Programmed Proteolysis of Chemotaxis Proteins in Sinorhizobium meliloti: Features in the C-Terminal Region Control McpU Degradation

Abstract

Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentusIMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen. Chemotaxis of S. meliloti cells toward alfalfa roots mediates this symbiosis. The present study establishes programmed proteolysis as a factor in the maintenance of the S. meliloti chemotaxis system. Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S. meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response. While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S. meliloti In addition, our study should alert about the cautionary use of epitope tags for protein quantification.

Keywords: ClpXP protease; alphaproteobacteria; cell cycle; chemotaxis; epitope tags.