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Antisense RNA Regulation by Stable Complex Formation in the Enterococcus faecalis Plasmid pAD1 par Addiction System. Keith E. Weaver, 2004.The par stability determinant, encoded by the Enterococcus faecalis plasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria . Because of the unique organization of the par locus, the par antisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity . The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex . The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost . After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I . The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required . Predictability of Vibrio cholerae in Chesapeake Bay. Valérie R. Louis, 2003.Vibrio cholerae is autochthonous to natural waters and can pose a health risk when it is consumed via untreated water or contaminated shellfish . The correlation between the occurrence of V . cholerae in Chesapeake Bay and environmental factors was investigated over a 3-year period . Water and plankton samples were collected monthly from five shore sampling sites in northern Chesapeake Bay (January 1998 to February 2000) and from research cruise stations on a north-south transect (summers of 1999 and 2000) . Enrichment was used to detect culturable V . cholerae, and 21.1% (n = 427) of the samples were positive . As determined by serology tests, the isolates, did not belong to serogroup O1 or O139 associated with cholera epidemics . A direct fluorescent-antibody assay was used to detect V . cholerae O1, and 23.8% (n = 412) of the samples were positive . V . cholerae was more frequently detected during the warmer months and in northern Chesapeake Bay, where the salinity is lower . Statistical models successfully predicted the presence of V . cholerae as a function of water temperature and salinity . Temperatures above 19°C and salinities between 2 and 14 ppt yielded at least a fourfold increase in the number of detectable V . cholerae . The results suggest that salinity variation in Chesapeake Bay or other parameters associated with Susquehanna River inflow contribute to the variability in the occurrence of V . cholerae and that salinity is a useful indicator . Under scenarios of global climate change, increased climate variability, accompanied by higher stream flow rates and warmer temperatures, could favor conditions that increase the occurrence of V . cholerae in Chesapeake Bay . Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated  54-Dependent Promoter in Acinetobacter sp . Strain ADP1.Rheinallt M. Jones, 2003.The areR gene in Acinetobacter sp . strain ADP1 regulates the expression of the areCBA genes, which determine growth on benzyl alkanoates . AreR is a member of the NtrC/XylR family of regulatory proteins as determined by sequence homology . Seventy-nine bases upstream of the start of transcription is a region carrying two overlapping inverted repeat (IR) sequences that we predict to be the AreR binding site, also known as the upstream activator site (UAS) . IR1 is a near-perfect (16 of 17 bp) repeat separated by 1 bp, and IR2 consists of 9- and 7-bp perfect repeats with a 3-bp gap, with the central bases of the two arms of the repeat separated by 44 and 22 bp . We report here a method for site-directed mutagenesis of chromosomal genes in ADP1 in which linear fragments generated by overlap extension PCR are used to transform ADP1 via its natural transformation system and recombinants are selected by a marker exchange-eviction strategy with a newly created sacB-Km cassette . This method was used to generate 38 strains with designed mutations in the putative UAS upstream of areCBA. The effects of the mutations on areCBA expression were measured by enzyme assays of benzyl alcohol dehydrogenase (AreB) and by reporter gene assays of lacZ inserted into areA . Substitutions or deletions in IR1 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of IR2, than when they were in its outer regions . By contrast, substitutions in the right arm of IR2 resulted in mutants with relatively high expression levels compared to that of the wild type . Effects of deletions in the right arm of IR2 were very dependent upon the length of the deletion, with 3- or 5-bp deletions reducing expression by >90% whereas an 11-bp deletion in the same area reduced the expression levels by only 50%, suggesting that alterations in the distance and the orientation of the UAS relative to the -24, -12 54 promoter are critical .
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