Bacterial predation transforms the landscape and community assembly of biofilmsHighlights d Biofilms with high cell packing are protected from bacterial predator access d The protection effect strongly alters biofilm microlandscapes after predation d Predation on the periphery of biofilms loosens their local architecture d These changes in biofilm structure allow other bacteria to invade prey biofilms

Collective behavior in spatially structured groups, or biofilms, is the norm among microbes in their natural environments. Though biofilm formation has been studied for decades, tracing the mechanistic and ecological links between individual cell morphologies and the emergent features of cell groups is still in its infancy. Here we use single-cell–resolution confocal microscopy to explore biofilms of the human pathogenVibrio choleraein conditions mimicking its marine habitat. Prior reports have noted the occurrence of cellular filamentation inV. cholerae, with variable propensity to filament among both toxigenic and nontoxigenic strains. Using a filamenting strain ofV. choleraeO139, we show that cells with this morphotype gain a profound competitive advantage in colonizing and spreading on particles of chitin, the material many marineVibriospecies depend on for growth in seawater. Furthermore, filamentous cells can produce biofilms that are independent of primary secreted components of theV. choleraebiofilm matrix; instead, filamentous biofilm architectural strength appears to derive at least in part from the entangled mesh of cells themselves. The advantage gained by filamentous cells in early chitin colonization and growth is countered in long-term competition experiments with matrix-secretingV. choleraevariants, whose densely packed biofilm structures displace competitors from surfaces. Overall, our results reveal an alternative mode of biofilm architecture that is dependent on filamentous cell morphology and advantageous in environments with rapid chitin particle turnover. This insight provides an environmentally relevant example of how cell morphology can impact bacterial fitness.

Vibrio cholerae, the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae, which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.

Small regulatory RNAs (sRNAs) acting in concert with the RNA chaperone Hfq are prevalent in many bacteria and typically act by base-pairing with multiple target transcripts. In the human pathogen Vibrio cholerae, sRNAs play roles in various processes including antibiotic tolerance, competence, and quorum sensing (QS). Here, we use RIL-seq (RNA-interaction-by-ligation-and-sequencing) to identify Hfq-interacting sRNAs and their targets in V. cholerae. We find hundreds of sRNA-mRNA interactions, as well as RNA duplexes formed between two sRNA regulators. Further analysis of these duplexes identifies an RNA sponge, termed QrrX, that base-pairs with and inactivates the Qrr1-4 sRNAs, which are known to modulate the QS pathway. Transcription of qrrX is activated by QrrT, a previously uncharacterized LysR-type transcriptional regulator. Our results indicate that QrrX and QrrT are required for rapid conversion from individual to community behaviours in V. cholerae.

No abstract

Bacteria are thought to often occupy environments with many other microbial species with which they interact extensively, including during biofilm formation. Adherence to surfaces and secretion of extracellular matrix is common in the microbial world, and we are still learning how the rich variety of ecological interactions that dictate biofilm structure and community ecology occur at the cellular scale. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacterium Bdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, including Vibrio cholerae and Escherichia coli. V. cholerae can protect itself from predation within highly packed biofilm structures that it creates, whereas E. coli biofilms are generally highly susceptible to predation by B. bacteriovorus. We were curious here how predator-prey dynamics might change if V. cholerae and E. coli are growing in biofilms together before exposure to B. bacteriovorus. We first find that in dual species prey biofilms, E. coli predation survival increases, whereas V. cholerae survival decreases. The benefit E. coli gains occurs when they become embedded within expanding groups of highly packed V. cholerae. But we find, interestingly, that normal, ordered and highly packed biofilm structure of V. cholerae can be disrupted if V. cholerae cells are directly adjacent to E. coli cells at the start of biofilm growth. When this occurs, the two species become entangled, and V. cholerae cannot coordinate its normal cell-cell alignment and matrix secretion that control the production of its normally ordered architecture. The resulting, disordered cell groups of the two species are viable and can grow into large groups, but they are no longer protected from predation; the loss of this portion of the V. cholerae population accounts for the decrease in predation protection they incur when in co-culture with E. coli. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure to B. bacteriovorus. Our study highlights the deep connections between the mechanics of biofilm architectural development, dispersal/colonization ecology, and population dynamics of predator-prey interaction in microbial systems, in addition to raising new important questions in each of these domains.

Numerous ecological interactions among microbes—for example, competition for space and resources, or interaction among phages and their bacterial hosts—are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons underlying this variability are not well understood, and how phage–host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we study how the cellular scale architecture of model 2-species biofilms impacts cell–cell and cell–phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms of Escherichia coli and Vibrio cholerae under exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms of E. coli can protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity, E. coli is highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups of V. cholerae in co-culture. This protection, in turn, is dependent on the cell packing architecture controlled by V. cholerae biofilm matrix secretion. In this manner, E. coli cells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation with V. cholerae can confer phage protection to E. coli, it comes at the cost of competing with V. cholerae and a disruption of normal curli-mediated protection for E. coli even in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages.

Numerous ecological interactions among microbes - for example, competition for space and resources, or interaction among phages and their bacterial hosts - are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons causing this variability are not well understood, and how phage-host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we explore how the microscale biofilm structure of a model 2-species biofilms impacts cell-cell and cell-phage interactions underlying larger population wide patterns. Our system consists of dual-culture biofilms of Escherichia coli and Vibrio cholerae under exposure to T7 phages or λ phages. In the absence of phages, the two species compete with each other for limited space and resources. As shown previously, sufficiently mature biofilms of E. coli can protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity, E. coli is highly susceptible to phages, however we show that these bacteria can gain lasting protection against phage exposure if they have become embedded within highly packed groups of V. cholerae in co-culture. In this manner, E. coli cells that are otherwise susceptible to T7 and λ phages can survive phage exposure in the absence of de novo resistance evolution. While co-culture growth allows for earlier protection from phages conferred by V. cholerae cells, it comes at the cost of competing with V. cholerae and a disruption of normal curli-mediated protection for E. coli even in dual species biofilms grown over long time scales.