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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.

Urease acts as an important acid resistance system and virulence factor that is widespread among microorganisms. RovM is a global regulator that regulates a series of genes and pathways including acid survival systems in the enteric bacterium Yersinia pseudotuberculosis (Yptb). However, whether RovM regulates the urease activity in Yptb was still unknown. In this study, by using qualitative and quantitative urease assays, we show that the urease expression responds to nutrient conditions and the RovM protein represses urease expression by binding to its promoter. A previously reported positive regulator OmpR activates urease activity but RovM plays a dominant role in different nutrient conditions. In addition, carbon storage regulator system A (CsrA), the upstream regulator of RovM, dramatically down-regulates urease activity possibly by its binding to the Shine-Dalgarno (SD) sequence of the mRNA encoding the urease. In conclusion, this study demonstrates that urease activity is strictly controlled by nutrient conditions and is down-regulated by the CsrA-RovM pathway.

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.

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.

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.

Summary 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.

Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H-2). Here, the authors expressed the four alpha beta gamma delta subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 degrees C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H-2 gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.

The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.

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.

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments., Author Summary One unique feature of type VI secretion system is the presence of multiple distinct systems in certain bacterial species. It is well established that some of these systems function to compete for their living niches among diverse bacterial species, whilst the activity of many such transporters remains unknown. Because metal ions are essential components to virtually all forms of life including bacteria, eukaryotic hosts have evolved complicated strategies to sequester metal ions, which constitute a major branch of their nutritional immunity. Therefore the ability to acquire metal ions is critical for bacterial virulence. This study reveals that the T6SS-4 of Yersinia pseudotuberculosis (Yptb) functions to import Zn2+ from the environment to mitigate the detrimental effects such as hydroxyl radicals induced by diverse stresses. Expression of the transporter is activated by multiple regulatory proteins, including OxyR and OmpR that sense diverse environmental cues. Zinc ion acquisition is achieved by translocating a Zn2+-binding substrate YezP, which is co-regulated with T6SS-4 by OxyR. Our results reveal a novel role for type VI secretion system, which is important in the study of the mechanism of metal ion acquisition by bacteria and the role of this process in bacterial pathogenesis and survival in detrimental environments.