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Construction and Characterization of a cheA Mutant of Treponema denticola. Renate Lux, 2002.The Treponema denticola cheA gene, encoding the central kinase of the general chemotaxis pathway, was analyzed for its role in chemotaxis and tissue penetration . The cheA gene was interrupted by insertion of an ermF-ermAM gene cassette . Reverse transcription-PCR confirmed that the other downstream chemotaxis genes within the same operon (cheW, cheX, and cheY) were still expressed in the cheA mutant strain . Lack of cheA resulted in decreased swarming on soft-agar swarm plates and failure to respond chemotactically to a mixture of nutrients . Behavioral analyses using video microscopy revealed that the cheA mutant exhibited coordinated cell movement . The cellular reversal frequency, however, was severely reduced, indicating that CheA in T . denticola mainly controls cellular reversal and that active chemotaxis signaling input is not required for coordination of flagellar rotation at both cell poles . Inducible Control of Virulence Gene Expression in Listeria monocytogenes: Temporal Requirement of Listeriolysin O during Intracellular Infection. Christina E. Dancz, 2002.We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes . An L . monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-ß-D-thiogalactopyranoside (IPTG)-dependent promoter . Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L . monocytogenes-containing phagosomes . Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture . Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG . We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth . Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line . Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent . The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed . Rambling and Scrambling in Bacterial Transformation— a Historical and Personal Memoir. Sanford A. Lacks, 2003. OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene. Marcos Fernández-Mora, 2004.The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily . It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S . enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins . A region of 316 bp, between nucleotides –413 and –97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S . enterica serovar Typhi wild-type strain . This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon . Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site . Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression . Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR . Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5' upstream regulatory region . Thus, the OmpR and LeuO regulators positively regulate ompS2 .
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