Growth phase-dependent gene regulation has recently been demonstrated to occur in

Growth phase-dependent gene regulation has recently been demonstrated to occur in is thought to have derived from a did not decrease in these later phases of growth. fimbriae, adenylate cyclase-hemolysin toxin, and dermonecrotic toxin (DNT), as well as a type III secretion system (TTSS) (5). Conversely, buy LSD1-C76 BvgAS is usually inactive during the Bvg? phase, resulting in the maximal expression of motility loci, virulence-repressed genes (genes), and genes required for the production of urease (2, 3, 26). Previous studies involving phase-locked and ectopic expression mutants demonstrated that the Bvg+ phase promotes respiratory tract colonization by and (1, 6, 7, 23, 27), while the Bvg? phase of promotes survival under conditions of nutrient deprivation (6, 7). Despite the close genetic relatedness and sharing a key pathogenic mechanism involving the BvgAS regulatory system, and differ in some interesting yet fundamental attributes of bacterial pathogens such as host range, pathologies, and persistence. Recently, growth phase-dependent gene expression changes have been reported to occur in is thought to have derived from a would occur in a manner analogous to that in and influence virulence factor expression and virulence-associated phenotypes. Additionally, the buy LSD1-C76 data arising from this comparative analysis may enhance our understanding of the molecular basis for the differences between these species. Using microarray analysis and quantitative real-time PCR (qRT-PCR), we demonstrate that growth phase-dependent gene regulation occurs in and results in large shifts in global gene expression during growth. Growth phase-dependent changes in two virulence phenotypes associated with these gene expression changes were tested. We found that the growth-dependent increase in expression of some TTSS genes led to a growth-dependent increase in a TTSS-dependent function, cytotoxicity. Additionally, while genes encoding adhesins previously shown to mediate adherence were decreased in late log and stationary phases, in contrast to (28), buy LSD1-C76 we found that adherence did not decrease in these later phases of growth. It has been previously shown that a Bvg+ phase-locked mutant failed to exhibit growth-dependent gene regulation, indicating that a BvgAS system capable of modulating is required for growth-dependent gene regulation (28). Thus, to broadly evaluate the role of the BvgAS regulatory system in growth phase-dependent gene regulation, the transcriptional profiles of both Bvg+ and Bvg? phase-locked mutants were assessed during growth. Microarray analysis revealed and qRT-PCR confirmed growth phase-dependent global shifts in gene expression occurring in both phase-locked mutants. Therefore, in contrast to can function independently from the BvgAS regulatory system. MATERIALS AND METHODS Bacterial strains and growth conditions. strains RB50, RB53 (a Bvgphase-locked derivative of RB50), RB54 (a Bvg? phase-locked derivative of RB50), and RB50(an isogenic mutant lacking the putative ATPase required for the function of the TTSS apparatus) have been previously described (6, 43). strains RB63 (strains were cultured on Bordet-Gengou (BG) agar (Difco, Sparks, MD) containing 10% defibrinated buy LSD1-C76 sheep’s blood for determination of colony morphology and hemolytic activity or in Stainer-Scholte (SS) broth (39) supplemented with 40 g/ml streptomycin. Beta-hemolysis on BG agar was verified following growth in buy LSD1-C76 liquid cultures to ensure that bacteria remained Bvgand were not spontaneous Bvg? mutants. For all those growth phase time course experiments, a single colony was inoculated in SS broth supplemented with 40 g/ml streptomycin at 37C with shaking. To ensure similar inocula, bacteria were then subcultured at a starting optical density at 600 nm (OD600) of 0.02 into a 250-ml flask containing 50 ml SS broth and grown at 37C with shaking at 275 rpm. This was repeated twice, resulting in three biological replicates for each strain. Measurement of growth by optical Rabbit Polyclonal to VEGFR1 density, colony counts, and DNA concentration. Growth was monitored by removing culture samples at 6-h intervals and measuring the OD600 and determining the number of CFU of per ml of culture by plating dilutions on BG plates containing 40 g/ml streptomycin. Additionally, growth was monitored by measuring the DNA concentration by absolute quantification of genomic DNA (gDNA). Absolute quantification of gDNA. RB50 gDNA was purified using the High Pure PCR template preparation kit (Roche Applied Science, Indianapolis, IN). A 53-bp qPCR amplicon of the 16S rRNA gene was amplified using the 16S rRNA forward and reverse primers (see data.