Conditioned mediums modulate expression of the RcsB-regulon genes in S. typhimurium

Abstract

The Rcs is an unusual phosphorelay system composed of the inner membrane proteins RcsC and RcsD as sensors, and the response regulator RcsB. At the present, the signals that lead to activate the Rcs phosphorelay system remain unknown. Even though, a wide range of conditions have been described as Rcs system activation state. The growth at low temperature or on solid surface; the polymyxin B exposition; the DjlA overproduction, the rcsC11 constitutive mutation; mutations that affect cell envelope like tolB and pmrA, or rcsB overexpression are some examples of this activation states. Previously, we reported that the rcsB gene is transcribed from two promoters: i) PrcsDB located upstream of rcsD, and ii) PrcsB located within the rcsD coding region. The first promoter is induced during the exponential growth phase while the last one it does at lower levels in stationary phase. We also reported that the RcsB overproduction repressed the rcsD expression by directly binding to the PrcsDB promoter. The repression, resulting in a differential rate of rcsD and rcsB genes expression levels, was also observed using rcsC11 mutant or polymyxin B to induce the system. Under these conditions an increased level of RcsB was observed when the bacteria reach the stationary growth phase and the regulator began to be also transcribed from PrcsB. In addition, the PrcsB is physiologically required to maintain the repression on swarming behavior. The subject of the present work was to determine if an Rcs stimulation factor is excreted to the supernatant of tolB and pmrA mutant culture. This finding would allow us to identify the Rcs system signal. Here, the supernatant obtained from the above mutants cultures, as ?conditioned mediums?, was used to determine the reporter expression levels. The cps and flhDC operons were the reporter of the factor presence in the conditioned mediums. We demonstrated that the cps and flhDC operons were modulated under the growth on these mediums. As RcsB regulator is expressed exponential and stationary phase and the above reporters are exponentially controlled, we looking for reporter RcsB-depended gene that are transcribed in stationary phase like bapA gene. Here we report that asr is a new member of the RcsB regulon, which was reported as an stationary phase expressed gene. Additionally, we studied the expression modulation of asr, bap, cps and flhDC using conditioned medium harvested from both different growth phase in order to relate with expression of RcsB regulator. Under this condition we observed a growth phase-dependent expression mediated by RcsB. These results increase the Rcs system knowledge on regulon and ligand identification issues.