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Expression and Regulation of a Silent Operon, hyf, Coding for Hydrogenase 4 Isoenzyme in Escherichia coli. William T. Self, 2004.On the basis of hyf-lacZ fusion studies, the hyf operon of Escherichia coli, noted for encoding the fourth hydrogenase isoenzyme (HYD4), is not expressed at a significant level in a wild-type strain . However, mutant FhlA proteins (constitutive activators of the hyc-encoded hydrogenase 3 isoenzyme) activated hyf-lacZ . HyfR, an FhlA homolog encoded by the hyfR gene present at the end of the hyf operon, also activated transcription of hyf-lacZ but did so only when hyfR was expressed from a heterologous promoter . The HYD4 isoenzyme did not substitute for HYD3 in H2 production . Optimum expression of hyf-lacZ required the presence of cyclic AMP receptor protein-cyclic AMP complex and anaerobic conditions when HyfR was the activator . Molecular and Physiological Analysis of the Role of Osmolyte Transporters BetL, Gbu, and OpuC in Growth of Listeria monocytogenes at Low Temperatures. Henrike H. Wemekamp-Kamphuis, 2004.Listeria monocytogenes is a ubiquitous food-borne pathogen found widely distributed in nature as well as an undesirable contaminant in a variety of fresh and processed foods . This ubiquity can be at least partly explained by the ability of the organism to grow at high osmolarity and reduced temperatures, a consequence of its ability to accumulate osmo- and cryoprotective compounds termed osmolytes . Single and multiple deletions of the known osmolyte transporters BetL, Gbu, and OpuC significantly reduce growth at low temperatures . During growth in brain heart infusion broth at 7°C, Gbu and OpuC had a more pronounced role in cryoprotection than did BetL . However, upon the addition of betaine to defined medium, the hierarchy of transporter importance shifted to Gbu > BetL > OpuC . Upon the addition of carnitine, only OpuC appeared to play a role in cryoprotection . Measurements of the accumulated osmolytes showed that betaine is preferred over carnitine, while in the absence of a functional Gbu, carnitine was accumulated to higher levels than betaine was at 7°C . Transcriptional analysis of the genes encoding BetL, Gbu, and OpuC revealed that each transporter is induced to different degrees upon cold shock of L . monocytogenes LO28 . Additionally, despite being transcriptionally up-regulated upon cold shock, a putative fourth osmolyte transporter, OpuB (identified by bioinformatic analysis and encoded by lmo1421 and lmo1422), showed no significant contribution to listerial chill tolerance . Growth of the quadruple mutant LO28  BCGB (betL
opuC
gbu
opuB) was comparable to the that of the triple mutant LO28BCGsoe (betL
opuC
gbu) at low temperatures . Here, we conclude that betaine and carnitine transport upon low-temperature exposure is mediated via three osmolyte transporters, BetL, Gbu, and OpuC .
Trypsin Mediates Growth Phase-Dependent Transcriptional Regulation of Genes Involved in Biosynthesis of Ruminococcin A, a Lantibiotic Produced by a Ruminococcus gnavus Strain from a Human Intestinal Microbiota. Ana Gomez, 2002.Ruminococcin A (RumA) is a trypsin-dependent lantibiotic produced by Ruminococcus gnavus E1, a gram-positive strict anaerobic strain isolated from a human intestinal microbiota . A 12.8-kb region from R . gnavus E1 chromosome, containing the biosynthetic gene cluster of RumA, has been cloned and sequenced . It consisted of 13 open reading frames, organized in three operons with predicted functions in lantibiotic biosynthesis, signal transduction regulation, and immunity . One unusual feature of the locus is the presence of three almost identical structural genes, all of them encoding the RumA precursor . In order to determine the role of trypsin in RumA production, the transcription of the rum genes has been investigated under inducing and noninducing conditions . Trypsin activity is needed for the growth phase-dependent transcriptional activation of RumA operons . Our results suggest that bacteriocin production by R . gnavus E1 is controlled through a complex signaling mechanism involving the proteolytic processing of a putative extracellular inducer-peptide by trypsin, a specific environmental cue of the digestive ecosystem . Regulation of fur Expression by RpoS and Fur in Vibrio vulnificus. Hyun-Jung Lee, 2003.In a proteomic analysis of rpoS-deficient Vibrio vulnificus versus the wild type, one of the down-regulated proteins in the rpoS mutant strain was identified as a Fur protein, a ferric uptake regulator . The expression of a fur::luxAB fusion was significantly influenced by sigma factor S, the rpoS gene product, and positively regulated by Fur under iron-limited conditions . Infectivity of RNA from Inactivated Poliovirus. Suphachai Nuanualsuwan, 2003.During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA . DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells . After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution . The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity . Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions . The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation . For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different . However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation . The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid .
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