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Diversity and Dynamics of a North Atlantic Coastal Vibrio Community. Janelle R. Thompson, 2004.Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains . Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation . A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period . Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE) . Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations . Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction . It further showed that CDCE populations corresponded in most cases to  98% rRNA similarity groups and suggested that the abundance of these follows temperature trends . Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples . Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend .
Multiple Pathways of Spx (YjbD) Proteolysis in Bacillus subtilis. Shunji Nakano, 2002.ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways . Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development of Bacillus subtilis . Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation . The spx (formally yjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred by clpP and clpX . The level of Spx in wild-type cells grown in competence medium is low, and that in clpP mutants is high . This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from the clpP mutation . In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx . Western blot analysis showed that Spx accumulated in clpX mutant to the same level as that observed in the clpP mutant . In contrast, a very low concentration of Spx was detected in a clpC mutant . An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH . However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx . Transcription of spx, as measured by expression of spx-lacZ, was slightly increased by the clpX mutation . To exclude a possible effect of clpX and clpP on spx transcription, the spx gene was placed under the control of the IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible Pspac promoter . In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx . Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP . The putative proteolysis by ClpXP might require another adapter protein . Spx probably is degraded by ClpCP under as yet unidentified conditions . This study suggests that the level of Spx is tightly controlled by two different ClpP proteases . Impact of Phosphorylation of Specific Residues in the Tyrosine Autokinase, Wzc, on Its Activity in Assembly of Group 1 Capsules in Escherichia coli. Anne Paiment, 2002.WzcCPS is a tyrosine autokinase essential for the assembly of a high-molecular-weight (HMW) group 1 capsular polysaccharide (CPS) in Escherichia coli . Homologues of Wzc participate in the formation of CPS and exopolysaccharides in a variety of gram-positive and gram-negative bacteria . Phosphorylation of tyrosine residues in the WzcCPS C terminus is essential for HMW CPS assembly . Overexpression of WzbCPS (phosphatase) in a wild-type background caused a 3.7-fold decrease in the amount of cell-associated K30 CPS produced, confirming the importance of WzcCPS phosphorylation for capsule assembly . In this study, the tyrosine-rich region was dissected in an attempt to identify residues critical for WzcCPS phosphorylation and/or capsule expression . Site-directed mutagenesis demonstrated that no single tyrosine residue in this region is sufficient for detectable phosphorylation of WzcCPS in vivo or for HMW CPS expression . Furthermore, no single tyrosine residue is essential for phosphorylation or capsule assembly, since removal of any one tyrosine residue has no detectable effect . Altering combinations of tyrosine residues (from two to five) led to WzcCPS derivatives that were still competent for phosphorylation but that could not support assembly of HMW CPS, showing that phosphorylation of Wzc per se is not an accurate measure of its ability to function in capsule assembly . One interpretation of these data is that the overall level of phosphorylation in this region, rather than the precise combination of residues accessible to phosphorylation, is important for the activity of WzcCPS . Tyrosine 569, a residue shown to modulate the in vitro phosphorylation of WzcCA from E . coli K-12, was also mutated . The derivative with this mutation still functioned in capsule assembly . Quantitation of K30CPS from this mutant revealed no difference in the amount of polymer produced . Finally, dithiobis(succinimidylpropionate) cross-linking was used to confirm that WzcCPS forms complexes in vivo, independent of the phosphorylation state of the protein . Subtractive Hybridization Reveals a Type I Polyketide Synthase Locus Specific to Mycobacterium ulcerans. Grant A. Jenkin, 2003.Mycobacterium ulcerans causes Buruli ulcer, the third most prevalent mycobacterial infection of immunocompetent humans after tuberculosis and leprosy . Recent work has shown that the production by M . ulcerans of mycolactone, a novel polyketide, may partly explain the pathogenesis of Buruli ulcer . To search for the genetic basis of virulence in M . ulcerans, we took advantage of the close genetic relationship between M . ulcerans and Mycobacterium marinum by performing genomic suppressive subtractive hybridization of M . ulcerans with M . marinum . We identified several DNA fragments specific to M . ulcerans, in particular, a type I polyketide synthase locus with a highly repetitive modular arrangement . We postulate that this locus is responsible for the synthesis of mycolactone in M . ulcerans . Isolation and Characterization of a Sulfur-Oxidizing Chemolithotroph Growing on Crude Oil under Anaerobic Conditions. Yumiko Kodama, 2003.Molecular approaches have shown that a group of bacteria (called cluster 1 bacteria) affiliated with the  subclass of the class Proteobacteria constituted major populations in underground crude-oil storage cavities . In order to unveil their physiology and ecological niche, this study isolated bacterial strains (exemplified by strain YK-1) affiliated with the cluster 1 bacteria from an oil storage cavity at Kuji in Iwate, Japan . 16S rRNA gene sequence analysis indicated that its closest relative was Thiomicrospira denitrificans (90% identity) . Growth experiments under anaerobic conditions showed that strain YK-1 was a sulfur-oxidizing obligate chemolithotroph utilizing sulfide, elemental sulfur, thiosulfate, and hydrogen as electron donors and nitrate as an electron acceptor . Oxygen also supported its growth only under microaerobic conditions . Strain YK-1 could not grow on nitrite, and nitrite was the final product of nitrate reduction . Neither sugars, organic acids (including acetate), nor hydrocarbons could serve as carbon and energy sources . A typical stoichiometry of its energy metabolism followed an equation: S2- + 4NO3-
 SO42- + 4NO2- ( G0 = -534 kJ mol-1) . In a difference from other anaerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 1% NaCl was negligible . When YK-1 was grown anaerobically in a sulfur-depleted inorganic medium overlaid with crude oil, sulfate was produced, corresponding to its growth . On the contrary, YK-1 could not utilize crude oil as a carbon source . These results suggest that the cluster 1 bacteria yielded energy for growth in oil storage cavities by oxidizing petroleum sulfur compounds . Based on its physiology, ecological interactions with other members of the groundwater community are discussed .
Recombinant Saccharomyces cerevisiae Expressing P450 in Artificial Digestive Systems: a Model for Biodetoxication in the Human Digestive Environment. S. Blanquet, 2003.The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs . A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1 . The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% ± 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% ± 2.7% [n = 3]) . For the first time, the ability of recombinant S . cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract . In the gastric-small intestinal system, 41.0% ± 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% ± 1.6% (n = 3) in the stomach, 13.8% ± 3.3% (n = 3) in the duodenum, 11.8% ± 3.4% (n = 3) in the jejunum, and 6.5% ± 1.0% (n = 3) in the ileum . In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified . These results suggest that S . cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action . In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment .
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