We found that in addition to activating tcpP, AphB was required for full expression of ToxR in V. cholerae stationary growth phase. AphB regulated toxR directly as purified recombinant AphB binds to the toxR promoter. This study HSP inhibitor suggests that V. cholerae may use this additional layer of activation to turn on virulence factor production efficiently in optimal conditions. Results and Discussion Examination of toxR expression under different in vitro conditions using a transcriptional fusion reporter ToxR is one of two proteins, along with TcpP, shown to activate the expression of ToxT,

the master virulence activator in V. cholerae (Fig. 1). The expression of tcpP has been shown to be induced by AphA and AphB [11, 19], while toxR has been thought to be constitutively expressed and only modulated by temperature [16, 18]. To measure toxR expression, we placed the toxR promoter upstream of the luxCDABE operon on a plasmid [20] and transformed into wild type V. cholerae. We then grew the resulting cells at 37°C or 22°C. Expression of P toxR -luxCDABE was significantly increased at 22°C (Fig. 2A), consistent with the previous report [18] that the expression of toxR

is modulated by temperatures. Since the availability of 3-Methyladenine price oxygen concentrations is different during V. cholerae infection, we also examined the expression of toxR under varying oxygen concentrations (Fig. 2B). The lux expression was similar under each condition, suggesting that oxygen levels do not regulate toxR expression. Figure 2

The expression of toxR in wild type under selleck different conditions using a P toxR -luxCDABE transcriptional reporter. (A). The reporter strain was grown at 22°C or 37°C, and at successive time points, luminescence was measured. Units are arbitrary light units/OD600. The results are the average of three experiments ± SD. (B). The reporter strain was grown at 37°C aerobically, in an anaerobic chamber (Mini MACS Anaerobic workstation, Microbiology International) or in a BBL CampyPak Microaerophilic Myosin System. At different time points, samples were withdrawn and luminescence was measured. Units are arbitrary light units/OD600. The results are the average of three experiments ± SD. Influence of virulence regulatory proteins on toxR expression To investigate molecular influences on toxR expression, we introduced the P toxR -lux construct into various strains of V. cholerae with mutations in virulence regulator genes. We also included a tcpA mutant because a previous study showed that TcpA, the major subunit of TCP pilin [2], affects cholera toxin gene expression in vivo but not in vitro [21]. We grew these strains at 37°C for 12 hours and measured luminescence (Fig. 3A). We found that ToxR and ToxS did not affect toxR expression, indicating that ToxR does not autoregulate.