Abstract
A variety of cell surface structures, including capsular polysaccharides (CPS), dictate the interactions between bacteria and elements of their environment including their viruses (bacteriophages). Members of the prominent human gut Bacteroidetes characteristically produce several phase-variable CPS, which have been demonstrated as key determinants in interacting with the host immune system. However, the contributions of Bacteroidetes CPS to bacteriophage interactions are unknown. We used engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the CPS they express, to isolate bacteriophages from two locations in the United States. Testing each of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of eight different single CPS, or no CPS at all, revealed that each infects only a subset of strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was sufficient to abolish infection for several individual bacteriophages.
Likewise, infection of wild-type B. thetaiotaomicron with one bacteriophage from this collection selected for a cell population expressing non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing by this bacteriophage, but surviving bacteria increased expression of a family of 9 phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins enhances resistance to this bacteriophage. Our results reveal distinct roles for Bacteroides CPS in mediating bacteriophage susceptibility. Beneath this vanguard protective layer, additional mechanisms exist to protect these bacteria from being eradicated by bacteriophage. Given the diversity of CPS and other phase-variable surface structures encoded by gut-dwelling Bacteroidetes, our results provide important insight into the adaptations that allow these bacteria to persist despite bacteriophage predation and hold important implications for using bacteriophages therapeutically to target gut symbionts.
Introduction
The community of cellular microorganisms in the human intestinal tract is dominated by a diverse population of bacteria, with hundreds of different species and even more strains typically present within one person1. In the face of frequent perturbations from a variety of environmental pressures, including diet changes, antibiotics and host immunity, the relative abundances of different bacteria may vary greatly within an individual over short time periods. However, the microbiota members within a host generally remain stable over long time periods2,3, suggesting that individual bacteria have evolved strategies that enable them to be resilient in the face of challenges. One potential adaptive mechanism that has clearly been diversified in gut resident Bacteroidetes is the ability of individual strains to alternately produce several different capsular polysaccharides (CPS), which are extracellular matrix components encoded by cps biosynthetic loci. Many cps loci in the Bacteroidetes are under the control of phase variable promoters, allowing for substantial phenotypic heterogeneity within an isogenic population. Furthermore, there is much broader diversification of cps loci among different strains within a species4–7. While a few studies have shown that these CPS take part in evasion or modulation of host immunity4,8–11, the sheer diversity and number of CPS synthesis loci in the Bacteroidetes suggests that they could also fill other fundamental roles4,6,12,13.
Bacterial viruses or bacteriophages (herein, phages), like the bacteria on which they prey, vary greatly across individual gut microbiomes and are even responsive to host dietary changes or disease states14–17. Compared to its bacterial counterpart, far less is understood about the human gut virome, especially the mechanisms governing phage-bacteria interactions. Specifically, while phages that target several species of Bacteroides (the prominent genus of the Bacteroidetes phylum in humans) have been shown to exhibit species-or strain-specificity18–21, little is known about the molecular interactions that drive bacterial susceptibility22 or the mechanisms by which these bacteria persist despite an abundance of phages in the gastrointestinal tract.
Given the observations that Bacteroides CPS are extremely variable, even within members of a single species4,12, and employ complex regulatory mechanisms that diversify expression in members of a population4,23, CPS are ideal candidates for modulating Bacteroides-phage interactions. Thus, we sought to test the hypothesis that CPS are direct positive or negative mediators of Bacteroides-phage interactions. To accomplish this, we employed a panel of engineered strains of the model symbiont Bacteroides thetaiotaomicron that each constitutively expresses a different single CPS or none at all. While our results clearly support the conclusion that specific CPS may either block or be required for phage infection, they also reveal that B. thetaiotaomicron possesses additional phage-evasion strategies that function in the absence of CPS. For one phage, CPS-independent survival involves altered expression of nutrient receptors and increased expression of phase-variable surface lipoproteins by the surviving bacteria. One hypothesis is that these latter functions also encode active resistance mechanisms. This idea is supported by locking on expression of one of these lipoproteins, which we show to be an additional determinant of phage tropism, conferring increased resistance to this phage. Our results provide a mechanistic glimpse into the intricacy of bacterial-phage interactions that exist in the human gut and provide a foundation for future work aimed at leveraging these interactions as a facet of targeted manipulation of the gut microbiome.
Results
Individual bacteriophages target B. thetaiotaomicron in a CPS-dependent fashion
To test the hypothesis that variable Bacteroides surface CPS mediate interactions with phages, we isolated phages that infect B. thetaiotaomicron VPI-5482 (ATCC 29148). To maximize our chances of collecting phages that could differ in their interactions with CPS, we used the wild-type strain of B. thetaiotaomicron that expresses 8 different, phase-variable CPS, each encoded by a different multi-gene cps locus; an isogenic panel of 8 single CPS-expressing strains (designated “cps1” through “cps8”)25; and an acapsular strain in which all cps genes from all 8 loci are deleted24 as independent hosts for phage isolation. Primary sewage effluent from two cities within the United States (Ann Arbor, Michigan and San Jose, California; separated by approximately 3,300 kilometers) was used as the source for these phages (for further details on phage isolation, see Methods and Table S1). All phages were plaque purified at least 3 times and high titer lysates were generated for each of the 71 phages. Plaque morphologies varied greatly among the strains, ranging in size from <1 mm to 3 mm or greater and in opacity from very turbid to clear (Figures 1, S1).
To determine if phages isolated on each individual host strain are restricted in their ability to infect other strains, we systematically tested each phage against each of the 10 B. thetaiotaomicron host strains that varied only in the CPS they are capable of expressing (n=3). Hierarchical clustering of the host infection profiles revealed a cladogram with 3 main branches that each encompasses phages from both collection sites, although substantial variation in host tropism exists for phages within each branch (Figure 1). Furthermore, individual phages within each branch displayed a range of plaque morphologies (Figures 1, S1), suggesting additional diversity in the collection that is not captured by this assay. Finally, host range assays were robust when performed by different experimenters at different research sites (Figure S2).
Phages in Branch 1 generally exhibited robust infection of the acapsular strain, although 3 of these phages did not form plaques on this host. Furthermore, phages in Branch 1 generally exhibited robust infection on strains expressing CPS7 or CPS8 alone, although a separate subset of 3 phages did not form plaques on the CPS8 expressing strain. Some Branch 1 phages also displayed less efficient infection of other strains with the exception of cps4, which was not infected by any phages in this group. Interestingly, ARB154 exclusively infected cps8, an uncommon CPS among B. thetaiotaomicron strains that appears to be contained in a mobile element4. Phages in Branch 2 generally exhibited robust infection of all strains except cps2, cps3 and cps4. However, subsets of this group were unable to infect cps1 or cps6. Finally, Branch 3 tended to exhibit strong infection of wild-type, cps1, cps2, and cps3, with some variations. Some Branch 3 phages also exhibited the ability to infect the cps7 and acapsular strains but were the only branch that poorly infected cps8. A subset of phages on Branches 1 and 3 failed to infect the acapsular strain, suggesting that they may require the presence of CPS for infection. Taken together, the observed variations in phage infection of B. thetaiotaomicron strains expressing different CPS, or none at all, provide initial support for our hypothesis that these surface structures are a key mediator of B. thetaiotaomicron-phage interactions.
Elimination of specific CPS subsets alters bacterial susceptibility to phages
One explanation for the differences in host infectivity described above is that there are distinct, CPS-dependent mechanisms of phage adsorption. For example, several phages robustly infect the acapsular strain, indicating that a capsule-independent cell surface receptor mediates infection for these examples. However, each of these phages also infect subsets of the single CPS-expressing strains, suggesting that some “non-permissive” CPS could block access to cell surface receptors, while other “permissive” CPS fail to do so. For phages that do not efficiently infect the acapsular strain, one or more CPS may serve as a direct phage receptor(s) or as a required co-receptor.
To further define the roles of specific CPS during phage infection, we investigated a subset of 6 phages (ARB72, ARB78, ARB82, ARB101, ARB105, and ARB25). All 6 of these phages can infect the wild-type B. thetaiotaomicron strain that variably expresses its 8 different CPS and 5 of them infect the acapsular strain poorly or not at all (Figure 1). We tested our hypothesis that some CPS are required as receptors or co-receptors by deleting only the subsets of CPS biosynthetic genes encoding permissive capsules based on our prior experiments with single CPS-expressing strains. For ARB72, which most robustly infects the CPS1-and CPS3-expressing strains, simultaneous elimination of both of these capsules from wild-type B. thetaiotaomicron reduced infection below the limit of detection (Figure 2A). Likewise, elimination of the most permissive CPS for four other phages (ARB78, ARB82, ARB101 and ARB105) significantly reduced B. thetaiotaomicron infection by these phages, in some cases in the presence of permissive CPS (Figure 2B-E).
For ARB25, which infected 7 of the 10 strains tested in our initial plaque assays (Figure 1), some single and compounded cps gene deletions significantly reduced infection rates or reduced them below the limit of detection. The most noteworthy of these was deletion of the cps4 (initially determined in Figure 1 to be non-permissive for ARB25) in combination with deleting the permissive cps1, which completely eliminated detectable infection (Figure 2F). While individual deletion of four other permissive CPS (cps1,6,7,8) led to partially reduced infection, so did single deletions of two CPS initially determined to be non-permissive (cps3 and cps4), suggesting the possibility of more complicated regulatory interactions, which are known to occur with Bacteroides CPS23,26. Interestingly, strains lacking either cps4 or cps1/cps4 together compensated by significantly increased relative expression of the non-permissive cps2 locus, which could contribute to ARB25 resistance (Figure 2G).
A strain expressing only two of the non-permissive CPS (CPS2 and CPS3) could not be detectably infected by ARB25 (Figure 2F, “2,3 only” condition). However, a strain expressing CPS2,3,4 regained some susceptibility (Figure 2F, “2,3,4 only” condition), indicating that CPS4 is capable of mediating some infection by this phage (addressed further below). In contrast to sole expression of cps2 and cps3 promoting resistance to ARB25, deletion of the cps2 and cps3 loci led to dominant expression of cps1 and cps4 genes, which increased infection efficiency and led to the production of clearer plaques (Figure 2F,H). Additional support for the idea that loss of cps4 alone modifies ARB25 susceptibility comes from plaque morphologies arising from infection of the Δcps4 strain, which produced smaller and more turbid plaques, demonstrating that when infection does occur it is less productive (Figure 2H). Thus, CPS4-expressing cells are in some cases susceptible to ARB25, and loss of the genes encoding this capsule result in increased expression of CPS2, perhaps through alleviation of the UpxZ transcription termination mechanism, as described for capsule regulation in B. fragilis23. Because this programmed shift in CPS expression may increase resistance to phage, it is possible that phase-variation can equip Bacteroides populations to survive phage predation through selection of individual cell sub-populations that are expressing non-permissive CPS.
Finally, we used ARB25 to test if purified, exogenous CPS could modify phage infection as has been shown for other phage-CPS interactions27. Given that strains expressing CPS1 and CPS2 show different susceptibility to infection by ARB25, we tested purified preparations of both CPS types, expecting that exogenous CPS2 might inhibit ARB25 infection of the acapsular strain if it is capable of directly interfering with recognition of a surface receptor. Arguing against this hypothesis, pre-incubation with purified preparations of CPS2 or CPS1 (a permissive capsule control) both resulted in no significant difference in plaquing efficiency on the acapsular strain (Figure S3A), suggesting that exogenous CPS cannot block infection by phage ARB25.
B. thetaiotaomicron acquires transient resistance to phage infection
Interestingly, we observed that liquid cultures of the various B. thetaiotaomicron strains infected with ARB25 did not show evidence of complete lysis after 36 hours of growth, as determined by optical density at 600 nm (OD600) (Figure 3). Previous reports demonstrated that B. fragilis20 and B. intestinalis21 exhibited transient resistance to phage infection that could be “reset” through removal of the phage from the culture, although the underlying mechanism of this transient resistance was not determined. Based on these observations, we questioned whether similar transient resistance occurs with B. thetaiotaomicron and whether this resistance could be dependent on CPS expression. Growth curves of each of the CPS-expressing strains inoculated with active or heat-killed ARB25 confirmed our initial host range assays, except that cultures containing the CPS4-expressing strain were sensitive to killing by this phage in liquid culture, while cultures of the CPS6-expressing strain had no significant decrease in OD600 (Figure 3). In these experiments, most strains deemed to be susceptible via plaque assay (Figure 1) exhibited an initial lag in growth or a drop in OD600. As expected, the cps2 and cps3 strains remained resistant to ARB25 infection for the duration of the experiments. However, after initial growth inhibition, the susceptible strains displayed either growth stagnation without complete loss of culture density (cps4, cps5, cps7, cps8) or resumption of growth (wild-type, acapsular, cps1) to near uninfected levels, the latter suggesting outgrowth of a resistant subpopulation of bacteria. Culture supernatants taken from ARB25 post-infected, wild-type B. thetaiotaomicron still contained high phage titers when exposed to naïve bacteria that had not been exposed to phage, excluding the possibility that the phages were inactivated (Figure S3B).
We next determined whether strains that had survived or proliferated after exposure to phage retained resistance after removal of phage. In order to isolate phage-free bacterial clones, we isolated individual colonies by sequentially streaking each twice from a subset of the cultures that gained resistance to ARB25 (WT, acapsular, cps1, and cps4) as well as the inherently ARB25-resistant cps2 strain. The majority of clones isolated using this process were free from detectable phage (see Methods). We then re-infected each clone with live ARB25 and monitored susceptibility by delayed growth or drop in the culture density as compared to infection with heat-killed phage. As expected, the cps2 strain remained resistant. On the other hand, the majority of clones (42/61 total, ~69%) of the other four strains regained susceptibility (Table 1), suggesting that resistance to this phage is not caused by a permanent genetic alteration in most cases.
Phage resistant wild-type B. thetaiotaomicron populations exhibit altered cps locus expression
Given that CPS type is correlated with resistance to phage infection (e.g., ARB25 fails to infect strains expressing CPS2 or CPS3 under all conditions tested), we hypothesized that wild-type B. thetaiotaomicron cells inherently expressing resistant capsules would be positively selected in the presence of phage. To test this, we infected wild-type B. thetaiotaomicron with ARB25 and monitored bacterial growth. For cells treated with a high MOI (MOI ≈ 1), culture turbidity increased very slightly, declined before 3 hours after infection, and finally increased again to ultimately achieve a high growth level as previously observed (Figure 4A).
Interestingly, bacterial cultures originating from different single colonies displayed variable growth kinetics and possibly resistance frequency to ARB25, with the growth of one clone barely delayed by treatment with live ARB25. Next, we measured if infection with ARB25 resulted in a change in CPS expression by the phage-resistant B. thetaiotaomicron population. In support of our hypothesis, B. thetaiotaomicron exposed to heat-killed phage predominantly expressed genes encoding CPS3 and CPS4, which we typically observe in in vitro culture. Treatment with live ARB25 resulted in a dramatic loss of cps1 and cps4 expression (capsules for which combined loss eliminates ARB25 infection, Figure 2F), with a concomitant increase of expression of the non-permissive cps3 (Figure 4B). Similar growth and expression phenotypes occurred in cultures treated with a low MOI (MOI ≈ 10−4), albeit with higher culture turbidity before a decline and subsequent resumption of growth (Figure S4). Dirichlet regression (see Methods) supported significant cps expression changes for cps1, cps3, and cps4 in response to ARB25 (p < 0.01 for experiments with both low and high MOI). Notably, the most resistant of the three bacterial clones (as evidenced by faster outgrowth post-infection) in each of the two experiments (low and high MOI) exhibited similar cps expression to the other clones after treatment with live phage, but expressed lower levels of permissive cps1 and cps4 and higher levels of non-permissive cps3 in heat-killed phage treatment groups (Figure S5). This alteration in expression of permissive and non-permissive CPS may contribute to the ability of these clones to resume growth more rapidly after phage challenge because it was already skewed towards expression of non-permissive CPS.
Multiple layers of phase-variable resistance functions equip B. thetaiotaomicron to survive phage predation
The results described above support a model in which some individual cells within a B. thetaiotaomicron population are pre-adapted to resist eradication by a single phage like ARB25 through expression of different CPS. Complex phase-variation mechanisms have already been described in controlling CPS expression in B. thetaiotaomicron and B. fragilis4,23,28 and we demonstrate the ability of CPS to dictate interactions between phages and B. thetaiotaomicron (Figures 1, 2, 3). However, in acapsular B. thetaiotaomicron infected with ARB25, we observed significant growth after initial lysis that frequently regained susceptibility when isolated from and subsequently re-challenged with ARB25 (Figure 3, Table 1), suggesting the emergence of a transiently phage-resistant subpopulation in the absence of CPS. To determine if additional phage resistance mechanisms are involved, we performed whole genome transcriptional profiling by RNA-sequencing (RNA-seq) to measure transcriptional differences between ARB25 post-infected and mock-infected B. thetaiotaomicron. In a wild-type B. thetaiotaomicron population, in which cells retain the ability to phase-vary expression of their eight different CPS, the transcriptional profiles of bacterial populations surviving after ARB25 infection (n=3) were largely characterized by decreased gene expression: among a total of 56 genes that exhibited significant expression differences >3-fold between B. thetaiotaomicron exposed to live and heat-killed ARB25, 51 genes were decreased in post-infected bacteria (Figure 5A, Table S2). Most of these genes with decreased transcription (44/51) belong to the loci encoding CPS1 and CPS4, consistent with our findings by qPCR that lower expression of these CPS occurs after ARB25 challenge (Figure 4B). Correspondingly, increased expression of genes encoding CPS2 and CPS3 was also apparent by RNA-seq, but did not reach statistical significance. Interestingly, two additional gene clusters encoding different outer-membrane “Sus-like systems”, which are well-described Bacteroidetes mechanisms for import and degradation of carbohydrates and other nutrients2,29, were also decreased in post-infected bacteria (6/7 remaining non-cps genes). The central features of these systems are outer membrane TonB-dependent transporters (similar to E. coli TonA, or T one phage receptor A; the first described phage receptor30), suggesting the possibility that the proteins encoded by these genes are part of the receptor for ARB25.
To further investigate CPS-independent mechanisms that allow B. thetaiotaomicron to avoid eradication by phage, we performed identical RNA-seq experiments on acapsular B. thetaiotaomicron populations exposed to heat-killed or live ARB25. In this case, we hypothesized that eliminating the ability to phase vary capsules would better reveal the mechanism(s) that allows the acapsular strain to survive ARB25 predation, possibly by further reducing expression of putative phage receptors like the Sus-like systems identified above. Expression of the same two Sus-like systems (BT2170-73, BT2365) that were decreased in ARB25-exposed wild-type were also decreased to similar levels in acapsular B. thetaiotaomicron (Figure 5B). Otherwise, the transcriptional profiles of acapsular B. thetaiotaomicron surviving after ARB25 infection (n=3) were largely characterized by increased gene expression: 71 of the 81 genes differentially regulated >3-fold between ARB25-infected and the heat-killed reference showed increased expression in ARB25 post-infected cells. All but 3 of these genes were unique to the post-infection transcriptome of acapsular B. thetaiotaomicron compared to wild-type B. thetaiotaomicron (Figure 5B, Table S2).
Among the 71 genes with increased expression in post-infected acapsular B. thetaiotaomicron, 24 genes were part of 8 different gene clusters that encode putative tyrosine recombinases along with pairs of outer membrane lipoproteins and OmpA β-barrel proteins (Figure 5C). One of these genes (BT1927) was previously characterized as encoding a phase-variable, S-layer protein, which organizes into a tessellated structure on the cell surface and when locked into the “on” orientation promoted increased B. thetaiotaomicron resistance to complement-mediated killing31. The remaining 7 gene clusters share both syntenic organization and homology to this original S-layer gene cluster. Closer scrutiny of the promoter regions upstream of the 7 newly identified gene clusters encoding putative lipoproteins revealed that each is also flanked by a pair of imperfect, 17 nucleotide palindromic repeats (Figure 5C). Three of these newly identified repeats are identical to the repeats known to mediate recombination at the BT1927 promoter31. The remaining 4 sequences only varied by the sequence of a trinucleotide located in the middle of each imperfect palindrome (colored blocks in Figure 5C middle). Finally, amplicon sequencing of each promoter supported the existence of the proposed recombination events in 5 of the 7 newly identified loci (Figure S6).
Among the remaining genes that were significantly up-or down-regulated in post ARB25-infected acapsular B. thetaiotaomicron, there was an additional signature of genes for which DNA recombination may be involved in re-organizing expression of cell surface proteins. Specifically, the expression of 3 of 4 genes in an operon (BT1042-45) previously implicated in utilization of host glycans32 were expressed an average of 6.9-fold less in ARB25-infected acapsular cells compared to heat-killed controls. Correspondingly, 5 genes in an adjacent operon (BT1046-51) with similar arrangement and predicted functions exhibited an average of 8.3-fold increased expression in acapsular B. thetaiotaomicron exposed to live ARB25. Both of these operons have been previously linked to transcriptional regulation by a nearby extra-cytoplasmic function sigma (ECF-σ), anti-σ factor pair, such that when the single ECF-σ coding gene (BT1053) is deleted, the ability to activate the adjacent operons is eliminated33. Based on 1) the ARB25-dependent shift in gene expression described above; 2) previously established common ECF-σ regulation of the BT1042-45 and BT1046-51 operons; 3) the observation that two genes encoding TonB-dependent transporters (BT1040, BT1046) appear to be truncated at their 5’ ends compared to BT1042 (Figure 4, S7A) and only the full-length BT1042 sequence harbors a required anti-σ contact domain33; and 4) annotation of a gene encoding a putative tyrosine recombinase (BT1041) located in the middle of this locus, we hypothesized that this gene cluster possesses the ability to undergo recombination and that specific combinatorial variants are selected under phage pressure.
To test this hypothesis, we designed PCR primer pairs (Figure 5D, green dumbbells) to detect both the originally annotated sequence orientation and 3 potential alternative recombination states derived from either moving the full-length 5’ end of BT1042 to one of two alternative susC-like genes or an internal rearrangement derived from recombination of two incomplete susC-like genes (Figure 5D, variants 1-3). In support of our hypothesis, we were able to detect by both PCR (Figure 5E) and amplicon sequencing (Figure S7B) the presence of all 5 predicted alternative recombination states (Figure 5D,E amplicons 2, 3, 4, 6, 7), plus the 3 expected from the originally published genome assembly34. In further support of our hypothesis, an insertion mutation in the associated tyrosine recombinase-coding gene (BT1041) locked the corresponding mutant into the native genomic architecture as determined by the presence of amplicon 1, but the absence of amplicons 2 and 3 (Figure 5E). Further sequence analysis and tracking of single nucleotide polymorphisms in the 5’ ends of the three recombinationally active susC-like genes narrowed the recombination site down to a 7 bp sequence that is flanked by an imperfect direct repeat encompassing over 132 additional downstream bp that may also influence recombination specificity (Figure S7B). Thus, three separate operons that are under the transcriptional control of a single ECF-σ regulator and are involved in utilization of host glycans, are also able to undergo recombinational shuffling via a tyrosine recombinase/direct repeat mediated mechanism to vary which of the three operons is expressed to produce its corresponding surface proteins. This strategy is similar to recombinational shufflons involving nutrient utilization functions that have been characterized in B. fragilis35,36, with the exception that in the example described here, recombination occurs between direct repeats instead of palindromes. One explanation for this phenomenon is that shufflons evolved to subvert phage infection by expressing alternate cell surface receptors for important nutrients, which are also targeted by phages. Contrary to this hypothesis that the proteins encoded by BT1042-45 might serve as a receptor for ARB25, elimination of the genes spanning BT1033-52 did not eliminate ARB25 infection in the acapsular strain, suggesting that an additional or different receptor(s) exists. Interestingly, the BT1033-52 mutant exhibited variable plaquing efficiency compared to the acapsular parent (Figure S8), suggesting that loss of these genes cold exert global effects that mediate susceptibility to ARB25.
Since the gene encoding the original outer membrane S-layer protein (BT1927), and its downstream gene (BT1926), were among the most highly activated (147-and 114-fold, respectively) in post-infected, acapsular B. thetaiotaomicron, and can be alternatively locked into the “on” or “off” orientations by mutating the recombination site upstream of the phase-variable promoter31, we chose to focus on the role of this single function in resisting phage infection. We re-engineered acapsular B. thetaiotaomicron to contain locked “on” and locked “off” versions of this promoter and evaluated sensitivity to ARB25 and another phage, SJC01, which has a similar infection profile on B. thetaiotaomicron (Figure 1). Consistent with the observation that S-layer is highly activated in acapsular B. thetaiotaomicron infected with ARB25, acapsular S-layer “off” cells were more effectively killed in the presence of live phage relative to acapsular S-layer “on” cells (Figure 6A). Interestingly, SJC01 showed the opposite effect, as it more effectively killed cells with S-layer locked “on” versus cells with S-layer locked “off” (Figure 6B). These data indicate that in addition to the 8 surface-exposed capsular polysaccharide types, S-layer lipoproteins can function as positive or negative determinants of phage tropism in B. thetaiotaomicron. Finally, the observation that CPS5,6 and homologous S-layer like functions are broadly represented in gut Bacteroidetes31, suggest that these two mechanisms help to diversify members of this phylum under phage-mediated selection.
Discussion
Production of multiple phase-variable CPS is a hallmark of human gut Bacteroidetes. Previous work has revealed the importance of Bacteroides CPS in interactions with the host immune system4,8,37,38. However, other biological roles for Bacteroides CPS remain relatively unexplored. Using a panel of B. thetaiotaomicron strains that express individual CPS, we tested a previously inaccessible hypothesis: that Bacteroides-targeting phage can be both inhibited and assisted by the repertoire of capsules expressed by their host bacteria. Our data clearly indicate that production of specific CPS is associated with alterations in phage susceptibility, which is underscored by the observation that none of the 71 phages characterized here infect every CPS-variable strain that we tested (Figure 1). Phage-mediated selection and interactions with the host immune system help to explain both the extensive diversification of CPS structures in gut-resident Bacteroidetes4,12 and their complex phase-variable regulation mechanisms within a given strain or species23,25. Surprisingly, our results also reveal that additional phase variable markers are expressed by B. thetaiotaomicron under phage-mediated selection, highlighting that other strategies exist in Bacteroides for surviving in the face of phage predation.
There are several mechanisms through which CPS could promote or prevent phage infection. First, CPS may sterically mask surface receptors to block phage binding, although additional specificity determinants must be involved because no individual phage that infects the acapsular strain is blocked by all single B. thetaiotaomicron CPS. These specificity determinants could be driven by CPS structure (physical depth on the cell surface, polysaccharide charge, permeability) or be actively circumvented by the presence of polysaccharide depolymerases on the phage particles, as has been described in other phage-bacterium systems (e.g., E. coli K1 and phiK1-539). Alternatively, certain permissive CPS could serve as obligate receptors40 (i.e., phage that do not infect acapsular) or more generally increase the affinity of a phage for the bacterial cell surface – similar to what has been proposed in the “bacteriophage adhering to mucus” model, whereby hypervariable domains on phage capsids facilitate adherence to mucus and increase the frequency of bacteria-phage interaction41. This latter type of adherence to CPS might increase the likelihood that a phage would contact its receptor by sustained interaction with the extracellular matrix. Some combination of these possibilities is likely to explain the host range infection profile for the majority of the phages in our collection. Collectively, our observations provide the foundation for future mechanistic work, beginning with phage genome sequencing, aimed at understanding the physical and chemical interactions that mediate infection of B. thetaiotaomicron and other Bacteroides by their phages.
Using ARB25 as a representative from our larger collection, we demonstrate that infection with this single phage does not fully eradicate presumably susceptible B. thetaiotaomicron populations. Rather, resistant cells grow, often quickly, after what we interpret to be an initial lytic event (Figure 3). Similar observations were previously made with ΦCrass001, a phage that infects B. intestinalis 21. Specifically, though ΦCrass001 robustly formed plaques on lawns of B. intestinalis, it failed to eradicate this bacterium in liquid culture. We hypothesize that ΦCrass001 undergoes lytic growth on a subset of B. intestinalis cells expressing permissive CPS. Subsequently, clones expressing resistant CPS would grow to dominate a culture after phage challenge, as we observed with ARB25.
Given the roles of CPS in mediating B. thetaiotaomicron-phage interactions, the outgrowth of a phage resistant sub-population was especially surprising in the context of acapsular B. thetaiotaomicron. While wild-type B. thetaiotaomicron primarily appears to survive continued ARB25 predation by pre-adaptive CPS variation or by shifting the population to express non-permissive CPS, the acapsular strain instead shifts to increased expression of phase-variable sets of surface proteins, at least one of which (BT1927-26) confers increased resistance to ARB25 when locked “on” in acapsular B. thetaiotaomicron. A previous study measured that only 1:1000 B. thetaiotaomicron cells in an unchallenged population express the S-layer encoded by BT1927 31. Given that ARB25 non-permissive CPS can comprise up to 40% of the expressed capsule population (e.g., CPS3 in Figure 4B), the rapid emergence of cells expressing alternative CPS could be explained by this frequency difference, in which case S-layer expressing wild-type bacteria might also emerge given longer phage exposure times. The original B. thetaiotaomicron S-layer study also demonstrated that locking the invertible promoter for the BT1927 S-layer into the “on” orientation facilitated survival against complement-mediated killing31, suggesting that orthogonal roles for this and related proteins exist in B. thetaiotaomicron and likely facilitate survival in the face of diverse environmental disturbances. Combined with our data on CPS-mediated phage tropism, our observations that the BT1927-encoded S-layer confers resistance to some phages, that 7 other homologous systems are also upregulated after exposure to ARB25, and that a shufflon harboring three recombinationally-variable nutrient acquisition operons exists in B. thetaiotaomicron, together reveal that there are at least 17 independent cell surface structures in B. thetaiotaomicron that could be altered in cells exposed to phages. The fact that almost all of these surface structures (14/17) are under the control of independent phase-variable promoters with associated tyrosine recombinases4,31,36 and that their products show altered expression after phage exposure speaks to the effectiveness of this strategy in pre-adapting some thetaiotaomicron cells within a population to thrive in the face of phage predation.
Phages are the most abundant biological entities in the gut microbiome41 and interest in the roles and identities of these gut-resident viruses is increasing as metagenomic sequencing approaches are unveiling a more comprehensive view of their dynamics during health and disease16,17,42. Although sequence-based approaches are powerful for describing the phages that are present, they do not generate information on the definitive hosts or the mechanisms of individual bacteria-phage interactions, which are likely to be elaborate. These limitations will prohibit full dissection of the ecological interactions that phage exert on bacterial populations in the gut. The approach taken here of isolating phages for a particular host of interest, with added layers of detail like systematic variation of surface CPS when possible, will be an essential complement to high throughput sequencing studies and will help build a foundation of mechanistic gut bacterium-phage interactions.
Our results, with a single strain of bacteria commonly found in human gut microbiomes, point to the existence of a very complex relationship between bacteria and phage in the gut microbiome. Considering the possibilities that these interactions could vary over time, differ by host species, and evolve differently within individuals or regionally distinct global populations, the landscape becomes even more complex. Given the diverse adaptive and counter-adaptive strategies that have apparently evolved in the successful gut symbiont B. thetaiotaomicron and its relatives, our findings hold important implications for the use of phages to intentionally alter the composition or function of the gut microbiota. While a cocktail of multiple phages could theoretically be harnessed together to elicit more robust alteration of target populations within a microbiome, the complexity of host tropisms and bacterial countermeasures that exist for B. thetaiotaomicron imply that a deliberate selection of complementary phage would be needed. If selections of effective phage cocktails need to be further tailored to individual microbiomes, or elicit resistance within individuals or populations the way antibiotics do, the prospects for effective gut microbiome-targeting phage therapy could indeed become very complicated. Given these considerations, our findings here contribute an important early step towards building a deep functional understanding of the bacterium-virus interactions that occur in the human gut microbiome. As such, this work contributes to the overall goal of understanding the ecology of this important microbial community and developing rational approaches to shape its physiology.
Methods
Bacterial strains and culture conditions
The bacterial strains used in this study are listed in Table S3. Frozen stocks of these strains were maintained in 25% glycerol at −80°C and were routinely cultured in an anaerobic chamber or in anaerobic jars (using GasPak EZ anaerobe container system sachets w/indicator, BD) at 37°C in Bacteroides Phage Recovery Medium (BPRM), as described previously43: per 1 liter of broth, 10 g meat peptone, 10 g casein peptone, 2 g yeast extract, 5 g NaCl, 0.5 g L-cysteine monohydrate, 1.8 g glucose, and 0.12 g MgSO4 heptahydrate were added; after autoclaving and cooling to approximately 55 °C, 10 ml of 0.22 µm-filtered hemin solution (0.1% w/v in 0.02% NaOH), 1 ml of 0.22 µm-filtered 0.05 g/ml CaCl2 solution, and 25 ml of 0.22µm-filtered 1 M Na2CO3 solution were added. For BPRM agar plates, 15 g/L agar was added prior to autoclaving and hemin and Na2CO3 were added as above prior to pouring the plates. For BPRM top agar used in soft agar overlays, 3.5 g/L agar was added prior to autoclaving. Hemin, CaCl2, and Na2CO3 were added to the top agar as above immediately before conducting experiments. Bacterial strains were routinely struck from the freezer stocks onto agar plates of Brain Heart Infusion supplemented with 10% horse blood (Quad Five, Rygate, Montana) (BHI-blood agar; or for the SJC phages used in Figure 1, on BPRM agar) and grown anaerobically for up to 3 days. A single colony was picked for each bacterial strain, inoculated into 5 mL BPRM, and grown anaerobically overnight to provide the starting culture for experiments.
For the experiment described in Figure 2G, liquid cultures of B. thetaiotaomicron were grown in BPRM using the pyrogallol method as described previously4. Briefly, a sterile cotton ball was burned and then pushed midway into the tube, after which 200 µl of saturated NaHCO3 and 200 µl of 35% pyrogallol solution were added to the cotton ball. A rubber stopper was used to seal the tubes, and tubes were incubated at 37 °C.
Bacteriophage isolation from primary wastewater effluent
The bacteriophages described in this study were isolated from primary wastewater effluent from two locations at the Ann Arbor, Michigan Wastewater Treatment Plant and from the San Jose-Santa Clara Regional Wastewater Treatment Facility. After collection, the primary effluent was centrifuged at 5,500 rcf for 10 minutes at room temperature to remove any remaining solids. The supernatant was then sequentially filtered through 0.45 µm and 0.22 µm polyvinylidine fluoride (PVDF) filters to yield “processed primary effluent.” Initial screening for plaques was done using a soft agar overlay method44 where processed primary effluent was combined with 1 part overnight culture to 9 parts BPRM top agar and poured onto a BPRM agar plate (e.g. 0.5 mL overnight culture and 4.5 mL BPRM top agar was used for standard circular petri dishes [100 mm × 15 mm]). Soft agar overlays were incubated anaerobically at 37 °C overnight. Phages were successfully isolated using three permutations of this assay: (1) Direct plating, where processed primary effluent was directly added to overnight culture prior to plating. (2) Enrichment, where 10 mL processed primary effluent was mixed with 10 mL 2XBPRM and 3 mL exponential phase B. thetaiotaomicron culture and grown overnight. The culture was centrifuged at 5500 rcf for 10 minutes and filtered through a 0.22 µm PVDF filter. (3) Size exclusion, where processed primary effluent was concentrated up to 500-fold via 30 or 100 kDa PVDF or polyethersulfone size exclusion columns. Up to 1 mL of processed primary effluent, enrichment, or concentrated processed primary effluent was added to the culture prior to adding BPRM top agar, as described above. To promote a diverse collection of phages, no more than 5 plaques from the same plate were plaque purified and a diversity of plaque morphologies were selected as applicable. When using individual enrichment cultures, only a single plaque was purified.
Single, isolated plaques were picked into 100 µL phage buffer (prepared as an autoclaved solution of 5 ml of 1 M Tris pH 7.5, 5 ml of 1 M MgSO4, 2 g NaCl in 500 ml with ddH2O). Phages were successfully plaque purified using one of two methods: (1) a standard full plate method, where the diluted phage samples were combined with B. thetaiotaomicron overnight culture and top agar and plated via soft agar overlay as described above or (2) a higher throughput 96-well plate-based method, where serial dilutions were prepared in 96-well plates and 1 µL of each dilution was spotted onto a solidified top agar overlay. This procedure was repeated at least 3 times to plaque purify each phage. For more details on the phages isolated in this work, see Figure S1.
High titer phage stocks were generated by flooding a soft agar overlay that plate yielded a “lacey” pattern of bacterial growth (near confluent lysis). Following overnight incubation of each plate, 5 ml of sterile phage buffer was added to the plate to resuspend the phage. After at least 2 hours of incubation at room temperature, the lysate was spun at 5,500 rcf for 10 minutes to clear debris and then filter sterilized through a 0.22 µm PVDF filter.
Quantitative host range assays
To accommodate the large number of phage isolates in our collection, we employed a spot titer assay for semi-quantitative comparisons of infectivity on each bacterial strain. High titer phage stocks were prepared on their “preferred host strain,” which was the strain that yielded the highest titer of phages in a pre-screen of phage host range. Lysates were then diluted to approximately 106 PFU/mL, were added to the wells of a 96-well plate, and further diluted to 105, 104, and 103 PFU/mL using a multichannel pipettor. One microliter of each of these dilutions was plated onto solidified top agar overlays containing single bacterial strains indicated in each figure. After spots dried, plates were incubated anaerobically for 15-24 hours prior to counting plaques. Phage titers were normalized to the bacterial strain that typically exhibited the highest phage titer, which was designated as the “preferred host strain”.
Images of phage plaques
To document the morphologies of plaques formed by the purified phages, two sets of plaque pictures were taken: the first set were taken with a Color QCount Model 530 (Advanced Instruments) with a 0.01 second exposure. Images were cropped to 7.5 mm2 but were otherwise unaltered. The second set of images were taken on a ChemiDoc Touch instrument (BioRad) with a 0.5 second exposure. Images were cropped to 7.5 mm2 and background unnaturally high pixels were removed (Image Lab, BioRad) to facilitate viewing of the plaques.
Incubation of phage with extracted CPS
Approximately 50-100 PFU of ARB25 in 50 µl phage buffer were mixed with an equal volume of H2O or capsule (2 mg/ml) extracted by the hot water-phenol method (as described in Reference 4) and incubated at 37 °C for 30 minutes. Samples were then plated on the acapsular strain, and plaques were counted after 15-24 hours anaerobic incubation at 37 °C. Counts from two replicates on the same day were then averaged, and the experiment was performed three times.
Growth curves
For growth curve experiments, 3 individual clones of each indicated strain were picked from agar plates and grown overnight in BPRM. Then, for experiments in Figures 3 and 6, each clone was diluted 1:100 in fresh BPRM and 100 µl was added to a microtiter plate. 10 µl of approximately 5*106 PFU/ml live or heat-killed phage were added to each well, plates were covered with an optically clear gas-permeable membrane (Diversified Biotech, Boston, MA) and optical density at 600 nm (OD600) values were measured using an automated plate reading device (BioTek Instruments). Phages were heat killed by heating to 95 °C for 30 minutes, and heat-killed phage had no detectable PFU/ml with a limit of detection of 100 PFU/ml.
In Figure S3B, wild-type B. thetaiotaomicron was infected with live or heat-killed ARB25, and bacterial growth was monitored via optical density at 600 nm (OD600) on an automated plate reader for 12 hours. At 0, 6.02, 8.36, and 11.7 hours post inoculation, replicate cultures were vortexed in 1:5 volume chloroform, centrifuged at 5,500 rcf at 4 °C for 10 minutes, and the aqueous phase was titered on the acapsular strain. No phages were detected in heat-killed controls.
Generation of phage-free bacterial isolates and determination of their phage susceptibility
To isolate phage-free bacterial clones from ARB25-infected cultures (Table 1), each culture was streaked on a BHI-blood agar plate. Eighteen individual colonies were picked from each plate, and each of these clones was re-streaked onto a new BHI-blood agar plate. One colony was picked from each of these secondary plates and was inoculated into 150 l BPRM broth and incubated anaerobically at 37 °C for 2 days. Only one of the clones (a cps4 isolate) failed to grow in liquid media. To determine whether cultures still contained viable phage, 50 µl of each culture was vortexed with 20 µl chloroform, then centrifuged at 5,500 rcf for 10 minutes. 10 µl of the lysate was spotted on BPRM top agar containing naïve acapsular bacteria and was incubated anaerobically overnight at 37 °C. Loss of detectable phage in the twice passaged clones was confirmed for most of the clones (79/89, 89%) by the absence of plaques on the naïve acapsular strain.
To determine whether the resultant phage-free plaques were resistant to ARB25 infection, each culture was diluted 1:100 in fresh BPRM, 100 µl was added to a microtiter plate, and 10 µl of either live or heat-killed ARB25 (approximately 5*106 PFU/ml) was added. Plates were incubated anaerobically at 37 °C for 48 hours, and OD600 was measured as described above. Cultures were determined to be susceptible to ARB25 by demonstration of delayed growth or drop in OD600, as compared to heat-killed controls.
Measurement of cps gene expression
For Figures 2G, 4, and S4, overnight cultures were subcultured into fresh BPRM to an OD600 of 0.01. For Figure 4B, 200 µL of approximately 2 × 108 PFU/mL live phage or heat killed phage were added to 5 mL of the diluted cultures. For Figure S4, 200 µL of approximately 2 × 105 PFU/mL live phage or heat killed phage were added to 5 mL of the diluted cultures. Bacterial growth was monitored by measuring OD600 every 15-30 minutes using a GENESYS 20 spectrophotometer (Thermo Scientific). Cultures were briefly mixed by hand before each measurement. For determination of relative cps gene expression, cultures were grown to OD600 0.6-0.8, were centrifuged at 7700 rcf for 2.5 minutes, the supernatant was decanted, and the pellet was immediately resuspended in 1 ml RNA-Protect (Qiagen). RNA-stabilized cell pellets were stored at −80 °C.
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) then treated with the TURBO DNA-free Kit (Ambion) followed by an additional isolation using the RNeasy Mini Kit. cDNA was then synthesized using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions using random oligonucleotide primers (Invitrogen). qPCR analyses for cps locus expression were performed on a Mastercycler ep realplex instrument (Eppendorf). Expression of each of the 8 cps synthesis loci was quantified using primers to a single gene in each locus (primers are listed in Table S4) and normalized to a standard curve of DNA from wild-type B. thetaiotaomicron. Relative abundance of expression for each locus was then calculated. A custom-made SYBR-based master mix was used for qPCR: 20 µl reactions were made with ThermoPol buffer (New England Biolabs), and containing 2.5 mM MgSO4, 0.125 mM dNTPs, 0.25 µM each primer, 0.1 µl of a 100 X stock of SYBR Green I (Lonza), and 500 U Hot Start Taq DNA Polymerase (New England Biolabs). 10 ng of cDNA was used for each sample, and samples were run in duplicate. A touchdown protocol with the following cycling conditions was used for all assays: 95 °C for 3 minutes, followed by 40 cycles of 3 seconds at 95 °C, 20 seconds of annealing at a variable temperature, and 20 seconds at 68 °C. The annealing temperature for the first cycle was 58 °C, then dropped one degree each cycle for the subsequent 5 cycles. The annealing temperature for the last 34 cycles was 52 °C. These cycling conditions were followed by a melting curve analysis to determine amplicon purity.
Transcriptomic analysis of B. thetaiotaomicron after phage infection
Whole genome transcriptional profiling of wild-type and acapsular B.thetaiotaomicron infected with live or heat-killed ARB25 was conducted using total bacterial RNA that was extracted the same as described above (Qiagen RNAEasy, Turbo DNA-free kit) and then treated with Ribo-Zero rRNA Removal Kit (Illumina Inc.) and concentrated using RNA Clean and Concentrator −5 kit (Zymo Research Corp, Irvine, CA). Sequencing libraries were prepared using TruSeq barcoding adaptors (Illumina Inc.), and 24 samples were multiplexed and sequenced with 50 base pair single end reads in one lane of an Illumina HiSeq instrument at the University of Michigan Sequencing Core. Demultiplexed samples were analyzed via Arraystar software (DNASTAR, Inc.) using RPKM normalization and the default parameters. Changes in gene expression in response to live ARB25 infection were determined by comparison to the heat-killed reference: retained were genes with > 3-fold expression changes up or down, Benjamini-Hochberg corrected P value < 0.05, and genes for which the lower value in the fold-change calculation was at least 1% of the mean RPKM-normalized value for all genes in the transcriptome. The latter cutoff was implemented to reduce the noise effects of changes in genes with very low expression values.
PCR and sequencing of phase variable B. thetaiotaomicron chromosomal loci
We found that each of the 8 chromosomal loci shown in Figure 5C had nearly identical 301 bp promoter sequences, including both of the imperfect palindromes that we predict to mediate recombination and the intervening sequence at each locus. While the 8 S-layer genes and the 7/8 of the upstream regions encoding putative tyrosine recombinases (all but the BT1927 region) shared significant nucleotide identity, we were able to design primers that were specific to regions upstream and downstream of each invertible promoter and used these to generate an amplicon for each locus that spanned the predicted recombination sites. After gel extracting a PCR product of the expected size for each locus, which should contain promoter orientations in both the “on” and “off” orientations, we performed a second PCR using a universal primer that lies within the 301 bp sequence of each phase-variable promoter and extended to unique primers that anneal within each S layer protein encoding gene. Bands of the expected size were excised from agarose gels, purified and sequenced using the primer that anneals within each S layer encoding gene to determine if the predicted recombined “on” promoter orientation can be detected. (Note that the assembled B. thetaiotaomicron genome architecture places all of these promoters in the proposed “off” orientation. We were able to detect 6/8 of these loci in the “on” orientation in ARB25-treated cells by this method, Figure S6.) A similar approach was used to determine the re-orientation of DNA fragments in the B. thetaiotaomicron PUL shufflon shown in Figure 5D, using PCR primer amplicons positioned according to the schematic followed by sequencing with the primer on the “downstream” end of each amplicon according to its position relative to the shuffled promoter sequence. For a list of primers used see Table S4.
Construction of acapsular B. thetaiotaomicron S-layer ‘ON’ and S-layer ‘OFF’ mutants
Acapsular B. thetaiotaomicron S-layer ‘ON’ and ‘OFF’ mutants (∆cps BT1927-ON and ∆cps BT1927-OFF, respectively) were created using the Δtdk allelic exchange system45. To generate homologous regions for allelic exchange, the primers BT_1927_XbaI-DR and BT_1927_SalI-UF were used to amplify the BT1927-ON and BT1927-OFF promoters from the previously-constructed BT1927-ON and BT1927-OFF strains31 via colony PCR using Q5 High Fidelity DNA polymerase (New England Biolabs). Candidate ∆cps BT1927-ON and ∆cps BT1927-OFF mutants were screened by PCR using the primer pair BT1927_Diagnostic_R and BT1927_Diagnostic_F and confirmed by Sanger sequencing using these diagnostic primers. All plasmids and primers are listed in Supplementary Tables S3 and S4, respectively.
Data representation and statistical analysis
The heatmaps for Figures 1 and S2 and the dendrogram for Figure 1 were generated in R using the “heatmap” function. Other graphs were created in Prism software (GraphPad Software, Inc., La Jolla, CA). Statistical significance in this work is denoted as follows unless otherwise indicated: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Statistical analyses other than Dirichlet regression were performed in Prism. Dirichlet regression was performed in R using the package “DirichletReg” (version 0.6-3), employing the alternative parameterization as used previously4,46. Briefly, the parameters in this distribution are the proportions of relative cps gene expression and the total cps expression, with cps7 expression used as a reference. The variable of interest used in Figure 2G is bacterial strain, whereas the variable of interest used in Figure 4B is phage viability (live versus heat-killed phage). Precision was allowed to vary by group given this model was superior to a model with constant precision, as determined by a likelihood ratio test at significance level p < 0.05.
Author Contributions
NTP, AJH, BDM, JOG, and SS performed the experiments. NTP, AJH, and ECM designed the experiments, and analyzed and interpreted the data. JLS and ECM provided tools and reagents. NTP, AJH and ECM prepared the display items. NTP, AJH and ECM wrote the paper. All authors edited the manuscript prior to submission.
Figure S1. Representative pictures of phage plaques for all phages from this study: A) phages from Ann Arbor (ARB); B) phages from San Jose (SJC). The top row of images for each phage are unaltered; background and saturated pixels were removed from images in the bottom row to facilitate viewing of the plaques. Scale bar = 2 mm
Figure S2. Replication of subset of host range assays of B. thetaiotaomicron-targeting phages on strains expressing different CPS types. Ten bacteriophages isolated and purified on the wild-type, acapsular, or the 8 single CPS-expressing strains were re-tested in a spot titer assay to determine phage host range. 10-fold serial dilutions of each phage ranging from approximately 106 to 103 plaque-forming units (PFU) / ml were spotted onto top agar plates containing the 10 bacterial strains. Plates were then grown overnight, and phage titers were calculated. Titers are normalized to the titer on the preferred host strain for each replicate. Each row in the heatmap corresponds to a replicate for an individual phage, whereas each column corresponds to one of the 10 host strains. One to three replicates of the assay were conducted for each phage by the two lead authors (AJH and NTP). Assays were carried out at the same time, and each author used the same set of cultures and phage stocks. For comparison, individual replicates from Figure 1 are included (marked with *)
Figure S3. A) Phage titers in infected cultures incubated with purified capsules. ARB25 was incubated with purified CPS1 or CPS2 (1 mg/ml) before plating on the acapsular strain, and plaques were counted after overnight incubation. Titers are normalized to mock (H2O) treatment. No significant differences in titers were found compared to mock treatment, as determined by Welch’s t test. B) Wild-type B. thetaiotaomicron was infected with live or heat-killed ARB25, and bacterial growth was monitored via optical density at 600 nm (OD600) on an automated plate reader for 12 hours. At 0, 6.02, 8.36, and 11.7 hours post inoculation, replicate cultures were removed and phage levels were titered. No phage were detected in heat-killed controls.
Figure S4. Infection of wild-type B. thetaiotaomicron at a low multiplicity of infection and subsequent effects on cps ene expression. (A) The wild-type (WT) strain was infected at a low multiplicity of infection (MOI = 1 × 10−4) of live or heat-killed ARB25, and bacterial growth was monitored via OD600. (B) RNA was harvested from cultures after reaching an OD of 0.6-0.7, cDNA was generated, and relative expression of the 8 cps loci was determined by qPCR.
Figure S5. Single replicates of cps expression in heat-killed versus live phage-treated B. thetaiotaomicron. Relative cps CPS transcript abundance in ARB25 infection experiments at high MOI (A) and low MOI (B). In the high MOI experiment, replicate 2 showed higher starting expression of the non-permissive CPS3 compared to others. In the low MOI experiment, replicate 3 showed higher starting expression of the non-permissive CPS3. In both experiments, post phage-exposed replicates displayed nearly identical CPS expression profiles characterized by high expression of CPS3.
Figure S6. Determination of phase-variable promoter switching for six loc encoding putative S-layer proteins. The hypothesis that the promoters associated with seven newly identified B. thetaiotaomicron S-layer like lipoproteins was validated using a PCR amplicon sequencing strategy. Because of high nucleotide identity in both the regions flanking the 7 new loci, a nested PCR approach was required to specifically amplify and sequence each site. In the first step, a primer lying in each S-layer gene (Table S3 “S-layer gene” primers) was oriented towards the promoter and used in a PCR extension to a primer in the upstream recombinase gene (Table S3 “recombinase gene 3” primer). The products of this PCR were purified without gel extraction and used in a second reaction with a nested primer that lies internal to the previous recombinase gene primer (Table S3 “recombinase 2” primer). The expected PCR products from this reaction, which are ~1 kb and span promoter sequences in both the ON and OFF orientations, were excised and used for an orientation-specific PCR using the original S-layer gene primer for each site and a universal primer (green schematic) that was designed for each promoter and is oriented to extend upstream of the S-layer gene (e.g., OFF orientation). Resulting products from this third reaction, which should correspond to the ON orientation if a promoter inversion has occurred in some cells, were obtained for 5/7 of the newly identified loci and the BT1927 S-layer locus as a control. In all cases in which an amplicon and sequence were obtained, the expected recombination occurred between the inverted repeat site proximal to the S-layer gene start (new DNA junction), which would orient the promoter to enable expression of the downstream S-layer gene. The sequences shown are the consensus between forward and reverse reads for each amplicon. The putative core promoter −7 sequence is shown in bold/red text, the coding region of each S-layer gene is shown in bold/blue text and the S-layer gene proximal recombination site is noted and highlighted in bold/gold text. Note that the 5’-end of the sequenced amplicon was not resolved for the BT2486 locus.
Figure S7. Recombination between the genes BT1040, BT1042, and BT1046. (A) Pfam domain schematics of the amino acid sequences of these three genes highlighting that BT1040 and BT1046, as originally assembled in the B. thetaiotaomicron genome sequence, lack additional N-terminal sequences that are present on BT1042. (B) Sequencing of the 8 PCR amplicons schematized in Figure 5C. Amplicons 1, 5 and 8 represent the original genome architecture, while the others represent inferred recombination events that are validated here by sequencing. The 5’ and 3’ ends of the BT1042, BT1040 and BT1046 genes are color-coded to assist in following their connectivity changes after recombination. A series of single-nucleotide polymorphisms (SNPs) present in BT1042, downstream of the proposed recombination site, are highlighted in yellow. The transfer of these SNPs to a fragment containing the 5’ end of BT1040 (Amplicon 4) was used to narrow the recombination region to the 7 nucleotide sequence highlighted in red. Additional SNPs that are specific to the regions upstream of this recombination site are shown in white text for each sequence.
Figure S8. The BT1033-52 locus does not affect susceptibility of acapsular B. thetaiotaomicron to ARB25. Ten-fold serial dilutions of ARB25 were spotted onto lawns of B. thetaiotaomicron ∆cps (n=5) and B. thetaiotaomicron ∆cps ∆BT1033-52 (n=5, n=3 independent clones each). Plaquing efficiency was determined by normalizing plaque counts on B. thetaiotaomicron ∆cps ∆BT1033-52 relative to plaque counts on B. thetaiotaomicron ∆cps for each replicate. Statistical significance was determined using the Mann-Whitney test.
Table S1. Phages used in this study and details on their isolation.
Table S2. Genes that are differentially regulated in post ARB25-infected wild-type and acapsular B. thetaiotaomicron.
Table S3. Bacterial strains and plasmids used in this study.
Table S4. Primers used in this study.
Acknowledgements
We thank Rey Honrada at the San Jose-Santa Clara Wastewater Treatment Plant and the staff at the Ann Arbor Wastewater treatment plant for assistance in collecting primary sewage effluent. This work was funded by NIH grants (GM099513 and DK096023 to E.C.M), an NIH postdoctoral NRSA (5T32AI007328 to A.J.H.), a Stanford University School of Medicine Dean’s Postdoctoral Fellowship (A.J.H.), and the NIH Cellular Biotechnology Training Program (N.T.P., T32GM008353).