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bioRxiv2019

Bactericidal Type IV Secretion System Homeostasis in Xanthomonas citri

Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized T4SS, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the entirety of structural genes of the T4SS in X. citri, is regulated by the global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citris aggressive posture when confronted by competitors.\n\nAuthor SummaryXanthomonas citri is a member of a family of phytopathogenic bacteria that can cause substantial losses in crops. At different stages of the infection cycle, these cells will encounter other bacterial species with whom they will have to compete for space and nutrients. One mechanism which improves a cells chance to survive these encounters is a type IV secretion system that transfers a cocktail of antimicrobial effector proteins into other Gram-negative bacteria in a contact-dependent manner. Here, we show that this system is constitutively produced at a low basal level, even during low nutrient conditions, despite representing a significant metabolic burden to the cell. The conserved global regulator, CsrA, provides a constant, nutrient-independent, repression on the production T4SS components, thereby holding production costs to a minimum while at the same time ensuring X. citris competitiveness during encounters with bacterial rivals.

microbiology↗

bioRxiv2019

The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. They have been long known to play a fundamental role in bacterial conjugation and virulence of several species. It was recently shown that a subtype of T4SS harboured by the plant pathogenic bacterium Xanthomonas citri transfers toxins into other bacteria cells resulting in cell death. In this study, we show that a similar T4SS from the multi-drug-resistant global opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain (XVIPCD) conserved in X. citri T4SS effectors identified eleven putative effectors secreted by the S. maltophilia T4SS. Six of these effectors have no recognizable domain except for the XVIPCD. We selected one of these new effectors (Smlt3024) and its cognate inhibitor (Smlt3025) for further characterization and confirmed that Smlt3024 is indeed secreted in a T4SS-dependent manner by S. maltophilia when in contact with a target bacterial species. Expression of Smlt3024 in the periplasm of E. coli resulted in greatly reduced growth rate and cell size, which could be counteracted by co-expression with its cognate periplasmic inhibitor, Smlt3025. This work expands our current knowledge about the diverse function of T4SSs subtypes and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition. Further elucidation of the mechanism of these antibacterial proteins could lead to the discovery of new antibacterial targets. The study also adds information about the molecular mechanisms possibly contributing to the establishment of S. maltophilia in different biotic and abiotic surfaces in both clinical and environmental settings. Author SummaryCompetition between microorganisms for nutrients and space determines which species will emerge and dominate or be eradicated from a specific habitat. Bacteria use a series of mechanisms to kill or prevent multiplication of competitor species. Recently, it was reported that a subtype of type IV secretion system (T4SS) works as a weapon to kill competitor bacterial species. In this study, we show that an important human opportunistic pathogen, Stenotrophomonas maltophilia, harbours a T4SS that promotes killing of competitor species. We also identified a series of new toxic proteins secreted by S. maltophilia via its T4SS to poison competitor species. We showed that two different bacterial species that harbour a bacteria-killing T4SS can kill each other; most likely due to differences in effector-immunity protein pairs. This work expands our current knowledge about the bacterial arsenal used in competitions with other species and expands the repertoire of antibacterial ammunition fired by T4SSs. In addition, the work contributes with knowledge on the possible mechanisms used by S. maltophilia to establish communities in different biotic and abiotic surfaces in both clinical and environmental settings.

microbiology↗