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Multiple Regulators Control Capsular Polysaccharide Production in Vibrio parahaemolyticus. Zehra Tüzün Güvener, 2003.Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies . Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR . CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein . The present study identifies cps genes and examines their regulation . Transposon insertions in the cps locus, which contains 11 genes, abolished opacity . Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay . Reporter fusions to cpsA, the first gene in the locus, showed  10-fold-enhanced transcription in the OP (opaR+) strain compared to a TR ( opaR) strain . Two additional transcriptional regulators were discovered . One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA
opaR strains . CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression . CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor . Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in TR strains and primarily OpaR dependent in OP strains . Thus, a network of positive and negative regulators modulates CPS production in V . parahaemolyticus .
Hemin Binding, Functional Expression, and Complementation Analysis of Pap 31 from Bartonella henselae. Rainer Zimmermann, 2003.Growth of Bartonella henselae is strongly heme dependent, and B . henselae is unable to synthesize heme itself . At least five outer membrane-associated proteins from B . henselae bind hemin, including the 31-kDa protein designated Pap31 . The gene of this protein was heterologously expressed in Escherichia coli M15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction . Complementation of the hemA-deficient mutant E . coli K-12 EB53 (aroB tsx malT hemA) with pap31 demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis of B . henselae . Identification of Cryptosporidium spp . Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay. R. A. B. Nichols, 2003.We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp . oocysts in natural mineral waters and drinking waters . Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA . The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion . Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels . DNA extracted from Cryptosporidium parvum oocysts, C . muris (RN 66), C . baileyi (Belgium strain, LB 19), human-derived C . meleagridis, C . felis (DNA from oocysts isolated from a cat), and C . andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI . Discrimination between C . andersoni and C . muris isolates was confirmed by a separate, subsequent digestion with DdeI . Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR . The Cryptosporidium species detected in these finished water samples was C . parvum genotype 1 . This method consistently and routinely detected >5 oocysts per sample .
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