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Flagellar Phase Variation in Salmonella enterica Is Mediated by a Posttranscriptional Control Mechanism. Heather R. Bonifield, 2003.Salmonella enterica has two antigenically distinct flagellin genes, fliC and fljB, that are alternatively expressed . The fljA gene is cotranscribed with fljB and encodes a protein that has been characterized as a transcriptional repressor of the unlinked fliC gene when FljB is expressed . In this study we report genetic evidence that FljA prevents the production of FliC protein through an interaction with the 5'-untranslated region of the fliC mRNA transcript . Studies with operon and gene fusions, Western analyses, and T2 RNase protection assays were performed for strains with the fljBA promoter locked in either the on or the off orientation . ß-Galactosidase assays of fliC transcriptional and translational fusions to the lac operon demonstrated that while FljA inhibits fliC transcription fivefold in the fljBAON orientation, it has a 200-fold effect on both fliC transcription and translation, indicating that the FljA inhibitor might act at both the transcriptional and translational level . T2 RNase protection assays also demonstrated a fivefold decrease in fliC transcript levels for cells locked in the fljBAON orientation compared to those in the fljBAOFF orientation, and an eightfold decrease in FliC protein levels was observed by Western analysis . This reduction in FliC protein levels is greater than the decrease observed for the transcript . These results are consistent with a new model whereby FljA inhibits FliC expression by an attenuation or translational control mechanism . Doripenem versus Pseudomonas aeruginosa In Vitro: Activity against Characterized Isolates, Mutants, and Transconjugants and Resistance Selection Potential. Shazad Mushtaq, 2004.Doripenem is a broad-spectrum parenteral carbapenem under clinical development in Japan and North America . Its activities against (i) Pseudomonas aeruginosa isolates with graded levels of intrinsic efflux-type resistance, (ii) mutants with various combinations of AmpC and OprD expression, (iii) PU21 transconjugants with class A and D ß-lactamases, and (iv) P . aeruginosa isolates with metallo-ß-lactamases were tested by the agar dilution method of the National Committee for Clinical Laboratory Standards . Selection of resistant P . aeruginosa mutants was investigated in single- and multistep procedures . Doripenem MICs for isolates without acquired resistance mostly were 0.12 to 0.5 µg/ml, whereas meropenem MICs were 0.25 to 0.5 µg/ml and imipenem MICs were 1 to 2 µg/ml . The MICs of doripenem, meropenem, ertapenem, and noncarbapenems for isolates with increased efflux-type resistance were elevated, whereas the MICs of imipenem were less affected . The MICs of doripenem were increased by the loss of OprD but not by derepression of AmpC; nevertheless, and as with other carbapenems, the impermeability-determined resistance caused by the loss of OprD corequired AmpC activity and was lost in OprD– mutants also lacking AmpC . The TEM, PSE, PER, and OXA enzymes did not significantly protect P . aeruginosa PU21 against the activity of doripenem, whereas MICs of  16 µg/ml were seen for clinical isolates with VIM and IMP metallo-ß-lactamases . Resistant mutants seemed to be harder to select with doripenem than with other carbapenems (or noncarbapenems), and the fold increases in the MICs were smaller for the resistant mutants . Single-step doripenem mutants were mostly resistant only to carbapenems and had lost OprD; multistep mutants had broader resistance, implying the presence of additional mechanisms, putatively including up-regulated efflux . Most mutants selected with aminoglycosides and quinolones had little or no cross-resistance to carbapenems, including doripenem .
Function of Oxygen Resistance Proteins in the Anaerobic, Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough. Marjorie Fournier, 2003.Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H2O2) reductase, were constructed . Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase . The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain . No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H2O2 resistance may also be conferred by two other rbr genes in the D . vulgaris genome . Inhibition of Sod activity by azide and H2O2, but not by cyanide, indicated it to be an iron-containing Sod . The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE) . A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D . vulgaris . Thus, Sor plays a key role in oxygen defense of D . vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm . Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets . Characterization of a Nucleotide-Binding Domain Associated with Neisserial Iron Transport. Gloria H. Y. Lau, 2004.The fbpABC operon in Neisseria gonorrhoeae encodes an ATP-binding cassette transporter required for iron uptake from the host ferric binding proteins . The gene for the nucleotide-binding domain (fbpC) expressed in Escherichia coli has intrinsic ATPase activity (0.5 mmol/min/mg) uncoupled from the iron transport process . The FbpC E164D mutant is found to have a 10-fold reduction in specific activity . FbpC is covalently modified by 8-azido-[  32P]ATP, indicating that FbpC is a functional ATPase that likely combines with FbpB to form a ferric iron transporter .
A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library. Mark R. Liles, 2003.We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil . A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes . The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil . The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus,  -Proteobacteria, and CFB groups; greater representation among the ß- and
 -Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea . In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives . The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation . This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division .
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