(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Multispecies biofilm architecture determines bacterial exposure to phages [1]

['James B. Winans', 'Department Of Biological Sciences', 'Dartmouth', 'Hanover', 'New Hampshire', 'United States Of America', 'Benjamin R. Wucher', 'Carey D. Nadell']

Date: 2023-01 

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.

Funding: This work was supported by the Simons Foundation (award number 826672 to CDN), the National Science Foundation (award number 2017879 to CDN; award number 1817352 to CDN), and the Human Frontier Science Program (award number RGY0077/2020 to CDN). JBW is supported by a GAANN Fellowship from Department of Biological Sciences at Dartmouth. BRW is supported by a Gillman Fellowship from the Department of Biological Sciences at Dartmouth. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we explore these open questions, studying how co-culture with V. cholerae influences matrix secretion and biofilm architecture of E. coli, and how these changes in turn influence the ability of E. coli to protect itself from phage attack in the midst of competition with V. cholerae for space and resources. We find that the patterns of phage infection among E. coli are qualitatively altered by the presence of a competing species, depending on cell group spatial structure.

Recent work has documented protection of biofilm-dwelling bacteria against phage exposure among several species, including V. cholerae, E. coli, Pseudomonas aeruginosa, and Pantoea stewartii [ 41 , 55 – 57 ]. In each of these cases, phage protection has either been directly or indirectly traced to biofilm architecture controlled by secreted matrix materials. Most pertinently, recent work in E. coli has shown that mature biofilms are able to block phage diffusion in a manner dependent on secreted curli polymers controlling cell–cell packing on the biofilm periphery [ 49 , 55 ]. Curli, along with the polysaccharide cellulose, are central elements of the E. coli biofilm matrix; both are commonly produced by environmental isolates of E. coli [ 52 , 53 , 58 ]. Phages trapped in the outer biofilm layers remain at least partially viable and can infect newly arriving susceptible bacteria colonizing the biofilm exterior [ 59 ]. In general, there is little known about how growing in a multispecies context alters biofilm matrix production and architecture relative to that found in mono-species contexts; likewise, there is little known about whether and how these potential changes in biofilm architecture influence the ability of phages to access their hosts.

Our model system comprises dual culture biofilms of Escherichia coli and Vibrio cholerae under exposure to T7 phages or λ phages. Beyond the experimental tractability that makes E. coli and V. cholerae excellent for controlled experiments, these species can be found in natural environments together: for example, residing in brackish water [ 32 , 33 ] and within surface-fouling biofilms in coastal waters near human populations [ 34 ]. Members of the Escherichia and Vibrio genera are also common components of zebrafish microbiota [ 35 , 36 ]. Their tractability make E. coli and V. cholerae together a superb platform for exploring principles of how multispecies biofilm structure could influence bacteria–phage interactions in fine detail. The cellular arrangement and secreted matrix architectures of V. cholerae have been explored in great detail in the last decade [ 37 – 47 ]. In V. cholerae, biofilm structure is characterized by tight cell packing coordinated by 4 matrix components: the proteins RbmA, RbmC, Bap1, and the polysaccharide VPS [ 46 ]. E. coli biofilms, likewise, have been dissected extensively [ 48 – 53 ]. T7 phages are obligately lytic and routinely isolated from the environment alongside E. coli [ 54 ]. T7 was used as our primary model phage, but we tested the generality of our core results with temperate phage λ as well.

Previous work has shown that dual species biofilm cultures can increase, decrease, or have no measurable effect on phage susceptibility of a target host species living alongside a different, phage-resistant species [ 23 – 31 ]. Why do some multispecies biofilms confer increased phage protection to susceptible host bacteria, while others appear to do the opposite? The details underlying this variability in outcome are not well understood. A common feature among many previous studies on this topic is the use of bulk assay colony-forming unit (CFU) and plaque-forming unit (PFU) plating techniques from microtiter dish cultures; these tools, while highly effective for experimental throughput, by their nature provide an average result over entire biofilm populations residing on microtiter well walls. The conditions within these wells—for example, as a function of distance from the air–liquid interface—can vary substantially. An important way to expand on the foundation set by prior work is to examine the cellular scale variability in biofilm structure that can clarify the cell–cell and cell–phage interactions giving rise to patterns at larger spatial scales. In this paper, we target this less-explored element of phage–host interaction in multispecies contexts.

Many organisms find refuge from threats within groups. This observation applies across scales from bird flocks and animal herds to fish schools and insect swarms [ 1 , 2 ]. Bacteria are no exception and routinely live as collectives either free-floating or adhered to surfaces. Usually termed biofilms, these bacterial communities are abundant in natural settings [ 3 – 10 ], as are the threats faced by biofilm-dwelling microbes, such as invading competitors [ 11 , 12 ], diffusible antimicrobial compounds [ 13 ], phages [ 14 , 15 ], and predatory bacteria [ 16 – 18 ]. While biofilms formed by just a single species do occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment [ 19 – 22 ]. How predator–prey dynamics might change within multispecies biofilms is not well known, particularly at the cellular spatial scale of interactions that underlie large-scale patterns in biofilm-dominated microbial communities.

Results

E. coli cells overgrown by and embedded within V. cholerae clusters are shielded from phage exposure V. cholerae N16961 (serogroup O1 El Tor) and E. coli AR3110 were engineered to constitutively produce the fluorescent proteins mKO-κ and mKate2, respectively, such that they could be distinguished by fluorescence microscopy. We note that the strain background of V. cholerae that we use here, N16961, does not antagonize E. coli via Type VI secretion activity in culture conditions used for this study, which are detailed below [60,61]. The 2 species were inoculated at a 2:1 ratio of V. cholerae and E. coli into microfluidic devices bonded to glass coverslips, allowed to attach to the glass surface for 45 min, and then incubated under continuous flow of M9 minimal medium with 0.5% glucose for 48 h. Within this time frame, biofilms begin to form; however, monoculture E. coli biofilms have not yet produced sufficient curli matrix to prevent phage entry. This time frame was established by prior work and confirmed in our own experiments described below [55,59]. T7 phages were then introduced to the system continuously at 104 per μL for 16 h; this strain of T7 contains a reporter construct causing infected hosts to produce sfGFP prior to lysis [55]. Changes in E. coli abundance and localization in the chamber were tracked and compared to those in equivalent biofilms without phage introduction. Prior to phage introduction, we noted considerable variation in biofilm structure and composition across the glass substrata of our flow devices. Depending on the initial surface distribution of V. cholerae and E. coli, different regions of the devices contained cell groups of E. coli mostly on its own, locally mixed with V. cholerae, or occasionally embedded in the bottom layers of highly packed, V. cholerae-dominated clusters. Shortly after phage introduction, most E. coli cells growing on their own quickly began reporting infection and then lysed (Fig 1A and S1 Movie). Over the next 16 h, E. coli cells embedded on the bottom layers of V. cholerae-dominated cell groups largely survived phage exposure, with scattered singleton E. coli cells elsewhere in the chambers. These single cells persisted for as long as we continued to track the system (up to 144 h) but did not appear to be actively replicating. After 16 h in the dual species biofilms, waves of T7 infection could be seen proceeding partially into groups of E. coli embedded within V. cholerae biofilms, but a fraction of E. coli most often survived (Fig 1A). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. E. coli embedded within V. cholerae cell groups can evade exposure to phages in the surrounding medium. (A) Time-lapse series of a dual culture biofilm of E. coli (yellow) and V. cholerae (purple), undergoing T7 phage exposure (infected E. coli cells reporting in cyan/white). The biofilm was grown for 48 h prior to continuous phage introduction thereafter. Time points noted in the upper right of each panel represent time since phage introduction was started. (B) The neighborhood biovolume fraction (biovolume fraction within a 6 μm around each segmented bacterium) of the merged biovolumes of both V. cholerae and E. coli for the first time point in panel A. (C) Mean V. cholerae fluorescence signal found around E. coli cells in biofilms with and without phage exposure (Mann–Whitney U test with n = 9). (D) E. coli biovolume normalized to biovolume prior to the introduction of phage in dual culture with V. cholerae and monoculture controls (Mann–Whitney U test with n = 9, n = 3). (E) Total biovolume of E. coli in dual culture and monoculture control biofilms with and without phage exposure at equivalent time points (Mann–Whitney U tests with n = 8, n = 8, n = 6, n = 7 from left to right). The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3001913.g001 To determine if the remaining E. coli survived in dual species biofilms because of de novo evolution of resistance to T7, we performed runs of this experiment after which all E. coli cells in the chamber were dispersed by agitation and tested for T7 resistance (see Methods). The frequency of T7 resistance in the surviving E. coli population was 10−5, roughly the same as the frequency of resistance prior to the introduction of T7 phages [62]. This outcome shows that there was little or no substantive population compositional shift due to selection for de novo phage resistance (S1C Fig). This is not particularly surprising, as the host and phage population sizes—and, most importantly, the extent of movement and contact events between hosts and phages—are dramatically lower in these experiments compared to those that are typical in well-mixed batch culture. Rather, these experiments suggest that T7-susceptible E. coli survives phage introduction in our biofilm culture conditions by avoiding exposure to them entirely when embedded in groups of V. cholerae. We confirmed that this outcome is specific to the biofilm context by replicating the same experiment in shaken liquid culture beginning with the same cell inoculum and phages introduced at equivalent multiplicity of infection (see Methods). In liquid co-culture conditions with V. cholerae, T7-susceptible E. coli gained no protection against phage exposure and infection (S1D Fig). In the biofilm context, the delay between the start of biofilm growth and phage introduction was important for the experimental outcome; if phages were introduced from the beginning of biofilm growth, rather than 48 h after biofilm growth, then the extent of E. coli protection was all but eliminated (S2 Fig). Our observations above suggested that in dual-species culture conditions, the majority of E. coli that survive phage introduction are the cell groups that have been overgrown and enveloped within the bottom layers of expanding, densely packed V. cholerae clusters. To test this idea quantitatively [16,63], we segmented and merged the cell volumes of E. coli and V. cholerae to calculate the joint neighborhood cell packing density for the 2 species throughout the imaged 3D space (Fig 1B). By visual inspection, regions in which E. coli survived contained a majority of V. cholerae and had relatively high cell packing (biovolume fraction >0.9), in comparison with other regions where E. coli tended to die of phage exposure and cell packing was lower (biovolume fraction = 0.3 to 0.6). We next measured the spatial association of V. cholerae with E. coli to see how this may change in the presence versus the absence of phage exposure. For this measurement, we segmented the E. coli population away from background, and then measured V. cholerae fluorescence in direct proximity within 2 μm of E. coli throughout all replicates with or without phages introduced. Compared to control experiments with no phages (Fig 1C), V. cholerae fluorescence was indeed significantly elevated in close proximity to E. coli after phage exposure, representing the surviving, protected portion of the E. coli population embedded within groups of V. cholerae. This protection effect could be replicated when introducing λ phages instead of T7 phages (S3 Fig), and in a parallel study, we show that the same effect occurs under predation by the bacterium B. bacteriovorus [64]. At the scale of the entire chamber community, E. coli showed higher survival rate in co-culture with V. cholerae than in monoculture on its own (Fig 1D). In absolute terms, the total population size of E. coli after phage exposure in co-culture with V. cholerae was not statistically different from the surviving population size after phage exposure in E. coli monoculture (Fig 1E). This result occurred because E. coli total abundance prior to phage introduction is lower in co-culture with V. cholerae, with which it is competing for space and nutrient resources, but due to embedding of many E. coli cells within V. cholerae clusters, their per-cell survival rate against phage exposure is substantially higher relative to a E. coli monoculture condition (Fig 1D). So, on short time scales after phage introduction (10 h), there is a significant increase in survival rate for E. coli growing in co-culture with V. cholerae, but not yet a significant difference in the absolute abundance of surviving E. coli relative to monoculture conditions. However, our experiments later in the paper demonstrate that on longer time scales (100+ h), the E. coli that survive phage exposure in co-culture within V. cholerae colonies maintain positive net growth and recover from the initial population decline, whereas the surviving E. coli cells from monoculture biofilms do not recover. Before elaborating on this point with longer time scale experiments detailed below, we first turn to the biofilm architectural mechanisms in co-culture biofilms of E. coli and V. cholerae that are responsible for the observations reported in Fig 1.

Protection within V. cholerae cell clusters depends on their packing structure After demonstrating that E. coli cells have reduced exposure to phages when embedded in clusters of V. cholerae, we explored the biofilm architectural features needed for protection to occur. As we have found previously that the extent of V. cholerae cell–cell packing can influence transport of phages and bacteria through biofilms, our first hypothesis based on prior work was that the high-density cell packing of V. cholerae biofilms was important for this protection mechanism [11,16,41]. Our other hypothesis, not mutually exclusive, was that phages may be sequestered away from E. coli by irreversible attachment to the surface of V. cholerae cells in close proximity. To distinguish between these mechanisms, or to estimate their relative contribution to E. coli protection within V. cholerae clusters, we performed new experiments manipulating V. cholerae cell packing in co-culture with E. coli and assessing the degree of attachment and neutralization of T7 phages on the surface of V. cholerae. To alter V. cholerae cell packing structure, we performed co-culture experiments similar to those in the previous section, but using a strain of V. cholerae (denoted ΔrbmA) with a clean deletion of the rbmA locus. This strain cannot produce the matrix protein RbmA, which is not essential for biofilm formation but is necessary for the tight cell packing that is characteristic of mature V. cholerae biofilms (S4 Fig) [44,47,65]. Biofilms without RbmA, in contrast with those of wild type (WT), can be invaded into their interior by planktonic competitor bacteria as well as predatory bacteria such as B. bacteriovorus [11,16]. If the high cell packing to which RbmA contributes is important to the protection of E. coli from phage exposure, we expect that in co-culture biofilms with V. cholerae ΔrbmA, E. coli will be more exposed to T7 phage predation and show different population dynamics relative to control co-cultures with WT V. cholerae. We grew E. coli and V. cholerae ΔrbmA in biofilm co-culture, introduced T7 phages after 48 h as above, and found that the E. coli grown in the presence of V. cholerae ΔrbmA does not exhibit population recovery after phage introduction as it does in co-culture with V. cholerae WT (Fig 2A). This outcome suggests that E. coli does not gain protection from phage exposure amidst V. cholerae ΔrbmA, and that the cell packing architecture of V. cholerae WT is in fact important for this protection effect. If E. coli is protected within V. cholerae WT clusters, but not within ΔrbmA clusters, then in a triculture experiment of E. coli, V. cholerae ΔrbmA, and V. cholerae WT, we expect a statistical shift of E. coli spatial association toward WT V. cholerae after introducing phages as E. coli associated with ΔrbmA V. cholerae are more often killed. We performed this triculture experiment, measuring the average distance between E. coli and V. cholerae WT, and that between E. coli and V. cholerae ΔrbmA, before and after phage introduction. Without the addition of phages into the triculture condition, E. coli cells are just as likely to be associated with WT V. cholerae (median distance: 0.88 μm) as they are with ΔrbmA V. cholerae (median distance: 0.69 μm) (Fig 2B and 2C). When phages are introduced, the remaining E. coli were significantly closer to WT V. cholerae (median distance: 0.59 μm) than they were to ΔrbmA V. cholerae (median distance: 3.46 μm) (Fig 2B). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. E. coli evasion of phages within V. cholerae biofilms depends on the high cell–cell packing produced by WT V. cholerae. (A) E. coli biovolume over time in dual culture conditions with either V. cholerae WT or V. cholerae ΔrbmA (n = 7, n = 8, n = 3–6, n = 3–8 from top to bottom in legend). (B) Average distance between E. coli cells and either V. cholerae WT or ΔrbmA in a triculture condition with or without phage exposure (Wilcoxon paired comparison tests with n = 9). (C, D) Representative images from the triculture condition with E. coli (yellow), V. cholerae WT (purple), and V. cholerae ΔrbmA (cyan) (C) without phage exposure and (D) after phage exposure. (E) PFU recovered after incubation of starting T7 phage inoculum with either no bacteria, V. cholerae, E. coli WT, or E. coli ΔtrxA over a 60-min time course. E. coli ΔtrxA allows for T7 phage attachment and genome ejection, but not for phage replication. Each trajectory shows the data for 1 run of each treatment (n = 3 for each treatment, giving 3 traces per treatment). (F) The neighborhood biovolume fraction of the merged biovolumes of both V. cholerae genotypes and E. coli from panel (D). The data underlying this figure can be found in S1 Data. PFU, plaque-forming unit; WT, wild type. https://doi.org/10.1371/journal.pbio.3001913.g002 The experiments above indicate that the packing architecture of V. cholerae WT biofilms is important for phage exposure protection of E. coli within them, as E. coli gains little if any protection from phage exposure in proximity to loosely packed V. cholerae ΔrbmA. These data do not exclude the possibility that this difference is due in part to sequestration of phages by attachment to V. cholerae cells, which could occur more often in WT clusters with higher density of available V. cholerae cell surface relative to clusters of the ΔrbmA strain. To help assess whether sequestration of phages by direct attachment to V. cholerae cell surface was important, we incubated V. cholerae, E. coli, and ΔtrxA E. coli with T7 phages in shaken liquid culture, tracking the ability to recover T7 phages every 5 min for 1 h (Fig 2E). In addition to a blank media control, the ΔtrxA E. coli strain was included because this strain can adsorb phages normally but undergoes abortive infection, preventing phage amplification [66]. As expected, with E. coli ΔtrxA incubation, T7 PFU recovery steadily decreased until saturation at 1 h. Incubated with T7-sensitive E. coli WT, T7 PFU recovery initially decreased as phage adsorption occurred, followed by a rapid increase as new phages were released. Another round of latency and amplification then occurred before the 1-h stop time. Incubated with V. cholerae, no change in T7 PFU recovery was observed, which was identical to the blank media control for the duration of the experiment. These data suggest that T7 phages are not sequestered by adsorption to the V. cholerae cell surface. Though T7 phages do not appear to adhere to V. cholerae cell surface in planktonic culture, within biofilm cell clusters V. cholerae surface properties may differ, and they are also embedded in matrix polysaccharide and protein components. With this in mind, we also performed experiments with fluorescently labeled T7 phages in biofilm growth conditions to determine if T7 is sequestered to V. cholerae cell groups in this context. Labeled phages were introduced to V. cholerae and E. coli dual culture biofilms and tracked over time, and we found that they localize strongly to unprotected E. coli cells and not to V. cholerae (S5 Fig). When V. cholerae monoculture biofilms were grown in flow devices with labeled phages added continuously in the media, we saw no accumulation of phages along the outer surface of V. cholerae cell clusters (S6 Fig). We found occasional phages within V. cholerae cell groups along the basal glass substrata, but not in the rest of their interior volume (S6 Fig). As phages were added from the beginning of biofilm growth onward in this experiment, the results suggest that V. cholerae biofilm colonies expanded over the top of initially glass-attached phages, rather than phages diffusing through biofilms to the basal layer. Taken all together, the data in the experiments above suggest that E. coli is unexposed to phages within WT V. cholerae biofilms due to their architectural features, with minimal if any sequestration of phages by direct adsorption to the surface of V. cholerae cells.

Cohabitation with V. cholerae alters E. coli matrix production As noted in the first Results section, E. coli accumulates less quickly in co-culture with V. cholerae than it does on its own, owing to competition for limited space and resources. Previous work has shown that, in monoculture, E. coli biofilms can protect themselves against phages once they begin to produce curli matrix proteins, which interrupt phage binding on the single-cell scale and contribute to biofilm architecture that blocks phage diffusion on the collective cell scale [55]. Curli production does not usually start until several days after beginning E. coli biofilm growth in microfluidic culture conditions [55], and we wondered if growing together with V. cholerae in dual culture might delay or disrupt curli formation. We note again that in the experiments in previous sections, biofilms were cultivated for too short a time for E. coli to begin producing curli matrix even in monoculture conditions. Here, we explored whether co-culture with V. cholerae impacts curli production on longer time scales, when E. coli on its own would ordinarily be able to protect itself against phage exposure via curli production. If curli production is reduced or disrupted by growth with V. cholerae as a competitor, we would expect no difference in phage exposure survival between E. coli WT and a strain lacking curli matrix in co-culture with V. cholerae. To explore this possibility, E. coli WT and an isogenic curli null deletion strain (denoted ΔcsgA) were grown either on their own or in co-culture with V. cholerae for 96 h. This cultivation period is twice as long as is normally required for monoculture E. coli WT biofilms to produce curli and block phage diffusion. Biofilms were imaged at 96 h, exposed to phages at 104 per μL under 0.1 μL/min flow for 16 h, and then imaged again to document population sizes of WT and ΔcsgA before and after phage introduction. As expected, the E. coli WT monoculture biofilms had the highest level of survival, with some replicates showing net increases in population size after the 16-h phage treatment. E. coli ΔcsgA monoculture biofilms, lacking any protection mechanism against phage exposure, had the lowest level of survival. In contrast, when in co-culture with V. cholerae, E. coli WT and ΔcsgA (Fig 3A) showed no substantial difference in survival to phage exposure (pairwise test not significant with Bonferroni correction), suggesting that curli production is no longer necessary for T7 exposure protection for WT E. coli in this context. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. E. coli biofilms’ normal production of curli matrix protein is interrupted in co-culture with V. cholerae to the extent that phage protection is no longer provided by E. coli biofilm matrix. (A) E. coli biovolume normalized to biovolume prior to phage introduction in dual culture and monoculture conditions for both E. coli WT and E. coli ΔcsgA (Mann–Whitney U tests with n = 7, n = 12). (B) Total E. coli biovolume with and without phage treatments at equivalent time points (Mann–Whitney U tests with n = 12). In these experiments, in contrast with Fig 1E, biofilms were grown for longer periods before phage addition such that E. coli WT on its own could produce protective curli matrix prior to phage addition. (C) Frequency distribution of csgBAC transcriptional reporter fluorescence around E. coli in monoculture and dual culture conditions. (D) Frequency distribution of curli immunofluorescence intensity in proximity to E. coli in monoculture and dual culture conditions. (E) Dual culture conditions of E. coli (yellow) and V. cholerae (purple) before phage exposure (top) and after 16 h of continuous phage exposure (bottom). (F) Monoculture conditions of E. coli before phage exposure (top) and after phage exposure (bottom). The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3001913.g003 To assess why curli-based phage protection was no longer operating for E. coli even in co-culture biofilms that had grown over 96 h, we repeated the experiments above with an E. coli WT strain harboring reporter fusions for monitoring csgBAC transcription and curli protein production. The transcriptional reporter was made previously by introducing mKate2 in single copy on the chromosome within the csgBAC operon encoding 2 subunits of curli fiber protein (CsgB baseplate and CsgA primary curli monomer) and CsgC, which inhibits improper aggregation of CsgA monomers [55]. The protein production reporter was also made previously by introducing a 6x-His fusion tag to csgA, which allowed for in situ immunostaining of curli fibers produced by E. coli during growth in monoculture and co-culture with V. cholerae. As noted previously, the total population size of E. coli in biofilms with V. cholerae is lower than that found in monoculture (Fig 3B and 3E and 3F). On a per cell basis over the entire chambers, csgBAC transcription and curli immunostaining were significantly higher for E. coli growing alone versus E. coli growing in co-culture with V. cholerae (Fig 3C and 3D). These patterns manifested at the scale of the whole chamber; on a smaller spatial scale, E. coli distance from V. cholerae in co-culture was not correlated with curli production (S7 Fig). Overall, these results suggest that E. coli curli production is substantially reduced when growing together with V. cholerae. It is not clear exactly why this is the case, but we speculate here that co-culture with V. cholerae alters one or a combination of nutrient availability, microenvironment osmolarity, and envelope stress experienced by E. coli, all of which influence the regulation of curli production [52]. The reduction in curli production may in turn contribute to the loss of curli-based protection against T7 phages even after long incubation periods over which E. coli normally develops curli-based phage protection on its own in monoculture. Together with the previous section, our experiments here also indicate that while E. coli has a lower ability to protect itself via curli matrix production when in co-culture, it can avoid phage exposure altogether when it been overgrown by and embedded within V. cholerae colonies.

[END]
---
[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001913

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/