The nucleoid occlusion protein SlmA is a direct transcriptional activator of chitobiose utilization inVibrio cholerae

Abstract

Chitin utilization by the cholera pathogen Vibrio cholerae is required for its persistence and evolution via horizontal gene transfer in the marine environment. Genes involved in the uptake and catabolism of the chitin disaccharide chitobiose are encoded by the chb operon. The orphan sensor kinase ChiS is critical for regulation of this locus, however, the mechanisms downstream of ChiS activation that result in expression of the chb operon are poorly understood. Using an unbiased transposon mutant screen, we uncover that the nucleoid occlusion protein SlmA is a regulator of the chb operon. SlmA has not previously been implicated in gene regulation. Also, SlmA is a member of the TetR family of proteins, which are generally transcriptional repressors. In vitro, we find that SlmA binds directly to the chb operon promoter, and in vivo, we show that this interaction is, surprisingly, required for transcriptional activation of this locus and for chitobiose utilization. Using point mutations that disrupt distinct functions of SlmA, we find that DNA-binding, but not nucleoid occlusion, is critical for transcriptional activation. This study identifies a novel role for SlmA as a transcriptional regulator in V. cholerae in addition to its established role as a cell division licensing factor.AUTHOR SUMMARYThe cholera pathogen Vibrio cholerae is a natural resident of the aquatic environment and causes disease when ingested in the form of contaminated food or drinking water. In the aquatic environment, the shells of marine zooplankton, which are primarily composed of chitin, serve as an important food source for this pathogen. The genes required for the utilization of chitin are tightly regulated in V. cholerae, however, the exact mechanism underlying this regulation is currently unclear. Here, we uncover that a protein involved in regulating cell division is also important for regulating the genes involved in chitin utilization. This is a newly identified property for this cell division protein and the significance of a common regulator for these two disparate activities remains to be understood.

ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program inVibrio cholerae

“…ChiS is required for activation of the chitin utilization program. To study ChiS activity, we focused on the expression of one activated locus in this genetic program, the chb operon (Pchb) (7). This operon is responsible for the uptake and degradation of the disaccharide of chitin, chitobiose.…”

“…ChiS is a hybrid HK and it was presumed to regulate gene expression in conjunction with a cognate RR tentatively named ChiR (5). However, no candidate for ChiR has ever been identified despite numerous attempts from our group and others (4,5,7). Additionally, ChiS is active when its kinase activity is ablated, making it unlikely that this HK passes a phosphate to a cognate RR.…”

“…In addition to ChiS, other DNA binding proteins, like SlmA, bind upstream of the CBSs and contribute to Pchb activation (7). To assess if upstream factors exhibit helical phasedependence for Pchb activation, we inserted or deleted 5 or 10 bp upstream of the CBSs (Fig.…”

“…Measuring GFP Fluorescence GFP fluorescence was determined essentially as previously described (7,41). Briefly, single colonies were picked and grown in LB broth at 30°C for 18 hours.…”

“…Growth was tested using M9 minimal medium supplemented with the indicated carbon source to a final concentration of 0.2%. V. cholerae can grow on chitobiose through both the activity of the chitobiose ABC transporter encoded within the chb operon and through a GlcNAc PTS transporter (7,42). ChiS is only required for regulation of the chitobiose ABC transporter (7).…”