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Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli. Christelle Richard, 2004.Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41 . To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain . The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form . The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 µg per 20 ml of culture) . This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E . coli cytoplasm . YmoA Negatively Regulates Expression of Invasin from Yersinia enterocolitica. Damon W. Ellison, 2003.inv encodes invasin, which is the primary invasion factor of Yersinia enterocolitica . inv expression in vitro is regulated in response to temperature, pH, and growth phase . In vitro, inv is maximally expressed at 26°C and repressed at 37°C at neutral pH but, when the pH of the media is adjusted to 5.5, levels of inv expression at 37°C are comparable to those at 26°C . A previous genetic screen for regulators of inv identified RovA, which was found to be required for activation of inv in vitro under all conditions tested as well as in vivo. Here we describe a screen that has identified a negative regulator of inv expression, ymoA . The ymoBA locus was identified by transposon mutagenesis as a repressor of inv expression in vitro at 37°C at neutral pH . This mutant shows increased inv expression at 37°C . The mutant can be fully complemented for inv expression by a plasmid expressing ymoA . These results indicate that YmoA plays a role in the negative regulation of inv . Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes. Ameur Cherif, 2003.Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the Bacillus cereus group showed that B . anthracis Davis TE702 and B . mycoides G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the B . cereus group . Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced . Two main types of ITS were found, either with two tRNA genes (tRNAIle and tRNAAla) or without any at all . Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNAIle and tRNAAla genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS . Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences . Neighbor-joining analysis of tDNA-containing long ITS indicated that B . cereus and B . thuringiensis represent a single clade . Three signature sequences discriminated B . anthracis from B . cereus and B . thuringiensis, indicating that the anthrax agent started evolving separately from the related clades of the B . cereus group . B . mycoides and B . weienstephanensis were very closely related, while B . pseudomycoides appeared the most distant species . Role of Ectoine in Vibrio cholerae Osmoadaptation. Kathryn J. Pflughoeft, 2003.Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community . To persist in the estuarine environment, V . cholerae must adjust to changes in ionic composition and osmolarity . These changes in the aquatic environment have been correlated with cholera epidemics . In this work, we study the response of V . cholerae to increases in environmental osmolarity . Optimal growth of V . cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl . However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed . During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth . We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay . We also demonstrate that high-osmolarity-adapted V . cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium . In contrast, low-osmolarity-adapted V . cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium . These results may have implications for V . cholerae population dynamics when seawater and freshwater and their attendant microbes mix .
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