The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.

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Bacterial pathogens use protein secretion systems to translocate virulence factors into the host and to control bacterial gene expression. The ESX-1 (ESAT-6 system 1) secretion system facilitates disruption of the macrophage phagosome during infection, enabling access to the cytoplasm, and regulates widespread gene expression in the mycobacterial cell. The transcription factors contributing to the ESX-1 transcriptional network during mycobacterial infection are not known. We showed that the EspM and WhiB6 transcription factors regulate the ESX-1 transcriptional networkin vitrobut are dispensable for macrophage infection byMycobacterium marinum. In this study, we used our understanding of the ESX-1 system to identify EspN, a critical transcription factor that controls expression of the ESX-1 genes during infection, but whose effect is not detectable under standard laboratory growth conditions. Under laboratory conditions, EspN activity is masked by the EspM repressor. In the absence of EspM, we found that EspN is required for ESX-1 function because it activates expression of thewhiB6transcription factor gene, and specific ESX-1 substrate and secretory component genes. Unlike the other transcription factors that regulate ESX-1, EspN is required forM. marinumgrowth within and cytolysis of macrophages, and for disease burden in a zebrafish larval model of infection. These findings demonstrate that EspN is an infection-dependent regulator of the ESX-1 transcriptional network, which is essential for mycobacterial pathogenesis. Moreover, our findings suggest that ESX-1 expression is controlled by a genetic switch that responds to host specific signals.

Chemotaxis and motility are important traits supporting 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 residue, and including common epitope tags such as 3xFLAG and 6xHis to 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 post-translational control together with regulatory pathways on the transcriptional level limit the chemotaxis machinery to early exponential growth. Our study identified parallels to cell-cycle dependent processes during asymmetric cell division in Caulobacter crescentus. IMPORTANCE 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 towards 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.