Civantos, C. | bioRxiv Science

bioRxiv2023

The type VI secretion system of Sinorhizobium fredii USDA257 is required for a successful symbiosis with Glycine max cv Pekin

For agriculture, the symbiosis carried out by rhizobia with legumes stands out as crucial for both economic and environmental reasons. In this process, the bacteria colonize the roots of the plants, inducing the formation of plant organs called nodules. Within these structures, rhizobia fix the environmental nitrogen into ammonia reducing the demand for this essential element required for plant growth. Various bacterial secretion systems (TXSS, Type X Secretion System) are involved in the establishment of this symbiosis, with the T3SS being the most extensively studied. The T6SS is a nanoweapon present in 25% of gram-negative bacteria, commonly used against other gram-negative bacteria, though some of them use it to manipulate eukaryotic cells. Interestingly, although T6SS is widely distributed among rhizobia, whether it has a specific role in symbiosis with legumes remains elusive. Sinorhizobium fredii USDA257 is a fast-growing rhizobium with the capacity to nodulate a great variety of legume plants. This strain harbors a single T6SS cluster, containing the genes encoding all the structural components of the system and two genes encoding potential effectors that could target the cell wall of the plants and/or be acting as a toxin/antitoxin system. We have demonstrated that this system is active and can be induced in poor culture media. In addition, we have seen by fluorescence microscopy that the T6SS is active in nodules. Competition assays between USDA257 and different preys have shown that USDA257 cannot kill any of them using its T6SS under tested conditions. By constract, nodulation assays demonstrated that USDA257 utilizes this protein secretion system to enhance nodulation and competitiveness with its host Glycine max cv Pekin.

microbiology↗

bioRxiv2023

The carboxyl-terminal processing proteases Prc and CtpA modulate cell-surface signalling activity and Pseudomonas aeruginosa virulence

Cell-surface signalling (CSS) is a signal transfer system of Gram-negative bacteria used to detect extracellular signals and modulate gene transcription in response. These three-protein systems are formed by an outer membrane receptor, a cytoplasmic membrane-embedded anti-{sigma} factor and a cytosolic extracytoplasmic function {sigma} factor ({sigma}ECF). In absence of an inducing signal, the anti-{sigma} factor binds to and keeps the {sigma}ECF factor sequestered, thus preventing its interaction with the RNA polymerase and the transcription of signal response genes. Presence of the signal triggers a signalling cascade that extends from the outer membrane to the cytosol and results in {sigma}ECF factor activation. Recently, we and others have reported that CSS {sigma}ECF factor activation requires the regulated and sequential proteolysis of the cognate anti-{sigma} factor, and the function of the Prc and RseP proteases. However, many features of this proteolytic cascade are still unclear. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. We show that both CtpA and the previously identified protease Prc control CSS activation by modulating the levels of the anti-{sigma} factor. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-{sigma} factor. Importantly, using zebrafish embryos and the A549 cell line as hosts, we show that mutants in the rseP and ctpA proteases of the human pathogen Pseudomonas aeruginosa are considerably attenuated in virulence while the prc mutation increases virulence likely by enhancing the production of outer membrane vesicles. Because proteases are druggable proteins, the identification of regulatory proteases involved in P. aeruginosa virulence holds promise for the development of novel strategies to fight this clinically relevant pathogen.

microbiology↗