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Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and Helix 3. Yinghua Chen, 2004.Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independentof its phosphorylation state, phosphorylation of PhoP may optimizeDNA binding or the interaction with RNA polymerase . We describealanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3region of PhoPC [Val190 to E214] and functional analysis of the mutated proteins . Eight residues important for DNA binding were clustered between Val202 and Arg210 . Using in vivo andin vitro functional analyses, we identified three classes ofmutated proteins . Class I proteins [PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A] were phosphorylation proficient and could dimerizebut could not bind DNA or activate transcription in vivo or in vitro . Class II proteins [PhoPH205A and PhoPV204A] were phosphorylationproficient and could dimerize but could not bind DNA prior tophosphorylation . Members of this class had higher transcriptionactivation in vitro than in vivo . The class III mutants, PhoPV202Aand PhoPD203A, had a reduced rate of phosphotransfer and coulddimerize but could not bind DNA or activate transcription invivo or in vitro . Seven alanine substitutions in PhoP [PhoPV190A,PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A]that specifically affected transcription activation were broadlydistributed throughout the transactivation loop extending fromVal190 to as far toward the C terminus as Arg200 . PhoPW191Aand PhoPR200A could not activate transcription, while the otherfive mutant proteins showed decreased transcription activationin vivo or in vitro or both . The mutagenesis studies may indicatethat PhoP has a long transactivation loop and a short alpha helix3, more similar to OmpR than to PhoB of Escherichia coli. Delineation of Upstream Signaling Events in the Salmonella Pathogenicity Island 2 Transcriptional Activation Pathway. Charles C. Kim, 2004.Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host . Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2) . SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells . A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors . While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium . In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P) . Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ . In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P . As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P . Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo . Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus. Michelle D. Glew, 2002.The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas . Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis . In this study, six vpma genes were characterized in the M . agalactiae type strain PG2 . All vpma genes clustered within an 8-kb region and shared highly conserved 5' untranslated regions, lipoprotein signal sequences, and short N-terminal sequences . Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5' untranslated region of all vpma genes . This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus . An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the  integrase family of site-specific xer recombinases . It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M . agalactiae .
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