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Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing. Xiaoming Zhan, 2004.We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity . The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell . Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain . Following chemical mutagenesis, a total of eight E . coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated . This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC . In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product . Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci . The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37°C in wild-type cells but was two- to threefold longer in some of the stronger mutants . These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression . Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in Escherichia coli. Kouya Shiraishi, 2002.We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation . The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination . On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination . It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation . Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination . Control of SXT Integration and Excision. Vincent Burrus, 2003.The Vibrio cholerae SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics . SXT integrates in a site-specific fashion at prfC and excises from the chromosome to form a circular but nonreplicative extrachromosomal form . Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int . Here we found that Int is necessary and sufficient for SXT integration and that int expression in recipient cells requires the SXT activators SetC and SetD . Although no xis-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision . We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration . Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs . Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors . In the absence of prfC, SXT integrated into several secondary attachment sites but preferentially into the 5' end of pntB . SXT excision and transfer from a donor containing pntB::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility . Prevalence of Pandemic Thermostable Direct Hemolysin-Producing Vibrio parahaemolyticus O3:K6 in Seafood and the Coastal Environment in Japan. Yukiko Hara-Kudo, 2003.Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples . In this study, we detected and enumerated tdh gene-positive V . parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method . The tdh gene was detected in 33 of 329 seafood samples (10.0%) . The number of tdh-positive V . parahaemolyticus ranged from <3 to 93/10 g . The incidence of tdh-positive V . parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V . parahaemolyticus . TDH-producing strains of V . parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster) . TDH-producing strains of V . parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan . Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques . The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment .
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