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IMP-1 and a Novel Metallo-ß-Lactamase, VIM-6, in Fluorescent Pseudomonads Isolated in Singapore. Tse Hsien Koh, 2004.Four carbapenem-resistant Pseudomonas spp . were isolated from patients in Singapore . One Pseudomonas putida isolate contained a blaIMP-1 identical to that first described in Japan . The sequence of a variant blaIMP-1 in Pseudomonas fluorescens contained four silent mutations compared with the original sequence . The remaining P . putida isolates contained blaVIM-6, a novel VIM gene variant . Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida. M. Manzanera , 2004.Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials . Bacteria are recovered by rehydration after physical disruption of the plastic . P . putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination . Regulation of the nfsA Gene in Escherichia coli by SoxS. E. Suzanne Paterson, 2002.In Escherichia coli, the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by the soxRS regulon . One member of the soxRS regulon, nfsA, encodes the major oxygen-insensitive nitroreductase in Escherichia coli which catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH . In this study we investigate the regulation of nfsA in response to the superoxide generating compound paraquat . The transcription start site (TSS) of nfsA was located upstream of the ybjC gene, a small open reading frame of unknown function located directly upstream of nfsA, suggesting that these two genes form an operon . The activity of the promoter associated with this TSS was confirmed with lacZ fusions and was shown to be inducible by paraquat . Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the -35 promoter sequence in the forward orientation, suggesting that the ybjC-nfsA promoter is a class I SoxS-dependent promoter . Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG. Takako Murakami, 2002.The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene . Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans . A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer . On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins . We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation . In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B . subtilis translocase homologues play an important role in maintaining the viability of the cell . This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth . spoIIIJ mutations have already been established to block sporulation at stage III . In contrast, disruption of yqjG did not interfere with sporulation . We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level . Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells . This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation . Different Evolutionary Constraints on Chemotaxis Proteins CheW and CheY Revealed by Heterologous Expression Studies and Protein Sequence Analysis. Gladys Alexandre, 2003.CheW and CheY are single-domain proteins from a signal transduction pathway that transmits information from transmembrane receptors to flagellar motors in bacterial chemotaxis . In various bacterial and archaeal species, the cheW and cheY genes are usually encoded within homologous chemotaxis operons . We examined evolutionary changes in these two proteins from distantly related proteobacterial species, Escherichia coli and Azospirillum brasilense . We analyzed the functions of divergent CheW and CheY proteins from A . brasilense by heterologous expression in E . coli wild-type and mutant strains . Both proteins were able to specifically inhibit chemotaxis of a wild-type E . coli strain; however, only CheW from A . brasilense was able to restore signal transduction in a corresponding mutant of E . coli . Detailed protein sequence analysis of CheW and CheY homologs from the two species revealed substantial differences in the types of amino acid substitutions in the two proteins . Multiple, but conservative, substitutions were found in CheW homologs . No severe mismatches were found between the CheW homologs in positions that are known to be structurally or functionally important . Substitutions in CheY homologs were found to be less conservative and occurred in positions that are critical for interactions with other components of the signal transduction pathway . Our findings suggest that proteins from the same cellular pathway encoded by genes from the same operon have different evolutionary constraints on their structures that reflect differences in their functions . Genome Organization and Molecular Analysis of the Temperate Bacteriophage MM1 of Streptococcus pneumoniae. Virginia Obregón, 2003.The genome of MM1 (40,248 bp), a temperate bacteriophage from the Spain23F-1 multiresistant epidemic clone of Streptococcus pneumoniae, is organized in 53 open reading frames (ORFs) and in at least five functional clusters . Bioinformatic and N-terminal amino acid sequence analyses enabled the assignment of possible functions to 26 ORFs . Analyses comparing the MM1 genome with those of other bacteriophages revealed similarities, mainly with genomes of phages infecting gram-positive bacteria, which suggest recent exchange of genes between species colonizing the same habitat . Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry. C. Sieiro, 2003.The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe . The optimum pH and temperature for the recombinant enzyme were 5 and 40°C, respectively, these being around 0.5 U higher and 5°C lower than those shown by the native enzyme . The Km value was about fourfold higher than that of the S . cerevisiae enzyme . The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme .
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