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Panel of Bacillus subtilis Reporter Strains Indicative of Various Modes of Action. Bernd Hutter, 2004.In a recent project, we collected the transcriptional profiles of Bacillus subtilis 168 after treatment with a large set of diverse antibacterial agents . One result of the data analysis was the identification of marker genes that are indicative of certain compounds or compound classes . We cloned these promoter regions in front of a luciferase reporter gene and reintroduced the constructs individually into the B . subtilis chromosome . Strains were analyzed for their responsiveness after treatment with a set of 37 antibacterials . Twelve functional reporter strains were generated that were selectively and significantly upregulated by the compounds . The selectivity of the reporter strains ranged from generic pathways like protein biosynthesis, cell wall biosynthesis, and fatty acid biosynthesis to compound classes (quinolones and glycopeptides) and individual compounds (rifampin, cycloserine, and clindamycin) . Five of the strains are amenable for high-throughput applications, e.g., pathway-specific screening . In summary, we successfully generated B . subtilis reporter strains that are indicative of the mechanisms of action of various classes of antibacterials . The set of reporter strains presented herein can be used for mode-of-action analyses and for whole-cell screening of compound libraries in a mode-of-action-specific manner . Dimethylsulfoniopropionate Metabolism by Pfiesteria-Associated Roseobacter spp.. Todd R. Miller, 2004.The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP) . In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP . DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined . Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 µM . Cultures of P . piscicida or Cryptoperidiniopsis sp . that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 µM DMSP (within 30 h), and the rate of catabolism was much higher for P . piscicida cultures than for P . shumwayae cultures . The community of bacteria from P . piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH) . Four DMSP-degrading bacteria were isolated from the P . piscicida cultures and found to be taxonomically related to Roseobacter species . All four isolates produced MMPA from DMSP . Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways . The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp . offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates . The PtlE Protein of Bordetella pertussis Has Peptidoglycanase Activity Required for Ptl-Mediated Pertussis Toxin Secretion. Amy A. Rambow-Larsen, 2002.Pertussis toxin of Bordetella pertussis is secreted by a type IV secretion system comprised of the products of the nine ptl (pertussis toxin liberation) genes . These proteins are believed to form a complex spanning both the inner and outer membranes and passing through the peptidoglycan layer . Peptidoglycan acts as a barrier for transport through the periplasm of large folded molecules . Assembled pertussis toxin and the secretion component proteins PtlC through PtlH are too large to diffuse through intact peptidoglycan . Therefore, we hypothesized that the Ptl system contains a peptidoglycanase activity . The PtlE protein was found to exhibit a sequence match to the active site of glycohydrolase enzymes . An N-terminally polyhistidine-tagged PtlE fusion protein, constructed and expressed in Escherichia coli and in B . pertussis, exhibited peptidoglycanase activity on activity gels . A fusion protein with alanine substitutions at the putative active site residues (aspartic acid at position 53 and glutamic acid at position 62) lacked peptidoglycanase activity . B . pertussis strains with the amino acid substitutions were deficient for pertussis toxin secretion . Based on these results, we concluded that PtlE is a peptidoglycanase responsible for the local removal or rearrangement of the peptidoglycan layer during Ptl secretion complex assembly . Bacteriophages of Erwinia amylovora. J. J. Gill, 2003.Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario . Forty-two phages survived the isolation, purification, and storage processes . The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms . Only five phages were isolated from infected aerial tissue in pear and apple orchards . To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes . Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered . Ten phage isolates were related to the previously characterized E . amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites . A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E . amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans . Representatives from the six molecular groups were studied by electron microscopy to determine their morphology . The phages exhibited distinct morphologies when examined by an electron microscope . Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages . Based on morphotypes, the bacteriophages of E . amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae . Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources. Sylvie Seurinck, 2003.Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources . Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows . We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E . coli isolates as tools to identify nonpoint fecal sources . The BOXA1R primer was used for rep-PCR analysis . A total of 267 E . coli isolates from different fecal sources were typed with both techniques . E . coli was found to be highly diverse . Only two candidate source-specific E . coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints . Similarly, there was only one candidate source-specific E . coli fingerprint for horse out of 59 BOX fingerprints . Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study . When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained . Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed . Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E . coli population of that source . The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days . In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E . coli isolates have the potential to identify nonpoint fecal sources . A fairly small number of isolates was needed to find candidate source-specific E . coli fingerprints that were stable under the simulated environmental conditions .
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