Weipeng Zhang

A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis.

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.

Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity

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.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning “plug-and-play” approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Modulation of a thermoregulated type VI secretion system by AHL-dependent Quorum Sensing in Yersinia pseudotuberculosis

The type VI secretion system (T6SS) is a novel secretion system found in many Gram-negative bacterial pathogens, which appears to be tightly regulated by different regulatory mechanisms. In the present study, we identified 4 T6SS clusters in Yersinia pseudotuberculosis and demonstrated that they were differentially thermoregulated. Among them, T6SS4 was preferentially expressed at 26°C, and its expression was growth phase dependent and subject to quorum sensing regulation. Both YpsI and YtbI AHL synthases contributed to the positive regulation of T6SS4, whereas YpsI synthase played the major role as T6SS4 expression was reduced strongly in the ypsI mutant strain but weakly in the ytbI mutant strain. Moreover, we provided evidence that exogenous addition of different synthetic AHLs complemented T6SS4 expression in different efficiencies in an ypsIytbI double mutant strain, suggesting C6-HSL had an antagonistic effect on T6SS4 expression. This is the first study demonstrating that the expression of T6SS is precisely regulated by temperature, growth phase, and AHL-dependent quorum sensing systems in Y. pseudotuberculosis.

A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition

Iron sequestration by host proteins contributes to the defence against bacterial pathogens, which need iron for their metabolism and virulence. A Pseudomonas aeruginosa mutant lacking all three known iron acquisition systems retains the ability to grow in media containing iron chelators, suggesting the presence of additional pathways involved in iron uptake. Here we screen P. aeruginosa mutants defective in growth in iron-depleted media and find that gene PA2374, proximal to the type VI secretion system H3 (H3-T6SS), functions synergistically with known iron acquisition systems. PA2374 (which we have renamed TseF) appears to be secreted by H3-T6SS and is incorporated into outer membrane vesicles (OMVs) by directly interacting with the iron-binding Pseudomonas quinolone signal (PQS), a cell–cell signalling compound. TseF facilitates the delivery of OMV-associated iron to bacterial cells by engaging the Fe(III)-pyochelin receptor FptA and the porin OprF. Our results reveal links between type VI secretion, cell–cell signalling and classic siderophore receptors for iron acquisition in P. aeruginosa.

Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin.

Colibactin represents an as-yet uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886 that uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block in its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal cancer-linked microbial chemical.

Adaptation of intertidal biofilm communities is driven by metal ion and oxidative stresses

Marine organisms in intertidal zones are subjected to periodical fluctuations and wave activities. To understand how microbes in intertidal biofilms adapt to the stresses, the microbial metagenomes of biofilms from intertidal and subtidal zones were compared. The genes responsible for resistance to metal ion and oxidative stresses were enriched in both 6-day and 12-day intertidal biofilms, including genes associated with secondary metabolism, inorganic ion transport and metabolism, signal transduction and extracellular polymeric substance metabolism. In addition, these genes were more enriched in 12-day than 6-day intertidal biofilms. We hypothesize that a complex signaling network is used for stress tolerance and propose a model illustrating the relationships between these functions and environmental metal ion concentrations and oxidative stresses. These findings show that bacteria use diverse mechanisms to adapt to intertidal zones and indicate that the community structures of intertidal biofilms are modulated by metal ion and oxidative stresses.

Post-translational modifications are enriched within protein functional groups important to bacterial adaptation within a deep-sea hydrothermal vent environment.

BackgroundPost-translational modification (PTM) of proteins is one important strategy employed by bacteria for environmental adaptation. However, PTM profiles in deep-sea microbes remain largely unexplored.ResultsWe provide here insight into PTMs in a hydrothermal vent microbial community through integration of metagenomics and metaproteomics. In total, 2919 unique proteins and 1306 unique PTMs were identified, whereas the latter included acetylation, deamination, hydroxylation, methylation, nitrosylation, oxidation, and phosphorylation. These modifications were unevenly distributed among microbial taxonomic and functional categories. A connection between modification types and particular functions was demonstrated. Interestingly, PTMs differed among the orthologous proteins derived from different bacterial groups. Furthermore, proteomic mapping to the draft genome of a Nitrospirae bacterium revealed novel modifications for proteins that participate in energy metabolism, signal transduction, and inorganic ion transport.ConclusionsOur results suggest that PTMs are enriched in specific functions, which would be important for microbial adaptation to extreme conditions of the hydrothermal vent. PTMs in deep-sea are highly diverse and divergent, and much broader investigations are needed to obtain a better understanding of their functional roles.

Species sorting during biofilm assembly by artificial substrates deployed in a cold seep system

Studies focusing on biofilm assembly in deep-sea environments are rarely conducted. To examine the effects of substrate type on microbial community assembly, biofilms were developed on different substrates for different durations at two locations in the Red Sea: in a brine pool and in nearby bottom water (NBW) adjacent to the Thuwal cold seep II. The composition of the microbial communities in 51 biofilms and water samples were revealed by classification of pyrosequenced 16S rRNA gene amplicons. Together with the microscopic characteristics of the biofilms, the results indicate a stronger selection effect by the substrates on the microbial assembly in the brine pool compared with the NBW. Moreover, the selection effect by substrate type was stronger in the early stages compared with the later stages of the biofilm development. These results are consistent with the hypotheses proposed in the framework of species sorting theory, which states that the power of species sorting during microbial community assembly is dictated by habitat conditions, duration and the structure of the source community. Therefore, the results of this study shed light on the control strategy underlying biofilm-associated marine fouling and provide supporting evidence for ecological theories important for understanding the formation of deep-sea biofilms.

The icmF3 locus is involved in multiple adaptation- and virulence-related characteristics in Pseudomonas aeruginosa PAO1

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria. Three separate T6SSs called H1-, H2-, and H3-T6SS have been discovered in Pseudomonas aeruginosa PAO1. Recent studies suggest that, in contrast to the H1-T6SS that targets prokaryotic cells, H2- and H3-T6SS are involved in interactions with both prokaryotic and eukaryotic cells. However, the detailed functions of T6SS components are still uncharacterized. The intracellular multiplication factor (IcmF) protein is conserved in type VI secretion systems (T6SS) of all different bacterial pathogens. Bioinformatic analysis revealed that IcmF3 in P. aeruginosa PAO1 is different from other IcmF homologs and may represent a new branch of these proteins with distinct functions. Herein, we have investigated the function of IcmF3 in this strain. We have shown that deletion of the icmF3 gene in P. aeruginosa PAO1 is associated with pleiotropic phenotypes. The icmF3 mutant has variant colony morphology and an hypergrowth phenotype in iron-limiting medium. Surprisingly, this mutant is also defective for the production of pyoverdine, as well as defects in swimming motility and virulence in a C. elegans worm model. The icmF3 mutant exhibits higher conjugation frequency than the wild type and increased biofilm formation on abiotic surfaces. Additionally, expression of two phenazine biosynthetic loci is increased in the icmF3 mutant, leading to the overproduction of pyocyanin. Finally, the mutant exhibits decreased susceptibility to aminoglycosides such as tobramycin and gentamicin. And the detected phenotypes can be restored completely or partially by trans complementation of wild type icmF3 gene. The pleiotropic effects observed upon icmF3 deletion demonstrate that icmF3 plays critical roles in both pathogenesis and environmental adaptation in P. aeruginosa PAO1.