Abstract
Collective behavior in spatially structured groups, or biofilms, is the norm among microbes in their natural environments. Though microbial physiology and biofilm formation have been studied for decades, tracing the mechanistic and ecological links between individual cell properties and the emergent features of cell groups is still in its infancy. Here we use single-cell resolution confocal microscopy to explore biofilm properties of the human pathogen Vibrio cholerae in conditions closely mimicking its marine habitat. We find that some – but not all – pandemic isolates produce filamentous cells than can be over 50 μm long. This filamentous morphotype gains a profound competitive advantage in colonizing and spreading on particles of chitin, the material many marine Vibrio species depend on for growth outside of hosts. Furthermore, filamentous cells can produce biofilms that are independent of all currently known secreted components of the V. cholerae biofilm matrix; instead, filamentous biofilm architectural strength appears to derive from the entangled mesh of cells themselves. The advantage gained by filamentous cells in early chitin colonization and growth is counter-balanced in longer term competition experiments with matrix-secreting V. cholerae variants, whose densely packed biofilm structures displace competitors from surfaces. Overall our results reveal a novel mode of biofilm architecture that is dependent on filamentous cell morphology and advantageous in environments with rapid chitin particle turnover. This insight provides concrete links between V. cholerae cell morphology, biofilm formation, marine ecology, and – potentially – the strain composition of cholera epidemics.
Introduction
Bacterial existence in the wild is predominated by life in spatially structured groups, termed biofilms (1, 2), which inhabit environments ranging from the rhizosphere (3), to chronic infections (4, 5), to the pipes of industrial and wastewater flow systems (6, 7), to the surfaces of marine snow (8–13). Although living in groups correlates with increased tolerance to many exogenous threats, including antibiotic exposure (14–16), biofilm-dwelling cells also experience intense competition for space and resources (2, 17, 18). Furthermore, cells in mature biofilms are generally non-motile and incur a trade-off between optimizing local competition versus dispersal to new environments (19–22). Balancing colonization, local growth, and dispersal is therefore a critical element of microbial fitness during biofilm formation, and understanding how bacteria have evolved to modulate this balance is a central challenge in microbial ecology. Here we study how variation in individual cell morphology impacts biofilm architecture and the competition/colonization tradeoff among different pandemic isolates of Vibrio cholerae.
V. cholerae is a notorious human pathogen responsible for the diarrheal disease cholera, but between epidemics, it persists as a common component of aquatic ecosystems, where it consumes chitin harvested from the exoskeletons of arthropods (23–25). To utilize this resource, V. cholerae must colonize and produce biofilms on chitin particles, creating a dense, resource-limited, and architecturally complex space for both inter-species and inter-strain competition (26–28). V. cholerae strains that are better adapted to colonize chitin surfaces, exploit the resources embedded in them, and spread to other particles are thus likely to be better represented in estuarine conditions. Although it remains poorly understood how V. cholerae makes the environmental-to-epidemic transition, emerging models implicate chitin-associated aggregates as disease vectors due to the high density of cells within them (24, 26, 29–33). Consequently, competition for access to space on chitin particles, and other substrates that form the basis for marine snow, may also impact which strains ultimately cause cholera pandemics (23, 34).
Many strains of V. cholerae can be found in the marine environment, and their relative abundances are continually in flux. This pathogen is characterized by its O-antigen, a glycan polymer on its outer membrane. While there are over two hundred O-antigen serotypes, only two have been known to cause major pandemics – serotype O1, comprised of both the Classical and El Tor biotypes, and serotype O139, which has arisen more recently alongside El Tor as the current leading cause of cholera in Southern Asia (35, 36). To better understand how competition to colonize biotic surfaces impacts the interactions between potentially co-occurring V. cholerae strains, we developed a microfluidic assay in which chitin particles are embedded in flow devices perfused with artificial sea water, and onto which cells could be readily inoculated to monitor colonization and biofilm growth.
We discovered that some pandemic isolates of V. cholerae, and in particular strain CVD112 of the O139 serogroup, filaments aggressively under nutrient-limited conditions, including on particles of chitin in sea water. Filamentation confers markedly altered chitin colonization and biofilm architecture relative to shorter cells. Differences in chitin colonization and biofilm architecture, in turn, strongly influence competition for space and resources, suggesting that normal-length and filamentous morphotypes are fundamentally adapted to different regimes of chitin particle turnover in the water column. Overall, our results highlight a novel mode of biofilm assembly and yield new insights into the fundamental roles of cell shape in the marine ecology of V. cholerae.
Results
Filamentation in conditions mimicking a marine habitat
We explored the biofilm architectures of different isolates of V. cholerae, focusing on N16961 (serogroup O1, El Tor biotype) and CVD112 (serogroup O139), representing the two major extant causative agents of the current cholera pandemic (36). The El Tor biotype has been used extensively in the study of biofilm formation and architecture (37–46); less is known about biofilms of the O139 serogroup (but see (47)). For visualization by microscopy, the far-red fluorescent protein-encoding locus mKate2 (48) was inserted in single copy under the control of a strong constitutive promoter on the chromosome of CVD112 and its derivatives, while an analogous construct encoding the orange fluorescent protein mKO-κ (49) was inserted onto the chromosome of N16961 and its derivatives. We have shown previously that insertion of these fluorescent protein constructs to the chromosome does not substantially influence growth rate or other biofilm-associated phenotypes (27, 43).
In preliminary experiments, CVD112 and N16961 were inoculated into separate microfluidic devices and perfused with M9 minimal media containing 0.5% glucose. After 24 hours, confocal imaging revealed dramatic differences in the biofilm architecture of the two strains (Figure 1). Consistent with prior reports, N16961 grew in well-separated microcolonies of cells about 2 μm long, with each biofilm colony appearing to descend from one progenitor on the glass surface (Figure 1A) (42, 43). In contrast, CVD112 formed large groups of filamented cells entangled with one another and loosely associated with the glass surface (Figure 1B).
We found that CVD112 filamentation occurs most strongly under nutrient-limited media environments, as CVD112 and N16961 cells were both observed exclusively as cells approximately 2 μm long when cultured in nutrient-rich lysogeny broth (LB) (Figure S1). In other circumstances, filamentation has been associated with an acute stress response prior to cell death, and since CVD112 filaments were seen in M9 media, but not LB, we first suspected that they might be in a disturbed physiological state. However, CVD112 showed no growth defect in either LB or M9 media relative to N16961 (Figure 1C).
In natural seawater environments, V. cholerae colonizes the exoskeletons of arthropods, where it consumes N-acetyl glucosamine (GlcNAc) released from chitin that Vibrio species digest by secretion of extracellular chitinases (26). We found that CVD112, but not N16961, filaments extensively in artificial seawater with 0.5% GlcNAc as the sole source of carbon and nitrogen (Figure S1). As was the case in LB and M9 media, CVD112 and N16961 growth curves are indistinguishable by CFU count in artificial seawater with GlcNAc, indicating similar rates of cell division (Figure 1C). Following this observation, we suspected that CVD112 produces more total biomass than N16961 per unit time in low-nutrient media (where CVD112 produces filaments, while N16961 produces cells of normal length), but not in high-nutrient media (where both strains produce cells of normal length). We tested this possibility by measuring the rate of total biomass production in liquid culture by optical density. As predicted, CVD112 produces biomass more quickly and to higher final density than N16961 in M9 media with glucose, and in artificial sea water with GlcNAc, but not in LB (Figure 1D). We visualized cells at regular time intervals over the course of their growth and confirmed that CVD112 begins filamenting in mid-exponential phase in sea water with GlcNAc, but not LB (Figure S1).
Filamentation of CVD112 in nutrient-limited conditions must derive in part from an increased cell elongation rate relative to cell division rate. This ratio is altered under nutrient-rich conditions such that CVD112 returns to a normal cell length, which we could observe by generating time-lapse image sets of CVD112 cells pre-filamented in low-nutrient media and placed under an agar pad made from LB. The filaments were found to septate rapidly, yielding progeny of the more conventionally observed 2 μm cell length for V. cholerae (Supplemental Movie 1). CVD112 thus appears to be healthy with respect to growth physiology, and it reversibly modulates its rates of elongation relative to division in low nutrient media so as to produce filaments. After inspecting other available isolates of V. cholerae in seawater with GlcNAc as the sole carbon and nitrogen source, we found that some strains of El Tor and O139 serogroups produce filaments in stationary phase, while others of each serogroup do not (Figure S2). Meanwhile, filamentation has previously been intimated by scanning electron microscopy in variants of MO10, another common model strain of V. cholerae O139 (50). Filamentation in seawater is thus found across a diversity of pandemic classes, and we hypothesized that filamentation is an adaptation – not specific to the O1 or O139 serogroups – that confers a fitness advantage in some natural conditions. As N16961 and CDV112 represent the strongest cases, respectively, of non-filamenting versus filamenting strains that we observed, we use these two for the remainder of the study as representative of their cell shape strategies in sea water conditions.
Filamentation promotes colonization of chitin particles and enables matrix-independent biofilm formation
We next explored the biofilm morphologies of filamenting CVD112 and non-filamenting N16961 V. cholerae, simulating the natural conditions they experience in marine environments. We inoculated these strains in microfluidic channels decorated with traps to immobilize chitin particles (shrimp shell), a natural substrate of V. cholerae for biofilm growth and nutrient consumption (23, 28, 51). The chambers were perfused with artificial sea water lacking any additional source of carbon or nitrogen (27).
In the process of inoculating chitin with V. cholerae under flow, we noticed that CVD112 filaments were particularly adept at colonizing shrimp shell surface, with single cells often wound around the contours of individual chitin particles (Figure S3). This suggests that single filaments can undergo large deformation in shear flow, despite the stiffness of the cell wall, and that these flexible filaments can wrap around objects of similar or smaller length scale than the cell body. To determine if these properties give CVD112 a colonization advantage on chitin, we measured the attachment rates of filamentous CVD112 and non-filamenting N16961 by flowing a 1:1 mixture of the two strains (normalized by biomass per volume) onto chitin particles in artificial sea water. Filamentous V. cholerae does indeed colonize chitin more rapidly than short cells (Figure 2A). This qualitative result holds when the two strains’ colonization rates are tested in monoculture (Figure S4A), and when cells dispersing directly from previously occupied chitin particles are flowed into new chambers containing fresh chitin (Figure S4B).
We next sought to determine whether the increased colonization ability of CVD112 was due to cell shape, as opposed to differences in other factors contributing to adhesion. To directly test the hypothesis that cell shape was responsible for increased chitin attachment, we treated N16961 cells with sub-inhibitory concentrations of cefalexin, which blocks cell division with minimal impact on cell viability or other aspects of V. cholerae morphology (52). N16961 pre-treated with cephalexin exhibited similar cell elongation and a chitin colonization rate statistically equivalent to that observed for CVD112 filaments (Figure 2B). These results indicate that filamentous morphology alone is sufficient to increase chitin surface colonization rate by an order of magnitude.
Following colonization, CVD112 produces biofilms that differ dramatically from those of N16961; they are composed of enmeshed cell filaments and lack the typical cell-cell packing and radial orientation associated with El Tor biofilm microcolonies (Figure 2C) (42, 44). The relative absence of tight cell-cell association prompted us to ask whether CVD112 was producing biofilm matrix, including Vibrio polysaccharide (VPS) and the adhesin RbmA, which interacts with the cell exterior and with VPS to hold neighboring cells in close proximity (40, 42–44, 53). To assess the contribution of matrix production to filamentous biofilms on chitin, we inoculated CVD112 and its isogenic ΔvpsL null mutant, which is unable to synthesize VPS or to accumulate any of the major matrix proteins (40). Biofilm production of wild type N16961, whose structure depends on matrix secretion (15, 40, 45), and its isogenic ΔvpsL null mutant were also measured for comparison. All strains contained a FLAG epitope inserted at the C-terminus of the native rbmA locus (40, 43). Fusion of a FLAG epitope to RbmA has been shown not to interfere with its function (40, 44), and allowed us to localize and quantify RbmA by immunostaining as a proxy for general matrix accumulation.
As expected, N16961 produced biofilms with abundant RbmA, while its isogenic ΔvpsL mutant was impaired for biofilm growth relative to the wild type parent and showed no matrix accumulation by RbmA staining (Figure 2D-F). The biomass and visible biofilm architecture of CVD112 and its ΔvpsL null mutant, on the other hand, were indistinguishable. CVD112 biofilms showed no detectable matrix accumulation, even in areas of dense growth (Figure 2D, G-H). Previous reports have suggested involvement of O-antigen and capsule polysaccharide in calcium-dependent biofilms of (non-filamentous) O139, but here we observed that filamentous biofilms did not rely on these factors: their biomass on chitin was unchanged in artificial sea water without calcium and in a DwbfR null background, which cannot synthesize O-antigen or capsule polysaccharide (Figure S5). The filamentous biofilm structures of CVD112 are thus independent of the currently known components of V. cholerae extracellular matrix. We infer that filamentous biofilm architecture can derive instead from entanglement of the cells themselves, which serve as both the actively growing biomass and the structural foundation of the community.
V. cholerae filamentation is advantageous in frequently disturbed environments
Once we had discovered that filamentation provides V. cholerae with an advantage during chitin attachment, we sought to understand the relative fitness effects this cell morphology on chitin in co-culture with cells or normal length. To explore this question we inoculated CVD112 and N16961 together on chitin in artificial seawater, and their subsequent biofilm compositions were measured by confocal microscopy daily for 12 days.
When biofilms were left unperturbed for the full duration of the experiment, filamenting CVD112 cells had an initial advantage consistent with our colonization and growth rate experiments, but non-filamenting N16961 eventually increased in frequency to become the overwhelming majority of the population (Figure 3A,C). As noted above, filamentous CVD112 biofilms are independent of known matrix components, while N16961 secretes copious extracellular matrix within chitin-bound biofilms. Though CVD112 is superior in its initial surface occupation, its eventual displacement by N16961 is consistent with our prior work demonstrating that matrix secretion and cellcell packing confer a pronounced local competitive advantage within biofilms of V. cholerae, as well as resistance to spatial invasion by other bacteria (19, 20, 43, 54, 55). As opposed to matrix-replete biofilms of V. cholerae, biofilms of filamentous CVD112 permit competing strains to invade their interior volume: they do not maintain a grip over the space they initially claim during the colonization and early growth phases of the experiment (Figure S6).
Although matrix-producing N16961 outcompetes CVD112 within a patch of chitin-attached biofilm over time, the latter’s colonization advantage led us to speculate that it could be successful when residence times on a given chitin particle are shorter. This could be the case, for example, under frequently-agitated water column conditions or when chitin particle sizes are smaller, such that they are depleted quickly. To assess this idea, we repeated our competition experiments, but instead of tracking biofilm growth within a single microfluidic chamber over 12 days, we periodically used the liquid effluent exiting the chitin chamber to inoculate a new chamber containing fresh chitin, where a new competition would resume (Figure S7). This process simulates a disturbance event in which dispersal is advantageous and increases representation on new chitin particles elsewhere in the water column. We implemented two disturbance regimes, re-colonizing fresh chitin chambers once every 72 hours, or once every 24 hours.
When dispersed cells were taken from the effluent and allowed to recolonize fresh chitin every 72 hours, filamenting CVD112 cells showed a protracted early increase in frequency, but once again were eventually outcompeted by matrix-secreting, non-filamented N16961 cells (Figure 3A-B, D). However, when effluent collection and recolonization of fresh chitin occurred every 24 hours, the CVD112 strain dominated co-cultures for the full duration of the 12-day experiment (Figure 3A-B, E). The trajectories of these population dynamics were remarkably consistent from one run of the experiment to the next in all dispersal/recolonization regimes. This demonstrates the strength of the effects of competition during colonization, biofilm growth, and dispersal, relative to the influence of stochastic factors such as orientation of chitin particles in the chambers, or local variation in flow regime.
Discussion
Individual cell morphology varies widely within and across bacterial species (56, 57), but in most cases it is not clear how cell shape relates to emergent structure and ecology of cell collectives such as biofilms. Here we have found that some isolates of V. cholerae produces long cell filaments under conditions closely matched to the natural marine environment. This cell morphology generates a pronounced advantage in chitin surface colonization and a matrix-independent biofilm architecture that permits rapid surface occupation, but also high dispersal rates. Filamentous cells’ superiority in early surface occupation, however, comes at a cost to long-term competition against other strains that invest in secretion of adhesive extracellular matrix. Filamentation is thus advantageous when patches of chitin turn over quickly, such that faster colonization and more easily reversible attachment are important components of fitness. Our results demonstrate that the shape of V. cholerae variants is a crucial factor controlling the relative investment into surface colonization, long-term biofilm robustness, and ease of dispersal back into the planktonic phase. These are fundamental elements of fitness for any biofilm-producing microbe, and they are especially important for the marine ecology of V. cholerae as it colonizes, consumes, and re-colonizes chitin in its environment outside of hosts.
Bacterial cell shape can serve a broad range of ecological functions (55–57). The curved shape of Caulobacter crescentus, for example, promotes the formation of biofilms as hydrodynamic forces reorient single cells to optimize daughter cell attachment (58); this process also nucleates clonal clusters under strong flow (59, 60). Simulations and experiments with engineered variants of E. coli suggest that rod-shaped bacteria can obtain a competitive advantage over spherical cells in colonies on agar plates, because rod-shaped cells burrow underneath spherical cells and spread more effectively to access fresh nutrients on the colony periphery (61). Filamentation has been observed in a wide variety of bacteria and eukaryotic microbes; this morphology is implicated in assisting spatial spread through soil or host tissue, and defense against phagocytosing ameboid predators (56, 62, 63). Here we have shown that filamentation can simultaneously alter surface colonization, biofilm architecture, and, as a result, the relative investment into rapid surface occupation versus long-term competitive success in a realistic environment.
The biophysical bases of our results are a topic of future work, but here we note that single filaments of V. cholerae can rapidly bend in shear flow, despite the stiffness of the bacterial cell wall (64). Our results demonstrate that sufficiently long bacteria can behave as elastic filaments (65); in analogy with the stretching behavior of polymers in flows that approach and split at the interface with an obstacle (i.e. extensional flows), we expect that filamentous bacteria experience shear that stretches them into alignment with stationary surfaces in the flow path (66–68). This process can increase the dwell time of filaments in proximity to obstacles in flow (Figure S8). We speculate that this process promotes attachment and wrapping of filaments around chitin particles (Figure S3), yielding a substantially augmented chitin colonization rate for filaments relative to shorter cells of V. cholerae.
Following surface attachment, filamentation allows the construction of biofilms in which cell-cell contacts generate a mesh network that is not dependent on currently known secreted components of the V. cholerae biofilm matrix. In this respect, filamentous biofilms may be analogous to a polymeric gel in which cell bodies are associated through physical entanglement rather than mutual attachment via secreted adhesives (69). Here, this cell network is more natively inclined to fast surface spreading and subsequent dispersal, but also porous and prone to physical invasion by competing strains or species. This strategy of rapid colonization and biomass accumulation but high reversibility of surface association is particularly well suited to fluctuating environments in which chitin particles are short-lived. This could be the case when particles are small and quickly consumed, or when disturbance events are common and destroy or disrupt chitin particles with high frequency.
The frequency of disturbance events and chitin particle size distribution are both likely to depend on the community ecology of planktonic organisms producing the chitin on which V. cholerae feeds in its aquatic environments. The preponderance and size of biofilm clusters has been strongly implicated in the ability of this pathogen to initiate infections that lead to epidemics: removing biofilm-like clusters from drinking water by filtration, for example, reduces the incidence of cholera infections by as much as 50% (70). Consequently, the differential competitive success of V. cholerae variants on chitin particles could be a significant determinant of which strains initiate disease outbreaks, which in turn often correlate with seasonal blooms of planktonic arthropod population growth (71). The size and shape of V. cholerae in its aquatic reservoir may therefore have consequences that emerge on large geographic scales in the extent and strain composition of cholera epidemics. More broadly, as we found that some but not all strains from both major pandemic serogroups produce filaments in seawater conditions, we expect that filamentation is a generic adaptation to rapid habitat turnover that may be found distributed quite widely in Vibrio spp. and other marine microbes for which particle surface attachment and biofilm growth are important fitness components.
Materials and Methods
Methods for microfluidic device assembly, growth conditions, strain construction, growth curve measurements, septation imaging, biofilm cultivation, matrix staining, chitin colonization, competition assays, confocal microscopy, image analysis, and statistical analysis can be found further described in the SI appendix, Supplemental Materials and Methods.
Author Contributions
CDN and BRW conceived the project. All authors contributed to experimental design. BRW performed strain construction, data collection, and image processing. BRW and CDN analyzed data and produced the figures. CDN and BRW wrote the manuscript with input from all authors.
Acknowledgements
We are grateful to Rob McClung, Mary Lou Guerinot, Daniel Schultz, Karen Skorupski, Ryan Calsbeek, and Fitnat Yildiz for helpful comments on earlier versions of this manuscript, and to Kai Papenfort, Knut Drescher, Matthew Bond, and Swetha Kasetty for comments on the project. BRW is supported by a GANN Fellowship from Dartmouth College. AP is supported by the Swiss National Science Foundation (Projects grant 31003A_169377) and the Giorgio Cavaglieri Foundation. CDN is supported by the National Science Foundation (MCB 1817342), a Burke Award from Dartmouth College, a pilot award from the Cystic Fibrosis Foundation (STANTO15RO), and NIH grant P20-GM113132 to the Dartmouth BioMT COBRE.