that the D . radiodurans RecD protein is a DNA helicase with 5'-3' polarity and low processivity.

Appl Environ Microbiol, 2004 Oct, 70(10), 6296 - 8
Aquatic snails, passive hosts of Mycobacterium ulcerans; Marsollier L et al.; Accumulative indirect evidence of the epidemiology of Mycobacterium ulcerans infections causing chronic skin ulcers (i.e., Buruli ulcer disease) suggests that the development of this pathogen and its transmission to humans are related predominantly to aquatic environments . We report that snails could transitorily harbor M . ulcerans without offering favorable conditions for its growth and replication . A novel intermediate link in the transmission chain of M . ulcerans becomes likely with predator aquatic insects in addition to phytophage insects . Water bugs, such as Naucoris cimicoides, a potential vector of M . ulcerans, were shown to be infected specifically by this bacterium after feeding on snails experimentally exposed to M . ulcerans.

Appl Environ Microbiol, 2004 Oct, 70(10), 6230 - 9
Spatiotemporal distribution of marine magnetotactic bacteria in a seasonally stratified coastal salt pond; Simmons SL et al.; The occurrence and distribution of magnetotactic bacteria (MB) were studied as a function of the physical and chemical conditions in meromictic Salt Pond, Falmouth, Mass., throughout summer 2002 . Three dominant MB morphotypes were observed to occur within the chemocline . Small microaerophilic magnetite-producing cocci were present at the top of the chemocline, while a greigite-producing packet-forming bacterium occurred at the base of the chemocline . The distributions of these groups displayed sharp changes in abundance over small length scales within the water column as well as strong seasonal fluctuations in population abundance . We identified a novel, greigite-producing rod in the sulfidic hypolimnion that was present in relatively constant abundance over the course of the season . This rod is the first MB that appears to belong to the gamma-Proteobacteria, which may suggest an iron- rather than sulfur-based respiratory metabolism . Its distribution and phylogenetic identity suggest that an alternative model for the ecological and physiological role of magnetotaxis is needed for greigite-producing MB.

Appl Environ Microbiol, 2004 Oct, 70(10), 5875 - 81
Chthoniobacter flavus gen . nov., sp . nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia; Sangwan P et al.; The phylum Verrucomicrobia is increasingly recognized as an environmentally significant group of bacteria, particularly in soil habitats . At least six subdivisions of the Verrucomicrobia are resolved by comparative analysis of 16S rRNA genes, mostly obtained directly from environmental samples . To date, only two of these subdivisions (1 and 4) have characterized pure-culture representatives . We have isolated and characterized the first known pure-culture representative of subdivision 2 . Strain Ellin428 is an aerobic heterotrophic bacterium that is able to grow with many of the saccharide components of plant biomass but does not grow with amino acids or organic acids other than pyruvate . Cells are yellow, rod-shaped, nonmotile, and gram-stain negative, and they contain peptidoglycan with direct cross-linkages of the A1 gamma meso-Dpm type . The isolate grows well at 25 degrees C on a variety of standard biological media, including some used in the routine cultivation of bacteria from soil . The pH range for growth is 4.0 to 7.0 . Low levels of menaquinones MK-10 and MK-11 were detected . The major cellular fatty acids are C(14:0), a-C(15:0), C(16:1 omega 7c), and/or 2OH i-C(15:0), and C(16:0) . The G+C content of the genomic DNA is 61 mol% . We propose a new genus and species, Chthoniobacter flavus gen . nov., sp . nov., with isolate Ellin428 as the type strain, and a new class for the subdivision to which it belongs, Spartobacteria classis nov . Environmental sequences indicate that the class Spartobacteria is largely represented by globally distributed, abundant, and active soil bacteria.

Plant Physiol, 2004 Oct, 136(2), 3159 - 76 Epub 2004 Oct 01.
Expression profiling in Medicago truncatula identifies more than 750 genes differentially expressed during nodulation, including many potential regulators of the symbiotic program; El Yahyaoui F et al.; In this study, we describe a large-scale expression-profiling approach to identify genes differentially regulated during the symbiotic interaction between the model legume Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti . Macro- and microarrays containing about 6,000 probes were generated on the basis of three cDNA libraries dedicated to the study of root symbiotic interactions . The experiments performed on wild-type and symbiotic mutant material led us to identify a set of 756 genes either up- or down-regulated at different stages of the nodulation process . Among these, 41 known nodulation marker genes were up-regulated as expected, suggesting that we have identified hundreds of new nodulation marker genes . We discuss the possible involvement of this wide range of genes in various aspects of the symbiotic interaction, such as bacterial infection, nodule formation and functioning, and defense responses . Importantly, we found at least 13 genes that are good candidates to play a role in the regulation of the symbiotic program . This represents substantial progress toward a better understanding of this complex developmental program.

J Bacteriol, 2004 Oct, 186(20), 6944 - 55
Use of fluorescent-protein tagging to determine the subcellular localization of mycoplasma pneumoniae proteins encoded by the cytadherence regulatory locus; Kenri T et al.; Mycoplasma pneumoniae lacks a cell wall but has internal cytoskeleton-like structures that are assumed to support the attachment organelle and asymmetric cell shape of this bacterium . To explore the fine details of the attachment organelle and the cytoskeleton-like structures, a fluorescent-protein tagging technique was applied to visualize the protein components of these structures . The focus was on the four proteins--P65, HMW2, P41, and P24--that are encoded in the crl operon (for "cytadherence regulatory locus"), which is known to be essential for the adherence of M . pneumoniae to host cells . When the P65 and HMW2 proteins were fused to enhanced yellow fluorescent protein (EYFP), a variant of green fluorescent protein, the fused proteins became localized at the attachment organelle, enabling visualization of the organelles of living cells by fluorescence microscopy . The leading end of gliding M . pneumoniae cells, expressing the EYFP-P65 fusion, was observed as a focus of fluorescence . On the other hand, when the P41 and P24 proteins were labeled with EYFP, the fluorescence signals of these proteins were observed at the proximal end of the attachment organelle . Coexpression of the P65 protein labeled with enhanced cyan fluorescent protein clearly showed that the sites of localization of P41 and P24 did not overlap that of P65 . The localization of P41 and P24 suggested that they are also cytoskeletal proteins that function in the formation of unknown structures at the proximal end of the attachment organelle . The fluorescent-protein fusion technique may serve as a powerful tool for identifying components of cytoskeleton-like structures and the attachment organelle . It can also be used to analyze their assembly.

Mol Microbiol, 2004 Oct, 54(1), 278 - 85
PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation; Narumi I et al.; The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double-strand breaks . By analysing the DNA damage repair-deficient mutant, KH311, a unique radiation-inducible gene (designated pprA) responsible for loss of radiation resistance was identified . Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double-stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases . These results suggest that D . radiodurans has a radiation-induced non-homologous end-joining repair mechanism in which PprA plays a critical role.

Mol Microbiol, 2004 Oct, 54(1), 223 - 38
New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii; Zaar A et al.; A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways . In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl-CoA, which is subsequently attacked by an oxygenase, followed by a non-oxygenolytic fission of the ring . We provide evidence for this hypothesis and show that benzoyl-CoA conversion in the bacterium Azoarcus evansii requires NADPH, O(2) and two protein components, BoxA and BoxB . BoxA is a homodimeric 46 kDa iron-sulphur-flavoprotein, which acts as reductase . In the absence of BoxB, BoxA catalyses the benzoyl-CoA stimulated artificial transfer of electrons from NADPH to O(2) via free FADH(2) to produce H(2)O(2) . Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron-protein that acts as benzoyl-CoA oxygenase . The product of benzoyl-CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA . This suggests that BoxBA act as a benzoyl-CoA dioxygenase/reductase . Unexpectedly, benzoyl-CoA transformation by BoxBA was greatly stimulated when another enoyl-CoA hydratase/isomerase-like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl-CoA . The benzoyl-CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.

Chembiochem, 2004 Oct 4, 5(10), 1401 - 22
Computational design of reduced metabolic networks; Holzhutter S et al.; Cellular functions are based on thousands of chemical reactions and transport processes, most of them being catalysed and regulated by specific proteins . Systematic gene knockouts have provided evidence that this complex reaction network possesses considerable redundancy, that is, alternative routes exist along which signals and metabolic fluxes may be directed to accomplish an identical output behaviour . This property is of particular importance in cases where parts of the reaction network are transiently or permanently impaired, for example, due to an infection or genetic alterations . Here we present a computational concept to determine enzyme-reduced metabolic networks that are still sufficient to accomplish a given set of cellular functions . Our approach consists of defining an objective function that expresses the compromise that has to be made between successive reduction of the network by omission of enzymes and its decreasing thermodynamic and kinetic feasibility . Optimisation of this objective function results in a linear mixed-integer program . With increasing weight given to the reduction of the number of enzymes, the total flux in the network increases and some of the reactions have to proceed in thermodynamically unfavourable directions . The approach was applied to two metabolic schemes: the energy and redox metabolism of red blood cells and the carbon metabolism of Methylobacterium extorquens . For these two example networks, we determined various variants of reduced networks differing in the number and types of disabled enzymes and disconnected reactions . Using a comprehensive kinetic model of the erythrocyte metabolism, we assess the kinetic feasibility of enzyme-reduced subnetworks . The number of enzymes predicted to be indispensable amounts to 14 (out of 28) for the erythrocyte scheme and 13 (out of 77) for the bacterium scheme, the largest group of enzymes predicted to be simultaneously dispensable amounts to 3 and 37 for these two systems . Our approach might contribute to identifying potential target enzymes for rational drug design, to rationalising gene-expression profiles of metabolic enzymes and to designing synthetic networks with highly specialised metabolic functions.

J Biol Chem, 2004 Dec 10, 279(50), 51908 - 14 Epub 2004 Dec 10.
Role of a bacterial organic hydroperoxide detoxification system in preventing catalase inactivation; Wang G et al.; In the gastric pathogen Helicobacter pylori, catalase (KatA) and alkyl hydroperoxide reductase (AhpC) are two highly abundant enzymes that are crucial for oxidative stress resistance and survival of the bacterium in the host . Here we report a connection unidentified previously between the two stress resistance enzymes . We observed that the catalase in ahpC mutant cells in comparison with the parent strain is inactivated partially (approximately 50%) . The decrease of catalase activity is well correlated with the perturbation of the heme environment in catalase, as detected by electron paramagnetic resonance spectroscopy . To understand the reason for this catalase inactivation, we examined the inhibitory effects of hydroperoxides on H . pylori catalase (either present in cell extracts or added to the purified enzyme) by monitoring the enzyme activity and the EPR signal of catalase . H . pylori catalase is highly resistant to its own substrate, without the loss of enzyme activity by treatment with a molar ratio of 1:3000 H2O2 . However, it inactivated is by lower concentrations of organic hydroperoxides (the substrate of AhpC) . Treatment with a molar ratio of 1:400 t-butyl hydroperoxide resulted in an inactivation of catalase by approximately 50% . UV-visible absorption spectra indicated that the catalase inactivation by organic hydroperoxides is caused by the formation of a catalytically incompetent compound II species . To further support the idea that organic hydroperoxides, which accumulate in the ahpC mutant cells, are responsible for the inactivation of catalase, we compared the level of lipid peroxidation found in ahpC mutant cells with that found in wild type cells . The results showed that the total amount of extractable lipid hydroperoxides in the ahpC mutant cells is approximately three times that in the wild type cells . Our findings reveal a novel role of the organic hydroperoxide detoxification system in preventing catalase inactivation.

Physiol Genomics, 2004 Dec 15, 20(1), 21 - 35 Epub 2004 Sep 28.
Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection; Rise ML et al.; Piscirickettsia salmonis is the intracellular bacterium that causes salmonid rickettsial septicemia, an infectious disease that kills millions of farmed fish each year . The mechanisms used by P . salmonis to survive and replicate within host cells are not known . Piscirickettsiosis causes severe necrosis of hematopoietic kidney . Microarray-based experiments with QPCR validation were used to identify Atlantic salmon macrophage and hematopoietic kidney genes differentially transcribed in response to P . salmonis infection . Infections were confirmed by microscopy and RT-PCR with pathogen-specific primers . In infected salmon macrophages, 71 different transcripts were upregulated and 31 different transcripts were downregulated . In infected hematopoietic kidney, 30 different transcripts were upregulated and 39 different transcripts were downregulated . Ten antioxidant genes, including glutathione S-transferase, glutathione reductase, glutathione peroxidase, and cytochrome b558 alpha- and beta-subunits, were upregulated in infected macrophages but not in infected hematopoietic kidney . Changes in redox status of infected macrophages may allow these cells to tolerate P . salmonis infection, raising the possibility that treatment with antioxidants may reduce hematopoietic tissue damage caused by this rickettsial infection . The downregulation of transcripts involved in adaptive immune responses (e.g., T cell receptor alpha-chain and C-C chemokine receptor 7) in infected hematopoietic kidney but not in infected macrophages may contribute to infection-induced kidney tissue damage . Molecular biomarkers of P . salmonis infection, characterized by immune-relevant functional annotations and high fold differences in expression between infected and noninfected samples, may aid in the development of anti-piscirickettsial vaccines and therapeutics.

Clin Immunol, 2004 Nov, 113(2), 140 - 4
Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis; Chierakul W et al.; Interferon (IFN)-gamma plays an important role in the induction of a type 1 immune response against intracellular pathogens . We compared the plasma levels of IFN-gamma and IFN-gamma-inducing cytokines in adult Thai patients with scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, and leptospirosis, caused by extracellular Leptospira interrogans . IFN-gamma, interleukin (IL)-18, and IL-15 levels were elevated only in patients with scrub typhus, whereas IL-12p40 and tumor necrosis factor-alpha concentrations were elevated in both patient groups, although more so in scrub typhus . These data suggest a role for a cell-mediated immune response in host defense against O . tsutsugamushi.

Trends Cell Biol, 2004 Oct, 14(10), 532 - 6
Bacterial cell polarity: a "swarmer-stalked" tale of actin; Li R et al.; The actin cytoskeleton is important for cell polarity and morphogenesis in eukaryotic organisms . A recent article describes an unexpected requirement for the actin-like protein MreB in the polarization of the bacterium Caulobacter crescentus . More surprisingly, the formation of a filamentous MreB structure that traverses the length of the cell is sufficient for randomized polar localization of cell-fate proteins . In this article, we discuss the significance of these findings and the possible mechanisms by which an actin-like cytoskeleton could mediate cell polarity in bacteria.

Biochemistry, 2004 Oct 5, 43(39), 12692 - 9
Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti; Ardissone S et al.; Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti . The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization . The three proteins contain heme b with high-spin Fe(III) . KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM) . KatA and KatC are acidic monofunctional homotetrameric catalases . Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM) . However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly . The kinetic parameters are in good agreement with the physiological role of these heme proteins . KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene . Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).

Biosci Biotechnol Biochem, 2004 Sep, 68(9), 1942 - 8
Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems; Shimada Y et al.; Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E . coli extracts . The alpha-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus . Both of the expression systems produced alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-apoprotein and the pigment molecule bacteriochlorophyll a . The expression product in E . coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100 . Circular dichroism measurement indicated that the expressed alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one . About two thirds of the alpha-apoprotein expressed in E . coli was found to have the amino terminal methionine residue modified by a formyl group . About one third of the alpha-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue . Functional expression of the alpha-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1837 - 43
Ultrastructure and phylogenetic analysis of 'Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks; Kawahara M et al.; A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan . Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells . Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R . norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R . norvegicus rats from China . The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R . norvegicus rats and Ixodes ovatus ticks in Japan . No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae . It is proposed that this new cluster of bacteria should be designated 'Candidatus Neoehrlichia mikurensis'.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1453 - 7
Methylobacillus pratensis sp . nov., a novel non-pigmented, aerobic, obligately methylotrophic bacterium isolated from meadow grass; Doronina NV et al.; Strain F31T was isolated from meadow grass (Poa trivialis L.) sampled from the city park in Helsinki . Analysis of phenotypic and genotypic properties showed the strain to be related to the group of obligately methylotrophic non-methane utilizing bacteria (methylobacteria) with the ribulose monophosphate pathway of formaldehyde assimilation . Phylogenetic analysis showed the strain to be closely related to the genus Methylobacillus, and analysis of fatty acid composition confirmed this association . Thus, on the basis of its genotypic and phenotypic properties, the isolate is proposed as a novel species of the genus Methylobacillus, Methylobacillus pratensis sp . nov., with F31T as the type strain (= VKM B-2247T = NCIMB 13994T).

Carbohydr Res, 2004 Oct 4, 339(14), 2371 - 80
Structural studies of the main exopolysaccharide produced by the deep-sea bacterium Alteromonas infernus; Roger O et al.; The structure of the extracellular polysaccharide produced by the mesophilic species, Alteromonas infernus, found in deep-sea hydrothermal vents and grown under laboratory conditions, has been investigated using partial depolymerization, methylation analysis, mass spectrometry and NMR spectroscopy . The repeating units of this polysaccharide is a nonasaccharide with the following structure: {carbohydrate: see text}.

Arch Environ Contam Toxicol, 2004 Aug, 47(2), 168 - 76
Degradation of aromatic hydrocarbons by Sphingomonas paucimobilis strain EPA505; Story SP et al.; To determine the substrate range capability of Sphingomonas paucimobilis strain EPA505, a number of aromatic compounds were tested as potential growth substrates . Strain EPA505 grew on phenanthrene, naphthalene, fluoranthene, toluene, benzoic acid, 2,3- and 3,4-dihydroxybenzoic acids, 1-chloro-2,4-dinitrobenzene, anthracene, 2-hydroxy-3-naphthoic acid and 1-hydroxy- 2-naphthoic acid, salicylic acid, and catechol . Strain EPA505 was unable to grow on coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, pyrene, benzo{b}fluoranthene, and fluorene . Catabolic products were not detected or identified when the bacterium was incubated with coumarine 3-carboxylic acid, naphthalene dicarboxylic acid, acenaphthene, chrysene, or benzo{b}fluoranthene . Dihydroxypyrene, the ortho ring fission product of pyrene, and 10-hydroxy-1- phenanthroic acid were detected when the bacterium was incubated with pyrene . The open rings of benzo{b}fluoranthene, hydroxyacephenanthroic acid, hydroxyacephenanthrene, and phenanthrene anhydride, catabolites of benzo{b}fluoranthene degradation, were detected with Tn5 mutants of EPA505 . With strain EPA505, both 9-fluorenone and an open ring fission product accumulated during incubation with fluorene . Other catabolites beyond the open ring of fluorene were detected, specifically dihydroxyfluorene, hydroxy-9-fluorenone, dihydroxy-9-fluorenone, hydroxyindane, and a putative glutathione-conjugated benzylanhydride . Benzylanhydride appeared to be a final end product of fluorene degradation by strain EPA505.

Curr Microbiol, 2004 Oct, 49(4), 295 - 9
Nutritional requirements of Allisonella histaminiformans, a ruminal bacterium that decarboxylates histidine and produces histamine; Garner MR et al.; Histamine is an inflammatory agent that contributes to bovine laminitis . Cattle fed silage-containing rations often have large populations of Allisonella histaminiformans, but this obligate histidine-decarboxylating bacterium could not be isolated from cattle fed timothy hay . The growth of A . histaminiformans was stimulated by yeast extract, protein hydrolysates, and water-soluble extracts of alfalfa or corn silage . Extracts of alfalfa were more potent than corn silage . Because growth and histamine production were not stimulated by Casamino Acids or a mixture of purified amino acids, it appeared that A . histaminiformans requires peptides . The idea that A . histaminiformans requires peptides is consistent with the observation that alfalfa silages often have a large amount of peptide nitrogen.

Curr Microbiol, 2004 Sep, 49(3), 208 - 14
Wolbachia pipientis in Australian spiders; Rowley SM et al.; Wolbachia pipientis is an endosymbiotic bacterium common to arthropods and filarial nematodes . This study presents the first survey and characterization of Wolbachia pipientis that infect spiders . All spiders were collected from Queensland, Australia during 2002-2003 and screened for Wolbachia infection using PCR approaches . The Wolbachia strains present in the spiders are diverse, paraphyletic, and for the most part closely related to strains that infect insects . We have also identified several spider Wolbachia strains that form a lineage outside the currently recognized six main Wolbachia supergroups (A-F) . Incongruence between spider and Wolbachia phylogenies indicates a history of horizontal transmission of the bacterium in these host taxa . Like other arthropods, spiders are capable of harboring multiple Wolbachia strains.

Infect Immun, 2004 Oct, 72(10), 5963 - 71
Magnetic resonance imaging of pulmonary lesions in guinea pigs infected with Mycobacterium tuberculosis; Kraft SL et al.; We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis . Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs . Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium . Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development . Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals . A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time . Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines.

Infect Immun, 2004 Oct, 72(10), 5910 - 8
Porphyromonas gingivalis strain-dependent activation of human endothelial cells; Walter C et al.; Porphyromonas gingivalis is an important bacterium involved in periodontal diseases . Colonization by periodontopathogens has been associated with severe local inflammatory reactions in the connective tissue . In this study we characterized P . gingivalis-mediated infection and activation of human umbilical vein endothelial cells by using two strains of different virulence capacities, strains ATCC 53977 and DSMZ 20709 . Both strains were able to adhere to and infect endothelial cells with an infection rate of 0.48% for ATCC 53977 and 0.007% for DSMZ 20709 . The triggering of two signal transduction pathways in P . gingivalis-infected endothelial cells was demonstrated for both strains, with a rapid increase of p38 mitogen-activated protein kinase phosphorylation and a more delayed degradation of IkappaBalpha, followed by nuclear translocation of NF-kappaB . In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1) . Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 DeltafimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT) . Thus, two P . gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation.

Infect Immun, 2004 Oct, 72(10), 5597 - 604
Humoral immunity through immunoglobulin M protects mice from an experimental actinomycetoma infection by Nocardia brasiliensis; Salinas-Carmona MC et al.; An experimental model of infection with Nocardia brasiliensis, used as an example of a facultative intracellular pathogen, was tested . N . brasiliensis was injected into the rear foot pads of BALB/c mice to establish an infection . Within 30 days, infected animals developed a chronic actinomycetoma infection . Batch cultures of N . brasiliensis were used to purify P61, P38, and P24 antigens; P61 is a catalase, and P38 is a protease with strong caseinolytic activity . Active and passive immunizations of BALB/c mice with these three purified soluble antigens were studied . Protection was demonstrated for actively immunized mice . However, immunity lasted only 30 days . Other groups of immunized mice were bled at different times, and their sera were passively transferred to naive recipients that were then infected with N . brasiliensis . Sera collected 5, 6, and 7 days after donor immunization conferred complete, long-lasting protection . The protective effect of passive immunity decreased when sera were collected 2 weeks after donor immunization . However, neither the early sera (1-, 2-, and 3-day sera) nor the later sera (30- or 45-day sera) prevented the infection . Hyperimmune sera with the highest levels of immunoglobulin G (IgG) to N . brasiliensis antigens did not protect at all . The antigens tested induced two IgM peaks . The first peak was present 3 days after immunization but was not antigen specific and did not transfer protection . The second peak was evident 7 days after immunization, was an IgM response, was antigen specific, and conferred protection . This results clearly demonstrate that IgM antibodies protect the host against a facultative intracellular bacterium.

J Immunol, 2004 Oct 1, 173(7), 4635 - 42
Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus; Giambartolomei GH et al.; Inflammation is a hallmark of brucellosis . Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known . We examined the mechanism whereby heat-killed B . abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages . Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1 . HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B . abortus LPS only stimulated cells from C3H/HeN mice . Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells . Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B . abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response . Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion . Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production . Together, these results entail a mechanism whereby B . abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis . We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.

Can J Microbiol, 2004 Jul, 50(7), 509 - 13
Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression; Jayaraj J et al.; Azospirillum is used extensively in rice and other cereal crops as a biofertilizer . There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms . Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities . So far there are no reports about transfer of plant genes into Azospirillum and their expression . The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense . A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter . The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating . The conjugation frequency was in the range of 35-40 x 10(-6) . The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro . However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type . Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody . The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants . The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

Biochem Biophys Res Commun, 2004 Oct 22, 323(3), 852 - 7
Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1; Calugay RJ et al.; A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis . The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed . The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue . Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon . Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore . Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type . These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M . magneticum AMB-1.

Proteomics, 2004 Oct, 4(10), 2831 - 42
Genome and proteome analysis of Chlamydia; Vandahl BB et al.; It has been difficult to study the molecular biology of the obligate intracellular bacterium Chlamydia due to lack of genetic transformation systems . Therefore, genome sequencing has greatly expanded the information concerning the biology of these pathogens . Comparing the genomes of seven sequenced Chlamydia genomes has provided information of the common gene content and gene variation . In addition, the genome sequences have enabled global investigation of both transcript and protein content during the developmental cycle of chlamydiae . During this cycle Chlamydia alternates between an infectious extracellular form and an intracellular dividing form surrounded by a phagosome membrane termed the chlamydial inclusion . Proteins secreted from the chlamydial inclusion into the host cell may interact with host cell proteins and modify the host cell's response to infection . However, identification of such proteins has been difficult because the host cell cytoplasm of Chlamydia infected cells cannot be purified . This problem has been circumvented by comparative proteomics.

Biomed Microdevices, 2004 Sep, 6(3), 223 - 9
Patterning and separating infected bacteria using host-parasite and virus-antibody interactions; Suh KY et al.; Bacteria were selectively deposited on substrates patterned with poly(ethylene glycol) (PEG) microstructures by using host-parasite and virus-antibody interactions . In this scheme viruses were used to attach onto a host bacterium, Escherichia coli (E . coli) . The E . coli expressing the virus were selectively adhered to the regions pretreated with an antibody against the virus proteins while E . coli without the virus showed no selectivity . Single or aggregated cell arrays were fabricated depending on the initial pattern size with respect to the size of E . coli . The current approach could be a general route to spatially positioning or controlling adhesion of other biological species that are not accessible by conventional methods and as a tool for separating and isolating specific cell populations based on host-parasite interactions.

Biochemistry (Mosc), 2004 Aug, 69(8), 890 - 6
Temporary stabilization of electron on quinone acceptor side of reaction centers from the bacterium Rhodobacter sphaeroides wild type and mutant SA(L223) depending on duration of light activation; Knox PP et al.; The dark reduction of photooxidized bacteriochlorophyll (P+) by photoreduced secondary quinone acceptor (QB-) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied . The kinetics of the dark reduction of P+ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium . In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P+ on duration of light was not observed . Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain . It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site . At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB .

Extremophiles . 2004 Sep 16; {Epub ahead of print}
Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH; Kamimura K et al.; The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH . This isolate specifically requires NaCl for growth . The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline- N-oxide . Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors . Thiosulfate-oxidizing activity was not inhibited by these uncouplers . Valinomycin did not inhibit the oxidation of sulfur compounds . NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts . The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity . Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur . The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na(+)-dependent . A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A . thiooxidans strain SH.

J Bacteriol, 2004 Oct, 186(19), 6667 - 70
Channel-forming (Porin) activity in Herpetosiphon aurantiacus Hp a2; Harwardt R et al.; Detergent extracts of cell envelopes of the gliding bacterium Herpetosiphon aurantiacus formed channels in lipid bilayers . Fast protein liquid chromatography across a HiTrap-Q cation-exchange column demonstrated that a 45-kDa protein forms the channel . The observation of a channel-forming protein suggests that Herpetosiphon aurantiacus Hp a2 has a permeability barrier on its surface.

J Bacteriol, 2004 Oct, 186(19), 6465 - 76
Insecticidal pilin subunit from the insect pathogen Xenorhabdus nematophila; Khandelwal P et al.; Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host . Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X . nematophila . Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X . nematophila isolated from that protein complex . The gene was amplified by PCR, cloned, and expressed in Escherichia coli . The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient . The protein oligomerized during in vitro refolding, forming multimers . Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation . The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase . The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives . The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein . The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H . armigera, causing extensive damage to the midgut epithelial membrane . To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

Asian Pac J Cancer Prev, 2004 Jul-Sep, 5(3), 246 - 52
Helicobacter pylori eradication as a preventive tool against gastric cancer; Hamajima N et al.; Helicobacter pylori (H . pylori), which increases the risk of gastric diseases, including digestive ulcers and gastric cancer, is highly prevalent in Asian countries . There is no doubt that eradication of the bacterium is effective as a treatment of digestive ulcer, but eradication aiming to reduce the gastric cancer risk is still controversial . Observational studies in Japan demonstrated that the eradication decreased the gastric cancer risk among 132 stomach cancer patients undergoing endoscopical resection (65 treated with omeprazol and antibiotics and 67 untreated) . In Columbia, 976 participants were randomized into eight groups in a three-treatment factorial design including H . pylori eradication, resulting in significant regression in the H . pylori eradication group . A recent randomized study in China also showed a significant reduction of gastric cancer risk among those without any gastric atrophy, intestinal metaplasia, and dysplasia . Efficacy of eradication may vary in extent among countries with different incidence rates of gastric cancer . Since the lifetime cumulative risk (0 to 84 years old) of gastric cancer in Japan is reported to be 12.7% for males and 4.8% for females (Inoue and Tominaga, 2003), the corresponding values for H . pylori infected Japanese can be estimated at 21.2% in males and 8.0% in females under the assumptions that the relative risk for infected relative to uninfected is 5 and the proportion of those infected is 0.5 . Both the fact that not all individuals are infected among those exposed and the knowledge that only a small percentage of individuals infected with the bacterium develop gastric cancer, indicate the importance of gene-environment interactions . Studies on such interactions should provide useful information for anti-H . pylori preventive strategies.

Funct Integr Genomics, 2004 Oct, 4(4), 219 - 30 Epub 2004 Jul 24.
Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry; Barsch A et al.; A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula . A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition . S . meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step . Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS . The different compounds were identified by comparison with the NIST 98 database and available standards . From about 200 peaks in each chromatogram 65 compounds have been identified so far . A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools . A principal component analysis (PCA) was able to distinguish S . meliloti cells grown on different carbon sources based on their metabolite profile . A comparison of the metabolite composition of a S . meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant . Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine . This finding further emphasises the importance of integrating metabolic data into post-genomic research.

J Biol Chem, 2004 Dec 3, 279(49), 51122 - 30 Epub 2004 Dec 3.
Function of a PscD subunit in a homodimeric reaction center complex of the photosynthetic green sulfur bacterium Chlorobium tepidum studied by insertional gene inactivation . Regulation of energy transfer and ferredoxin-mediated NADP+ reduction on the cytoplasmic side; Tsukatani Y et al.; The PscD subunit in the homodimeric "type I" photosynthetic reaction center (RC) complex of the green sulfur bacterium Chlorobium tepidum was disrupted by insertional mutagenesis of its relevant pscD gene . This is the first report on the use of the direct mutagenic approach into the RC-related genes in green sulfur bacteria . The RC complex of C . tepidum is supposed to form a homodimer of two identical PscA subunits together with three other subunits: PscB (FA/FB-containing protein), PscC (cytochrome cz), and PscD . PscD shows a relatively low but significant similarity in its amino acid sequence to PsaD in the photosystem I of plants and cyanobacteria . We studied the biochemical and spectroscopic properties of a mutant lacking PscD in order to elucidate its unknown function . 1) The RC complex isolated from the mutant cells showed no band corresponding to PscD on SDS-PAGE analysis . 2) The growth rate of the PscD-less mutant was slower than that of the wild-type cells at low light intensities . 3) Time-resolved fluorescence spectra at 77 K revealed prolonged decay times of the fluorescence from bacteriochlorophyll c on the antenna chlorosome and from bacteriochlorophyll a on the Fenna-Matthews-Olson antenna protein in the mutant cells . The loss of PscD led to a much slower energy transfer from the antenna pigments to the special pair bacteriochlorophyll a (P840) . 4) The mutant strain exhibited slightly less activity of ferredoxin-mediated NADP+ photoreduction compared with that in the wild-type strain . The extent of suppression, however, was less significant than that reported in the PsaD-less mutants of cyanobacterial photosystem I . The evolutionary relationship between PscD and PsaD was also discussed based on a structural homology modeling of the former.

Rev Environ Contam Toxicol, 2003, 179, 37 - 71
Spinosad toxicity to pollinators and associated risk; Mayes MA et al.; Spinosad is a natural insecticide derived from an actinomycete bacterium species, Saccharopolyspora spinosa (Mertz and Yao 1990), that displays the efficacy of a synthetic insecticide . It consists of the two most active metabolites, designated spinosyn A and D . Both spinosyns are readily degraded in moist aerobic soil, and field dissipation, which is quite rapid (half-life, 0.3-0.5 d) can be attributed to photolysis or a combination of metabolism and photolysis . Spinosad causes neurological effects in insects that are consistent with the general activation of nicotinic acetylcholine receptors but by a mechanism that is novel among known insecticide compounds . Spinosad has a high level of efficacy for lepidopteran larvae, as well as some Diptera, Coleoptera, Thysanoptera, and Hymenoptera, but has limited to no activity to other insects and exhibits low toxicity to mammals and other wildlife . Although spinosad has low toxicity to most beneficial insects, initial acute laboratory tests indicated that spinosad is intrinsically toxic to pollinators . The hazard of spinosad to bees was evaluated using a tiered approach . Initial acute laboratory exposures were conducted, followed by toxicity of residues of spinosad on treated foliage, greenhouse studies to assess acute as well as chronic toxicity, confined field assessments, and finally full field studies using a variety of crops under typical use conditions . These data were used to assess the potential of adverse effects on foraging bees following the use of spinosad . This research has clearly demonstrated that spinosad residues that have been allowed to dry for 3 hr are not acutely harmful to honeybees when low-volume and ultralow-volume sprays are used . Further, glasshouse and semifield studies have demonstrated that dried residues are not acutely toxic, and although pollen and nectar from sprayed plants may have transient effects on brood development, the residues do not overtly affect hive viability of either the honeybee or the bumblebee . Field studies in which typical application methods of spinosad were used on a variety of crops have demonstrated that spinosad has low risk to adult honeybees and has little or no effect on hive activity and brood development . The collective evidence from these studies indicates that once spinosad residues have dried on plant foliage, generally 3 hr or less, the risk of spinosad to honeybees is negligible.

Rev Med Interne, 2004 Sep, 25(9), 659 - 62
{Violacein: a molecule of biological interest originating from the soil-borne bacterium Chromobacterium violaceum}; Dessaux Y et al.; INTRODUCTION: Soil micro-organisms have evolved functions that allow them to withstand the strong competition for survival that characterizes most of their habitats . The production of antibiotic or antifungal compounds is one of these mechanisms . The relevant molecules often exhibit valuable therapeutic properties . EXEGESIS: Chromobacterium violaceum is a soil-borne bacterium producing a characteristic antibiotic termed violacein . It is part of a series of compounds released by C . violaceum to oppose competitors and predators in the soil and water environments . Violacein, and one of these compounds, i.e . structure FR901228, exhibit antiparasitic and antitumoral activities of potential medical interest . Genes involved in the synthesis of these compounds are available, the genome sequence of C . violaceum (strain ATCC 12472) being published . CONCLUSIONS: The above example, involving Chromobacterium, is not an exception: soil constitutes a reservoir of molecules, enzymatic activities and micro-organisms of biological interest, the study of which will undoubtedly lead to developments in fields as diverse as agronomy or animal and human therapeutics.

Braz J Infect Dis, 2004 Apr, 8(2), 184 - 5 Epub 2004 Sep 08.
Mycobacterium tuberculosis bacteremia diagnosed in an HIV-negative patient in Brazil: a rare or an under-reported event?
Hadad DJ, Pignatari AC, Machado MA, Telles MA.
A case of Mycobacterium tuberculosis bacteremia in an HIV negative immunodepressed patient was described using the BACTEC 460 TB system . This bacterium should be investigated in the blood of immunodepressed non-HIV infected patients with prolonged fever.

PLoS Biol . 2004 Oct;2(10):e304 . Epub 2004 Sep 07.
Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1; Harris DR et al.; The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair . The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D . radiodurans DNA repair system . DR0423 is a homologue of the eukaryotic Rad52 protein . Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation . When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D . radiodurans reassembles its genome while the mutant lacking DR0423 function does not . In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3' ends, and protects those ends from nuclease degradation . We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation . We designate the DR0423 protein as DNA damage response A protein.

Helicobacter, 2004 Oct, 9(5), 429 - 35
Melanoidin, a food protein-derived advanced maillard reaction product, suppresses Helicobacter pylori in vitro and in vivo; Hiramoto S et al.; BACKGROUND: Extracellular urease proteins located on the surface of Helicobacter pylori are gastric mucin-targeted adhesins, which play an important role in infection and colonization to the host . In this study we have determined the inhibitory activity of a variety of melanoidins, protein-derived advanced Maillard reaction products, ubiquitously found in heat-treated foods, on urease-gastric mucin adhesion . In addition, we have determined the anticolonization effect of melanoidin I, prepared by the Maillard reaction between casein and lactose, in an animal model and in human subjects infected with this bacterium . METHODS: The inhibitory activity of each compound was determined by a competitive binding assay of labeled gastric mucin to plate-immobilized urease . Melanoidin I was used in an in vivo trial using euthymic hairless mice as an infection model . Melanoidin I was consumed for 8 weeks by subjects infected with H . pylori . The {(13)C} urease breath test and H . pylori-specific antigen in the stool (HpSA) test were performed on subjects at week 0 and week 8 . RESULTS: A variety of food protein-derived melanoidins strongly inhibited urease-gastric mucin adhesion in the concentration range of 10 micro g/ml to 100 micro g/ml . In particular, melanoidin I significantly (p <.05) suppressed colonization of H . pylori in mice when given for 10 weeks via the diets . Eight weeks daily intake of 3 g melanoidin I significantly (p <.05) decreased the optical density of HpSA in subjects . CONCLUSION: Foods containing protein-derived melanoidins may be an alternative to antibiotic-based therapy to prevent H . pylori that combines safety, ease of administration and efficacy.

Clin Diagn Lab Immunol, 2004 Sep, 11(5), 963 - 8
CXCR2 blockade influences Anaplasma phagocytophilum propagation but not histopathology in the mouse model of human granulocytic anaplasmosis; Scorpio DG et al.; Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils and causes human granulocytic anaplasmosis . Infection induces neutrophil secretion of interleukin-8 or murine homologs and perpetuates infection by recruiting susceptible neutrophils . We hypothesized that antibody blockade of CXCR2 would decrease A . phagocytophilum tissue load by interrupting neutrophil recruitment but would not influence murine hepatic pathology . C3H-scid mice were treated with CXCR2 antiserum or control prior to or on day 14 after infection . Quantitative PCR and immunohistochemistry for A . phagocytophilum were performed and severity of liver histopathology was ranked . Control mice had more infected cells in tissues than the anti-CXCR2-treated group . The histopathological rank was not different between treated and control animals . Infected cells of control mice clustered in tissue more than in treated mice . The results support the hypothesis of bacterial propagation through chemokine induction and confirm that tissue injury is unrelated to A . phagocytophilum tissue load.

Chemosphere, 2004 Oct, 57(2), 81 - 90
Reducing bioassay variability by identifying sources of variation and controlling key parameters in assay protocol; Ren S et al.; Reducing bioassay variability by identifying sources of variation and controlling important parameters in assay protocol was demonstrated in this study . The variability of a bioassay based on a luminescent bacterium was examined as an example . This assay involved the growth of cells, storage at a low temperature, activation, and exposure to a test sample, and the assay response was bioluminescence inhibition . After determining that measurement error was small and negligible, the total assay variability was decomposed in an initial variance components study into between-batch, between-vial, and between-tube variations . Results indicated that between-vial variations accounted for the majority of the total observed variability and that reducing this type of variation would be beneficial . Five parameters in the assay protocol were determined as factors that potentially affected assay variability significantly . A split-plot design was employed to investigate the effects of these factors and some of their interactions on the assay response . One of the five factors, i.e., activation temperature, turned out to have a significant effect . The variance components study was repeated with better control of activation temperature as well as other parameters . Results indicated that the total variability of the bioassay was reduced by approximately 85%.

Annu Rev Microbiol . 2004 Jun 16; {Epub ahead of print}
Bacterial Iron Sources: From Siderophores to Hemophores; Wandersman C et al.; Iron is an essential element for most organisms, including bacteria . The oxidized form is insoluble, and the reduced form is highly toxic for most macromolecules and, in biological systems, is generally sequestrated by iron- and heme-carrier proteins . Thus, despite its abundance on earth, there is practically no free iron available for bacteria whatever biotope they colonize . To fulfil their iron needs, bacteria have multiple iron acquisition systems, reflecting the diversity of their potential biotopes . The iron/heme acquisition systems in bacteria have one of two general mechanisms . The first involves direct contact between the bacterium and the exogenous iron/heme sources . The second mechanism relies on molecules (siderophores and hemophores) synthesized and released by bacteria into the extracellular medium; these molecules scavenge iron or heme from various sources . Recent genetic, biochemical and crystallization studies have allowed substantial progress in describing molecular mechanisms of siderophore and hemophore interactions with the outer membrane receptors, transport through the inner membrane, iron storage, and regulation of genes encoding biosynthesis and uptake proteins . Expected online publication date for the Annual Review of Microbiology Volume 58 is September 8, 2004 . Please see for revised estimates.

J Mol Biol, 2004 Sep 24, 342(4), 1171 - 86
Regulation of hem gene expression in Rhodobacter capsulatus by redox and photosystem regulators RegA, CrtJ, FnrL, and AerR; Smart JL et al.; Biosynthetic pathways for heme and chlorophyll share common intermediates from 5-aminolevulinic acid through protoporphyrin IX . To obtain a better understanding of how photosynthetic organisms coordinate heme and chlorophyll biosynthesis, we have undertaken detailed analysis of the expression pattern of numerous heme biosynthesis genes in the purple photosynthetic bacterium Rhodobacter capsulatus . beta-Galactosidase reporter assays demonstrated that expression of hemA, hemB, hemC, hemE and hemZ genes is elevated under conditions that give rise to elevated bacteriochlorophyll synthesis . Heme gene expression is shown to be affected by mutations in previously identified transcriptional regulators RegA, FnrL, CrtJ, and AerR, which also control expression of genes involved in bacteriochlorophyll and carotenoid synthesis, and synthesis of the apoprotein subunits of the photosynthetic and electron transport apparatus . High-resolution primer extension analysis of hem mRNA reveals the presence of numerous putative RegA, FnrL and CrtJ binding sites in several hem promoter regions.

Appl Environ Microbiol, 2004 Sep, 70(9), 5621 - 7
Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease; Orchard SS et al.; The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects . Presently, it is not known what nutrients the bacterium uses to thrive in these host environments . In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X . nematophila in laboratory or host environments . We identified an X . nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF . Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease . We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts . We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects . However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions . These data demonstrate that the opp genes allow X . nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X . nematophila in either of its host niches . To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.

C R Biol, 2004 Jul, 327(7), 687 - 94
Growth and cellular fatty-acid composition of a sulphate-reducing bacterium, Desulfatibacillum aliphaticivorans strain CV2803T, grown on n-alkenes; Cravo-Laureau C et al.; The anaerobic degradation of n-alkenes by a sulphate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T was investigated . Results suggest that enzymes required for alkene degradation are inducible . Moreover, total cellular fatty acids of strain CV2803T were predominantly C-odd when the strain was grown on C-odd substrates and C-even when grown on C-even substrates . In addition to classical bacterial fatty acids, unusual 4-Me-17:1delta11 and 4-Me-18:1delta11 fatty acids and their saturated homologues were detected when strain CV2803T was grown on 1-pentadecene and 1-hexadecene, respectively . These methyl-branched monounsaturated fatty acids could constitute specific metabolites of n-alkene degradation by sulphate-reducing bacteria.

Bioorg Khim, 2004 May-Jun, 30(3), 247 - 53
{The release of flavin adenine dinucleotide upon a local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase}; The auxiliary protein HypX provides oxygen tolerance to the soluble {NiFe}-hydrogenase of ralstonia eutropha H16 by way of a cyanide ligand to nickel; Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, NL-1018 TV Amsterdam, The NetherlandsThe hypX gene of the facultative lithoautotrophic bacterium Ralstonia eutropha is part of a cassette of accessory genes (the hyp cluster) required for the proper assembly of the active site of the {NiFe}-hydrogenases in the bacterium . A deletion of the hypX gene led to a severe growth retardation under lithoautotrophic conditions with 5 or 15% oxygen, when the growth was dependent on the activity of the soluble NAD+ -reducing hydrogenase . The enzymatic and infrared spectral properties of the soluble hydrogenase purified from a HypX-negative strain were compared with those from an enzyme purified from a HypX-positive strain . In activity assays under anaerobic conditions both enzyme preparations behaved the same . Under aerobic conditions, however, the mutant enzyme became irreversibly inactivated during H2 oxidation with NAD+ or benzyl viologen as the electron acceptor . Infrared spectra and chemical determination of cyanide showed that one of the four cyanide groups in the wild-type enzyme was missing in the mutant enzyme . The data are consistent with the proposal that the HypX protein is specifically involved in the biosynthetic pathway that delivers the nickel-bound cyanide . The data support the proposal that this cyanide is crucial for the enzyme to function under aerobic conditions.

J Bacteriol, 2004 Sep, 186(18), 6050 - 8
Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein; Rawat M et al.; Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates . Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH . In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis . The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S . Introduction of M . tuberculosis mca epichromosomally or introduction of M . smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates . The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin . Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin . Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.

Mol Microbiol, 2004 Sep, 53(6), 1721 - 30
Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans; Lecointe F et al.; DNA polymerases of the X family have been implicated in a variety of DNA repair processes in eukaryotes . Here we show that Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced double-stranded DNA breaks, expresses a DNA polymerase belonging to the X family . This novel bacterial polymerase, named PolX(Dr), was identified as the product of the Deinococcal DR0467 gene . The purified PolX(Dr) protein possesses a DNA polymerase activity that is stimulated by MnCl2, a property of the X family DNA polymerases . Antibodies raised against PolX(Dr) recognized human pol lambda, rat pol beta and yeast Pol4 and, conversely, antibodies raised against these proteins recognized PolX(Dr) . This immunological cross-reactivity suggests a high degree of structural conservation among the polymerases of the X family . Lack of PolX(Dr) reduced the rate of repair of double-stranded DNA breaks and increased cell sensitivity to gamma-rays . PolX(Dr) thus appears to play an important role in double-stranded DNA break repair in D . radiodurans.

Arch Microbiol, 2004 Oct, 182(2-3), 165 - 74 Epub 2004 Aug 31.
The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR; Prange A et al.; During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules . The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC . Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A . vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules . Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes . The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions . Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the "main proteins" of the sulfur globule envelope . Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules . This indicates a role of SgpC for globule expansion . A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.

Lancet Infect Dis, 2004 Sep, 4(9), 575 - 83
Molecular survival strategies of the Lyme disease spirochete Borrelia burgdorferi; Singh SK et al.; Lyme disease is a tick-transmitted disease caused by the spirochete Borrelia burgdorferi . The bacterium adopts different strategies for its survival inside the immunocompetent host from the time of infection until dissemination in different parts of body tissues . The success of this spirochete depends on its ability to colonise the host tissues and counteract the host's defence mechanisms . During this process borrelia seems to maintain its vitality to ensure long-term survival in the host . Borrelia's proteins are encoded by plasmid and chromosomal genes . These genes are differentially regulated and expressed by different environmental factors in ticks as well as in the mammalian host during infection . In addition, antigenic diversity enables the spirochete to escape host defence mechanisms and maintain infection . In this review we focus on the differential expression of proteins and genes, and further molecular mechanisms used by borrelia to maintain its survival in the host . In light of these pathogenetic mechanisms, further studies on spirochete host interaction are needed to understand the complex interplay that finally lead to host autoimmunity.

Dig Liver Dis, 2004 Aug, 36(8), 505 - 12
Duodenal alkaline secretion: its mechanisms and role in mucosal protection against gastric acid; Konturek PC et al.; Duodenal mucosa, especially its proximal portion, is exposed to intermittent pulses of gastric acid (H+) . This review summarises the mechanisms of duodenal bicarbonate (HCO3-) secretion and their role in protecting duodenal epithelium against gastric H+ . Duodenal epithelium is a leaky barrier against gastric H+, which diffuses into duodenocytes, but fails to damage them due to: (a) an enhanced expression of cyclooxygenase, producing protective prostaglandins and expression of nitric oxide synthase, releasing nitric oxide, both stimulating duodenal HCO3- secretion and (b) the release of several neurotransmitters also stimulating HCO3- secretion such as vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, acetylcholine and melatonin . At the apical duodenocyte membrane, several HCO3-/Cl- anion exchangers operate in response to luminal H+ to extrude HCO3- into duodenal lumen . In baso-lateral duodenocyte membrane, both non-electrogenic and electrogenic Na+-HCO3- cotransporters are activated after exposure of duodenum to gastric H+, causing inward movement of HCO3- from extracellular fluid to duodenocytes . There are also at least three Na+/H+ exchangers, eliminating H+ which diffused into these cells . The Helicobacter pylori infection with gastric metaplasia in the duodenum and bacterium inoculation results in the inhibition of HCO3- secretion by its endogenous inhibitor dimethyl arginine, resulting in ulcerogenesis.

J Biol Chem, 2004 Dec 3, 279(49), 50654 - 61 Epub 2004 Dec 3.
An RNA ligase from Deinococcus radiodurans; Martins A et al.; Although DNA repair pathways have been the focus of much attention, there is an emerging appreciation that distinct pathways exist to maintain or manipulate RNA structure in response to breakage events . Here we identify an RNA ligase (DraRnl) from the radiation-resistant bacterium Deinococcus radiodurans . DraRnl seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA: DNA hybrid, but it cannot seal 3'-OH/5'-PO4 DNA nicks . The specificity of DraRnl arises from a requirement for RNA on the 3'-OH side of the nick . DraRnl is a 342-amino acid monomeric protein with a distinctive structure composed of a C-terminal adenylyltransferase domain linked to an N-terminal module that resembles the OB-fold of phenylalanyl-tRNA synthetases . RNA sealing activity was abolished by mutation of the predicted lysine adenylylation site (Lys-165) in the C-terminal domain and was reduced by an order of magnitude by deletion of the N-terminal OB module . Our findings highlight the existence of an RNA repair capacity in bacteria and support the hypothesis that contemporary DNA ligases, RNA ligases, and RNA capping enzymes evolved by the fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.

Huan Jing Ke Xue, 2004 Jan, 25(1), 49 - 52
{Electrofusion of building coupling system for degrading anthracene and fusants' choices}; Zhang JY et al.; Some researches have been carried out for the electro fusion of building coupling system for degrading anthracene and the choices of fusants . During the experiments, total rate of fusion stable in alternation current frequency 500 kHz, impulse intensity 300 V/cm, impulse width 5 micros, amount of impulse 2 and alternation current intensity 20-40 V/cm were found . The resistant plate with erythromycin and ampicillin can screen the fusant of autochthonous bacterium and surfactant-producing bacterium . The resistant plate with anthracene and karnamicin can screen the fusant of autochthonous bacterium and efficient anthracene-degradation bacterium . The new fusants were found to have stable fluorescent characteristics.

Environ Monit Assess, 2004 Aug-Sep, 96(1-3), 53 - 83
Toxicity of surficial sediments from Sydney Harbour and vicinity, Australia; McCready S et al.; The toxicological responses of three species to 103 surficial saltwater sediment samples from Sydney Harbour, and coastal lakes and estuaries on the south-east coast of New South Wales, Australia, were tested in a battery of four to six laboratory toxicity tests . This is the first large-scale toxicological study of sediments in Australia, the objective of which is to assess the protective and predictive abilities of North American biological effects-based sediment quality guidelines, recently adopted in Australia . Amphipods were exposed to whole sediments in survival and reburial tests, sea urchin fertilisation and larval development tests were conducted on porewaters, and bacterial bio-luminescence (Microtox) tests were conducted on organic solvent extracts and porewaters . Local indigenous species were used for the amphipod and sea urchin tests (Corophium sp . and Heliocidaris tuberculata, respectively) . A wide range of responses, from <25 to 100% of negative controls were observed in all tests . Mean control-adjusted responses ranged from 46 to 96% for all tests . The percentages of highly toxic samples ranged from 11 to 83% in the various tests . The order of test sensitivity was: amphipod survival < Microtox test of porewaters < amphipod reburial < sea urchin larval development < sea urchin fertilisation < Microtox test of solvent extracts . Concordance between toxicity tests in classifying samples as highly toxic or not, ranged from 47 to 79%, indicating some similarities between test results, but not complete equivalence . Combined toxicity test results showed that the incidence of highly toxic responses occurring in the majority of tests (75-100% of tests) was low (5% of samples), but a large percentage of samples had highly toxic results in at least one test (76% of samples) . Toxicity was more pervasive in the Sydney region than in coastal lakes and estuaries south of Sydney . The current study demonstrated the utility of indigenous invertebrate species and the Microtox bacterium in a sediment toxicity test battery for Australian saltwater sediments.

Int Immunol, 2004 Oct, 16(10), 1431 - 7 Epub 2004 Aug 23.
Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis; Hashimoto M et al.; Lipopolysaccharide (LPS) preparations from the periodontopathic bacterium Porphyromonas gingivalis (Pg-LPS) are thought to require Toll-like receptor (TLR)2 rather than TLR4, a receptor of Escherichia coli LPS (Ec-LPS), for activation of immune cells . However, we previously reported that P . gingivalis lipid A, an immunostimulatory principal component of LPS, and its synthetic counterpart activate cells through a TLR4-dependent pathway but not via TLR2 . In the present study, a lipoprotein from Pg-LPS (Pg-LP) was shown to be a principal component for TLR2-mediated cell activation . Pg-LP was separated by hydrophobic interaction chromatography followed by preparative electrophoresis and identified by internal peptide sequencing as PG1828, a putative lipoprotein encoded in the P . gingivalis genome . The N-terminal structure was characterized as a triacylated lipopeptide using mass spectrometry . Pg-LP, as well as Ec-LPS, was potent in inducing IL-8 production in human gingival fibroblasts . From our results, we propose that Pg-LP is a powerful inflammatory factor of P . gingivalis.

Biochemistry, 2004 Aug 31, 43(34), 11126 - 34
Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus; Turina P et al.; The proton-pumping and the ATP hydrolysis activities of the ATP synthase of Rhodobacter capsulatus have been compared as a function of the ADP and P(i) concentrations . The proton pumping was measured either with the transmembrane pH difference probe, 9-amino-6-chloro-2-methoxyacridine, or with the transmembrane electric potential difference probe, bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol, obtaining consistent results . The comparison indicates that an intrinsic uncoupling of ATP synthase is induced when the concentration of either ligand is decreased . The half-maximal effect was found in the submicromolar range for ADP and at about 70 microM for P(i) . It is proposed that a switch from a partially uncoupled state of ATP synthase to the coupled state is induced by the simultaneous binding of ADP and P(i).

Extremophiles . 2004 Aug 19; {Epub ahead of print}
Marinobacter alkaliphilus sp . nov., a novel alkaliphilic bacterium isolated from subseafloor alkaline serpentine mud from Ocean Drilling Program Site 1200 at South Chamorro Seamount, Mariana Forearc; Takai K et al.; Novel alkaliphilic, mesophilic bacteria were isolated from subseafloor alkaline serpentine mud from the Ocean Drilling Program (ODP) Hole 1200D at a serpentine mud volcano, South Chamorro Seamount in the Mariana Forearc . The cells of type strain ODP1200D-1.5(T) were motile rods with a single polar flagellum . Growth was observed between 10 and 45-50 degrees C (optimum temperature: 30-35 degrees C, 45-min doubling time), between pH 6.5 and 10.8-11.4 (optimum: pH 8.5-9.0), and between NaCl concentrations of 0 and 21% (w/v) (optimum NaCl concentration: 2.5-3.5%) . The isolate was a facultatively anaerobic heterotroph utilizing various complex substrates, hydrocarbons, carbohydrates, organic acids, and amino acids . Nitrate or fumarate could serve as an electron acceptor to support growth under anaerobic conditions . The G+C content of the genomic DNA was 57.5 mol% . Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Marinobacter and was the most closely related to M . aquaeolei strain VT8(T) and M . hydrocarbonoclasticus strain SP.17(T), while DNA-DNA hybridization demonstrated that the new isolate could be genetically differentiated from the previously described species of Marinobacter . Based on the physiological and molecular properties of the new isolate, we propose the name Marinobacter alkaliphilus sp . nov., type strain: ODP1200D-1.5(T) (JCM12291(T) and ATCC BAA-889(T)).

Microbiol Immunol, 2004, 48(8), 561 - 9
Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies; Murakami Y et al.; Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis . We have previously identified major outer membrane proteins from P . gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin . Fimbriae, another important virulence factor, exist on the cell surface . In this study, we identified major supernatant proteins . They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains . Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures . To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors . In general, localization of major virulence factors did not change at the various growth temperatures used . Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant . Gingipains were found in both the envelope fraction and supernatant . More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant . Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction . When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased . However, temperature had almost no effect on expression of putative porin . The tendency for expression of major virulence factors to decrease at higher temperatures may enable P . gingivalis to survive under hostile conditions.

Infect Immun, 2004 Sep, 72(9), 5530 - 3
Bordetella pertussis adenylate cyclase-hemolysin induces interleukin-6 secretion by human tracheal epithelial cells; Bassinet L et al.; After interaction with tracheal epithelial cells, Bordetella pertussis induces the secretion of interleukin-6 . This secretion is dependent on the expression of adenylate cyclase-hemolysin by the bacterium but not on the expression of other characterized bacterial toxins or adhesins . This finding confirms the important role of adenylate cyclase-hemolysin in the pathogenicity of the bacterium.

Infect Immun, 2004 Sep, 72(9), 5392 - 401
Neutrophil NADPH oxidase is reduced at the Anaplasma phagocytophilum phagosome; IJdo JW et al.; The intracellular organism Anaplasma phagocytophilum causes human granulocytic ehrlichiosis and specifically infects and multiplies in neutrophilic granulocytes . Previous reports have suggested that, for its survival, this bacterium suppresses the neutrophil respiratory burst . To investigate the mechanism of survival, we first assessed the kinetics of A . phagocytophilum entry into neutrophils by using double-labeling confocal microscopy . At 30, 60, 120, and 240 min of incubation, 25, 50, 55, and 70% of neutrophils contained bacteria, respectively . The neutrophil respiratory burst in the presence of A . phagocytophilum was assessed by a kinetic cytochrome c assay and by measurement of oxygen consumption . Neutrophils in the presence of A . phagocytophilum did not produce a significant respiratory burst, but A . phagocytophilum did not inhibit the neutrophil respiratory burst when phorbol myristate acetate was added . Immunoelectron microscopy of neutrophils infected with A . phagocytophilum or Escherichia coli revealed that NADPH oxidase subunits gp91(phox) and p22(phox) were significantly reduced at the A . phagocytophilum phagosome after 1 and 4 h of incubation . In neutrophils incubated simultaneously with A . phagocytophilum and E . coli for 30, 60, and 90 min, gp91(phox) was present on 20, 14, and 10% of the A . phagocytophilum phagosomes, whereas p22(phox) was present in 11, 5, and 4% of the phagosomes, respectively . Similarly, on E . coli phagosomes, gp91(phox) was present in 62, 64, and 65%, whereas p22(phox) was detected in 54, 48, and 48% . We conclude that A . phagocytophilum does not suppress a global respiratory burst and that, under identical conditions in the same cells, A . phagocytophilum, but not E . coli, significantly reduces gp91(phox) and p22(phox) from its phagosome membrane.

J Mol Biol, 2004 Aug 27, 341(5), 1215 - 26
Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface; Bjork A et al.; The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface . Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface . Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C) . The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures . The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface . Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced . Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains . Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 415 - 23
The Mycobacterium tuberculosis sigJ gene controls sensitivity of the bacterium to hydrogen peroxide; Hu Y et al.; Sigma factors are important global regulators which control bacterial gene expression during growth and in response to stress . Previous work showed that mRNA of the sigJ gene was up-regulated in late stationary-phase and after rifampicin treatment . In order to verify the function of SigJ, we constructed a Mycobacterium tuberculosis mutant lacking the sigJ gene . In a microaerophilic stationary-phase model, the sigJ mutant showed the same growth pattern as the wild-type strain . In an immune stasis murine model in which the bacterial number plateaued between the second and the 15th week, the mutant showed a similar growth curve to the wild-type strain . However, the sigJ mutant was more susceptible to killing by H2O2 than its parental strain . The parental level of sensitivity to H2O2 was recovered in the sigJ complemented strain . These data suggest that the SigJ protein is not essential for survival in long-term stationary phase or in bacterial stasis in mice . However, the sigJ gene may control an alternative H2O2 resistance pathway.

J Bacteriol, 2004 Sep, 186(17), 5842 - 55
Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae; McLeod MP et al.; Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts . Here we present the complete genome sequence of R . typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R . prowazekii and R . conorii . We identified 877 genes in R . typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts . In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system . The three rickettsial genomes share 775 genes: 23 are found only in R . prowazekii and R . typhi, 15 are found only in R . conorii and R . typhi, and 24 are unique to R . typhi . Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R . typhi, compared to R . prowazekii and R . conorii . In addition, we found a 124-kb R . typhi-specific inversion, starting 19 kb from the origin of replication, compared to R . prowazekii and R . conorii . Inversions in this region are also seen in the unpublished genome sequences of R . sibirica and R . rickettsii, indicating that this region is a hot spot for rearrangements . Genome comparisons also revealed a 12-kb insertion in the R . prowazekii genome, relative to R . typhi and R . conorii, which appears to have occurred after the typhus (R . prowazekii and R . typhi) and spotted fever (R . conorii) groups diverged . The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.

J Bacteriol, 2004 Sep, 186(17), 5834 - 41
Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation; Berleman JE et al.; Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients . In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle . Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R . centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle . These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability . Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators . Several of the disrupted genes also have sequence similarity to che-like signal transduction components.

J Bacteriol, 2004 Sep, 186(17), 5808 - 18
Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis; Johansson A et al.; The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism . We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci . In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F . tularensis isolates, including representatives from each of the four subspecies . The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95 . Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F . tularensis isolates, including accurate subspecies identification . F . tularensis subsp . tularensis (type A) isolates showed great diversity at VNTR loci, while F . tularensis subsp . holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence . The resolution of two distinct clades within F . tularensis subsp . tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies . F . tularensis subsp . holarctica appears to have recently spread globally across continents from a single origin, while F . tularensis subsp . tularensis has a long and complex evolutionary history almost exclusively in North America . The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain . Significant linkage disequilibrium was detected among VNTR loci of F . tularensis consistent with a clonal population structure . Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F . tularensis isolates.

J Bacteriol, 2004 Sep, 186(17), 5685 - 91
CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110; Yoshizawa Y et al.; Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle . Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx) . In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators . In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides . We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities . Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed . When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM . In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed . In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed . Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level . Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration . The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.

Shock, 2004 Sep, 22(3), 270 - 7
Persistent HIF-1alpha activation in gut ischemia/reperfusion injury: potential role of bacteria and lipopolysaccharide; Koury J et al.; In both animal models of hemorrhagic shock and clinical settings, shock-induced gut ischemia has been implicated in the development of the systemic inflammatory response syndrome and distant organ injury, yet the factors transducing these events remain to be fully determined . Because hypoxia-inducible factor (HIF-1), a transcription factor composed of oxygen-labile HIF-1alpha and constitutive HIF-1beta subunits, regulates the physiologic/pathophysiologic response to hypoxia and ischemia, we examined the HIF-1 response in two rat models of gut ischemia-reperfusion . We found that ileal nuclear HIF-1alpha protein levels were induced in rats subjected to trauma (laparotomy) plus hemorrhagic shock for 90 min relative to their trauma sham-shock and naive counterparts and that this trauma hemorrhagic shock-induced mucosal HIF-1alpha protein response persisted after 1 h and 3 h of reperfusion . Likewise, in a model of isolated gut ischemia-reperfusion injury, where the superior mesenteric artery was occluded for 45 min, nuclear HIF-1alpha were induced in the gut mucosa relative to their sham counterparts and persisted after 1 h and 3 h or reperfusion . Similar to the in vivo response, in vitro hypoxia induced HIF-alpha expression in three different enterocyte cell lines (rat IEC-6 and human Caco-2 and HT-29 cell lines) . However, in contrast to the in vivo response, HIF-1 expression rapidly disappeared on subsequent reoxygenation . Because in vivo enterocytes are exposed to bacteria, we tested whether the in vitro HIF-1alpha response would persist on reoxygenation if the enterocytes were cocultured with bacteria . P . aeruginosa, an enteric bacterium, markedly induced enterocyte HIF-1alpha protein levels under normoxic conditions . Furthermore, the addition of P . aeruginosa during either the hypoxic or reoxygenation phase prevented the degradation of HIF-1alpha protein levels . Moreover, the observation that lipopolysaccharide induced HIF-1alpha expression in a time-dependent manner in IEC-6 cells indicated that the induction of HIF-1 by exposure to P . aeruginosa is not dependent on bacterial viability . In conclusion, these results suggest that HIF-1alpha activation is an early reperfusion-independent event in models of gut ischemia-reperfusion and that this HIF-1alpha response is potentiated by the presence of P . aeruginosa or lipopolysaccharide.

Proc R Soc Lond B Biol Sci, 2004 Sep 7, 271(1550), 1777 - 82
Convergent coevolution in the domestication of coral mushrooms by fungus-growing ants; Munkacsi AB et al.; Comparisons of phylogenetic patterns between coevolving symbionts can reveal rich details about the evolutionary history of symbioses . The ancient symbiosis between fungus-growing ants, their fungal cultivars, antibiotic-producing bacteria and cultivar-infecting parasites is dominated by a pattern of parallel coevolution, where the symbionts of each functional group are members of monophyletic groups . However, there is one outstanding exception in the fungus-growing ant system, the unidentified cultivar grown only by ants in the Apterostigma pilosum group . We classify this cultivar in the coral-mushroom family Pterulaceae using phylogenetic reconstructions based on broad taxon sampling, including the first mushroom collected from the garden of an ant species in the A . pilosum group . The domestication of the pterulaceous cultivar is independent from the domestication of the gilled mushrooms cultivated by all other fungus-growing ants . Yet it has the same overall assemblage of coevolved ant-cultivar-parasite-bacterium interactions as the other ant-grown fungal cultivars . This indicates a pattern of convergent coevolution in the fungus-growing ant system, where symbionts with both similar and very different evolutionary histories converge to functionally identical interactions.

Mikrobiologiia, 2004 May-Jun, 73(3), 364 - 7
{The chemotactic properties of Bradyrhizobium japonicum in the presence of natural fine-grained minerals}; Chuiko NV et al.; The natural argillaceous minerals montmorillonite and palygorskite were found to enhance the motility of Bradyrhizobium japonicum cells and to slow down their chemotactic motion to glucose . The latter effect of the minerals is probably due to the adsorption of mineral particles on the cell surface and the blockade of the receptors that are responsible for the chemotactic behavior of the bacterium.

J Med Microbiol, 2004 Sep, 53(Pt 9), 861 - 8
Molecular cloning of the Chlamydophila abortus groEL gene and evaluation of its protective efficacy in a murine model by genetic vaccination; Hechard C et al.; The immunogenicity and protective effect of a DNA vaccine encoding the heat-shock protein (Hsp) GroEL of Chlamydophila abortus AB7, an obligate intracellular bacterium that causes abortion in sheep, was evaluated in pregnant and non-pregnant mouse models . The C . abortus groEL gene was cloned by screening a genomic library constructed in lambdaFIX II arms with a nucleic acid probe corresponding to the central portion of the groEL gene from C . abortus . Sequence analysis of a positive clone revealed an open reading frame of 1632 bp encoding a 544 amino acid polypeptide with a predicted molecular mass of 58 256 Da and highly similar to GroEL of Chlamydia trachomatis (93 %) and Chlamydophila pneumoniae (94 %) . As observed in other sequenced chlamydial genomes, the groEL gene belongs to an operon comprising another gene encoding the Hsp GroES . OF1 outbred mice were immunized intramuscularly with plasmid DNA carrying the groEL gene three times at 3 week intervals and challenged 2 weeks after the last DNA injection . In pregnant mice, no reduction in abortion was observed and the DNA vaccination failed to reduce the bacterial infection in the placenta and spleen of mice . Nevertheless, partial protection of fetuses was obtained . Immunization of non-pregnant mice with the groEL gene resulted in a specific humoral response with the predominant IgG2a isotype, suggesting a Th1-type immune response . The anti-GroEL antibodies showed no neutralizing effect in vitro on C . abortus infectivity . Although the DNA vaccine induced a delayed-type hypersensitivity response, it failed to elicit an efficient cellular immune response since the mice were not protected against bacterial challenge.

J Mol Biol, 2004 Sep 3, 342(1), 155 - 69
Structural basis for catalytic racemization and substrate specificity of an N-acylamino acid racemase homologue from Deinococcus radiodurans; Wang WC et al.; N-acylamino acid racemase (NAAAR) catalyzes the racemization of N-acylamino acids and can be used in concert with an aminoacylase to produce enantiopure alpha-amino acids, a process that has potential industrial applications . Here we have cloned and characterized an NAAAR homologue from a radiation-resistant ancient bacterium, Deinococcus radiodurans . The expressed NAAAR racemized various substrates at an optimal temperature of 60 degrees C and had Km values of 24.8 mM and 12.3 mM for N-acetyl-D-methionine and N-acetyl-L-methionine, respectively . The crystal structure of NAAAR was solved to 1.3 A resolution using multiwavelength anomalous dispersion (MAD) methods . The structure consists of a homooctamer in which each subunit has an architecture characteristic of enolases with a capping domain and a (beta/alpha)7 beta barrel domain . The NAAAR.Mg2+ and NAAAR.N-acetyl-L-glutamine.Mg2+ structures were also determined, allowing us to define the Lys170-Asp195-Glu220-Asp245-Lys269 framework for catalyzing 1,1-proton exchange of N-acylamino acids . Four subsites enclosing the substrate are identified: catalytic site, metal-binding site, side-chain-binding region, and a flexible lid region . The high conservation of catalytic and metal-binding sites in different enolases reflects the essentiality of a common catalytic platform, allowing these enzymes to robustly abstract alpha-protons of various carboxylate substrates efficiently . The other subsites involved in substrate recognition are less conserved, suggesting that divergent evolution has led to functionally distinct enzymes.

Biochem Biophys Res Commun, 2004 Sep 10, 322(1), 347 - 54
Genome-scale identification of conditionally essential genes in E . coli by DNA microarrays; Tong X et al.; Identifying the genes required for the growth or viability of an organism under a given condition is an important step toward understanding the roles these genes play in the physiology of the organism . Currently, the combination of global transposon mutagenesis with PCR-based mapping of transposon insertion sites is the most common method for determining conditional gene essentiality . In order to accelerate the detection of essential gene products, here we test the utility and reliability of a DNA microarray technology-based method for the identification of conditionally essential genes of the bacterium, Escherichia coli, grown in rich medium under aerobic or anaerobic growth conditions using two different DNA microarray platforms . Identification and experimental verification of five hypothetical E . coli genes essential for anaerobic growth directly demonstrated the utility of the method . However, the two different DNA microarray platforms yielded largely non-overlapping results after a two standard deviations cutoff and were subjected to high false positive background levels . Thus, further methodological improvements are needed prior to the use of DNA microarrays to reliably identify conditionally essential genes on genome-scale.

Cancer Lett, 2004 Sep 15, 213(1), 1 - 10
Helicobacter pylori and gastric diseases: a dangerous association; De Luca A et al.; The bacterium Helicobacter pylori is linked to the appearance of several gastric diseases and in particular is associated with a progression to gastric cancer . Thistrun -1 bacterium colonizes the gastric mucosa directly interacting with epithelial cells . It is well known that H . pylori is associated with alterations in the gastric epithelial cell cycle, and apoptosis, higher levels of mononuclear and neutrophilic infiltrates, more severe atrophy and intestinal metaplasia . In last years, two mechanisms that interact with each other or not have been proposed: the hyperproliferation of gastric cells and oxidative damage of stomach mucosa . In particular, cell cycle alterations induce mitogenic signals and proto-oncogene expression that may trigger the development of cancer . Contemporary, H . pylori is able to induce polymorphonuclear and mononuclear cells that produce oxygen free radicals that could cause DNA damage to the adjacent cells leading to cancer development . Due to dangerous infection of this bacterium, the scientific community must point out its attention on the development of detection and prevention strategies.

Hybrid Hybridomics, 2004 Jun, 23(3), 183 - 6
Inhibition of hemagglutinating activity by monoclonal antibody against Porphyromonas gingivalis 40-kDa outer membrane protein; Tagawa H et al.; Periodontitis is a chronic inflammatory disease of periodontal tissues that results in alveolar bone loss, and Porphyromonas gingivalis, which has a high hemagglutinating activity, has been implicated as an important pathogen in the development of periodontitis . This bacterium has a high hemagglutinating activity . We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P . gingivalis 381 . Although recombinant (r) 40-kDa OMP itself did not show hemagglutinating activity, its polymeric form, constructed with a cross-linking reagent, significantly expressed that activity . Furthermore, an affinity-purified antibody against r40-kDa OMP inhibited the hemagglutinating activity of P . gingivalis vesicles . In the present study, in order to clarify the pathological role of 40-kDa OMP and develop passive immunotherapy, we examined the inhibitory effect of monoclonal antibodies (MAbs) against r40-kDa OMP on the hemagglutinating activity of P . gingivalis vesicles . The MAbs reacted with r40-kDa OMP, the outer membrane fraction, vesicles, and P . gingivalis cell extracts, and significantly inhibited the hemagglutinating activities of the polymeric r40-kDa OMP as well as of P . gingivalis vesicles . These findings suggest that MAbs against 40-kDa OMP may be useful for the development of passive immunotherapy and for assessing treatment for periodontal diseases caused by P . gingivalis infection.

J Evol Biol, 2004 Sep, 17(5), 985 - 93
When mutualists are pathogens: an experimental study of the symbioses between Steinernema (entomopathogenic nematodes) and Xenorhabdus (bacteria); Sicard M et al.; In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila) . We made experimental combinations on an insect host where nematodes were associated with non-native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria . All non-native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature . We show that some of the non-native bacterial strains are pathogenic for S . carpocapsae . When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode . Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted . These results suggest that co-evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission . The pathogenicity of some non-native bacterial strains against S . carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.

Am J Perinatol, 2004 Aug, 21(6), 319 - 23
Identification and sequencing of bacterial rDNAs in culture-negative amniotic fluid from women in premature labor; Gardella C et al.; Our objective was to identify bacterial species present in culture-negative but 16S rDNA-positive amniotic fluid samples from women in preterm labor . Amniotic fluid from 69 women in preterm labor was cultured and examined for the proinflammatory cytokine interleukin-6 (IL-6) . Polymerase chain reaction technology was used to detect highly conserved bacterial ribosomal DNA sequences (16S rDNAs) . As previously reported, 16S rDNAs were identified in 15 (94%) of 16 culture-positive amniotic fluid samples, in 5 (36%) of 14 culture-negative samples with elevated IL-6, and in 1 (3%) of 39 culture-negative samples with low IL-6 levels . Direct sequencing was performed of 16S rDNAs from the 5 culture-negative amniotic fluid specimens with elevated IL-6, followed by database searches and phylogenetic analyses . The bacterial sequences identified included: two Leptotrichia sanguinegens, one human oral bacterium A33, one Fusobacterium nucleatum, and one Ureaplasma urealyticum . Identification and sequencing of 16S rDNAs in amniotic fluid is a promising technique to identify bacterial species associated with elevated IL-6 levels in culture-negative amniotic fluid that may contribute to the etiology of premature labor.

J Neurosurg, 2004 Aug, 101(2), 336 - 9
Whipple disease confined to the central nervous system presenting as a solitary frontal tumor . Case report; Lohr M et al.; Whipple disease is a rare infection caused by the bacterium Tropheryma whippelii . Patients usually present with gastrointestinal symptoms or migratory arthralgias . Although symptomatic central nervous system (CNS) involvement frequently occurs, Whipple disease confined to the CNS is rare . The authors present the case of a 40-year-old man who was surgically treated for a symptomatic left frontal tumor that had the neuroimaging features of a low-grade glioma (LGG) . A histopathological investigation revealed a perivascular accentuated inflammation with macrophages harboring PAS-positive diastase-resistant rods, which are distinctive features of cerebral Whipple disease . The patient received cotrimoxazole for 1 year postoperatively and recovered well . This case is exceptional because it represents an isolated cerebral manifestation of Whipple disease that presented as a solitary frontal tumor, thus raising the differential diagnosis of LGG . A review of diagnostic and therapeutic options in suspected cases is presented.

Appl Microbiol Biotechnol . 2004 Aug 12; {Epub ahead of print}
Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila; Sheng GP et al.; Among the four methods for extracting extracellular polymeric substances (EPS) from Rhodopseudomonas acidophila (EDTA, NaOH, H(2)SO(4), heating/centrifugation), EDTA extraction was found to be the most effective . The contents of the major components of EPS from R . acidophila, i.show $132#e., carbohydrate, protein and nucleic acid, were 6.5, 58.4 and 5.4 mg g(-1) dry cells, respectively . The optimum extraction time was 1-3 h and the EDTA dosage was approximately 2.8 g g(-1) dry cells . Under these conditions, no cell lysis was observed . The EPS content and the percentage of the three main components were greatly dependent on the extraction method . The intensity of absorption peaks for photosynthetic pigments in the UV-visible spectrum of bacteria remained unchanged prior to and after EDTA extraction; and no pigment peaks appeared in the EPS spectrum . This suggests that few cells were destroyed and lysis did not occur . UV-visible spectrum analysis, an easy and rapid technique, could be used to monitor cell lysis during EPS extraction from R . acidophila.

J Pediatr (Rio J), 2004 Jul-Aug, 80(4), 321 - 5
{Clinical and histological features of duodenal ulcer in children and adolescents}; Kawakami E et al.; OBJECTIVE: To evaluate clinical and histological features of duodenal ulcer in children and adolescents . METHODS: Forty-three children with duodenal ulcer were prospectively and consecutively evaluated in a 6-year period (7.2 patients per year) . Evaluation included clinical questionnaire focused on dyspeptic symptoms, physical examination, and digestive endoscopy with gastric biopsies for histological examination and Helicobacter pylori detection . RESULTS: Diagnostic age ranged from 4 years and 8 months to 17 years and 4 months (mean age: 12 years and 4 months) . Abdominal pain was the main symptom (39/43, 90.7%), which was epigastric in 31/39, periumbilical in 7/39, and nocturnal in 27/39 . Other symptoms were loss of appetite (32/43, 74.4%), vomiting (30/43, 69.8%), postprandial fullness (23/43, 53.5%), weight loss (22/43, 51.2%), and abdominal tenderness (19/43, 44.2%) . Upper gastrointestinal bleeding occurred in 19/43 (44.2%), whereas anemia occurred in (21/43, 48.8%) . Helicobacter pylori infection was detected in 41/43 (95.3%) . All infected patients presented acute chronic gastritis in antrum, with lymphomononuclear infiltrate predominance in 92% of them . Eradication of the bacterium occurred in 68.3% . Ulcer healing occurred in all eradicated patients and in 89% of non-eradicated patients . CONCLUSION: Duodenal ulcer was associated with chronic gastritis due to Helicobacter pylori in the majority of patients . Many complications occurred, especially upper digestive bleeding.

Am J Gastroenterol, 2004 Aug, 99(8), 1539 - 43
Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR; Ryan P et al.; OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease . While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects . The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease . METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined . Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E . coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene . RESULTS: E . coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001) . Also, E . coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections . CONCLUSIONS: E . coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas . This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells . In light of previous detection of M . paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease .

Proc R Soc Lond B Biol Sci, 2004 Jun 7, 271(1544), 1175 - 83
Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna; Mucklow PT et al.; Estimates of phenoloxidase (PO) activity have been suggested as a useful indicator of immunocompetence in arthropods, with the idea that high PO activity would indicate high immunocompetence against parasites and pathogens . Here, we test for variation in PO activity among clones of the planktonic crustacean Daphnia magna and its covariation with susceptibility to infections from four different microparasite species (one bacterium and three microsporidia) . Strong clonal variation in PO activity was found within and among populations of D . magna, with 45.6% of the total variation being explained by the clone effect . Quantitative measures of parasite success in infection correlated negatively with PO activity when tested across four host populations . However, these correlations disappeared when the data were corrected for population effects . We conclude that PO activity is not a useful measure of resistance to parasites or of immunocompetence within populations of D . magna . We further tested whether D . magna females that are wounded to induce PO activity are more resistant to infections with the bacterium Pasteuria ramosa than non-wounded controls . We found neither a difference in susceptibility nor a difference in disease progression between the induced group and the control group . These results do not question the function of the PO system in arthropod immune response, but rather suggest that immunocompetence cannot be assessed by considering PO activity alone . Immune response is likely to be a multifactorial trait with various host and parasite characteristics playing important roles in its expression.

Proc R Soc Lond B Biol Sci, 2004 Jul 7, 271(1546), 1421 - 6
Use of Wolbachia to drive nuclear transgenes through insect populations; Sinkins SP et al.; Wolbachia is an inherited intracellular bacterium found in many insects of medical and economic importance . The ability of many strains to spread through populations using cytoplasmic incompatibility, involving sperm modification and rescue, provides a powerful mechanism for driving beneficial transgenes through insect populations, if such transgenes could be inserted into and expressed by Wolbachia . However, manipulating Wolbachia in this way has not yet been achieved . Here, we demonstrate theoretically an alternative mechanism whereby nuclear rather than cytoplasmic transgenes could be driven through populations, by linkage to a nuclear gene able to rescue modified sperm . The spread of a 'nuclear rescue construct' occurs as long as the Wolbachia show imperfect maternal transmission under natural conditions and/or imperfect rescue of modified sperm . The mechanism is most efficient when the target population is already infected with Wolbachia at high frequency, whether naturally or by the sequential release of Wolbachia-infected individuals and subsequently the nuclear rescue construct . The results provide a potentially powerful addition to the few insect transgene drive mechanisms that are available.

Environ Microbiol, 2004 Sep, 6(9), 990 - 8
Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH); Leveau JH et al.; We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes . To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015 . Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations . Twelve out of 768 E . coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015 . This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA) . Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C . fungivorans harbours at least two 16S rRNA genes . For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

Appl Biochem Biotechnol, 1999 Dec, 82(3), 199 - 208
Biosynthesis of Poly-beta-hydroxybutyrate and Exopolysaccharides on Azotobacter chroococcum Strain 6B Utilizing Simple and Complex Carbon Sources; Quagliano JC et al.; Coproduction of poly-beta-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples . The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate . Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose . Protein was nearly twofold higher in ammonium-grown cultures . Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells . Sucrose almost did not produce EPS . Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt) . When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L) . On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h) . PHB fractions of total intra- and extracellular biopolymers were calculated . Sucrose-modified Burk's medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A . chroococcum 6B.

Appl Biochem Biotechnol, 2004 Jun, 117(3), 143 - 54
Hydrogen production from propionate by Rhodopseudomonas capsulata; Shi XY et al.; Hydrogen production from propionate at various concentrations by Rhodopseudomonas capsulata, a purple nonsulfur bacterium, was studied at a temperature of 31 degrees C, a pH of 7.0, and an illumination intensity of 3000 Lux . Among the six levels of propionate, 3.84 g/L was found to be the optimum propionate concentration for H2 production in terms of substrate utilization efficiency, H2 percentage, cumulative H2 production, and H2 yield . A modified Gompertz equation was able to describe properly the production of H2 from propionate . A comparative study of H2 production with acetate, propionate, and butyrate at 40 mM showed that, as a substrate for H2 production by R . capsulata, propionate was better than butyrate, but less favorable than acetate . Copryright 2004 Humana Press Inc.

Appl Biochem Biotechnol, 1999 Spring, 77-79, 511 - 20
Bioconversion of fumarate to succinate using glycerol as a carbon source; Ryu HW et al.; In this study, a facultative bacterium that converts fumarate to succinate at a high yield was isolated . The yield of bioconversion was enhanced about 1.2 times by addition of glucose into culture medium at an initial concentration of 6 g/L . When the initial cell density was high (2 g/L), the succinate produced at pH 7.0 for initial fumarate concentrations of 30, 50, 80, and 100 g/L were 29.3, 40.9, 63.6, and 82.5 g/L, respectively, showing an increase with the initial fumarate concentration . The high yield of 96.8%/mole of fumarate in just 4 h was obtained at the initial fumarate concentration of 30 g/L . Comparing these values to those obtained with low cell culture (0.2 g/L), we found that the amount of succinate produced was similar, but the production rate in the high cell culture was about three times higher than was the case in the low cell culture . This strain converted fumarate to succinate at a rate of 3.5 g/L . h under the sparge of CO2.

Nat Med, 2004 Sep, 10(9), 935 - 41 Epub 2004 Aug 08.
Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin; Temmerman S et al.; Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides . Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice . Methylated HBHA provides high levels of protection against M . tuberculosis challenge in mice, whereas nonmethylated HBHA does not . Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guerin, the only available anti-tuberculosis vaccine . Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Acta Crystallogr D Biol Crystallogr, 1997 Jul, 53(Pt 4), 461 - 3
Crystallization and preliminary X-ray crystallographic analysis of the electron-transferring flavoprotein from Megasphaera elsdenii; Sharkey CT; Electron-transferring flavoprotein from the rumen bacterium Megasphaera elsdenii is a heterodimer (M(r) = 75 kDa) containing FAD as cofactor and functioning solely to mediate electron transfer between the prosthetic groups of other proteins . The enzyme was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as precipitant . The crystals obtained belong to the space group P2(1)2(1)2(1) with unit-cell dimensions of a = 58.75, b = 61.77 and c = 122.27 A . Interestingly the crystals exhibit a low solvent content . Crystals diffracted to beyond 2.5 A using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 1997 Nov, 53(Pt 6), 798 - 801
Crystallization and preliminary X-ray analysis of methionine aminopeptidase from the hyperthermophilic bacterium Pyrococcus furiosus; Tahirov TH; Methionine aminopeptidase (MAP) from Pyrococcus furiosus (Pfu) has been crystallized in four different forms (A, B, C and D) . Form A crystals belong to space group P2(1) with unit-cell dimensions a = 54.18, b = 85.72, c = 72.84 A, beta = 108.34 degrees . Forms B, C and D belong to space group P6(2(4)) with unit-cell dimensions a = 139.1, c = 63.7 A for form B, a = 198.6, c = 243.8 A for form C, and a = 111.0, c = 125.0 A for form D . Forms A and D diffract to 2.9 A, form B diffracts to 3.5 A, and form C crystals diffract to 4.5 A . Form A contains two molecules of MAP-Pfu per asymmetric unit . The binuclear metal center positions and a non-crystallographic twofold symmetry matrix has been determined for the form A crystals.

Acta Crystallogr D Biol Crystallogr, 1996 Jan, 52(Pt 1), 194 - 6
Preliminary crystallographic studies of dimethylsulfoxide reductase from Rhodobacter capsulatus; Bailey S; Dimethylsulfoxide reductase from the photosynthetic bacterium Rhodobacter capsulatus has been crystallized in two similar forms which are suitable for X-ray structure determination . Both crystals forms belong to space group P4(1)22 or P4(3)22, with cell dimensions a = b = 80.81, c = 229.75 A (type I crystals) or a = b = 89.30, c = 230.05 A (type II crystals) and one molecule in the asymmetric unit . Diffraction has been observed to at least 2.0 A in type I crystals and to 2.6 A in type II crystals . Dimethylsulfoxide reductase from Rhodobacter is the simplest molybdenum oxotransferase known and this makes it an ideal model to study the structure and function of this class of enzymes.

Acta Crystallogr D Biol Crystallogr, 1995 May, 51(Pt 3), 368 - 79
Structure of the photochemical reaction centre of a spheroidene-containing purple-bacterium, Rhodobacter sphaeroides Y, at 3 A resolution; Arnoux B; The crystal structure of the photochemical reaction centre from Rhodobacter sphaeroides Y, a carotenoid-containing wild-type purple bacterium, has been determined at 3 A resolution . This membrane complex consists of three subunits (281, 307 and 260 residues, respectively) and ten cofactors . It was crystallized in presence of beta-D-octylglucoside . The crystals are orthorhombic with unit-cell dimensions, a = 143.7, b = 139.8, c = 78.7 A, space group P2(1)2(1)2(1) with four molecules in the unit cell . Refinement of the structure by X-PLOR and manual reconstructions yielded an R value of 22.1% for 19630 reflections between 7 and 3 A . The secondary structure is highly homologous to those determined for Rhodopseudomonas viridis (Protein Data Bank entry 1PRC) and Rhodobacter sphaeroides R26 (Protein Data Bank entry 4RCR) reaction centres . In the latter two structures one Fe(2+) ion located between the two quinones is coordinated by four histidines and one glutamic acid . In the Rhodobacter sphaeroides Y structure, Mn(2+) occupies the same position with identical ligands and geometry . The carotenoid conformation which is a non-planar 15-15'-cis spheroidene molecule in our structure differs from the 13-14-cis 2,4-dihydroneusporene in the Rhodopseudomonas viridis structure.

Biophys J, 2004 Aug, 87(2), 1165 - 72
Lamellar organization of pigments in chlorosomes, the light harvesting complexes of green photosynthetic bacteria; Psencik J et al.; Chlorosomes of green photosynthetic bacteria constitute the most efficient light harvesting complexes found in nature . In addition, the chlorosome is the only known photosynthetic system where the majority of pigments (BChl) is not organized in pigment-protein complexes but instead is assembled into aggregates . Because of the unusual organization, the chlorosome structure has not been resolved and only models, in which BChl pigments were organized into large rods, were proposed on the basis of freeze-fracture electron microscopy and spectroscopic constraints . We have obtained the first high-resolution images of chlorosomes from the green sulfur bacterium Chlorobium tepidum by cryoelectron microscopy . Cryoelectron microscopy images revealed dense striations approximately 20 A apart . X-ray scattering from chlorosomes exhibited a feature with the same approximately 20 A spacing . No evidence for the rod models was obtained . The observed spacing and tilt-series cryoelectron microscopy projections are compatible with a lamellar model, in which BChl molecules aggregate into semicrystalline lateral arrays . The diffraction data further indicate that arrays are built from BChl dimers . The arrays form undulating lamellae, which, in turn, are held together by interdigitated esterifying alcohol tails, carotenoids, and lipids . The lamellar model is consistent with earlier spectroscopic data and provides insight into chlorosome self-assembly.

J Clin Microbiol, 2004 Aug, 42(8), 3823 - 6
Analysis of p51, groESL, and the major antigen P51 in various species of Neorickettsia, an obligatory intracellular bacterium that infects trematodes and mammals; Rikihisa Y et al.; The p51 gene that encodes the major antigenic 51-kDa protein in Neorickettsia risticii was identified in strains of Neorickettsia sennetsu and the Stellantchasmus falcatus agent but not in Neorickettsia helminthoeca, suggesting that p51-based diagnosis would be useful to distinguish among them . groESL sequencing results delineated the phylogenic relationships among Neorickettsia spp.

J Clin Microbiol, 2004 Aug, 42(8), 3675 - 80
Population genetic analysis of Bartonella bacilliformis isolates from areas of peru where Carrion's disease is endemic and epidemic; Hambuch TM et al.; Carrion's disease is caused by infection with the alpha-proteobacterium Bartonella bacilliformis . Distribution of the disease is considered coincident with the distribution of its known vector, the sand fly Lutzomyia verrucarum . Recent epidemics of B . bacilliformis infections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes . Phylogenetic relationships and genomic diversity of 18 B . bacilliformis isolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellin gltA and ialB genes . A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction of B . bacilliformis.

Appl Environ Microbiol, 2004 Aug, 70(8), 4499 - 504
Expression of a temperature-sensitive esterase in a novel chaperone-based Escherichia coli strain; Ferrer M et al.; A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4 degrees C was experimentally evaluated . This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8(T), that allow E . coli to grow at high rates at 4 degrees C (maximum growth rate, 0.28 h(-1)) . The expression of a temperature-sensitive esterase in this host at 4 to 10 degrees C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E . coli strain grown at 37 degrees C (32,380 versus 190 micromol min(-1) g(-1)) . We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones . This is the first report of successful use of a chaperone-based E . coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E . coli strain (7.5 degrees C).

SAR QSAR Environ Res, 2004 Jun, 15(3), 169 - 90
QSARS for toxicity to the bacterium Sinorhizobium meliloti; Lessigiarska I et al.; In the present study, structure-activity relationship (QSAR) models for the prediction of the toxicity to the bacterium Sinorhizobium meliloti have been developed, based on a data set of 140 compounds . The data set is highly heterogeneous both in terms of chemistry and mechanisms of toxic action . For deriving QSARs, chemicals were divided into groups according to mechanism of action and chemical structure . The QSARs derived are considered to be of moderate statistical quality . A baseline effect (relationship between the toxicity and logP), which can be related to non-polar narcosis, was observed . To explain toxicity greater than the baseline toxicity, other structural descriptors were used . The development of models for non-polar and polar narcosis had some success . It appeared that the toxicity of compounds acting by more specific mechanisms of toxic action is difficult to predict . A global QSAR was also developed, which had square of the correlation coefficient r2 = 0.53 . A QSAR with reasonable statistical parameters was developed for the aliphatic compounds in the data set (r2 = 0.83) . QSARs could not be obtained for the aromatic compounds as a group.

Proc Natl Acad Sci U S A, 2004 Aug 17, 101(33), 12306 - 11 Epub 2004 Aug 03.
A eukaryotic BLUF domain mediates light-dependent gene expression in the purple bacterium Rhodobacter sphaeroides 2.4.1; Han Y et al.; The flavin-binding BLUF domain functions as a blue-light receptor in eukaryotes and bacteria . In the photoreceptor protein photo-activated adenylyl cyclase (PAC) from the flagellate Euglena gracilis, the BLUF domain is linked to an adenylyl cyclase domain . The PAC protein mediates a photophobic response . In the AppA protein of Rhodobacter sphaeroides, the BLUF domain is linked to a downstream domain without similarity to known proteins . AppA functions as a transcriptional antirepressor, controlling photosynthesis gene expression in the purple bacterium R . sphaeroides in response to light and oxygen . We fused the PACalpha1-BLUF domain from Euglena to the C terminus of AppA . Our results show that the hybrid protein is fully functional in light-dependent gene repression in R . sphaeroides, despite only approximately 30% identity between the eukaryotic and the bacterial BLUF domains . Furthermore, the bacterial BLUF domain and the C terminus of AppA can transmit the light signal even when expressed as separated domains . This finding implies that the BLUF domain is fully modular and can relay signals to completely different output domains.

J Bacteriol, 2004 Aug, 186(16), 5543 - 6
The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens; Nunez C et al.; Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure . Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron . This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments.

J Mol Biol, 2004 Aug 13, 341(3), 713 - 21
Class II photolyase in a microsporidian intracellular parasite; Slamovits CH et al.; Photoreactivation is the repair of DNA damage induced by ultraviolet light radiation using the energy contained in visible-light photons . The process is carried out by a single enzyme, photolyase, which is part of a large and ancient photolyase/cryptochrome gene family . We have characterised a photolyase gene from the microsporidian parasite, Antonospora locustae (formerly Nosema locustae) and show that it encodes a functional photoreactivating enzyme and is expressed in the infectious spore stage of the parasite's life cycle . Sequence and phylogenetic analyses show that it belongs to the class II subfamily of cyclobutane pyrimidine dimer repair enzymes . No photolyase is present in the complete genome sequence of the distantly related microsporidian, Encephalitozoon cuniculi, and this class of photolyase has never yet been described in fungi, the closest relatives of Microsporidia, raising questions about the evolutionary origin of this enzyme . This is the second environmental stress enzyme to be found in A.locustae but absent in E.cuniculi, and in the other case (catalase), the gene is derived by lateral transfer from a bacterium . It appears that A.locustae spores deal with environmental stress differently from E.cuniculi, these results lead to the prediction that they are more robust to environmental damage.

Lett Appl Microbiol, 2004, 39(3), 278 - 83
Isolation of salt-sensitive mutants from Sinorhizobium meliloti and characterization of genes involved in salt tolerance; Wei W et al.; AIMS: The purpose of our research is to isolate salt-sensitive mutants and to study the genes involved in salt tolerance of the salt-tolerant bacterium Sinorhizobium meliloti 042BM . METHODS: Wild type S . meliloti 042BM bacteria are able to grow at a NaCl concentration of 0.6 mol l(-1) . A transposon Tn5-1063a mutagenesis library of S . meliloti 042BM was constructed and eight salt-sensitive mutants were isolated, which were unable to growth on FY plates containing 0.4 mol l(-1) NaCl . SIGNIFICANCE: Our interest is to provide information about the mechanism of salt tolerance in bacteria by studying the genes involved in salt tolerance . Here, seven different genes were identified . These genes include omp10 encoding a cell outer membrane protein, relA encoding (p)ppGpp synthetase, greA encoding a transcription cleavage factor, nuoL encoding NADH dehydrogenase I chain L transmembrane protein, a putative nuclease/helicase gene and two unknown genes . Based on these findings, we suggest that the regulation of salt tolerance of S . meliloti 042BM is complex and on several levels.

Mol Biochem Parasitol, 2004 May, 135(1), 57 - 67
Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi; McGarry HF et al.; The human filarial nematode Brugia malayi contains an endosymbiotic bacterium, Wolbachia . We used real-time quantitative polymerase chain reaction (QPCR) and microscopy to investigate the population dynamics of the bacterium-nematode association . Two Wolbachia (wsp and ftsZ) and one nematode (gst) genes were amplified from all life-cycle stages of B . malayi and results expressed as gene copies per worm and as Wolbachia/nematode ratios . Since the genes were single copy and there was one genome per Wolbachia, the gene copy numbers were equivalent to the numbers of bacteria . These were similar in microfilariae and the mosquito-borne larval stages (L2 and L3), with the lowest ratios of Wolbachia/nematode DNA . However, within 7 days of infection of the mammalian host, bacteria had increased 600-fold and the bacteria/worm ratio was the highest of all life-cycle stages . The rapid multiplication continued throughout L4 development, so that the major period of bacterial population growth occurred within 4 weeks of infection of the definitive host . Microscopy confirmed that there were few bacteria in mosquito-derived L3 but many, in large groups, in L4 collected 9 and 21 days after infection . In adult male worms up to 15 months of age, the bacterial populations were maintained, whilst in females, bacteria numbers increased as the worms matured and as the ovary and embryonic larval stages became infected . These results support the hypothesis that the bacteria are essential for larval development in the mammalian host and for the long-term survival of adult worms.

Annu Rev Phytopathol, 2004, 42, 107 - 34
Lessons learned from the genome analysis of ralstonia solanacearum; Genin S et al.; Ralstonia solanacearum is a devastating plant pathogen with a global distribution and an unusually wide host range . This bacterium can also be free-living as a saprophyte in water or in the soil in the absence of host plants . The availability of the complete genome sequence from strain GMI1000 provided the basis for an integrative analysis of the molecular traits determining the adaptation of the bacterium to various environmental niches and pathogenicity toward plants . This review summarizes current knowledge and speculates on some key bacterial functions, including metabolic versatility, resistance to metals, complex and extensive systems for motility and attachment to external surfaces, and multiple protein secretion systems . Genome sequence analysis provides clues about the evolution of essential virulence genes such as those encoding the Type III secretion system and related pathogenicity effectors . It also provided insights into possible mechanisms contributing to the rapid adaptation of the bacterium to its environment in general and to its interaction with plants in particular.

J Appl Microbiol, 2004, 97(3), 504 - 11
A putative new endophytic nitrogen-fixing bacterium Pantoea sp . from sugarcane; Loiret FG et al.; AIMS: To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers . METHODS AND RESULTS: Two N2-fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18 . Both isolates showed acetylene reduction and H2 production in nitrogen-free media . Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037) . The nifH gene segment was amplified from both isolates using specific primers . Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis . CONCLUSIONS: Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus . For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H2 production (up to 67.7 nmol H2 h(-1) 10(10) cell(-1)) . Isolate T2 was a strain of Gluconacetobacter diazotrophicus . SIGNIFICANCE AND IMPACT OF THE STUDY: A new N2-fixing endophyte, i.e . Pantoea, able to produce H2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized . The strain with these attributes may well be valuable for agriculture .

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1227 - 34
Woodsholea maritima gen . nov., sp . nov., a marine bacterium with a low diversity of polar lipids; Abraham WR et al.; Two cauliform bacteria (CM243T and CM251) isolated by J . Poindexter from the Atlantic Ocean were characterized by 16S rRNA gene sequencing, TaqI restriction fragment length polymorphism and single-strand conformation polymorphism analyses of the internally transcribed 16S-23S rDNA spacer (ITS1) region, analysis of fatty acids from cellular lipids, mass spectrometry of polar lipids and physiological properties . The two strains showed very low diversity of polar lipids with diacyl-sulfoquinovosyl glycerols as the predominant lipids . The two bacterial strains were observed to have nearly identical 16S rRNA gene sequences and could not be differentiated by their ITS1 regions . The isolates differed from species of the genus Maricaulis by their 16S rRNA gene sequences, polar lipids and fatty acid patterns . On the basis of the genotypic analyses and estimations of phylogenetic similarities, physiological and chemotaxonomic characteristics, it is proposed that the isolates represent a new genus and species, for which the name Woodsholea maritima gen . nov., sp . nov . (type strain CM243T=VKM B-1512T=LMG 21817T) is proposed.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1191 - 6
Methylobacterium populi sp . nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides x nigra DN34); Van Aken B et al.; A pink-pigmented, aerobic, facultatively methylotrophic bacterium, strain BJ001T, was isolated from internal poplar tissues (Populus deltoidesxnigra DN34) and identified as a member of the genus Methylobacterium . Phylogenetic analyses showed that strain BJ001T is related to Methylobacterium thiocyanatum, Methylobacterium extorquens, Methylobacterium zatmanii and Methylobacterium rhodesianum . However, strain BJ001T differed from these species in its carbon-source utilization pattern, particularly its use of methane as the sole source of carbon and energy, an ability that is shared with only one other member of the genus, Methylobacterium organophilum . In addition, strain BJ001T is the only member of the genus Methylobacterium to be described as an endophyte of poplar trees . On the basis of its physiological, genotypic and ecological properties, the isolate is proposed as a member of a novel species of the genus Methylobacterium, Methylobacterium populi sp . nov . (type strain, BJ001T=ATCC BAA-705T=NCIMB 13946T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1173 - 6
Arenibacter certesii sp . nov., a novel marine bacterium isolated from the green alga Ulva fenestrata; Nedashkovskaya OI et al.; The taxonomic position of a novel, marine, heterotrophic, aerobic, pigmented, non-motile bacterium that was isolated from a green alga, Ulva fenestrata, inhabiting the Sea of Japan, was determined . 16S rRNA gene sequence analysis revealed that the strain, KMM 3941T, is a member of the genus Arenibacter . The results of DNA-DNA hybridization experiments, supported by phenotypic and chemotaxonomic data, showed that the isolate represents a novel species of the genus Arenibacter, for which the name Arenibacter certesii sp . nov . is proposed . The type strain is KMM 3941T (=KCTC 12113T=CCUG 48006T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1083 - 7
Shewanella pacifica sp . nov., a polyunsaturated fatty acid-producing bacterium isolated from sea water; Ivanova EP et al.; Six marine bacterial strains, KMM 3597T, KMM 3775, KMM 3590, KMM 3772, KMM 3605 and KMM 3601, that produce polyunsaturated fatty acids were isolated from sea water samples collected from different locations and depths in Chazhma Bay (Sea of Japan, Pacific Ocean) and characterized to clarify their taxonomic position . The DNA G+C contents of these strains were 39.5-40.3 mol% . The level of DNA hybridization between these strains was conspecific (83-96%), indicating that they represent a single genospecies . 16S rRNA gene sequence-based phylogenetic analysis of the novel strains revealed that Shewanella japonica KMM 3299T was the closest relative (99% similarity) . However, DNA-DNA hybridization experiments demonstrated only 45-50% binding with DNA of S . japonica . The novel organisms grew between 4 and 33 degrees C, were neutrophilic and haemolytic, and were able to degrade starch, gelatin, agar and Tween 80 . The predominant fatty acids were (%+/-sd): i13 : 0 (9.3+/-1.1); i15 : 0 (33.9+/-1.5); 16 : 0 (8.9+/-1.6); and 16 : 1omega7 (14.8+/-1.1) . The fatty acid 20 : 5omega3, formed at 28 degrees C, was present at up to 5.3% total fatty acids . The major isoprenoid quinones were Q7 (21-41%) and Q8 (50-59%) . The phylogenetic, genetic and physiological properties of the six strains placed them within a novel species, Shewanella pacifica sp . nov., the type strain of which is R10SW1T (=KMM 3597T=CIP 107849T).

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1031 - 6
Thiocapsa marina sp . nov., a novel, okenone-containing, purple sulfur bacterium isolated from brackish coastal and marine environments; Caumette P et al.; Four marine, phototrophic, purple sulfur bacteria (strains 5811T, 5812, BM-3 and BS-1) were isolated in pure culture from different brackish to marine sediments in the Mediterranean Sea, the White Sea and the Black Sea . Single cells of these strains were coccus-shaped, non-motile and did not contain gas vesicles . The colour of cell suspensions that were grown in the light was purple-red . Bacteriochlorophyll a and carotenoids of the okenone series were present as photosynthetic pigments . Photosynthetic membrane systems were of the vesicular type . Hydrogen sulfide, thiosulfate, elemental sulfur and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions; carbon dioxide was utilized as the carbon source . During growth on sulfide, elemental sulfur globules were stored inside the cells . In the presence of hydrogen sulfide, several organic substances could be photoassimilated . Comparative 16S rDNA sequence analysis revealed an affiliation of these four strains to the genus Thiocapsa . Both phylogenetic analysis and the results of DNA-DNA hybridization studies revealed that these strains formed a separate cluster within the genus Thiocapsa . Thus, according to phenotypic characteristics and mainly the carotenoid composition, 16S rDNA sequence analysis and DNA-DNA hybridization data, it is proposed that these strains should be classified as a novel species, Thiocapsa marina sp . nov., with strain 5811T (=DSM 5653T=ATCC 43172T) as the type strain.

J Econ Entomol, 2004 Jun, 97(3), 970 - 5
Effect of sucrose octanoate on survival of nymphal and adult Diaphorina citri (Homoptera: Psyllidae); McKenzie CL et al.; Asian citrus psylla, Diaphorina citri Kuwayama (Homoptera: Psyllidae) was detected for the first time in the United States near Delray Beach, FL, on 2 June 1998 and is continuing to spread and multiply throughout southern Florida . This psyllid is the vector of Liberobacter asiaticum, a phloem-limited bacterium that causes citrus greening disease . This pathogen has not been found in the Western Hemisphere to date . Furthermore, high infestation levels of D . citri can impact citrus plant health, fruit quality, or yield . Replicated laboratory and spray booth bioassays were conducted to determine the insecticidal activity of a synthetic analog of natural sugar esters found in leaf trichomes of wild tobacco, Nicotiatna gossei Domin, to nymphal and adult D . citri . Field trials were initiated in Fort Pierce, FL, in 2000 to determine activity of the sugar ester formulation (sucrose octanoate) on D . citri and other citrus pests, including immature Asian citrus leafminer, Phyllocnistis citrella Stainton and mites . Sucrose octanoate rates tested ranged from 400 to 8000 ppm (0.1-2% formulated product) . Our data suggest that both nymphal and adult D . citri as well as the mite complex tested would be equally controlled to levels of >90% at the higher concentrations of sucrose octanoate and that good coverage is key to efficacy.

Funct Integr Genomics . 2004 Jul 24; {Epub ahead of print}
Comprehensive metabolite profiling of Sinorhizobium meliloti using gas chromatography-mass spectrometry; Barsch A et al.; A metabolite analysis of the soil bacterium Sinorhizobium meliloti was established as a first step towards a better understanding of the symbiosis with its host plant Medicago truncatula . A crucial step was the development of fast harvesting and extraction methods for the bacterial metabolites because of rapid changes in their composition . S . meliloti 1021 cell cultures grown in minimal medium were harvested by centrifugation, filtration or immediate freezing in liquid nitrogen followed by a lyophilisation step . Bacteria were lysed mechanically in methanol and hydrophilic compounds were analysed after methoxymation and silylisation via GC-MS . The different compounds were identified by comparison with the NIST 98 database and available standards . From about 200 peaks in each chromatogram 65 compounds have been identified so far . A comparison of the different extraction methods giving the metabolite composition revealed clear changes in several amino acids and amino acid precursor pools . A principal component analysis (PCA) was able to distinguish S . meliloti cells grown on different carbon sources based on their metabolite profile . A comparison of the metabolite composition of a S . meliloti leucine auxotrophic mutant with the wild type revealed a marked accumulation of 2-isopropylmalate in the mutant . Interestingly, the accumulated metabolite is not the direct substrate of the mutated enzyme, 3-isopropylmalate dehydrogenase, but the substrate of isopropylmalate isomerase, which acts one step further upstream in the biosynthetic pathway of leucine . This finding further emphasises the importance of integrating metabolic data into post-genomic research.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11448 - 53 Epub 2004 Jul 26.
The SapB morphogen is a lantibiotic-like peptide derived from the product of the developmental gene ramS in Streptomyces coelicolor; Kodani S et al.; SapB is a morphogenetic peptide that is important for aerial mycelium formation by the filamentous bacterium Streptomyces coelicolor . Production of SapB commences during aerial mycelium formation and depends on most of the genes known to be required for the morphogenesis of aerial hyphae . Furthermore, the application of purified SapB to mutants blocked in morphogenesis restores their capacity to form aerial hyphae . Here, we present evidence that SapB is a lantibiotic-like peptide that is derived by posttranslational modification from the product of a gene (ramS) in the four-gene ram operon, which is under the control of the regulatory gene ramR . We show that the product of another gene in the operon (ramC) contains a region that is similar to enzymes involved in the biosynthesis of lantibiotics, suggesting that it might be involved in the posttranslational processing of RamS . We conclude that SapB is derived from RamS through proteolytic cleavage and the introduction of four dehydroalanine residues and two lanthionine bridges . We provide an example of a morphogenetic role for an antibiotic-like molecule.

Trends Microbiol, 2004 Aug, 12(8), 361 - 5
Morphological and functional asymmetry in alpha-proteobacteria; Hallez R et al.; The release of an increasing number of complete bacterial genomic sequences allows the evolutionary analysis of processes such as regulatory networks . CtrA is a response regulator of the OmpR subfamily, belonging to a complex regulatory network in the dimorphic bacterium Caulobacter crescentus . It coordinates the cell cycle with an asymmetric division, which is part of the adaptation of Caulobacter to poor-nutrient environments . CtrA is only found in alpha-proteobacteria, a group of bacteria encompassing genera with very distinct lifestyles, including host-associated bacteria . Analyses of CtrA regulatory networks and morphological examinations of some alpha-proteobacteria are presented . Our observations suggest that the core of the CtrA regulation network is conserved and that alpha-proteobacteria divide asymmetrically . We propose that the two daughter cells might be differentiated bacteria, each one displaying specific functions.

Rev Med Interne, 2004 Aug, 25(8), 573 - 81
{MALT gastric lymphomas}; Ruskone-Fourmestraux A; INTRODUCTION: The stomach is the most common site involved in primary gastrointestinal lymphoma . Gastric lymphoma originates from the mucosa-associated lymphoid tissue so called MALT . It comprises a group of distinctive clinicopathological entities which are important to consider for clinical management . CURRENT KNOWLEDGE AND KEY POINTS: In recent years, new diagnostic tools and new treatment strategies have improved the overall prognosis . One of the most exciting recent discoveries is the hypothesis that an infection by a bacterium, Helicobacter pylori has a decisive role in gastric lymphoma . FUTURE PROSPECTS AND PROJECTS: Recent advances, essentially due to molecular biology and cytogenetic studies may emerge with the understanding of pathogenesis and new prognostic factors of these different types of gastric lymphomas . It is the aim of our oncoming studies together with the evaluation of the new therapeutic options such as radiotherapy and monoclonal antibodies in prospective studies.

Parasitol Today, 1997 Jul, 13(7), 267 - 70
Borrelia burgdorferi genes selectively expressed in ticks and mammals; De Silva AM et al.; Lyme disease is a tick-borne infection caused by the spirochete Borrelia burgdorferi . Recent studies have focused on how the Lyme disease bacterium overcomes the challenges faced by an organism that depends on a vector-borne life style . These studies indicate that the spirochete expresses different surface proteins at different stages of its life . Here, Aravinda de Silva and Erol Fikrig review the evidence for differential gene expression and discuss the implications of these findings for the Lyme disease vaccine that is currently being tested in human trials.

Biochemistry, 2004 Aug 3, 43(30), 9909 - 17
Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis; Pessanha M et al.; The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes . Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 9.6 kDa . This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)) . In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G . sulfurreducens triheme cytochrome c(7) (PpcA) . NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure {Pokkuluri, P . R., Londer, Y . Y., Duke, N . E . C., Long, W . C., and Schiffer, M . (2004) Biochemistry 43, 849-859} using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2 . Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA . The results obtained showed that PpcA and Dac(7) have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac(7) . The differences observed in the redox behavior of PpcA and Dac(7) may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions . The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.

Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11293 - 7 Epub 2004 Jul 23.
Watching the photosynthetic apparatus in native membranes; Scheuring S et al.; Over the last 9 years, the structures of the various components of the bacterial photosynthetic apparatus or their homologues have been determined by x-ray crystallography to at least 4.8-A resolution . Despite this wealth of structural information on the individual proteins, there remains an urgent need to examine the architecture of the photosynthetic apparatus in intact photosynthetic membranes . Information on the arrangement of the different complexes in a native system will help us to understand the processes that ensure the remarkably high quantum efficiency of the system . In this work we report images obtained with an atomic force microscope of native photosynthetic membranes from the bacterium Rhodospirillum photometricum . Several proteins can be seen and identified at molecular resolution, allowing the analysis and modeling of the lateral organization of multiple components of the photosynthetic apparatus within a native membrane . Analysis of the distribution of the complexes shows that their arrangement is far from random, with significant clustering both of antenna complexes and core complexes . The functional significance of the observed distribution is discussed.

Infect Immun, 2004 Aug, 72(8), 4416 - 23
Induction of maturation and cytokine release of human dendritic cells by Helicobacter pylori; Kranzer K et al.; Helicobacter pylori causes a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease . Despite an intensive proinflammatory response, the immune system is not able to clear the organism . However, the immune escape mechanisms of this common bacterium are not well understood . We investigated the interaction between H . pylori and human dendritic cells . Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system . Stimulation of DCs with different concentrations of H . pylori for 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production . Lipopolysaccharide (LPS) from Escherichia coli, a known DC maturation agent, was used as a positive control . The cytokine release after stimulation with LPS was comparable to that induced by H . pylori except for IL-12 . After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced by H . pylori . We further investigated the potential of H . pylori to induce maturation of DCs . Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation with H . pylori or LPS . We found no significant differences between H . pylori seropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules . These data clearly demonstrate that H . pylori induces a strong activation and maturation of human immature DCs.

J Colloid Interface Sci, 2004 Aug 15, 276(2), 323 - 32
Selective separation of arsenopyrite from pyrite by biomodulation in the presence of Acidithiobacillus ferrooxidans; Chandraprabha MN et al.; Effective methods for selective separation using flotation or flocculation of arsenopyrite from pyrite by biomodulation using Acidithiobacillus ferrooxidans are presented here . Adhesion of the bacterium to the surface of arsenopyrite was very slow compared to that to pyrite, resulting in a difference in surface modification of the minerals subsequent to interaction with cells . The cells were able to effectively depress pyrite flotation in presence of collectors like potassium isopropyl xanthate and potassium amyl xanthate . On the other hand the flotability of arsenopyrite after conditioning with the cells was not significantly affected . The activation of pyrite by copper sulfate was reduced when the minerals were conditioned together, resulting in better selectivity . Selective separation could also be achieved by flocculation of biomodulated samples.

Helicobacter, 2004 Aug, 9(4), 342 - 6
Helicobacter pylori infection and immune thrombocytopenic purpura: an update; Franchini M et al.; Data are accumulating on the association between Helicobacter pylori infection and idiopathic thrombocytopenic purpura (ITP) and the significant increase in platelet count after bacterial eradication . The aim of this review was to consider the studies so far published on H . pylori infection and ITP in order to evaluate a possible correlation between these two conditions . A review of the literature showed that 278 out of the 482 ITP patients investigated (58%) were positive for H . pylori infection and that the bacterium was eradicated in 88% of cases . Eradication therapy was accompanied by a complete or partial platelet response in approximately half the cases . Overall, these data show that H . pylori eradication in patients with ITP is effective in increasing platelet count . However, because the studies so far published are few, are sometimes controversial and involve small series of patients, further studies on larger numbers of patients with longer follow-up are needed to confirm these preliminary findings.

Biotechnol Lett, 2004 Jun, 26(11), 947 - 50
Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b; Lee SG et al.; Methylosinus trichosporium OB3b oxidized methane to methanol in the presence of a high concentration of Cu2+ . Further oxidation of methanol to formaldehyde was prevented by adding 200 mM NaCl which acted as a methanol dehydrogenase H inhibitor . The bacterium, 0.6 mg dry cell ml(-1), in methane/air (1:4, v/v) at 25 degrees C in 12.9 mM phosphate buffer (pH 7) containing 20 mM sodium formate and 200 mM NaCl accumulated 7.7 mM methanol over 36 h.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 139 - 45
Effect of uncouplers on endogenous respiration and ferrous iron oxidation in a chemolithoautotrophic bacterium Acidithiobacillus (Thiobacillus) ferrooxidans; Chen Y et al.; Oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+) with oxygen (O2) by Acidithiobacillus (Thiobacillus) ferrooxidans is considered to be inhibited by uncouplers . Oxidation of the endogenous substrates (presumably NADH) with O2 or Fe3+, on the other hand, was stimulated by uncouplers, 2,4-dinitrophenol (DNP) and carbonylcyanide-m-chlorophenyl-hydrazone (CCCP), as expected in respiratorily controlled mitochondria or heterotrophic bacteria . Amytal and rotenone were inhibitory . Fe3+ reduction by endogenous substrates was studied extensively and was found to be stimulated by a permeable anion, SCN- and weak acids, as well as the above uncouplers . Proton translocating properties of some of these stimulators were shown by following a pH change in the cell suspension . It was concluded that any compounds that destroy proton electrochemical gradient, Deltap, stimulated endogenous respiration . Stimulation of Fe2+ or ascorbate oxidation by lower concentrations of uncouplers was successfully demonstrated by shortening the reaction time, but only to a small extent . Uncouplers at concentrations stimulatory to endogenous respiration inhibited Fe2+ oxidation if present before Fe2+ addition . The inhibition by 10 microM CCCP was reversed by washing the cells in a buffer . Complex I inhibitors, atabrine, rotenone and amytal inhibited Fe2+ oxidation, more strongly in the presence of 0.1 mM DNP . It is proposed that Fe2+ oxidation required Deltap perhaps to climb an energetically uphill reaction or to reduce NAD+ to NADH by reversed electron flow for CO2 fixation . The latter interpretation implies some obligatory coupling between Fe2+ oxidation and NAD+ reduction.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 119 - 26
A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp . paratuberculosis in water and milk; Rodriguez-Lazaro D et al.; A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp . paratuberculosis has been developed . It targets and amplifies sequences from the dnaA gene which are specific for this bacterium . The assay includes an internal amplification control, to allow identification of inhibited reactions . The assay was tested against 18 isolates of M . avium subsp . paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets . The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions) . Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M . avium subsp . paratuberculosis cells in 20 ml artificially contaminated drinking water . With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M . avium subsp . paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk . The assay will be a useful addition to the range of diagnostic tools available for the study of M . avium subsp . paratuberculosis.

Curr Biol, 2004 Jul 27, 14(14), 1256 - 61
The ERK MAP kinase cascade mediates tail swelling and a protective response to rectal infection in C . elegans; Nicholas HR et al.; The nematode Caenorhabditis elegans is proving to be an attractive model organism for investigating innate immune responses to infection . Among the known pathogens of C . elegans is the bacterium Microbacterium nematophilum, which adheres to the nematode rectum and postanal cuticle, inducing swelling of the underlying hypodermal tissue and causing mild constipation . We find that on infection by M . nematophilum, an extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade mediates tail swelling and protects C . elegans from severe constipation, which would otherwise arrest development and cause sterility . Involvement in pathogen defense represents a new role for ERK MAP kinase signaling in this organism.

J Biol Chem, 2004 Sep 24, 279(39), 40437 - 44 Epub 2004 Jul 20.
The role of active site glutamate residues in catalysis of Rhodobacter capsulatus xanthine dehydrogenase; Leimkuhler S et al.; Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD+ as the electron acceptor . R . capsulatus XDH forms an (alphabeta)2 heterotetramer and is highly homologous to homodimeric eukaryotic xanthine oxidoreductases . Here we first describe reductive titration and steady state kinetics on recombinant wild-type R . capsulatus XDH purified from Escherichia coli, and we then proceed to evaluate the catalytic importance of the active site residues Glu-232 and Glu-730 . The steady state and rapid reaction kinetics of an E232A variant exhibited a significant decrease in both kcat and kred as well as increased Km and Kd values as compared with the wild-type protein . No activity was determined for the E730A, E730Q, E730R, and E730D variants in either the steady state or rapid reaction experiments, indicating at least a 10(7) decrease in catalytic effectiveness for this variant . This result is fully consistent with the proposed role of this residue as an active site base that initiates catalysis .

Eur J Biochem, 2004 Aug, 271(15), 3093 - 102
Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont; Minic Z et al.; Riftia pachyptila (Vestimentifera) is a giant tubeworm living around the volcanic deep-sea vents of the East Pacific Rise . This animal is devoid of a digestive tract and lives in an intimate symbiosis with a sulfur-oxidizing chemoautotrophic bacterium . This bacterial endosymbiont is localized in the cells of a richly vascularized organ of the worm: the trophosome . These organisms are adapted to their extreme environment and take advantage of the particular composition of the mixed volcanic and sea waters to extract and assimilate inorganic metabolites, especially carbon, nitrogen, oxygen and sulfur . The high molecular mass hemoglobin of the worm is the transporter for both oxygen and sulfide . This last compound is delivered to the bacterium which possesses the sulfur oxidizing respiratory system, which produces the metabolic energy for the two partners . CO2 is also delivered to the bacterium where it enters the Calvin-Benson cycle . Some of the resulting small carbonated organic molecules are thus provided to the worm for its own metabolism . As far as nitrogen assimilation is concerned, NH3 can be used by the two partners but nitrate can be used only by the bacterium . This very intimate symbiosis applies also to the organization of metabolic pathways such as those of pyrimidine nucleotides and arginine . In particular, the worm lacks the first three enzymes of the de novo pyrimidine biosynthetic pathways as well as some enzymes involved in the biosynthesis of polyamines . The bacterium lacks the enzymes of the pyrimidine salvage pathway . This symbiotic organization constitutes a very interesting system to study the molecular and metabolic basis of biological adaptation.

Rev Biol Trop, 2003 Jun, 51 Suppl 4, 47 - 55
Monitoring the coral disease, plague type II, on coral reefs in St . John, U.S . Virgin Islands; Miller J et al.; In July 1997, conspicuous white patches of necrotic tissue and bare skeleton began to appear on scleractinian corals in several bays around St . John, US Virgin Islands . Analysis of diseased coral tissue from five different species confirmed the presence of a Sphingomonas-like bacterium, the pathogen for plague type II . To date, 14 species of hard corals have been affected by plague type II around St . John . This disease was monitored at Haulover and Tektite Reefs at depths of 7-12 meters . The study site at Tektite Reef has > 50% cover by scleractinian corals with 90% of hard corals being composed of Montastraea annularis . Monthly surveys at Tektite Reef from December 1997 to May 2001 documented new incidence of disease (bare white patches of skeleton) every month with associated loss of living coral and 90.5% of all disease patches occurred on M . annularis . The frequency of disease within transects ranged from 3 to 58%, and the area of disease patches ranged from 0.25 to 9000 cm2 . The average percent cover by the disease within 1 m2 ranged from 0.01% (+/- 0.04 SD) to 1.74% (+/- 9.08 SD) . Photo-monitoring of 28 diseased corals of 9 species begun in September 1997 at Haulover Reef revealed no recovery of diseased portions with all necrotic tissue being overgrown rapidly by turf algae, usually within less than one month . Most coral colonies suffered partial mortality . Very limited recruitment (e.g., of Agaricia spp., Favia spp . and sponges) has been noted on the diseased areas . This coral disease has the potential to cause more loss of live coral on St . John reefs than any other stress to date because it targets the dominant reef building species, M . annularis.

J Invertebr Pathol, 2004 Jul, 86(3), 65 - 71
An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta; Park Y et al.; The entomopathogenic bacterium, Xenorhabdus nematophila, induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts . A hypothesis has been raised that the bacteria inhibit eicosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts . Here, we show direct evidence that X . nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eicosanoid-biosynthesis pathway . Inhibition of PLA2 was dependent on both incubation time with X . nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes . While living bacteria inhibited PLA2 activity, heat-killed X . nematophila rather increased PLA2 activity . X . nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium . The PLA2 inhibitory activity of the organic extract was lost after heat treatment . These results clearly indicate that X . nematophila inhibits PLA2 activity, and thereby inhibits eicosanoid biosynthesis which leads to immunodepression of the infected hosts.

Trans R Soc Trop Med Hyg, 2003 Jul-Aug, 97(4), 382 - 6
Prevalence and risk factors associated with Helicobacter pylori infection in native populations from Brazilian Western Amazon; Almeida Cunha RP et al.; We evaluated the prevalence of, and factors associated with, Helicobacter pylori infection in 222 subjects from 3 distinct communities of native populations (Uru-Eu-Wau-Wau Indians and 2 riverine communities living on the banks of the Machado river and in Portuchuelo) living in isolation in the rainforest of Brazilian Western Amazon . The overall prevalence was 78.8% (95% CI 72.7-83.9) . The prevalence was higher in the Machado river community compared with Portuchuelo (chi2 = 3.84, P = 0.05), but no significant difference was observed between the Machado river community and the Uru-Eu-Wau-Wau Indians . Logistic regression showed that residential crowding and age were factors associated with the presence of H . pylori infection . Acquisition of the bacterium started early in life and by the age of 2 years 50% of children were infected . The prevalence increased with age, reaching near universal levels during adulthood (97.9%) . Residential crowding was high with a global index of 3.3 persons/room (SD = 1.8), varying significantly between the 3 communities (P = 0.001) . These data provide further evidence supporting direct person-to-person spread of the bacterium.

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