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Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6. Agneta Backman, 2004.Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C . In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil . A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil . In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system ("viable cells") whereas propidium iodide (PI) was used to stain cells with damaged membranes ("dead cells") . Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves . When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity . In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions . When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active . A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments . These results make A . chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates . Characterization of an ADP-Ribosyltransferase Toxin (AexT) from Aeromonas salmonicida subsp . salmonicida. Martin Braun, 2002.An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A . salmonicida subsp . salmonicida, the etiological agent of furunculosis in fish, was characterized . Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis . AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species . The aexT gene was detected in all of the 12 A . salmonicida subsp . salmonicida strains tested but was absent from all other Aeromonas species . Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity . Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A . salmonicida subsp . salmonicida and cross-reacted with ExoS and ExoT of P . aeruginosa . AexT toxin could be detected in a wild type (wt) strain of A . salmonicida subsp . salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells . Under these conditions, the AexT protein was found to be intracellular or tightly cell associated . No AexT was found when A . salmonicida subsp . salmonicida was incubated in cell culture medium in the absence of RTG-2 cells . Upon infection with wt A . salmonicida subsp . salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes . These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation . An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production . Hence AexT is directly involved in the toxicity of A . salmonicida subsp . salmonicida for RTG-2 fish cells . Transcriptional Regulation in the Streptococcus pneumoniae rlrA Pathogenicity Islet by RlrA. David L. Hava, 2003.The proper temporal expression of virulence genes during infection is crucial to the infectious life cycle of microbial pathogens, particularly in pathogens that encounter a multitude of environments in eukaryotic hosts . Streptococcus pneumoniae normally colonizes the nasopharynges of healthy adults but can cause a range of diseases at a variety of host sites . Transcriptional regulators that are essential for full virulence of S . pneumoniae in different animal models have been identified . One such regulator, rlrA, is required for colonization of the nasopharynx and lung infection but is dispensable for systemic infection . Previous work has shown that rlrA lies in a 12-kb pathogenicity islet, divergently opposed to three putative sortase-anchored surface proteins and three sortase enzymes . In addition to rlrA, one of the putative surface proteins and one of the sortases have also been shown to be essential for lung infection . In this work, we demonstrate that RlrA is a positive regulator of all seven genes in the rlrA pathogenicity islet, with transcriptional activation occurring at four different promoters in the islet with AT-rich sequences . These promoters direct the expression of rlrA itself, the three sortases, rrgA, and rrgBC . These data are consistent with the model whereby the rlrA pathogenicity islet acts in an autonomous manner to alter the bacterial surface components that interact with the pulmonary and nasopharyngeal environments . Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease. Mary M. Brinig, 2003.Members of the uncultivated bacterial division TM7 have been detected in the human mouth, but little information is available regarding their prevalence and diversity at this site . Human subgingival plaque samples from healthy sites and sites exhibiting various stages of periodontal disease were analyzed for the presence of TM7 bacteria . TM7 ribosomal DNA (rDNA) was found in 96% of the samples, and it accounted for approximately 0.3%, on average, of all bacterial rDNA in the samples as determined by real-time quantitative PCR . Two new phylotypes of this division were identified, and members of the division were found to exhibit filamentous morphology by fluorescence in situ hybridization . The abundance of TM7 rDNA relative to total bacterial rDNA was higher in sites with mild periodontitis (0.54% ± 0.1%) than in either healthy sites (0.21% ± 0.05%, P < 0.01) or sites with severe periodontitis (0.29% ± 0.06%, P < 0.05) . One division subgroup, the I025 phylotype, was detected in 1 of 18 healthy samples and 38 of 58 disease samples . These data suggest that this phylotype, and the TM7 bacterial division in general, may play a role in the multifactorial process leading to periodontitis .
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