vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae

Vibrio cholerae is the causative agent of the diarrheal disease cholera. By an incompletely understood developmental process, V. cholerae forms complex surface-associated communities called biofilms. Here we show that quorum sensing-deficient mutants of V. cholerae produce thicker biofilms than those formed by wild-type bacteria. Microarray analysis of biofilm-associated bacteria shows that expression of the Vibrio polysaccharide synthesis (vps) operons is enhanced in hapR mutants. CqsA, one of two known autoinducer synthases in V. cholerae, acts through HapR to repress vps gene expression. Vibrio biofilms are more acid resistant than planktonic cells. However, quorum sensing-deficient biofilms have lower colonization capacities than those of wild-type biofilms, suggesting that quorum sensing may promote cellular exit from the biofilm once the organisms have traversed the gastric acid barrier of the stomach. These results shed light on the relationships among biofilm development, quorum sensing, infectivity, and pathogenesis in V. cholerae.     

Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective mechanism, developmental stage and protozoan feeding mode

In a previous study we identified microcolony formation and inhibitor production as the major protective mechanisms of Pseudomonas aeruginosa biofilms against flagellate grazing. Here we compared the efficacy of these two key protective mechanisms by exposing biofilms of the non-toxic alginate overproducing strain PDO300 and the wild-type toxic strain PAO1 to a range of feeding types commonly found in the succession of protozoans associated with natural biofilms. Alginate-mediated microcolony formation conferred effective protection for strain PDO300 against the suspension feeding flagellate Bodo saltans and, as reported earlier, the surface feeding flagellate Rhynchomonas nasuta, both of which are considered as early biofilm colonizers. However, microcolonies of mature PDO300 biofilms were highly susceptible to late biofilm colonizers, the surface-feeding amoeba Acanthamoeba polyphaga and the planktonic ciliate Tetrahymena sp., resulting in a significant reduction of biofilm biomass. Mature biofilms of strain PAO1 inhibited growth of flagellates and A. polyphaga while the grazing activity of Tetrahymena sp. remained unaffected. Our findings suggest that inhibitor production of mature P. aeruginosa biofilms is effective against a wider range of biofilm-feeding predators while microcolony-mediated protection is only beneficial in the early stages of biofilm formation.     

Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Protozoan grazing is a major mortality factor faced by bacteria in the environment. Vibrio cholerae, the causative agent of the disease cholera, is a natural inhabitant of aquatic ecosystems, and its survival depends on its ability to respond to stresses, such as predation by heterotrophic protists. Previous results show that grazing pressure induces biofilm formation and enhances a smooth to rugose morphotypic shift, due to increased expression of Vibrio polysaccharide (VPS). In addition to negatively controlling vps genes, the global quorum sensing (QS) regulator, HapR, plays a role in grazing resistance as the ΔhapR strain is efficiently consumed while the wild type (WT) is not. Here, the relative and combined contributions of VPS and QS to grazing resistance were investigated by exposing VPS and HapR mutants and double mutants in VPS and HapR encoding genes at different phases of biofilm development to amoeboid and flagellate grazers. Data show that the WT biofilms were grazing resistant, the VPS mutants were less resistant than the WT strain, but more resistant than the QS mutant strain, and that QS contributes to grazing resistance mainly in mature biofilms. In addition, grazing effects on biofilms of mixed WT and QS mutant strains were investigated. The competitive fitness of each strain in mixed biofilms was determined by CFU and microscopy. Data show that protozoa selectively grazed the QS mutant in mixed biofilms, resulting in changes in the composition of the mixed community. A small proportion of QS mutant cells which comprised 4% of the mixed biofilm biovolume were embedded in grazing resistant WT microcolonies and shielded from predation, indicating the existence of associational protection in mixed biofilms.     

Quantification of individual flagellate - bacteria interactions within semi-natural biofilms

Here we present a new approach to quantify food-web interactions within semi-natural biofilms by combining the establishment of biofilms from natural rivers in flow cells with video microscopy. In a first application of this approach, we focused on the surface-gliding heterotrophic flagellates (HF) Neobodo designis, Rhynchomonas nasuta and Planomonas sp. It was shown that the three HF generally ingested single biofilm-associated bacteria whereas bacteria within microcolonies were attacked but not ingested. However, grazing strategies differed considerably. While the kinetoplastids N. designis and R. nasuta displayed long search and short handling times, Planomonas sp. showed the opposite grazing characteristics. The latter behaviour resulted in a high relative predation success of 80% (precent of attacked prey ingested), whereas the relative predation success of the two kinetoplastids was only 20%. However, the two contrasting strategies resulted in similar ingestion rates for Planomonas sp. and N. designis of 0.5 to 0.6 ingestions flagellates(-1) minute(-1), respectively. Our results showed distinct differences in the feeding behaviour of three flagellates having similar life forms and provide direct evidence that microcolony formation in biofilms protects bacteria from grazing by HF in situ. The new approach provides individual-based insights into the complex food web interactions within biofilms.     

The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.     

Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing

This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing. We tested P. aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface-feeding flagellate Rhynchomonas nasuta. Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated. Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type. Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella-deficient cells. The alginate-overproducing strain PDO300 developed larger microcolonies in response to grazing. These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing. More mature biofilms of the wild type exhibited acute toxicity to the flagellate R. nasuta. Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms. Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P. aeruginosa biofilms to resist protozoan grazing and to persist in the environment.     

Trophic regulation of Vibrio cholerae in coastal marine waters

Cholera disease, caused by the bacterium Vibrio cholerae, afflicts hundreds of thousands worldwide each year. Endemic to aquatic environments, V. cholerae's proliferation and dynamics in marine systems are not well understood. Here, we show that under a variety of coastal seawater conditions V. cholerae remained primarily in a free-living state as opposed to attaching to particles. Growth rates of free-living V. cholerae (micro: 0.6-2.9 day(-1)) were high (similar to reported values for the bacterial assemblages; 0.3-2.5 day(-1)) particularly in phytoplankton bloom waters. However, these populations were subject to heavy grazing-mortality by protozoan predators. Thus, grazing-mortality counterbalanced growth, keeping V. cholerae populations in check. Net population gains were observed under particularly intense bloom conditions when V. cholerae proliferated, overcoming grazing pressure terms in part via rapid growth (> 4 doublings day(-1)). Our results show V. cholerae is subject to protozoan control and capable of utilizing multiple proliferation pathways in the marine environment. These findings suggest food web effects play a significant role controlling this pathogen's proliferation in coastal waters and should be considered in predictive models of disease risk.     

The PhoB regulatory system modulates biofilm formation and stress response in El Tor biotype Vibrio cholerae

The PhoBR regulatory system is required for the induction of multiple genes under conditions of phosphate limitation. Here, we examine the role of PhoB in biofilm formation and environmental stress response in Vibrio cholerae of the El Tor biotype. Deletion of phoB or hapR enhanced biofilm formation in a phosphate-limited medium. Planktonic and redispersed biofilm cells of the Δ phoB mutant did not differ from wild type for the expression of HapR, suggesting that PhoB negatively affects biofilm formation through an HapR-independent pathway. The Δ phoB mutant exhibited elevated expression of exopolysaccharide genes vpsA and vpsL compared with the wild type. Deletion of hapR enhanced the expression of the positive regulator vpsT , but had no effect on the expression of vpsR . In contrast, deletion of phoB enhanced the expression of the positive regulator vpsR , but had no effect on the expression of hapR and vpsT . The Δ phoB mutant was more sensitive to hydrogen peroxide compared with the wild type and with an isogenic Δ rpoS mutant. Conversely, the Δ phoB mutant was more resistant to acidic conditions and high osmolarity compared with the wild type and with an isogenic Δ rpoS mutant. Taken together, our data suggest that phosphate limitation induces V. cholerae to adopt a free-swimming life style in which PhoB modulates environmental stress response in a manner that differs from the general stress response regulator RpoS.     

Environmental determinants of Vibrio cholerae biofilm development

Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V. cholerae biofilm development in Luria-Bertani broth. In this work, we showed that V. cholerae forms vps-dependent biofilms and vps-independent biofilms. The vps-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment.     

Quorum sensing contributes to natural transformation of Vibrio cholerae in a species-specific manner

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.     

Minimal increase in genetic diversity enhances predation resistance

The importance of species diversity to emergent, ecological properties of communities is increasingly appreciated, but the importance of within‐species genetic diversity for analogous emergent properties of populations is only just becoming apparent. Here, the properties and effects of genetic variation on predation resistance in populations were assessed and the molecular mechanism underlying these emergent effects was investigated. Using biofilms of the ubiquitous bacterium Serratia marcescens, we tested the importance of genetic diversity in defending biofilms against protozoan grazing, a main source of mortality for bacteria in all natural ecosystems. S. marcescens biofilms established from wild‐type cells produce heritable, stable variants, which when experimentally combined, persist as a diverse assemblage and are significantly more resistant to grazing than either wild type or variant biofilms grown in monoculture. This diversity effect is biofilm‐specific, a result of either facilitation or resource partitioning among variants, with equivalent experiments using planktonic cultures and grazers resulting in dominance by a single resistant strain. The variants studied are all the result of single nucleotide polymorphisms in one regulatory gene suggesting that the benefits of genetic diversity in clonal biofilms can occur through remarkably minimal genetic change. The findings presented here provide a new insight on the integration of genetics and population ecology, in which diversity arising through minimal changes in genotype can have major ecological implications for natural populations.