Cross-species activation of hydrogen cyanide production by a promiscuous quorum-sensing receptor promotes Chromobacterium subtsugae competition in a dual-species model.

Many saprophytic bacteria have LuxR-I-type acyl-homoserine lactone (AHL) quorum-sensing systems that may be important for competing with other bacteria in complex soil communities. LuxR AHL receptors specifically interact with cognate AHLs to cause changes in expression of target genes. Some LuxR-type AHL receptors have relaxed specificity and are responsive to non-cognate AHLs. These promiscuous receptors might be used to sense and respond to AHLs produced by other bacteria by eavesdropping. We are interested in understanding the role of eavesdropping during interspecies competition. The soil saprophyte Chromobacterium subtsugae has a single AHL circuit, CviR-I, which produces and responds to N-hexanoyl-HSL (C6-HSL). The AHL receptor CviR can respond to a variety of AHLs in addition to C6-HSL. In prior studies we have utilized a coculture model with C. subtsugae and another soil saprophyte, Burkholderia thailandensis. Using this model, we previously showed that promiscuous activation of CviR by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Here, we show that B. thailandensis AHLs activate transcription of dozens of genes in C. subtsugae, including the hcnABC genes coding for production of hydrogen cyanide. We show that hydrogen cyanide production is population density-dependent and demonstrate that the cross-induction of hydrogen cyanide by B. thailandensis AHLs provides a competitive advantage to C. subtsugae. Our results provide new information on C. subtsugae quorum sensing and are the basis for future studies aimed at understanding the role of eavesdropping in interspecies competition.

Plasmid-Encoded H-NS Controls Extracellular Matrix Composition in a Modern Acinetobacter baumannii Urinary Isolate.

Acinetobacter baumannii is emerging as a multidrug-resistant (MDR) nosocomial pathogen of increasing threat to human health worldwide. The recent MDR urinary isolate UPAB1 carries the plasmid pAB5, a member of a family of large conjugative plasmids (LCPs). LCPs encode several antibiotic resistance genes and repress the type VI secretion system (T6SS) to enable their dissemination, employing two TetR transcriptional regulators. Furthermore, pAB5 controls the expression of additional chromosomally encoded genes, impacting UPAB1 virulence. Here, we show that a pAB5-encoded H-NS transcriptional regulator represses the synthesis of the exopolysaccharide PNAG and the expression of a previously uncharacterized three-gene cluster that encodes a protein belonging to the CsgG/HfaB family. Members of this protein family are involved in amyloid or polysaccharide formation in other species. Deletion of the CsgG homolog abrogated PNAG production and chaperone-usher pathway (CUP) pilus formation, resulting in a subsequent reduction in biofilm formation. Although this gene cluster is widely distributed in Gram-negative bacteria, it remains largely uninvestigated. Our results illustrate the complex cross-talks that take place between plasmids and the chromosomes of their bacterial host, which in this case can contribute to the pathogenesis of Acinetobacter. IMPORTANCE The opportunistic human pathogen Acinetobacter baumannii displays the highest reported rates of multidrug resistance among Gram-negative pathogens. Many A. baumannii strains carry large conjugative plasmids like pAB5. In recent years, we have witnessed an increase in knowledge about the regulatory cross-talks between plasmids and bacterial chromosomes. Here, we show that pAB5 controls the composition of the bacterial extracellular matrix, resulting in a drastic reduction in biofilm formation. The association between biofilm formation, virulence, and antibiotic resistance is well documented. Therefore, understanding the factors involved in the regulation of biofilm formation in Acinetobacter has remarkable therapeutic potential.

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen.

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model.

Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities.IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.

Bacterial Quorum Sensing and Microbial Community Interactions.

Many bacteria use a cell-cell communication system called quorum sensing to coordinate population density-dependent changes in behavior. Quorum sensing involves production of and response to diffusible or secreted signals, which can vary substantially across different types of bacteria. In many species, quorum sensing modulates virulence functions and is important for pathogenesis. Over the past half-century, there has been a significant accumulation of knowledge of the molecular mechanisms, signal structures, gene regulons, and behavioral responses associated with quorum-sensing systems in diverse bacteria. More recent studies have focused on understanding quorum sensing in the context of bacterial sociality. Studies of the role of quorum sensing in cooperative and competitive microbial interactions have revealed how quorum sensing coordinates interactions both within a species and between species. Such studies of quorum sensing as a social behavior have relied on the development of "synthetic ecological" models that use nonclonal bacterial populations. In this review, we discuss some of these models and recent advances in understanding how microbes might interact with one another using quorum sensing. The knowledge gained from these lines of investigation has the potential to guide studies of microbial sociality in natural settings and the design of new medicines and therapies to treat bacterial infections.

Quorum-sensing control of antibiotic resistance stabilizes cooperation in Chromobacterium violaceum.

Many Proteobacteria use quorum sensing to regulate production of public goods, such as antimicrobials and proteases, that are shared among members of a community. Public goods are vulnerable to exploitation by cheaters, such as quorum sensing-defective mutants. Quorum sensing- regulated private goods, goods that benefit only producing cells, can prevent the emergence of cheaters under certain growth conditions. Previously, we developed a laboratory co-culture model to investigate the importance of quorum-regulated antimicrobials during interspecies competition. In our model, Burkholderia thailandensis and Chromobacterium violaceum each use quorum sensing-controlled antimicrobials to inhibit the other species' growth. Here, we show that C. violaceum uses quorum sensing to increase resistance to bactobolin, a B. thailandensis antibiotic, by increasing transcription of a putative antibiotic efflux pump. We demonstrate conditions where C. violaceum quorum-defective cheaters emerge and show that in these conditions, bactobolin restrains cheaters. We also demonstrate that bactobolin restrains quorum-defective mutants in our co-culture model, and the increase in antimicrobial-producing cooperators drives the C. violaceum population to become more competitive. Our results describe a mechanism of cheater restraint involving quorum control of efflux pumps and demonstrate that interspecies competition can reinforce cooperative behaviors by placing constraints on quorum sensing-defective mutants.

Nutritional stress induces exchange of cell material and energetic coupling between bacterial species.

Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell-cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner (D. vulgaris) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction.

LEGO® bricks as building blocks for centimeter-scale biological environments: the case of plants.

LEGO bricks are commercially available interlocking pieces of plastic that are conventionally used as toys. We describe their use to build engineered environments for cm-scale biological systems, in particular plant roots. Specifically, we take advantage of the unique modularity of these building blocks to create inexpensive, transparent, reconfigurable, and highly scalable environments for plant growth in which structural obstacles and chemical gradients can be precisely engineered to mimic soil.

A simple and versatile 2-dimensional platform to study plant germination and growth under controlled humidity.

We describe a simple, inexpensive, but remarkably versatile and controlled growth environment for the observation of plant germination and seedling root growth on a flat, horizontal surface over periods of weeks. The setup provides to each plant a controlled humidity (between 56% and 91% RH), and contact with both nutrients and atmosphere. The flat and horizontal geometry of the surface supporting the roots eliminates the gravitropic bias on their development and facilitates the imaging of the entire root system. Experiments can be setup under sterile conditions and then transferred to a non-sterile environment. The system can be assembled in 1-2 minutes, costs approximately 8.78$ per plant, is almost entirely reusable (0.43$ per experiment in disposables), and is easily scalable to a variety of plants. We demonstrate the performance of the system by germinating, growing, and imaging Wheat (Triticum aestivum), Corn (Zea mays), and Wisconsin Fast Plants (Brassica rapa). Germination rates were close to those expected for optimal conditions.