Biochim Biophys Acta, 2004 Jan 14, 1696(1), 59 - 65 Cold-active esterase from Psychrobacter sp . Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability; Kulakova L et al.; The gene encoding an esterase (PsyEst) of Psychrobacter sp . Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli . PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9 . A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme . Thus, we introduced the Gly(244)-->Pro substitution into the enzyme . Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme . The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type . Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters . All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme. Di Yi Jun Yi Da Xue Xue Bao, 2004 Jan, 24(1), 117 - 20 {An easier method for generating recombinant adenovirus containing CD/TK fusion gene driven by vascular endothelial growth factor promoter}; Huang YY et al.; OBJECTIVE: To efficiently construct the recombinant adenovirus containing CD/TK fusion gene driven by vascular endothelial growth factor (VEGF) promoter using improved homologous recombination in bacteria . METHODS: Chemical transformation, instead of electroporation, of the plasmid pAdEasy-1 into E.coli BJ5183 strain was performed to prepare BJ5183pAdEasy-1 as the competent bacterium, in which pAdEasy-1 and pAdtrack-VEGFP-CDglyTK were recombined . Finally, the recombinant adenovirus of AdVEGFP-CDglyTK was constructed by transfecting 293 cells with linearized adenoviral genomes of pAdEasy-VEGFP-CDglyTK . RESULTS: This new transformation procedure generated recombinant plasmids in a yield of 60% (6/10), and the adenovirus AdVEGFP-CDglyTK was harvested 7-12 d after transfection . CONCLUSION: The improved homologous recombination in bacteria is efficient, convenient and easy to be carried out. Scand J Infect Dis, 2003, 35(11-12), 858 - 62 High hepatocyte growth factor levels in faeces during acute infectious gastroenteritis; Nayeri F et al.; Hepatocyte growth factor (HGF) is a potent mitogen of mature epithelial cells which is produced after organ injuries and acts as a trigger for regeneration in the impaired organ . The aim of the present study was to investigate local production of HGF during infectious gastroenteritis . We measured the concentration of HGF in serum and faeces in 49 patients with acute infectious gastroenteritis (bacterium = 30, virus = 10, amoebae = 1, and probable infection = 8) at the time of referral to hospital and at convalescence (n = 31) . The values were compared with normal healthy vaccination volunteers (n = 11) as well as patients with acute non-infectious diarrhoea (n = 10) . The presence of HGF in faeces was confirmed by ELISA and Western immunoblot . HGF concentrations in faeces was significantly higher in the patients with infectious gastroenteritis compared to the control groups (p < 0.0001) . Using a cut-off concentration of 20 ng/g, the overall sensitivity of faeces HGF to distinguish infectious gastroenteritis (bacterial, viral, probable infection) was 98% with a specificity of 100% . At convalescence all patients had normal values . There was no significant correlation between HGF concentrations in faeces and serum . Determination of faeces HGF may identify cases of transmittable diarrhoea requiring isolation at an early stage. Biochemistry, 2004 Jan 20, 43(2), 437 - 45 Electron transfer from HiPIP to the photooxidized tetraheme cytochrome subunit of Allochromatium vinosum reaction center: new insights from site-directed mutagenesis and computational studies; Venturoli G et al.; The kinetics of electron transfer from reduced high-potential iron-sulfur protein (HiPIP) to the photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center (RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redox conditions by flash absorption spectroscopy . At ambient redox potential Eh = +200 mV, where only the high-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HP heme(s) follows second-order kinetics with rate constant k = (4.2 +/- 0.2) 10(5) M(-1) s(-1) at low ionic strength . Upon increasing the ionic strength, k increases by a maximum factor of ca . 2 at 640 mM KCl . The role of Phe48, which lies on the external surface of HiPIP close to the {Fe4S4} cluster and presumably on the electron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis . Substitution of Phe48 with arginine, aspartate, and histidine completely prevents electron donation . Conversely, electron transfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate is decreased by more than 1 order of magnitude . These results suggest that Phe48 is located on a key protein surface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobic residue on the putative electron-transfer pathway plays a critical role . This conclusion was supported by protein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involves a docking site close to the LP heme farthest from the bacteriochlorophyll special pair. Microbiol Res, 2003, 158(4), 337 - 44 Molecular analysis of a perchlorate reductase from a perchlorate-respiring bacterium Perc1ace; Okeke BC et al.; Perchlorate (ClO4-) is a major ground water pollutant of public health concern . ClO4- reductase is the key enzyme in the pathway of ClO4- breakdown . ClO4- reductase from cell-free extracts of the ClO4- -respiring bacterium perc lace was purified 10-fold by ion-exchange and molecular exclusion fast protein liquid chromatography (FPLC) . The ClO4- reductase catalyzed the reduction of ClO4- at a Vmax and Km of 4.8 U mg protein(-1) and 34.5 microM, respectively . ClO4- reduction was achieved in the temperature range of 20 to 40 degrees C and with optimum activity at 25 degrees C to 30 degrees C and pH 7.5 to 8.0 . Molecular masses of two subunits of ClO4- reductase were determined by SDS-PAGE to be 35 kDa and 75 kDa . MALDI-TOF/MS analysis of a trypsin digest of the 35 kDa subunit, revealed several tryptic peptides . Amino acid sequences of 22 tryptic peptides of the 35 kDa ClO4- reductase subunit were obtained by electrospray mass spectrometry . GenBank protein Blast analysis of the amino acid sequences revealed relevant similarity to reductases, dehydrogenases and heme proteins . Data obtained are useful towards the identification of the overall genetic determinants of ClO4- reduction and specific in situ detection of ClO4- as well as NO3-reducing bacteria in ground water. Microbiol Res, 2003, 158(4), 309 - 15 During stationary phase, Beijerinckia derxii shows nitrogenase activity concomitant with the release and accumulation of nitrogenated substances; Miyasaka NR et al.; Beijerinckia derxii, a free-living nitrogen-fixing bacterium, maintained an increasing nitrogenase specific activity during the stationary growth phase . To verify the destination of the nitrogen fixed during this phase, intra and extracellular nitrogenated contents were analyzed . Organic nitrogen and amino acids were detected in the supernatant of the cultures . An increase in intracellular content of both nitrogen and protein occurred . Cytoplasmic granules indicated the presence of arginine . The ability of a non-diazotrophic bacterium (E . coli) to use B . derxii proteins as a source of nitrogen was observed concomitantly with E . coli growth . There is a suggestion that B . derxii contributes to the environment by both releasing nitrogenated substances and accumulating substances capable of being consumed after its death. J Clin Microbiol, 2004 Jan, 42(1), 384 - 6 Helicobacter species in the intestinal mucosa of patients with ulcerative colitis; Oliveira AG et al.; In a search for Helicobacter species in the intestinal mucosae of 42 patients with ulcerative colitis (UC) and 74 without UC, only H . pylori was found . Although the bacterium was detected in UC patients by culture (7.1%) and nested PCR (19.0%), its presence was not associated with the disease (P = 0.13). Appl Environ Microbiol, 2004 Jan, 70(1), 363 - 9 Saturable, energy-dependent uptake of phenanthrene in aqueous phase by Mycobacterium sp . strain RJGII-135; Miyata N et al.; The mechanism of uptake of phenanthrene by Mycobacterium sp . strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced) . Washed cells were suspended in aqueous solutions of {9-(14)C}phenanthrene, and then the cells were collected by filtration . Low-level steady-state (14)C concentrations in uninduced cells were achieved within the first 15 s of incubation . This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone . These results indicated that phenanthrene enters rapidly into the cells by passive diffusion . However, induced cells showed cumulative uptake over several minutes . The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K(t)) of 26 +/- 3 nM (mean +/- standard deviation) . Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors . Analysis of cell-associated (14)C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism . These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells . Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K(d)) of 41 +/- 21 nM (mean +/- standard deviation) . Given the low values of K(t) and K(d), Mycobacterium sp . strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase. Appl Environ Microbiol, 2004 Jan, 70(1), 340 - 5 Degradation of benzo{a}pyrene by Mycobacterium vanbaalenii PYR-1; Moody JD et al.; Metabolism of the environmental pollutant benzo{a}pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined . This organism initially oxidized benzo{a}pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12 . The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses . The major intermediates of benzo{a}pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo{a}pyrene (benzo{a}pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo{a}pyrene (benzo{a}pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo{a}pyrene (benzo{a}pyrene trans-11,12-dihydrodiol), 10-oxabenzo{def}chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo{a}pyrene . The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo{a}pyrene cis-4,5-dihydrodiol . Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo{a}pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry . Benzo{a}pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity . The enantiomeric composition of benzo{a}pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules . The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo{a}pyrene . Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo{a}pyrene in M . vanbaalenii PYR-1. Appl Environ Microbiol, 2004 Jan, 70(1), 248 - 54 Generation and utilization of polyclonal antibodies to a synthetic C-terminal amino acid fragment of divercin V41, a class IIa bacteriocin; Richard C et al.; Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized C-terminal part of divercin V41 (DvnCt) conjugated to the carrier protein keyhole limpet hemocyanin (KLH) . The sensitivity and reactivity of the DvnCt-KLH-generated antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) using supernatant from cultures of 13 representative lactic acid bacterium strains, and specificity was confirmed by Western blot analysis . Anti-DvnCt-KLH antibodies were able to recognize not only divercin V41 but also enterocin P and piscicocin V1b, two other members of the class IIa bacteriocins . Production and activity of DvnV41 were evaluated by ELISA and activity tests during the growth of Carnobacterium divergens V41 in MRS medium containing or not containing Tween 80 . Divercin V41, enterocin P, and piscicocin V1b were therefore purified by a single-step immunoaffinity chromatography method using a Sepharose matrix CNBr-activated directed binding of anti-DvnCt-KLH polyclonal antibodies. Plasmid, 2004 Jan, 51(1), 37 - 40 Plasmid curing of Oenococcus oeni; Mesas JM et al.; Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures . Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2) . Sublethal temperature together with acridine orange only generated cured strains lacking pRS3 . These results suggest that acriflavine is a better curing agent than acridine orange for O . oeni, and that pRS3 is the most sensitive to these curing agents . We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation . The ability to cure O . oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium. EMBO Rep, 2004 Jan, 5(1), 104 - 10 Involvement of the intermediate filament protein cytokeratin-18 in actin pedestal formation during EPEC infection; Batchelor M et al.; While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria . The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474 . Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein . Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization . This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection. Biosens Bioelectron, 2004 Feb 15, 19(7), 727 - 36 Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis; Ramachandran A et al.; A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis . Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets . The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences . Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e . located in the stem or loop regions) . Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination . The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used . In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution. Klin Lab Diagn, 2003 Nov, (11), 47 - 8 {The specificity of interaction between the complement and the lipopolysaccharide with a low activity of the complement (hypothesis)}; Es'kov AP et al.; Interactions between the complement with a low activity and the lipopolysacharide (LPS) with an extensive concentration range were experimentally investigated in vitro . A model was suggested that provides an explanation to the concentration-related dependence . The complement and LPS were shown, under certain conditions, to form the membrane-attacking complexes, which are not bound with the bacterium membrane and which can lyse any cells . Damages made by the above complexes to endothelial cells of the vessels could result in the onset of the syndrome of disseminated intravascular coagulation (DIC) . The results can be used to develop a method applicable to evaluating a concentration of endotoxin and to creating anti-tumor drugs. Biotechnol Bioeng, 2003 Dec 30, 84(7), 855 - 63 Prediction of transcriptional profiles of Synechocystis PCC6803 by dynamic autoregressive modeling of DNA microarray data; Schmitt WA Jr et al.; Time-series profiles of gene expression generated by DNA microarrays possess sufficient information for building dynamic models of transcriptional behavior . This, however, requires properly designed experiments and sufficient independent data to validate such models . Here we report the use of AutoRegressive with eXogenous input (ARX) models to fit dynamic gene expression data obtained by subjecting cultures of the photosynthetic bacterium Synechocystis PCC6803 to consecutive light-to-dark transitions . Autoregressive with exogenous input models of appropriate complexity were selected by applying Akaike's information criterion (AIC) such as to maximize agreement between model predictions with experimental data without overfitting . These models were subsequently used to design the experimental profile of an optimal validating data set . Predictions from these models were tested in a second experiment and were found to match well with the validation data . Additionally, the models with the least error in predicting the expression profiles of the validation data set exactly match the model complexity predicted by AIC . Such models offer insights into cellular responses to environmental conditions and form the basis for hypothesizing and quantifying relationships that are presently poorly understood at the level of fundamental mechanisms . J Immunol, 2004 Jan 15, 172(2), 1177 - 85 Expression of signaling lymphocytic activation molecule-associated protein interrupts IFN-gamma production in human tuberculosis; Pasquinelli V et al.; Production of the Th1 cytokine IFN-gamma by T cells is considered crucial for immunity against Mycobacterium tuberculosis infection . We evaluated IFN-gamma production in tuberculosis in the context of signaling molecules known to regulate Th1 cytokines . Two populations of patients who have active tuberculosis were identified, based on their T cell responses to the bacterium . High responder tuberculosis patients displayed significant M . tuberculosis-dependent T cell proliferation and IFN-gamma production, whereas low responder tuberculosis patients displayed weak or no T cell responses to M . tuberculosis . The expression of the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) on cells from tuberculosis patients was inversely correlated with IFN-gamma production in those individuals . Moreover, patients with a nonfunctional SAP gene displayed immune responses to M . tuberculosis similar to those of high responder tuberculosis patients . In contrast to SAP, T cell expression of SLAM was directly correlated with responsiveness to M . tuberculosis Ag . Our data suggest that expression of SAP interferes with Th1 responses whereas SLAM expression contributes to Th1 cytokine responses in tuberculosis . The study further suggests that SAP and SLAM might be focal points for therapeutic modulation of T cell cytokine responses in tuberculosis. Biotechnol Bioeng, 2004 Jan 5, 85(1), 1 - 19 Fundamental Escherichia coli biochemical pathways for biomass and energy production: identification of reactions; Carlson R et al.; Cells grow by oxidizing nutrients using a complex network of biochemical reactions . During this process new biological material is produced along with energy used for maintaining cellular organization . Because the metabolic network is highly branched, these tasks can be accomplished using a wide variety of unique reaction sequences . However, evolutionary pressures under carbon-limited growth conditions likely select organisms that utilize highly efficient pathways . Using elementary-mode analysis, we demonstrate that the metabolism of the bacterium Escherichia coli contains four unique pathways that most efficiently convert glucose and oxygen into new cells and maintenance energy under any level of oxygen limitation . Observed regulatory patterns and experimental findings suggest growing cells use these highly efficient pathways . It is predicted that five knockout mutations generate a strain that supports growth using only the most efficient reaction sequence . The analysis approach should be generally useful for predicting metabolic capabilities and efficient network designs based on only genomic information . Nat Biotechnol, 2004 Jan, 22(1), 55 - 61 Epub 2003 Dec 14. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris; Larimer FW et al.; Rhodopseudomonas palustris is among the most metabolically versatile bacteria known . It uses light, inorganic compounds, or organic compounds, for energy . It acquires carbon from many types of green plant-derived compounds or by carbon dioxide fixation, and it fixes nitrogen . Here we describe the genome sequence of R . palustris, which consists of a 5,459,213-base-pair (bp) circular chromosome with 4,836 predicted genes and a plasmid of 8,427 bp . The sequence reveals genes that confer a remarkably large number of options within a given type of metabolism, including three nitrogenases, five benzene ring cleavage pathways and four light harvesting 2 systems . R . palustris encodes 63 signal transduction histidine kinases and 79 response regulator receiver domains . Almost 15% of the genome is devoted to transport . This genome sequence is a starting point to use R . palustris as a model to explore how organisms integrate metabolic modules in response to environmental perturbations. Genetics, 2003 Dec, 165(4), 1651 - 60 A conservative test of genetic drift in the endosymbiotic bacterium Buchnera: slightly deleterious mutations in the chaperonin groEL; Herbeck JT et al.; The obligate endosymbiotic bacterium Buchnera aphidicola shows elevated rates of sequence evolution compared to free-living relatives, particularly at nonsynonymous sites . Because Buchnera experiences population bottlenecks during transmission to the offspring of its aphid host, it is hypothesized that genetic drift and the accumulation of slightly deleterious mutations can explain this rate increase . Recent studies of intraspecific variation in Buchnera reveal patterns consistent with this hypothesis . In this study, we examine inter- and intraspecific nucleotide variation in groEL, a highly conserved chaperonin gene that is constitutively overexpressed in Buchnera . Maximum-likelihood estimates of nonsynonymous substitution rates across Buchnera species are strikingly low at groEL compared to other loci . Despite this evidence for strong purifying selection on groEL, our intraspecific analysis of this gene documents reduced synonymous polymorphism, elevated nonsynonymous polymorphism, and an excess of rare alleles relative to the neutral expectation, as found in recent studies of other Buchnera loci . Comparisons with Escherichia coli generally show patterns predicted by their differences in N(e) . The sum of these observations is not expected under relaxed or balancing selection, selective sweeps, or increased mutation rate . Rather, they further support the hypothesis that drift is an important force driving accelerated protein evolution in this obligate mutualist. J Bacteriol, 2004 Jan, 186(2), 356 - 65 Helicobacter acinonychis: genetic and rodent infection studies of a Helicobacter pylori-like gastric pathogen of cheetahs and other big cats; Dailidiene D et al.; Insights into bacterium-host interactions and genome evolution can emerge from comparisons among related species . Here we studied Helicobacter acinonychis (formerly H . acinonyx), a species closely related to the human gastric pathogen Helicobacter pylori . Two groups of strains were identified by randomly amplified polymorphic DNA fingerprinting and gene sequencing: one group from six cheetahs in a U.S . zoo and two lions in a European circus, and the other group from a tiger and a lion-tiger hybrid in the same circus . PCR and DNA sequencing showed that each strain lacked the cag pathogenicity island and contained a degenerate vacuolating cytotoxin (vacA) gene . Analyses of nine other genes (glmM, recA, hp519, glr, cysS, ppa, flaB, flaA, and atpA) revealed a approximately 2% base substitution difference, on average, between the two H . acinonychis groups and a approximately 8% difference between these genes and their homologs in H . pylori reference strains such as 26695 . H . acinonychis derivatives that could chronically infect mice were selected and were found to be capable of persistent mixed infection with certain H . pylori strains . Several variants, due variously to recombination or new mutation, were found after 2 months of mixed infection . H . acinonychis ' modest genetic distance from H . pylori, its ability to infect mice, and its ability to coexist and recombine with certain H . pylori strains in vivo should be useful in studies of Helicobacter infection and virulence mechanisms and studies of genome evolution. J Biol Chem, 2004 Mar 12, 279(11), 9892 - 8 Epub 2003 Dec 29. Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus; Borges N et al.; Rhodothermus marinus responds to fluctuations in the growth temperature and/or salinity by accumulating mannosylglycerate (MG) . Two alternative pathways for the synthesis of MG have been identified in this bacterium: a single-step pathway and a two-step pathway . In this work, the genetic and biochemical characterization of the two-step pathway was carried out with the goal of understanding the function of the two pathways and their regulatory mechanisms . Mannosyl-3-phosphoglycerate synthase (MPGS) of the two-step pathway was purified from R . marinus . Sequence information led to the isolation of two contiguous genes, mpgs (encoding MPGS) and mpgp (encoding mannosyl-3-phosphoglycerate phosphatase) . The recombinant MPGS had a low specific activity compared with other homologous MPGSs and contained approximately 30 additional residues at the C terminus . Truncation of this extension produced a protein with a 10-fold higher specific activity . Moreover, the activity of the complete MPGS was enhanced upon incubation with R . marinus cell extracts, and protease inhibitors abolished activation . Therefore, the C-terminal peptide of MPGS was identified as a regulatory site for short term control of MG synthesis in R . marinus . The control of gene expression by heat and osmotic stress was also studied; the level of mannosylglycerate synthase involved in the single-step pathway was selectively enhanced by heat stress, whereas MPGS was overproduced in response to osmotic stress . The concomitant changes in the level of MG were assessed as well . We conclude that the two alternative pathways for the synthesis of MG are differently regulated at the level of expression to play specific roles in the adaptation of R . marinus to two different types of aggression . This is the only example of pathway multiplicity being rationalized in terms of the need to respond efficiently to distinct environmental stresses. J Protein Chem, 2003 Jul, 22(5), 457 - 61 Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino)-cinnamaldehyde binding to the active site; Piersma SR et al.; Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry . This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm . Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data. Infect Immun, 2004 Jan, 72(1), 537 - 45 Helicobacter pylori disrupts STAT1-mediated gamma interferon-induced signal transduction in epithelial cells; Mitchell DJ et al.; Infection with Helicobacter pylori is chronic despite a vigorous mucosal immune response characterized by gastric T-helper type 1 cell expansion and gamma interferon (IFN-gamma) production . IFN-gamma signals by activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1); however, the effect of H . pylori infection on IFN-gamma-STAT1 signaling is unknown . We infected human gastric (MKN45 and AGS) and laryngeal (HEp-2) epithelial cell lines with type 1 and type 2 H . pylori strains and then stimulated them with IFN-gamma . Western blotting of whole-cell protein extracts revealed that infection with live, but not heat-killed, H . pylori time-dependently decreased IFN-gamma-induced STAT1 tyrosine phosphorylation . Electrophoretic mobility shift assay of nuclear protein extracts demonstrated that H . pylori infection reduced IFN-gamma-induced STAT1 DNA binding . STAT1 was unable to translocate from the cytoplasm to the nucleus in H . pylori-infected HEp-2 cells examined by immunofluorescence, and reverse transcription-PCR showed that IFN-gamma-induced interferon regulatory factor 1 expression was inhibited . These effects were independent of the cagA, cagE, and VacA status of the infecting H . pylori strain . Furthermore, neither H . pylori culture supernatants nor conditioned medium from H . pylori-infected MKN45 cells inhibited IFN-gamma-induced STAT1 tyrosine phosphorylation, suggesting that inhibition is independent of a soluble epithelial or bacterial factor but is dependent on bacterial contact with epithelial cells . H . pylori disruption of IFN-gamma-STAT1 signaling in epithelial cells may represent a mechanism by which the bacterium modifies mucosal immune responses to promote its survival in the human host. Infect Immun, 2004 Jan, 72(1), 159 - 67 Delayed clearance of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice; Ganta RR et al.; Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis . To examine the role of helper T cells in host resistance to this macrophage-tropic bacterium, we assessed E . chaffeensis infections in three mouse strains with differing functional levels of helper T cells . Wild-type, C57BL/6J mice resolved infections in approximately 2 weeks . Major histocompatibility complex class II (MHCII) knockout, B6.129-Abb(tm1) mice lacking helper T cells developed persistent infections that were not resolved even after several months . CD4+ T-cell-deficient, B6.129S6-Cd4(tm1Knw) mice cleared the infection, but the clearance took 2 weeks longer than it did for wild-type mice . C57BL/6J mice resolved infection more rapidly following a second experimental challenge, but B6.129S6-Cd4(tm1Knw) mice did not . The B6.129S6-Cd4(tm1Knw) mice also developed active E . chaffeensis-specific immunoglobulin G responses that were slightly lower in concentration and slower to develop than that observed in C57BL/6J mice . E . chaffeensis-specific cytotoxic T cells were not detected following a single bacterial challenge in any mouse strain, including wild-type C57BL/6J mice . However, the cytotoxic T-cell activity developed in all three mouse strains, including the MHCII and CD4+ T-cell knockouts, when challenged with a second E . chaffeensis infection . The data reported here suggest that the cell-mediated immunity, orchestrated by CD4+ T cells is critical for conferring rapid clearance of E . chaffeensis. Microb Pathog, 2004 Feb, 36(2), 93 - 108 Rapid and transient activation of gene expression in peripheral blood mononuclear cells from Johne's disease positive cows exposed to Mycobacterium paratuberculosis in vitro; Coussens PM et al.; Mycobacterium avium subspecies paratuberculosis (M . paratuberculosis) is the causative agent of Johne's disease in ruminants . M . paratuberculosis is a slow-growing intracellular bacterium and infections with M . paratuberculosis can persist in a subclinical state for several years . An early and appropriate T cell-mediated cytotoxic response (Th1-like) to M . paratuberculosis infection is often replaced with an antibody or Th 2-like response as infected animals move toward a progressively more clinical state . The reasons for this shift in immune response are unknown . Recent studies suggest that in vitro exposure of peripheral blood mononuclear cells (PBMCs) from Johne's disease positive cows to M . paratuberculosis for 18-24 h results in suppressed expression of numerous immune cell genes . This effect appears at odds with the notion that immune cells from infected cows would respond to M . paratuberculosis-specific antigens in a vigorous and positive manner . In this report, we detail experiments designed to test the hypothesis that many positive changes in PBMC gene expression induced by M . paratuberculosis in vitro are transient, being rapidly suppressed by as yet unknown mechanisms . Our results demonstrate that, indeed, in vitro stimulation with M . paratuberculosis induces rapid changes in infected cow PBMC gene expression (within 2-4 h of exposure) and that many of these changes are no longer evident by 8-16 h of exposure to M . paratuberculosis . Although precise mechanisms responsible for rapid M . paratuberculosis-mediated activation of PBMC gene expression and the loss thereof remain to be determined, our novel results suggest that PBMCs from Johne's disease positive cows are indeed capable of vigorously responding to M . paratuberculosis and that, for many genes, this response is tempered within 8 h of exposure. Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 181 - 3 Epub 2003 Dec 18. Crystallization and preliminary X-ray diffraction analysis of levansucrase (LsdA) from Gluconacetobacter diazotrophicus SRT4; Martinez-Fleites C et al.; The endophytic bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA; EC 2.4.1.10), which converts sucrose to fructo-oligosaccharides and levan . Fully active LsdA was purified to high homogeneity by non-denaturing reversed-phase HPLC and was crystallized at room temperature by the hanging-drop vapour-diffusion method using ammonium sulfate and ethanol as precipitants . The crystals are extremely sensitive, but native data have been collected to 2.5 A under cryogenic conditions using synchrotron radiation . LsdA crystals belong to the orthorhombic space group P22(1)2(1) or P2(1)2(1)2, with unit-cell parameters a = 53.80, b = 119.39, c = 215.10 A. Curr Protein Pept Sci, 2003 Dec, 4(6), 443 - 50 The biphasic virulence activities of gingipains: activation and inactivation of host proteins; Imamura T et al.; Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis . Rgps (HRgpA and RgpB) and Kgp are specific for -Arg-Xaa- and -Lys-Xaa- peptide bonds, respectively . HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the cata-lytic subunit of HRgpA . The multiple virulence activities of gingipains are reviewed in view of the biphasic mechanisms: activation and inactivation of host proteins . Rgps enhanced vascular permeability through prekallikrein activation or direct bradykinin release in combination with Kgp . This Rgp action is potentially associated with gingival edema and crevicular fluid production . Rgps activate the blood coagulation system, leading to progression of inflammation and consequent alveolar bone loss in the periodontitis site . Rgps also activate protease-activated receptors and induce platelet aggregation, which, together with the coagulation-inducing activity, may explain an emerging link between periodontitis and cardiovascular disease . Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains in human plasma, being involved in the bleeding tendency at the diseased gingiva . Gingipains stimulate expression of matrix metalloproteinases (MMPs) in fibroblasts and activate secreted latent MMPs that can destroy periodontal tissues . Gingipains degrade cytokines, components of the complement system and several receptors, including macrophage CD14, T cell CD4 and CD8, thus perturbing the host-defense systems and thereby facilitating sustained colonization of P . gingivalis . Gingipains are potent virulence factors of P . gingivalis, and in many regards their pathogenic activities constitute new mechanisms of bacterial virulence. Curr Protein Pept Sci, 2003 Dec, 4(6), 397 - 407 Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes; Potempa J et al.; Gingipains, extracellular cysteine proteinases of Porphyromonas gingivalis, constitute the major virulence factor of this periodontopathogenic bacterium . They are the product of three genes, two coding for an Arg-specific (RgpA and RgpB) and one for a Lys-specific proteinase (Kgp) . Proteinase domains of RgpA and RgpB are virtually identical; however, the gene encoding the former enzyme is missing a large segment coding for hemaglutinin / adhesin (HA) domains . The latter domains are present also in Kgp . The tertiary structure of RgpB revealed that the proteinase domain of gingipains has a protein fold referred to as the caspase-hemoglobinase fold . On this basis, they are also evolutionary related to other highly specific proteinases including clostripain, caspases, legumains and separase (clan CD of cysteine peptidases) . Gingipains are produced as large preproproteins and are subject to elaborate, not yet fully understood, secretion, glycosylation, activation, and maturation processes . How they traverse the outer membrane is unknown, although it can be hypothesized that they use an autotransporter pathway . Apparently during transport through the periplasm the LPS-like glycan moiety is added at the conserved C-terminal portion of progingipains . At the cell surface pro-gingipains fold into partially active, single-chain zymogens and undergo autocatalytic, intermolecular processing . Two sequential cleavages within the profragment domain enhance zymogen activity and in the case of RgpA and Kgp are followed by excision of the individual HA domains . These domains are further truncated at the C-terminus by concerted action of Kgp and carboxypeptidase and form a non-covalent multidomain, multifunctional complex anchored into the outer membrane by the glycated, C-terminal HA domain . This hypothetical scenario is a reasonable explanation for the occurrence of many forms of gingipains. Mikrobiologiia, 2003 Sep-Oct, 72(5), 600 - 8 {Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii}; Podkopaeva DA et al.; The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined . The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen . This resulted in a twofold decline of the growth yield coefficient . Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein) . The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown . It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase . The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield. J Bacteriol, 2004 Jan, 186(1), 212 - 25 Characterization of the 101-kilobase-pair megaplasmid pKB1, isolated from the rubber-degrading bacterium Gordonia westfalica Kb1; Broker D et al.; The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined . Plasmid pKB1 harbors 105 open reading frames . The predicted products of 46 of these are significantly related to proteins of known function . Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element . Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome . The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera . The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase . Expression of cadA in E . coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host . When G . westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance . Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1. J Bacteriol, 2004 Jan, 186(1), 29 - 34 Influence of growth temperature on lipid and phosphate contents of surface polysaccharides from the antarctic bacterium Pseudoalteromonas haloplanktis TAC 125; Corsaro MM et al.; The chemical structural variations induced by different growth temperatures in the lipooligosaccharide and exopolysaccharide components extracted from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125 are described . The increase in phosphorylation with the increase in growth temperature seems to be general, because it happens not only for the lipooligosaccharide but also for the exopolysaccharide . Structural variations in the lipid components of lipid A also occur . In addition, free lipid A is found at both 25 and 4 degrees C but not at 15 degrees C, which is the optimal growth temperature, suggesting a incomplete biosynthesis of the lipooligosaccharide component under the first two temperature conditions. Genes Genet Syst, 2003 Oct, 78(5), 319 - 27 Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans; Islam SM et al.; The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized . IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion . The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group . There were seven loci completely identical with the IS8301 sequence in the published D . radiodurans R(1) genome sequence . The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D . radiodurans were investigated using genomic hybridization analysis . At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301 . In strain MR1, a different wild-type isolate, six strong hybridization signals were detected . In spite of the identification of seven copies of IS8301 in the published D . radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection . Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D . radiodurans genome were identified . Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases. Arch Environ Contam Toxicol, 2003 Oct, 45(3), 317 - 23 Energization of Comamonas testosteroni ATCC 17454 for indicating toxic effects of chlorophenoxy herbicides; Loffhagen N et al.; The toxicity of chlorophenoxy herbicides to a bacterium, strongly related to the well-known species Delftia (formerly Comamonas) acidovorans that are able to detoxify these xenobiotics, was investigated . The oxidation of n-hexanol via alcohol dehydrogenases, coupled with the generation of ATP by electron transport phosphorylation (ETP), was used as an indicator for energy-toxic effects on the growth of Comamonas testosteroni ATCC 17454 . Uncoupling--reductions in ATP synthesis accompanied by increased respiration--was found to be induced by 1 mM of the classic uncoupler 2,4-dinitrophenol (2,4-DNP) at pH 7.0 and 8.0 . At pH 5.4 and 6.0, the ATP synthesis and respiration were strongly inhibited by both 2,4-DNP and the chlorophenoxy herbicides tested . In contrast, 5 mM of 2,4-dichlorophenoxyacetic acid (2,4-D) and of 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DCPP) were required for detectable uncoupling effects--reduction of the P/O ratios by about 30%--at pH 7.0 . These chemicals may have less uncoupling power because the concentration of their protonated (undissociated) forms (pKa values 2.7 and 3.0) is an order of magnitude lower than that of 2,4-DNP (pKa = 4.0) at this pH value . Strong uncoupling accompanied by increased respiration, like that induced by 1 mM 2,4-DNP, was also caused by 5 mM 4-(2,4-dichlorophenoxy)-butyric acid (2,4-DCPB), which correlates with its high pKa value of 4.6 . The order of toxicity of the chlorophenoxy herbicides (2,4-D < 2,4-DCPP < 2,4-DCPB) to the ETP, which correlates well with the lipophilicity of their undissociated forms (log P 2.7 < 3.4 < 3.5, respectively), was confirmed by measuring their capacity to inhibit the growth of Comamonas testosteroni ATCC 17454 . The results show that energization via alcohol dehydrogenases can be used as an indicator for investigating energy-toxic effects of organics on the ETP and growth of chlorophenoxy herbicide-detoxifying bacteria. J Mol Biol, 2004 Jan 9, 335(2), 425 - 35 Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation; Bae JB et al.; sigmaR is a sigma factor for transcribing genes to defend cells against oxidative stresses in the antibiotic-producing bacterium Streptomyces coelicolor . The availability of sigmaR is regulated by RsrA, an anti-sigma factor, whose sigmaR-binding activity is regulated by redox changes in the environment, via thiol-disulfide exchange . We found that reduced RsrA contains zinc in a stoichiometric amount, whereas oxidized form has very little: 1 mol of zinc per mol of RsrA was released upon oxidation as monitored by a chromogenic Zn-chelator, 4-(2-pyridylazo)-resorcinol (PAR) . Measurement of zinc bound in several RsrA mutants of various cysteine and histidine substitutions suggested that C3, H7, C41, and C44 serve as zinc-binding sites . The zinc-binding and sigmaR-binding activities of mutant proteins did not coincide, suggesting that zinc might not be absolutely required for the anti-sigma activity of RsrA . Zn-free apo-RsrA bound sigmaR and inhibited sigmaR-dependent transcription in vitro . Compared with Zn-RsrA, the anti-transcription activity of apo-RsrA was about threefold lower and its sigmaR-binding affinity decreased by about ninefold when measured by surface plasmon resonance analysis . Apo-RsrA was more sensitive to protease, suggesting that zinc allows RsrA to maintain a more compact structure, optimized for binding sigmaR . The cysteine pairs that form disulfide bonds were determined by MALDI-TOF mass spectrometry, revealing formation of the critical disulfide bond between C11 and one of the essential cysteine residues C41 or 44, most likely C44 . An improved model for the mechanism of redox-modulation of RsrA was presented. Ann Biol Clin (Paris), 2003 Sep-Oct, 61(5), 541 - 8 {West African tick-borne relapsing fever}; Lecompte Y et al.; West African tick-borne relapsing fever is an endemic disease due to Borrelia crocidurae . The tick Alectorobius sonrai is the only known vector of this bacterium . Several species of rodents and insectivores may be reservoir for this spirochete . The geographic distribution of Borrelia crocidurae is not well known . The zone where the presence of the vector has been recorded is situated in Sahelian regions, from Mauritania and northern Senegal up to Chad . In Senegal, it has been shown that the persistence of drought is responsible for a considerable spread of tick-borne relapsing fever to the south . Few epidemiological data are available about West African tick-borne relapsing fever . In Senegal, epidemiological investigations indicate that Borrelia crocidurae is a major cause of morbidity (annual incidence rate of 5.1%) . The relapsing nature of tick-borne borreliosis depends on Borrelia's antigenic variability . Except relapsing febrile episodes, this illness presents no pathognomonic signs . Borrelia crocidurae relapsing fever is generally benignant but neurologic or ocular complications can occur . The diagnosis of tick-borne relapsing fever is made by demonstrating the presence of Borrelia in peripheral blood in thick smear, by intraperitoneal inoculation of mice or more recently with quantitative buffy coat method (QBC test) . The best treatment for relapsing fever is tetracycline or doxycycline . When tetracyclines are contraindicated, the alternative is erythromycin . In neurologic complications, the effective treatment is intravenous penicillin G or ceftriaxone . West African tick-borne relapsing fever must be systematically mentioned in case of fever in a patient returning from the endemic area. Biochim Biophys Acta, 2003 Dec 8, 1607(2-3), 141 - 51 Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli; Schultz A et al.; The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells . Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis . Several of these mutations resulted in an almost complete inactivation of the enzyme . Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping . Two of these mutations, R176K and E584D, caused increased tolerance to salt . In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction . These results and their possible implications for the enzyme function are discussed. J Am Chem Soc, 2003 Dec 17, 125(50), 15352 - 8 Protein film voltammetry of Rhodobacter capsulatus xanthine dehydrogenase; Aguey-Zinsou KF 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 XDHs . The crystal structures of bovine XDH and R . capsulatus XDH showed that the two proteins have highly similar folds; however, R.capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form . Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode . Furthermore, we have determined all redox potentials of the four cofactors (Mo(VI/V), Mo(V/IV), FAD/FADH, FADH/FADH(2) and two distinct {2Fe-2S}(2+/+) clusters) using a combination of potentiometric and voltammetric methods . A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca . 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor . This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis. Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15918 - 23 Epub 2003 Dec 08. Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon; Shimono N et al.; An estimated one-third of the world's population is latently infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis . Here, we demonstrate that, unlike wild-type M . tuberculosis, a strain of M . tuberculosis disrupted in the mce1 operon was unable to enter a stable persistent state of infection in mouse lungs . Instead, the mutant continued to replicate and killed the mice more rapidly than did the wild-type strain . Histological examination of mouse lungs infected with the mutant strain revealed diffusely organized granulomas with aberrant inflammatory cell migration . Murine macrophages infected ex vivo with the mutant strain were reduced in their ability to produce tumor necrosis factor alpha, IL-6, monocyte chemoattractant protein 1, and nitric oxide (NO), but not IL-4 . The mce1 mutant strain complemented with the mce1 genes stimulated tumor necrosis factor alpha and NO production by murine macrophages at levels stimulated by the wild-type strain . These observations indicate that the mce1 operon mutant is unable to stimulate T helper 1-type immunity in mice . The hypervirulence of the mutant strain may have resulted from its inability to stimulate a proinflammatory response that would otherwise induce organized granuloma formation and control the infection without killing the organism . The mce1 operon of M . tuberculosis may be involved in modulating the host inflammatory response in such a way that the bacterium can enter a persistent state without being eliminated or causing disease in the host. Microbiology, 2003 Dec, 149(Pt 12), 3449 - 59 The LysR-type transcriptional regulator CysB controls the repression of hslJ transcription in Escherichia coli; Jovanovic M et al.; The LysR-type transcriptional regulator (LTTR) CysB is a transcription factor in Escherichia coli cells, where as a homotetramer it binds the target promoter regions and activates the genes involved in sulphur utilization and sulphonate-sulphur metabolism, while negatively autoregulating its own transcription . The hslJ gene was found to be negatively regulated by CysB and directly correlated with novobiocin resistance of the bacterium . cysB mutants showed upregulation of the hslJ : : lacZ gene fusion and exhibited increased novobiocin resistance . In this study the hslJ transcription start point and the corresponding putative sigma(70) promoter were determined . The hslJ promoter region was defined by employing different hslJ-lacZ operon fusions, and transcription of the hslJ gene was shown to be subject to both repression imposed by the CysB regulator and direct or indirect autogenous negative control . These two regulations compete to some extent but they are not mutually exclusive . CysB acts as a direct repressor of hslJ transcription and binds the hslJ promoter region that carries the putative CysB repressor site . This CysB binding, apparently responsible for repression, is enhanced in the presence of the ligand N-acetylserine (NAS), hitherto considered to be a positive cofactor in CysB-mediated gene regulations . Interallelic complementation of characterized CysB mutants I33N and S277Ter partially restored the repression of hslJ transcription and the consequent novobiocin sensitivity, but did not complement the cysteine auxotrophy. Theriogenology, 2004 Jan 15, 61(2-3), 595 - 601 Diagnosis and treatment of four stallions, carriers of the contagious metritis organism--case report; Kristula MA et al.; Contagious Equine Metritis (CEM), a venereal disease of horses caused by the bacterium Taylorella equigenitalis, was first diagnosed in 1977 and subsequently spread to many nations {Proc 24th AM Assoc Equine Pract (1979) 287} . The disease was confirmed in the United States in 1978 {Proc Am Assoc Equine Pract (1983) 295} . Specific regulatory procedures for this disease have been established in the United States and 37 other countries . From 1999 through 2001, four of 120 imported European stallions tested positive for CEM at a quarantine facility in Darlington, MD, USA . Two stallions were identified by positive bacterial cultures for T . equigenitalis on arrival . The other two positive stallions were negative on initial bacterial cultures, but were identified as CEM carriers when test mares (that they had mated) were culture-positive for T . equigenitalis . Since T . equigenitalis, is a fastidious slow-growing coccobacillus, additional sets of samples taken over a interval might be required to ensure positive stallions are detected before mating test mares . Likewise, additional sets of samples taken over a long interval after treatment of a stallion for CEM might be required to ensure that positive stallions treated for CEM are detected before mating test mares . Aggressive systemic antibiotic therapy accompanied by routine topical therapy might be required to treat some CEM-positive stallions. Appl Environ Microbiol, 2003 Dec, 69(12), 7447 - 52 Biological traits of Xylella fastidiosa strains from grapes and almonds; Almeida RP et al.; Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS) . PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X . fastidiosa isolated from these host plants . We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X . fastidiosa caused ALS and overwintered within almonds after mechanical inoculation . Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD . However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains . Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X . fastidiosa in grapes than in almonds . In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes . We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines . Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media. J Mol Biol, 2004 Jan 2, 335(1), 261 - 74 X-ray structures of the maltose-maltodextrin-binding protein of the thermoacidophilic bacterium Alicyclobacillus acidocaldarius provide insight into acid stability of proteins; Schafer K et al.; Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems . We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose . Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues . Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior . As a consequence, this protein has a highly positive surface charge . Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual . These features appear to be correlated with the acidostability of the A . acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one . Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of beta-turns, properties that are also probably related to thermostability . Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor. J Econ Entomol, 2000 Feb, 93(1), 88 - 92 Effects of temperature on the flight activity of Graphocephala atropunctata (Hemiptera: Cicadellidae); Feil H et al.; Graphocephala atropunctata (Signoret) is the principal vector of Xylella fastidiosa (Wells, Raju, Hung, Weisberg, Mandelco-Paul and Brenner), the bacterium that causes Pierce's disease of grapevine in coastal California . Monitoring the activity of C . atropunctata in the early spring is important for timing insecticide sprays and assessing the potential for disease spread to adjacent vineyards . Trapping studies with yellow sticky traps over 3 yr in Napa Valley, CA, established a significant correlation between early spring trap catch and temperature . Sticky trap catches of G . atropunctata occurred in the springs of 1996-1998 only when temperature was greater than or equal to 14.5 degrees C . In 1997 and 1998, the degree-hours (> 14.5 degrees C) per day from sunrise to sunset during March and April, but not in May, correlated significantly with trap catches . The temperature threshold of 14.5 degrees C in the early spring can be used to improve the timing of insecticidal applications aimed at reducing C . atropunctata populations in vineyards affected by Pierce's disease. Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2045 - 8 Sphingobium amiense sp . nov., a novel nonylphenol-degrading bacterium isolated from a river sediment; Ushiba Y et al.; A nonylphenol-degrading bacterial strain (YT(T)) was isolated previously from a river sediment sample obtained in Ami-machi, Ibaraki, Japan, and identified as a Sphingomonas species . In this study, the taxonomic relationship between strain YT(T), a recently described nonylphenol-degrading strain, Sphingomonas cloacae, and Sphingobium yanoikuyae, which is phylogenetically related, was examined . Their phenotypic characteristics were compared and levels of DNA-DNA relatedness between these strains were determined . Based on the results of physiological and biochemical tests and DNA-DNA hybridization, it is proposed that strain YT(T) (=IAM 15006(T)=JCM 11777(T)=CIP 107839(T)) represents a novel species of the genus Sphingobium, Sphingobium amiense sp . nov. Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1985 - 9 Psychrobacter okhotskensis sp . nov., a lipase-producing facultative psychrophile isolated from the coast of the Okhotsk Sea; Yumoto I et al.; A facultatively psychrophilic bacterium, strain MD17(T), which hydrolyses lipids at 5 degrees C, was isolated from the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan, when ice carried by the cold current came to the area . The isolate is an aerobic, non-motile coccobacillus that reduces nitrate to nitrite and hydrolyses Tweens 20, 40, 60 and 80, but not gelatin, DNA or alginic acid . The isolate grows at 0 degrees C, but not at temperatures higher than 36 degrees C; its optimum growth temperature is 25 degrees C . It grows in the presence of 0-10 % NaCl . Its major isoprenoid quinone is ubiquinone-8 (Q-8) and its DNA G+C content is 46.7 mol% . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD17(T) is closely related to Psychrobacter glacincola DSM 12194(T) (99.0 % similarity) and Psychrobacter immobilis DSM 7229(T) (98.7 % similarity) . DNA-DNA hybridization revealed 45.9 % relatedness between strain MD17(T) and P . immobilis ATCC 43116(T) and 33.4 % between strain MD17(T) and P . glacincola ATCC 700754(T) . Based on physiological and biochemical characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Psychrobacter okhotskensis sp . nov . is proposed . The type strain is MD17(T) (=NCIMB 13931(T)=JCM 11840(T)). Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1801 - 5 Sulfurimonas autotrophica gen . nov., sp . nov., a novel sulfur-oxidizing epsilon-proteobacterium isolated from hydrothermal sediments in the Mid-Okinawa Trough; Inagaki F et al.; A novel mesophilic, sulfur- and thiosulfate-oxidizing bacterium, strain OK10(T), was isolated from deep-sea sediments at the Hatoma Knoll in the Mid-Okinawa Trough hydrothermal field . Cells of strain OK10(T) were short rods, each being motile by means of a single polar flagellum . The isolate grew at 10-40 degrees C (optimum 25 degrees C) and pH 4.5-9.0 (optimum pH 6.5) . It grew chemolithoautotrophically with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor . Molecular hydrogen did not support growth . The G+C content of the genomic DNA of strain OK10(T) was 35.2 mol% . Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolate belonged to the epsilon-Proteobacteria . On the basis of its physiological and molecular characteristics, strain OK10(T) (=ATCC BAA-671(T)=JCM 11897(T)) represents the sole species of a new genus, Sulfurimonas, for which the name Sulfurimonas autotrophica is proposed. Nucleic Acids Res, 2003 Dec 15, 31(24), 7208 - 15 A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel; Long KS et al.; The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light . The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA . The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel . The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome. Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 1099 - 103 Leptin suppresses Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis; Slomiany BL et al.; Leptin, a multifunctional hormone produced predominantly by adipocytes but also identified throughout the glandular tissue of alimentary tract, including salivary glands and oral mucosa, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection . In this study, we report that leptin prevents (up to 88.4%) the reduction in mucin synthesis evoked in mucous cells of sublingual salivary gland by LPS of periodontopathic bacterium, Porphyromonas gingivalis . The effect of leptin, moreover, was reflected in a marked decrease in the LPS-induced apoptosis, expression of TNF-alpha, caspase-3 activity, and NO generation . The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, which also obviated the inhibitory effect of leptin on the LPS-induced upregulation in apoptosis, caspase-3 activity, and NO generation . A potentiation in the impedance by leptin of the LPS-induced apoptosis, caspase-3 activity, and NO generation was, however, attained with NOS-2 inhibitor, 1400W, that also caused further enhancement in the impedance by leptin of the LPS detrimental effect on mucin synthesis . Taken together, our data are the first to demonstrate the nature of the involvement of leptin in countering the pathological consequences of P . gingivalis infection on the synthesis of salivary mucins. Mol Microbiol, 2004 Jan, 51(1), 135 - 47 Is modification sufficient to protect a bacterial chromosome from a resident restriction endonuclease? Makovets S, Powell LM, Titheradge AJ, Blakely GW, Murray NE. It has been generally accepted that DNA modification protects the chromosome of a bacterium encoding a restriction and modification system . But, when target sequences within the chromosome of one such bacterium (Escherichia coli K-12) are unmodified, the cell does not destroy its own DNA; instead, ClpXP inactivates the nuclease, and restriction is said to be alleviated . Thus, the resident chromosome is recognized as 'self' rather than 'foreign' even in the absence of modification . We now provide evidence that restriction alleviation may be a characteristic of Type I restriction-modification systems, and that it can be achieved by different mechanisms . Our experiments support disassembly of active endonuclease complexes as a potential mechanism . We identify amino acid substitutions in a restriction endonuclease, which impair restriction alleviation in response to treatment with a mutagen, and demonstrate that restriction alleviation serves to protect the chromosome even in the absence of mutagenic treatment . In the absence of efficient restriction alleviation, a Type I restriction enzyme cleaves host DNA and, under these conditions, homologous recombination maintains the integrity of the bacterial chromosome. Mol Microbiol, 2004 Jan, 51(1), 7 - 13 Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter; Jacobs-Wagner C; Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes . Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles . Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle . This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria . Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2306 - 9 Epub 2003 Nov 27. Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris; Chatake T et al.; Dissimilatory sulfite reductase D (DsrD) from Desulfovibrio vulgaris has been crystallized for a neutron diffraction study . The initial crystals obtained were too small for the neutron experiment . In order to obtain a larger crystal (>1 mm3), a combination of two techniques was developed to determine the optimum crystallization conditions: a crystallization phase diagram was obtained, followed by crystal-quality assessment via X-ray diffraction . Using conditions determined in this manner, a large single crystal (1.7 mm3) of DsrD protein was subsequently grown in D(2)O solution by the macroseeding technique . A neutron diffraction experiment was carried out using the BIX-3 diffractometer at the Japan Atomic Energy Research Institute (JAERI), collecting data to 2.4 A resolution from an optimized crystal. Acta Crystallogr D Biol Crystallogr, 2003 Dec, 59(Pt 12), 2265 - 8 Epub 2003 Nov 27. Preliminary X-ray diffraction analysis of octaprenyl pyrophosphate synthase crystals from Thermotoga maritima and Escherichia coli; Guo RT et al.; Octaprenyl pyrophosphate synthase (OPPs) catalyzes the condensation of five isopentenyl pyrophosphates with farnesyl pyrophosphate to generate C(40) octaprenyl pyrophosphate . The enzymes from the hyperthermophilic bacterium Thermotoga maritima and from the mesophilic Escherichia coli were expressed in E . coli and the recombinant proteins were purified and crystallized . The T . maritima OPPs crystals belong to space group P42(1)2, with unit-cell parameters a = b = 151.53, c = 69.72 A . The E . coli OPPs crystals belong to space group C222(1), with unit-cell parameters a = 247.66, b = 266.10, c = 157.93 A . Diffraction data were collected at 100 K using synchrotron radiation and an in-house X-ray source . Structure determination of T . maritima OPPs has been carried out using MIR data sets at 2.8 A resolution . The asymmetric unit contains one dimer . An initial model with 280 residues per subunit has been built and refined to 2.28 A resolution . It shows mostly helical structure and resembles that of avian farnesyl pyrophosphate synthase. J Bacteriol, 2003 Dec, 185(24), 7120 - 8 Propane monooxygenase and NAD+-dependent secondary alcohol dehydrogenase in propane metabolism by Gordonia sp . strain TY-5; Kotani T et al.; A new isolate, Gordonia sp . strain TY-5, is capable of growth on propane and n-alkanes with C(13) to C(22) carbon chains as the sole source of carbon . In whole-cell reactions, significant propane oxidation to 2-propanol was detected . A gene cluster designated prmABCD, which encodes the components of a putative dinuclear-iron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced . A mutant with prmB disrupted (prmB::Kan(r)) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane . These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium . Downstream of the prm genes, an open reading frame (adh1) encoding an NAD(+)-dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized . The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan(r)) indicate that Adh1 plays a major role in propane metabolism . Two additional NAD(+)-dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation . On the basis of these results, we conclude that Gordonia sp . strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol. J Bacteriol, 2003 Dec, 185(24), 7111 - 9 Interaction between the H2 sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodobacter capsulatus; Elsen S et al.; The photosynthetic bacterium Rhodobacter capsulatus contains two {NiFe}hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV . The synthesis of HupSL is specifically activated by H(2) through a signal transduction cascade comprising three proteins: the H(2)-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR . Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes . To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His(6))-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system . The two proteins copurified as a His(6)-HupUHupV complex present in dimeric and tetrameric forms, both of which had H(2) uptake activity . We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel . Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues . As expected, H(2) affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT . Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants . Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H(2) regulation. Biophys J, 2003 Dec, 85(6), 3558 - 74 Model of bacterial band formation in aerotaxis; Mazzag BC et al.; Aerotaxis is a particular form of "energy taxis" . It is based on a largely elusive signal transduction machinery . In aerotaxis, oxygen dissolved in water plays the role of both attractant (at moderate concentrations) and repellent (at high and low concentrations) . Cells swimming from favorable oxygen concentrations into regions with unfavorable concentrations increase the frequency of reversals, turn back into the favorable domain, and become effectively trapped there . At the same time, bacteria consume oxygen, creating an oxygen gradient . This behavior leads to a pattern formation phenomenon: bacteria self-organize into a dense band at a certain distance from the air-water interface . We incorporate experimental observations of the aerotactic bacterium, Azospirillum brasilense, into a mathematical model . The model consists of a system of differential equations describing swimming bacterial cells and diffusing oxygen . The cells' frequency of reversals depends on the concentration of oxygen and its time derivative while oxygen is depleted by the bacteria . We suggest a hypothetical model of energy sensing mediated by aerotactic receptors Aer and Tsr . Computer simulations and analysis of the model equations allow comparisons of theoretical and experimental results and provide insight into the mechanisms of bacterial pattern formation and underlying signal transduction machinery . We make testable predictions about position and density of the bacterial band. Microb Pathog, 2004 Jan, 36(1), 51 - 7 Chemokine and cytokine production during Orientia tsutsugamushi infection in mice; Koh YS et al.; Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus which is histopathologically characterized by inflammatory manifestations, indicating that rickettsiae induce mechanisms that amplify the inflammatory response . To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production after infection with O . tsutsugamushi in mice . The mRNAs that were upregulated included lymphotactin, RANTES (regulated upon activation, normal T-cell expressed and secreted), macrophage inflammatory proteins 1alpha/beta (MIP-1alpha/beta), MIP-2, monocyte chemoattractant protein 1, lymphotoxin beta, tumor necrosis factor alpha, interleukin-6, gamma-interferon, transforming growth factor beta1, and migration inhibition factor . Peak expression of these chemokines and cytokines was observed between 4 and 8 days after infection . Gene induction was followed by the secretion of chemokine and cytokine proteins . Chemokine profile in infected mice was well correlated with kinetics of inflammatory cell infiltration . Thus, O . tsutsugamushi appears to be a strong inducer of chemokines and cytokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation observed in scrub typhus. Res Microbiol, 2003 Dec, 154(10), 681 - 7 Cloning and characterization of a thermostable intracellular alpha-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8; Lim WJ et al.; The gene encoding an intracellular alpha-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli . The AmyB enzyme hydrolyzed alpha-1,4 starch linkage . The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50187 Da) . The molecular weight of the enzyme was estimated to be 50000 Da by SDS-PAGE after starch-nondenaturing-PAGE . The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among alpha-amylases . The AmyB alpha-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 degrees C . Like the alpha-amylases of many other organisms, the thermostability of T . maritima MSB8 alpha-amylase, AmyB expressed in E . coli was enhanced in the presence of Ca(2+) (10 mM). Bioelectrochemistry, 2003 Oct, 61(1-2), 73 - 84 Energetics and mechanisms of high efficiency of charge separation and electron transfer processes in Rhodobacter sphaeroides reaction centers; Paschenko VZ et al.; Effects of environmental changes due to D(2)O/H(2)O substitution and cryosolvent addition on the energetics of the special pair and the rate constants of forward and back electron transfer reactions in the picosecond-nanosecond time domain have been studied in isolated reaction centers (RC) of the anaxogenic purple bacterium Rhodobacter sphaeroides . The following results were obtained: (i) . replacement of H(2)O by D(2)O or addition of either 70% (v/v) glycerol or 35% (v/v) DMSO do not influence the absorption spectra; (ii) . in marked contrast to this invariance of absorption, the maxima of fluorescence spectra are red shifted relative to control by 3.5, 6.8 and 14.5 nm for RCs suspended in glycerol, D(2)O or DMSO, respectively; (iii) . D(2)O/H(2)O substitution or DMSO addition give rise to an increase of the time constants of charge separation (tau(e)), and Q(A)(-) formation (tau(Q)) by a factors of 2.5-3.1 and 1.7-2.5, respectively; (iv) . addition of 70% glycerol is virtually without effect on the values of tau(e) and tau(Q); (v) . the midpoint potential E(m) of P/P(+) is shifted by about 30 and 45 mV towards higher values by addition of 70% glycerol and 35% DMSO, respectively, but remains invariant to D(2)O/H(2)O exchange; and (vi) . an additional fast component with tau(1)=0.5-0.8 ns in the kinetics of charge recombination P(+)H(A)(-)-->P*(P)H(A) emerges in RC suspensions modified either by D(2)O/H(2)O substitution or by DMSO treatment.The results have been analysed with special emphasis on the role of deformations of hydrogen bonds for the solvation mechanism of nonequilibrium states of cofactors . Reorientation of hydrogen bonds provides the major contribution of the very fast environmental response to excitation of the special pair P . The Gibbs standard free energy gap between 1P* and P(+)B(A)(-) due to solvation is estimated to be approximately 70, 59 and 48 meV for control, D(2)O- and DMSO-treated RC samples, respectively. Environ Microbiol, 2003 Nov, 5(11), 1053 - 63 The significance of organic carbon compounds for in situ metabolism and chemotaxis of phototrophic consortia; Glaeser J et al.; The significance of organic carbon substrates for the chemotaxis and physiology of phototrophic consortia was investigated in a dense chemocline community of Pelochromatium roseum . For the first time, the monopolar monotrichous flagellation of the central bacterium could be visualized . In situ, intact motile P . roseum consortia were strongly attracted by sulphide and 2-oxoglutarate, which indicated a potential role of these compounds in the metabolism of P . roseum . In chemocline water samples, 2-{14C(U)}-oxoglutarate was utilized at nanomolar concentrations (half saturation constant of uptake Kt < or = 10-40 nM), and at a maximum uptake rate of Vmax approximately 6 nM h-1 . The calculated turnover of 2-oxoglutarate at in situ concentrations was approximately 6 h . Microautoradiography of chemocline water samples revealed that 87.5% of the P . roseum consortia incorporated 2-oxoglutarate when both light and sulphide were present, whereas uptake was detected in less than 1.4% of the consortia if either light or sulphide were absent . Because the green sulphur bacterial epibionts in P . roseum have been shown to grow autotrophically, 2-oxoglutarate most likely is taken up and utilized by the central bacterium . Thus, our results indicate that incorporation of 2-oxoglutarate by the central bacterium is regulated by the metabolic state of the green sulphur bacterial epibionts. Lakartidningen, 2003 Nov 6, 100(45), 3596 - 7 {Buruli ulcer--Africa's latest mycobacterial scourge}; Roupe G; Buruliulcer is an extensive ulceration usually on the extremities . The ulcer can spread to subcutaneous fat, muscle and even bone causing osteomyelitis and death . It is the the third most common mycobacterial disease in humans after tuberculosis and leprosy . The bacterium grows in still standing water and infects children through small ulcerations in their skin . Mycobacterium ulcerans may also be transmitted by the bite of aquatic bugs (Naucordiae), which harbor the bacterium in their salivary glands . The disease affects poor people in rural, tropical areas where deforestation has led to flooding rivers, stagnant bodies of water and marsh . Benin, Cote d'Ivoire and Ghana in West Africa are seriously hit . Skin transplantation is the treatment of choice . Treatment with antibiotics has been disappointing. Infect Immun, 2003 Dec, 71(12), 6933 - 42 Mitogenic effect of Bartonella bacilliformis on human vascular endothelial cells and involvement of GroEL; Minnick MF et al.; Bartonellae are bacterial pathogens for a wide variety of mammals . In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana . The results of this study show that Bartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at >or=72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls . The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls . The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B . The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis . The Bartonella mitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium . In contrast, soluble cell lysate fractions from closely related B . henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers . Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity . A B . bacilliformis strain containing the intact groES-groEL operon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r(2) = 0.85) . Antiserum to GroEL significantly inhibited mitogenicity of the lysate . Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted by B . bacilliformis. Antimicrob Agents Chemother, 2003 Dec, 47(12), 3982 - 4 Efficacy of sulforaphane in eradicating Helicobacter pylori in human gastric xenografts implanted in nude mice; Haristoy X et al.; Sulforaphane, an isothiocyanate abundant in the form of its glucosinolate precursor in broccoli sprouts, has shown in vitro activity against Helicobacter pylori . We evaluated the effect of sulforaphane in vivo against this bacterium by using human gastric xenografts in nude mice . H . pylori was completely eradicated in 8 of the 11 sulforaphane-treated grafts . This result suggests that sulforaphane might be beneficial in the treatment of H . pylori-infected individuals. Biochem Biophys Res Commun, 2003 Dec 12, 312(2), 303 - 8 Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization; Khairnar NP et al.; Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization . Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E . coli promoter . The transgenic E . coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E . coli, suggesting the synthesis of an active protein . The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species . The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E . coli through an unknown mechanism . The study elucidates a hitherto unknown possible function of PQQ in bacteria. Crit Rev Microbiol, 2003, 29(4), 351 - 9 Helicobacter pylori and coronary heart disease: which directions for future studies? Pellicano R, Fagoonee S, Rizzetto M, Ponzetto A. Classical risk factors explain the pathogenesis of coronary heart disease (CHD) in only a proportion of cases; therefore, the need to investigate the possible role of "new" agents has incited intense research . Since 1994, a number of studies regarding the possible involvement of Helicobacter pylori (H . pylori) have been published with conflicting results . Establishing a causal link between this infection and CHD would be of major public health importance, since the eradication of the bacterium is easy and much less expensive than long-term treatment of the other risk factors . The main cause of this discordance was the vast heterogeneity of such studies: sufficiently powerful design was found only in few investigations, CHD was defined with a low degree of homogeneity, biases were obvious in the control groups, thus giving room for large variation in the adjustment of potential confounding factors . The present paper attempts to highlight the future directions towards which research should be headed in this area to establish a causal role of H . pylori infection in the pathogenesis of CHD . Future research should take three directions: 1) prospective population-based studies in which the incidence or the recurrence of CHD be evaluated in correlation with H . pylori status, 2) intervention trials, focusing separately on the chronic and acute phases of coronary heart disease and, 3) studies of physiopathology (both in the animal model and humans) to understand the potential biological plausibility. Helicobacter, 2003 Dec, 8(6), 608 - 12 Detection of a putative novel Wolinella species in patients with squamous cell carcinoma of the esophagus; Bohr UR et al.; BACKGROUND: Certain regions of South Africa exhibit an extraordinarily high incidence of esophageal carcinoma that develops via an esophagitis-dysplasia-carcinoma sequence . Bacteria belonging to the family Helicobacteraceae are candidates for involvement in the initiation of the esophagitis . We investigated patients with esophageal carcinoma for the occurrence of Helicobacter-related species . METHODS: Biopsies from tumor and nonlesional tissue of the esophagus from nine patients with squamous cell carcinoma were investigated for Helicobacteraceae using a PCR-based method targeting the 16S rRNA gene . RESULTS: Four out of nine patients tested negative, while samples from the other five patients revealed an infection by different Helicobacter species . Sequence analysis of the PCR fragments led to the identification of a hitherto unknown bacterium in three of these patients . Phylogenetically, this bacterium was assigned to the genus Wolinella within the family of Helicobacteraceae . Helicobacter pylori was identified in three patients, and one revealed a coinfection with the novel Wolinella species . CONCLUSIONS: Helicobacteraceae were detected in approximately 50% of South African patients with esophageal carcinoma . Furthermore, a novel bacterium was identified that might be linked to the enhanced incidence of esophagitis and subsequent malignant disease in South Africa. Mol Ecol, 2003 Nov, 12(11), 3057 - 68 The distribution and evolutionary history of Wolbachia infection in native and introduced populations of the invasive argentine ant (Linepithema humile); Tsutsui ND et al.; Wolbachia pipientis is a maternally transmitted bacterium that often alters the life history of its insect host to maximize transmission to subsequent generations . Here we report on the frequency and distribution of Wolbachia infection in a widespread invasive species, the Argentine ant (Linepithema humile) . We screened 1175 individual Argentine ants from 89 nests on five continents and several islands, including numerous locations in both the native (South American) and introduced ranges . We detected Wolbachia in four of 11 native populations, but only one of 21 introduced populations was infected . In the Argentine ant's native range, the distribution of Wolbachia supergroups A and B was nonoverlapping . By coupling infection frequency data with behaviourally defined colony boundaries, we show that infected and uninfected colonies are often adjacent to one another, supporting the proposition that little female-mediated gene flow occurs among Argentine ant colonies . We also conduct a phylogenetic analysis, and show that the Wolbachia infecting both native and introduced populations of Argentine ants belong to two lineages that appear to be specialized on infecting New World ants . One other lineage of Wolbachia has undergone frequent, recent episodes of horizontal transmission between distantly related, introduced insect hosts. Proteomics, 2003 Oct, 3(10), 1874 - 82 Proteome of Caulobacter crescentus cell cycle publicly accessible on SWICZ server; Vohradsky J et al.; Here we present the Swiss-Czech Proteomics Server (SWICZ), which hosts the proteomic database summarizing information about the cell cycle of the aquatic bacterium Caulobacter crescentus . The database provides a searchable tool for easy access of global protein synthesis and protein stability data as examined during the C . crescentus cell cycle . Protein synthesis data collected from five different cell cycle stages were determined for each protein spot as a relative value of the total amount of {(35)S}methionine incorporation . Protein stability of pulse-labeled extracts were measured during a chase period equivalent to one cell cycle unit . Quantitative information for individual proteins together with descriptive data such as protein identities, apparent molecular masses and isoelectric points, were combined with information on protein function, genomic context, and the cell cycle stage, and were then assembled in a relational database with a world wide web interface , which allows the database records to be searched and displays the recovered information . A total of 1250 protein spots were reproducibly detected on two-dimensional gel electropherograms, 295 of which were identified by mass spectroscopy . The database is accessible either through clickable two-dimensional gel electrophoretic maps or by means of a set of dedicated search engines . Basic characterization of the experimental procedures, data processing, and a comprehensive description of the web site are presented . In its current state, the SWICZ proteome database provides a platform for the incorporation of new data emerging from extended functional studies on the C . crescentus proteome. J Biol Chem, 2004 Feb 6, 279(6), 4686 - 95 Epub 2003 Nov 18. IcmR-regulated membrane insertion and efflux by the Legionella pneumophila IcmQ protein; Dumenil G et al.; Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease . Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane . Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems . We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000 . Both membrane insertion and pore formation were inhibited by IcmR . Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains . The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux . The C-terminal domain was necessary for efficient association of the protein with lipid bilayers . IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity . Localization of IcmQ on the surface of the L . pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium. J Exp Med, 2003 Nov 17, 198(10), 1563 - 72 A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to legionnaires' disease; Hawn TR et al.; Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood . We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria . Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5392STOP) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila, a flagellated bacterium . We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells . Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response. Arch Biochem Biophys, 2003 Dec 1, 420(1), 130 - 41 Common evolutionary origin of mitochondrial and rickettsial respiratory chains; Emelyanov VV; Comprehensive phylogenetic analysis of the subunits of respiratory chain was carried out using a variety of mitochondrial and bacterial sequences including those from all unfinished alpha-proteobacterial genomes known to date . Maximum likelihood, neighbor-joining, and maximum parsimony consensus trees, based on four proton-translocating complexes, placed mitochondria as a sister group to the order Rickettsiales of obligate endosymbiotic bacteria to the exclusion of free-living alpha-proteobacteria . Thus, phylogenetic relationship of most eukaryotic respiratory enzymes conforms to canonical pattern of mitochondrial ancestry, prior established in analyses of ribosomal RNAs, which are encoded by residual mitochondrial genomes . These data suggest that mitochondria may have derived from a reduced intracellular bacterium and that respiration may be the only evolutionary novelty brought into eukaryotes by mitochondrial endosymbiont. Eur J Biochem, 2003 Dec, 270(23), 4595 - 605 Kinetics of the quinone binding reaction at the QB site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes; Milano F et al.; Transmembrane proton translocation in the photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides is driven by light and performed by two transmembrane complexes; the photosynthetic reaction center and the ubiquinol-cytochrome c oxidoreductase complex, coupled by two mobile electron carriers; the cytochrome and the quinone . This paper focuses on the kinetics and thermodynamics of the interaction between the lipophylic electron carrier ubiquinone-10 and the photosynthetic enzyme reconstituted in liposomes . The collected data were simulated with an existing recognized kinetic scheme and the kinetic constants of the uptake (7.2 x 107 M(-1) x s(-1)) and release (40 s(-1)) processes of the ligand were inferred . The results obtained for the quinone release kinetic constant are comparable to the rate of the charge recombination reaction from the state D(+)QA(-) . Values for the kinetic constants are discussed as part of the overall photocycle, suggesting that its bottleneck may not be the quinone uptake reaction in agreement with a previous report. Acc Chem Res, 2003 Nov, 36(11), 825 - 31 Biochemical and biophysical properties of the CO-sensing transcriptional activator CooA; Aono S; Studies of heme-containing gas sensor proteins have revealed a novel function for heme, which acts as an active site for sensing the corresponding gas molecule of a physiological effector . Heme-based O(2), NO, and CO sensor proteins have now been described in which these gas molecules act as a signaling factor that regulates the functional activity of the sensor proteins . CooA is a CO-sensing transcriptional activator found in the photosynthetic bacterium Rhodospirillum rubrum . The binding of CO to the heme group stimulates the transcriptional activator activity of CooA . The mechanisms of CO sensing by CooA and CO-dependent activation of CooA have now been analyzed by both molecular biological and spectroscopic studies and are discussed in this Account. Biofouling, 2003 Jun, 19(3), 197 - 204 Induction of larval settlement in the serpulid polychaete Hydroides elegans (Haswell): role of bacterial extracellular polymers; Lau SC et al.; Larval settlement in the marine polychaete Hydroides elegans is effectively mediated upon contact with the surface of marine bacterial films . Using the bacterium Roseobacter litoralis as a model strain, the effect of bacterial extracellular polymers (exopolymers) on larval settlement of H . elegans was investigated . Bioassays with exopolymer fractions dissociated from bacterial films evoked the initial stages of the larval settlement process, i.e . larvae slowed down, secreted a mucous thread and crawled over the surface . This response is typical of larvae that encounter an attractive bacterial film . In contrast, bioassays with exopolymers in association with UV-irradiated, metabolically inactive bacterial films evoked complete settlement . However, the percentage of responding larvae was negatively correlated with the magnitude of UV-dosage . Since UV energy crosslinks both intra- and extracellular proteinaceous components, it could not be distinguished whether the decrease in larval settlement was due to a modification of proteinaceous components of exopolymers or due the elimination of cellular activity . Nevertheless, the results ascribe bacterial exopolymers the role of an indicator of substratum suitability and provide evidence that the polysaccharide moiety of exopolymers does not complement this effect. Helicobacter, 2003, 8 Suppl 1, 61 - 7 Helicobacter pylori infection in pediatrics; Wewer V et al.; A high prevalence and early colonization of Helicobacter pylori infection in childhood was described again this year in developing countries in contrast to developed ones . Upper gastrointestinal endoscopy including gastric biopsies remains the diagnostic gold standard method for this infection . Also noninvasive tests have been studied in children, including serology, 13C-urea breath test and stool antigen test, showing good results in the different age groups as compared to the gold standard . However, the infection often remains asymptomatic in children and the role of this bacterium in gastric manifestations is the subject of conflicting reports . Extra-digestive manifestations are also reported in the course of this infection . The treatment of H . pylori infection is influenced by resistance of the bacteria to the antibiotics used . We suggest that eradication of H . pylori should take place only after susceptibility testing . The association of a proton pump inhibitor and two antibiotics for 1 or 2 weeks gives the best eradication rates . The crucial question to elucidate is whether asymptomatic children should be treated to prevent cancer in the future. Biosens Bioelectron, 2003 Dec 15, 19(4), 345 - 52 Amperometric cytochrome c3-based biosensor for chromate determination; Michel C et al.; The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability . The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied . Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode) . Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited . The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)) . No interference was observed with As(V), As(III) and Fe(III) . Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor. Arch Microbiol, 2003 Dec, 180(6), 417 - 26 Epub 2003 Nov 11. Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium Chloronema sp . strain UdG9001; Gich F et al.; The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp . strain UdG9001 were analyzed . The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with gamma-carotene as the main carotenoid . HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17 . BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%) . Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm . This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome . Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence . Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes . Several minor polypeptides were also detected but not identified . These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations. J Microbiol Methods, 2003 Dec, 55(3), 829 - 39 Cell behavior and cell-cell communication during fruiting body morphogenesis in Myxococcus xanthus; Jelsbak L et al.; Formation of spatial patterns of cells from a mass of initially identical cells is a recurring theme in developmental biology . The dynamics that direct pattern formation in biological systems often involve morphogenetic cell movements . An