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RtsA Coordinately Regulates DsbA and the Salmonella Pathogenicity Island 1 Type III Secretion System. Craig D. Ellermeier, 2004.Salmonella serovars cause a wide variety of diseases ranging from mild gastroenteritis to life-threatening systemic infections . An important step in Salmonella enterica serovar Typhimurium infection is the invasion of nonphagocytic epithelial cells, mediated by a type III secretion system (TTSS) encoded on Salmonella pathogenicity island 1 (SPI1) . The SPI1 TTSS forms a needle complex through which effector proteins are injected into the cytosol of host cells, where they promote actin rearrangement and engulfment of the bacteria . We previously identified the Salmonella-specific regulatory protein RtsA, which induces expression of hilA and, thus, the SPI1 genes . Here we show that the hilA regulators RtsA, HilD, and HilC can each induce transcription of dsbA, which encodes a periplasmic disulfide bond isomerase . RtsA induces expression of dsbA independent of either the SPI1 TTSS or the only known regulator of dsbA, the CpxRA two-component system . We show that DsbA is required for both the SPI1 and SPI2 TTSS to translocate effector proteins into the cytosol of host cells . DsbA is also required for survival during the systemic stages of infection . We also present evidence that production of SPI1 effector proteins is coupled to assembly of the TTSS . This feedback regulation is mediated at either the transcriptional or posttranscriptional level, depending on the particular effector . Loss of DsbA leads to feedback inhibition, which is consistent with the hypothesis that disulfide bond formation plays a role in TTSS assembly or function . Salmonella Genomic Island 1 Multidrug Resistance Gene Clusters in Salmonella enterica Serovar Agona Isolated in Belgium in 1992 to 2002. Benoît Doublet, 2004.Salmonella genomic island 1 (SGI1) harbors a multidrug resistance (MDR) gene cluster which is a complex class 1 integron . Variant SGI1 MDR gene clusters conferring different MDR profiles have also been identified in several Salmonella enterica serovars and classified as SGI1-A to -F . A retrospective study was undertaken to characterize MDR regions from serovar Agona strains harboring SGI1 isolated from poultry in Belgium between 1992 and 2002 . A total of 171 serovar Agona strains, displaying resistance to at least one antibiotic, were studied for the presence of SGI1 . SGI1 was detected in 94 serovar Agona strains . The most prevalent variant was SGI1-A (85%), which harbors within the SGI1 complex class 1 integron a common region (CR1) containing orf513, a putative transposase gene, adjacent to the dfrA10 trimethoprim resistance gene . A new variant SGI1 named SGI1-G was identified in two strains . It consisted of the pse-1 gene cassette, as in SGI1-B, but with additional insertion of the orf513/dfrA10 region structure . Seven strains displaying the typical SGI1 MDR profile (Ap Cm Ff Sm Sp Su Tc) showed genetic variation at the 3' end of SGI1 . These strains harbored the insertion of the CR1 containing orf513 as in SGI1-A, -D, and -G . However, downstream the right end of CR1, they presented different 7.4- to 8.5-kb deletions of the SGI1 3' end that extended to the chromosomal genes yieE and yieF . These results suggest a possible role of CR1 in deletion formation, as has been reported for some insertion sequences . Pulsed-field gel electrophoresis analysis showed that all the serovar Agona SGI1-carrying strains belonged to a single clone . Thus, SGI1 is largely encountered in serovar Agona strains isolated from poultry in Belgium, the most prevalent variant being SGI1-A . SGI1 MDR region undergoes recombinational events resulting in a diversity of MDR gene clusters . Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126. Gerard M. Gibbs, 2004.Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes . The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography . Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography . Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126 . This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E . coli culture . The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane . Cloning and Characterization of Archaeal Type I Signal Peptidase from Methanococcus voltae. Sandy Y. M. Ng, 2003.Archaeal protein trafficking is a poorly characterized process . While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity . In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay . A truncated, His-tagged form of the M . voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity . With M . voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M . Processing of the M . voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product . Although E . coli signal peptidase was able to correctly process the signal peptide during overexpression of the M . voltae S-layer protein in vivo, the contribution of the E . coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E . coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays . In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E . coli signal peptidase I), efficient processing of the substrate was observed only when the E . coli membranes contained overexpressed M . voltae signal peptidase . This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product . Repression of Escherichia coli PhoP-PhoQ Signaling by Acetate Reveals a Regulatory Role for Acetyl Coenzyme A. Joseph A. Lesley, 2003.The PhoP-PhoQ two-component system regulates the transcription of numerous genes in response to changes in extracellular divalent cation concentration and pH . Here we demonstrate that the Escherichia coli PhoP-PhoQ two-component system also responds to acetate . Signaling by the E . coli PhoP-PhoQ system was repressed during growth in acetate (  25 mM) in a PhoQ-dependent manner . The periplasmic sensor domain of PhoQ was not required for acetate to repress signaling . Acetate-mediated repression of the PhoP-PhoQ system was not related to changes in the intracellular concentration of acetate metabolites such as acetyl-phosphate or acetyladenylate . Genetic analysis of acetate metabolism pathways suggested that a perturbation of acetyl coenzyme A turnover was the cause of decreased PhoP-PhoQ signaling during growth in acetate . Consistent with this hypothesis, intracellular acetyl coenzyme A levels rose during growth in the presence of exogenous acetate . Acetyl coenzyme A inhibited the autokinase activity of PhoQ in vitro, suggesting that the in vivo repressing effect may be due to a direct inhibition mechanism .
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