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Cloning Serratia entomophila Antifeeding Genes—a Putative Defective Prophage Active against the Grass Grub Costelytra zealandica. Mark R. H. Hurst, 2004.Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand . Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death . A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms . Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP . Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae . Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp . laumondii TTO1, a species which also has sep gene homologues . Noninvasive Optical Imaging Method To Evaluate Postantibiotic Effects on Biofilm Infection In Vivo. Jagath L. Kadurugamuwa, 2004.Eradication of Staphylococcus aureus biofilms after rifampin treatment was tested in a mouse model of device-related infection by using biophotonic imaging . Following treatment, the bioluminescent signals decreased to undetectable levels, irrespective of the age of the biofilm . After the final treatment, the signals rebounded in a time-dependent manner and reached those for the untreated mice . Readministration of rifampin was unsuccessful in eradicating reestablished infections, with the rifampin MICs for such bacteria being increased and with the bacteria having point mutations in the rpoB gene . Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli. Debra W. Jackson, 2002.The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms . Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites . While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown . Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions . Ectopic expression of the E . coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens . A csrA knockout mutation enhanced biofilm formation in E . coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation . In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant . Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism . Finally, the expression of a chromosomally encoded csrA'-'lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor . We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E . coli biofilm development . Molecular Organization of Intrinsic Restriction and Modification Genes BsuM of Bacillus subtilis Marburg. Hideyuki Ohshima, 2002.Transcriptional analysis and disruption of five open reading frames (ORFs), ydiO, ydiP, ydiR, ydiS, and ydjA, in the prophage 3 region of the chromosome of Bacillus subtilis Marburg revealed that they are component genes of the intrinsic BsuM restriction and modification system of this organism . The classical mutant strain RM125, which lacks the restriction and modification system of B . subtilis Marburg, lacks the prophage 3 region carrying these five ORFs . These ORFs constitute two operons, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon, both of which are expressed during the logarithmic phase of growth . The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases . The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known . Disruption of the ydiR-ydiS-ydjA operon resulted in enhanced transformation by plasmid DNA carrying multiple BsuM target sequences . Disruption of ydiO or ydiP function requires disruption of at least one of the following genes on the chromosome: ydiR, ydiS, and ydjA . The degrees of methylation of the BsuM target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with XhoI (an isoschizomer of BsuM) of DNAs extracted from the disruptant strains . Six XhoI (BsuM) sites were examined . XhoI digested at the XhoI sites in the DNAs from disruptants with disruptions in both operons, while XhoI did not digest at the XhoI sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the ydiR-ydiS-ydjA operon . Therefore, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon are considered operons that are responsible for BsuM modification and BsuM restriction, respectively . Evaluation of a Rapid, Quantitative Real-Time PCR Method for Enumeration of Pathogenic Candida Cells in Water. Nichole E. Brinkman, 2003.Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C . albicans, C . tropicalis, C . krusei, C . parapsilosis, C . glabrata and C . lusitaniae) in water . Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis . The assay's sensitivities were between one and three cells per filter . The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level) . In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy . Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species . Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms . With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution .
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