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The transition between the planktonic state and the biofilm-associated state is a key developmental decision for pathogenic bacteria. Biofilm formation by Yersinia pestis is regulated by hmsHFRS genes (β-1, 6-N-acetyl-D-glucosamine synthesis operon) in its flea vector and in vitro. However, the mechanism of biofilm formation in Yersinia pseudotuberculosis remains elusive. In this study, we demonstrate that the LysR-type regulator RovM inversely regulates biofilm formation and motility in Y. pseudotuberculosis by acting as a transcriptional regulator of these two functions. RovM is strongly induced during growth in minimal media but strongly repressed in complex media. On one hand, RovM enhances bacterial motility by activating the expression of FlhDC, the master regulator of flagellar genes, via the recognition of an operator upstream of the flhDC promoter. On the other hand, RovM represses β-GlcNAc production under nutrition-limited conditions, negatively regulating hmsHFRS expression by directly binding to the −35 element of its promoter. Compared to wild-type bacteria, the rovM mutant established denser biofilms and caused more extensive mortality in mice and silkworm larvae. These results indicate that RovM acts as a molecular switch to coordinate the expression of genes involved in biofilm formation and motility in response to the availability of nutrients.

The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.

RpoS (σ(S)), the stationary phase/stress σ factor, controls the expression of a large number of genes involved in cellular responses to a variety of stresses. However, the role of RpoS appears to differ in different bacteria. While RpoS is an important regulator of flagellum biosynthesis, it is associated with biofilm development in Edwardsiella tarda. Biofilms are dense communities formed by bacteria and are important for microbe survival under unfavorable conditions. The type VI secretion system (T6SS) discovered recently is reportedly associated with several phenotypes, ranging from biofilm formation to stress sensing. For example, Vibrio anguillarum T6SS was proposed to serve as a sensor for extracytoplasmic signals and modulates RpoS expression and stress response. In this study, we investigated the physiological roles of RpoS in Yersinia pseudotuberculosis, including bacterial survival under stress conditions, flagella formation, biofilm development and T6SS expression. We found that RpoS is important in resistance to multiple stressors-including H2O2, acid, osmotic and heat shock-in Y. pseudotuberculosis. In addition, our study showed that RpoS not only modulates the expression of T6SS but also regulates flagellum formation by positively controlling the flagellar master regulatory gene flhDC, and affects the formation of biofilm on Caenorhabditis elegans by regulating the synthesis of exopolysaccharides. Taken together, these results show that RpoS plays a central role in cell fitness under several adverse conditions in Y. pseudotuberculosis.

Coordinated regulation of various acid survival systems in response to environmental stimuli is crucial for the adaptation of enteropathogenic bacteria to acidic environments such as the stomach. In this study, we demonstrated that the RovM protein, a central regulator of the CsrABC-RovM-RovA cascade, conversely regulates the expression of two acid survival systems in Yersinia pseudotuberculosis by acting as a dual transcriptional regulator. RovM activated the expression of T6SS4, which is essential for bacterial survival under mild acidic conditions, by binding upstream of the T6SS4 promoter. On the contrary, RovM repressed the expression of a functional arginine-dependent acid resistance system (AR3), which is crucial for bacterial survival under strong acidic conditions, by directly binding to the -35 element in the AR3 promoter. Consistent with previous findings that rovM expression responds to the availability of nutrients, the expression of T6SS4 and AR3 was differentially regulated by nutritional status. Based on these results, a dynamic model whereby RovM coordinately regulates the expression of AR3 and T6SS4 in response to the availability of nutrients in the environment was proposed.

Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity., IMPORTANCE Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells, thus allowing the detection of protein-protein interactions in live bacterial cells. This BRET system added another useful tool to address important questions in microbiological studies.

The FlgM-FliA regulatory circuit plays a central role in coordinating bacterial flagellar assembly. In this study, we identified multiple novel binding partners of FlgM using bacterial two-hybrid screening. Among these binding partners, FliS, the secretion chaperone of the filament protein FliC, was identified to compete with FliA for the binding of FlgM. We further showed that by binding to FlgM, FliS protects it from secretion and degradation, thus maintaining an intracellular pool of FlgM reserved as the FliS-FlgM complex. Consequently, we found that the flagellar late-class promoter activities are significantly increased in the fliS deletion mutant. The fliS mutant is weakly motile and shows significantly increased biofilm formation on biotic surface. Based on the results obtained, we established for the first time the regulatory role of the flagellin chaperone FliS to fine-tune late flagellar assembly by modulating FlgM activity.

Type VI secretion systems (T6SSs) which widely distributed in Gram-negative bacteria have been primarily studied in the context of cell interactions with eukaryotic hosts or other bacteria. We have recently identified a thermoregulated T6SS4 in the enteric pathogen Yersinia pseudotuberculosis. Here we report that OmpR directly binds to the promoter of T6SS4 operon and regulates its expression. Further, we observed that the OmpR-regulated T6SS4 is essential for bacterial survival under acidic conditions and that its expression is induced by low pH. Moreover, we showed that T6SS4 plays a role in pumping H(+) out of the cell to maintain intracellular pH homeostasis. The acid tolerance phenotype of T6SS4 is dependent on the ATPase activity of ClpV4, one of the components of T6SS4. These results not only uncover a novel strategy utilized by Y. pseudotuberculosis for acid resistance, but also reveal that T6SS, a bacteria secretion system known to be functional in protein transportation has an unexpected function in H(+) extrusion under acid conditions.