Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the “flagellar switch” upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5’ leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Introduction were shown to undergo inversion, and the sequence upstream of the cmrRST operon was confirmed to regulate expression of the downstream genes in a manner consistent with phase variation [44,45]. We subsequently showed that site-specific recombination also mediates phase variation of flagella and toxins by inversion of a genetic sequence called the "flagellar switch" [33]. The 154 bp flagellar switch is flanked by imperfect inverted repeats and is located upstream of the flgB operon. In one orientation, the flagellar switch is permissive for expression of the flgB operon, resulting in "flg ON" bacteria exhibiting flagellum biosynthesis, swimming motility, and toxin production. Conversely, the inverse orientation reduces flgB operon transcription, yielding "flg OFF" bacteria that are aflagellate, non-motile, and attenuated for toxin production. Inversion of the flagellar switch requires the site-specific tyrosine recombinase RecV. Mutation of recV leads to genotypically and phenotypically "phase locked" strains that no longer undergo phase variation [33]. In contrast to these phase locked mutants, wildtype C. difficile R20291 in broth culture consists of a heterogeneous population of flg ON and OFF bacteria. Notably, RecV is also required for inversion of the cwpV switch, as well as two of the other identified invertible sequences including one shown to impact multiple phenotypes including virulence [40,44,45].
The canonical regulatory mechanism of phase variation by site-specific recombination involves an invertible promoter element that, when correctly oriented, promotes gene transcription in cis. For example, production of fimbriae by Escherichia coli and related species is regulated by the invertible fimbrial switch fimS, which contains a promoter for the adjacent fimA gene that encodes the fimbrial subunit [46,47]. The flagellar switch in C. difficile lies in the 5' untranslated region of the mRNA, between the previously identified σ A -dependent promoter and the flgB coding sequence. Previous work showed that the flagellar switch does not contain a functional promoter [33]. Instead, regulation occurs post-transcription initiation and involves an unidentified trans-acting posttranscriptional regulator specific to C. difficile.
In this study, we sought to identify the mechanism by which the flagellar switch controls expression of flagellum and toxin genes. Specifically, how does the orientation of the flagellar switch mediate the phase variable expression of the flgB operon? To answer this question, we used a non-motile recV phase-locked OFF strain in a suppressor analysis to identify factor(s) involved in inhibiting flgB operon transcription, swimming motility, and virulence characteristics. We recovered suppressor mutants that retained the flagellar switch in the OFF orientation but gained extragenic mutations that restored swimming motility, toxin production, and expression of flagellum and toxin genes. The extragenic mutations conferring the suppressor phenotypes mapped to rho, which encodes the transcription termination factor Rho. Using a series of reporter fusions in C. difficile with or without an intact rho allele, we determined that Rho inhibits transcription from the 5' leader sequence of flgB mRNA with the flagellar switch in the OFF orientation but not the ON orientation. These results suggest that Rho can discriminate between flg ON and flg OFF mRNA to selectively inhibit transcription in flagellar phase OFF variants, and reveal a role for Rho-mediated transcription termination phase variation of C. difficile flagella and toxins. Further phenotypic characterization of rho mutants additionally linked the loss of Rho to altered sporulation and ability to colonize the intestine in a murine model of infection, indicating a broader role for Rho-mediated transcription termination in C. difficile physiology.

Motile suppressors arise from a non-motile recV flg OFF mutant
In C. difficile strain R20291 (ribotype 027), the DNA recombinase RecV is required for flagellar switch inversion, and loss of recV leads to phenotypically-locked bacteria [33]. These recV locked ON (recV flg ON, RT1702) and locked OFF (recV flg OFF, RT1693) strains are phenotypically distinct. As previously shown, recV flg ON bacteria are motile in soft agar, while recV flg OFF bacteria typically remain non-motile even after 72 hours (Fig 1A); in a subset of experiments, we observed spreading of the recV flg OFF mutant after prolonged incubation in soft agar (�72 hours) (Fig 1A, right panel) [33]. In contrast to the uniform motility exhibited by WT and recV flg ON bacteria, these 'motile flares' were irregular and asymmetrical. The same phenomenon was observed for an independent recV flg OFF mutant, RT1694, which differs from RT1693 in the orientation of the invertible sequence upstream of cwpV, another phase variable locus (S1 Fig) [33,48]. To recover the motile bacteria, which we term motile suppressors (MS) hereafter, we collected growth from the outer edge of the motile flares and subcultured on BHIS agar to obtain isolated colonies. When inoculated into soft agar, these MS showed motility similar to that of WT bacteria; an example is shown in Fig 1B. A total of 14 MS isolated from RT1693 and RT1694 were assayed for swimming motility in soft agar. All 14 MS regained swimming motility to the level of the recV flg ON strain, while the recV flg OFF and non-motile sigD-null control remained non-motile (S2 Fig); the results for 6 representative MS are shown in Fig 1C. Consistent with these phenotypes, production of the flagellin FliC was restored in these 6 representative MS, with FliC levels equivalent to or higher than recV flg ON ( Fig 1D). FliC was undetectable in the recV flg OFF parental strain.
This observation suggested that the recV flg OFF mutant is capable of recovering motility. One possible explanation is that a recombinase other than recV catalyzed inversion of the flagellar switch. To test this possibility, we used orientation-specific polymerase chain reaction (OS-PCR), which employs primer pairs that specifically amplify one orientation of an invertible sequence or the other (S1 Table) [33]. Wild-type C. difficile R20291 grown in BHIS broth consists of a heterogeneous population. Accordingly, both OFF and ON flagellar switch orientations were detected by OS-PCR ( Fig 1E). As determined previously, the recV flg OFF and recV flg ON strains yielded 280bp and 375bp product sizes, respectively, which correspond to the known flagellar switch orientations in these mutants [33]. The 14 MS contained the flagellar switch exclusively in the OFF orientation, with a representative 6 MS shown in Fig 1D. These results indicate that flagellar switch inversion did not occur, and the MS retain the flg OFF genotype despite their motile phenotype (Fig 1B). We also sequenced the promoter region and 5' UTR of the flgB operon and did not find any mutations in the MS compared to the parental strains. Together, these results suggest that additional mechanisms are involved in inhibiting motility in C. difficile when the flagellar switch is in the OFF orientation.

Identification of Rho as a negative regulator of flagellar motility
Soft agar swimming motility assays provide a strong selective pressure for bacterial self-propulsion to enable access to nutrients as they become depleted locally. The results above suggest that extragenic mutations arose in the MS that alleviated the negative effect of the flg OFF genotype on swimming motility in this assay. We postulated that a suppressor mutation occurred in a gene(s) involved in inhibiting flagellar gene expression in recV flg OFF bacteria. We thus performed whole genome sequencing of seven motile suppressors (MS 1-7) to identify single nucleotide polymorphisms (SNPs) compared to the R20291 wild type reference genome (detailed in S2 Table, S1 Data). Compared to the recV flg OFF strain, five of seven MS carried SNPs upstream of CDR20291_1465, which encodes a putative Mn 2+ -containing catalase. The same five MS (MS 2-6) also carried SNPs in the region between CDR20291_1414 and 1415, which are convergently transcribed and encode the putative acetolactate synthase subunit IlvB and a phage-associated integrase, respectively. Additional SNPs appeared within the inverted repeats of switches upstream of cwpV and the flgB operon, in accordance with

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Rho factor mediated phase variation these strains containing inversions in the cwpV and flg switches relative to the R20291 reference genome. Similarly, SNPs were also present in the inverted repeats flanking the invertible sequence upstream of CDR20291_0685, which was previously shown to be heterogeneous compared to the R20291 reference [44,45].
A single locus, CDR20291_3324, contained SNPs in MS 1-7 compared to the allele in R20291 (Table 1, S1 Data); SNPs in this gene did not appear in either recV flg OFF parent. These SNPs showed a high frequency (89% or greater) in the sequencing reads. In lieu of whole genome sequencing, we Sanger sequenced CDR20291_3324 in MS 8-14 and identified additional SNPs in all of them. CDR20291_3324 encodes Rho factor, a transcriptional terminator known to act at the 3' ends of genes or operons as an alternative mechanism to the use of an intrinsic terminator [49]. Rho binds nascent transcripts at Rho utilization (rut) sites, cytosine-rich sequences with poor consensus [49]. Hexameric Rho then uses ATPase activity to translocate 5' to 3' along the RNA. Although the mechanism of Rho-dependent transcription termination is not fully understood, it is thought to occur when Rho reaches the RNA polymerase, e.g. at an RNAP pause site, and forces its movement on template DNA without nucleotide addition, leading to destabilization of the transcription complex and mRNA release [50]. The SNPs identified in rho mapped to different domains of the Rho protein, including an N-terminal insertion domain (NID), primary binding site (PBS), and the C-terminal ATPase domain (Fig 2A). Mutations included substitutions (e.g. R206I, G284E) as well as nonsense mutations resulting either from a SNP creating a stop codon (e.g. E113-Stop) or a frameshift (e.g. N66-FS, resulting in a stop codon at residue 73).
Rho is essential for viability in gram-negative bacteria but dispensable in gram-positive bacteria studied to date, such as Bacillus subtilis and Staphylococcus aureus [51,52]. Based on a transposon mutagenesis screen, Rho is not essential for viability in C. difficile [53]. However, loss of Rho can cause growth defects potentially due to inappropriate, pervasive read-through transcription [54][55][56]. Therefore, we assessed the growth of the 14 motile suppressors in BHIS broth. All motile suppressors reached a lower final optical density (OD 600 ) compared to recV flg OFF (S3 Fig). While doubling times during exponential growth for the recV flg ON and

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Rho factor mediated phase variation OFF strains were 56.5 ± 6.0 and 63.6 ± 3.5, respectively, growth rates for the MS ranged from 71.8 to 129.9 minutes, and 10 of the MS showed significant attenuation of doubling time ( Table 2).

Rho factor mediated phase variation
Because the rho mutations restored motility to the recV flg OFF mutants, we next tested the effect of expressing the wild-type rho allele in rho-null strains. We were unsuccessful in generating an in-frame deletion of rho, likely because of the associated growth defects. In lieu of a targeted mutant, we utilized MS5 and MS10, which contain stop codons early in the rho coding sequence: N66-FS (stop codon at position 73) and E113-St, respectively (Fig 2A). A plasmid carrying wild-type rho under control of an inducible promoter, pRho, was introduced into these MS, and the resulting strains were assayed for swimming motility. The vector control strains showed the expected swimming motility behaviors after 48 hours (Fig 2B and 2C). Expression of rho in MS5 and MS10 significantly inhibited swimming motility compared to the respective vector controls, effectively complementing the effects of the SNPs in rho. Consistent with these phenotypes, expression of rho led to 3-to 4-fold decreases in FliC levels in MS5 and MS10 compared to vector controls ( Fig 2D). To examine whether changes in protein production and swimming were due to changes in flagellar gene transcription, we used quantitative reverse transcriptase PCR (qRT-PCR) to measure the transcript abundance of flgB, the first gene of the operon controlled by the flagellar switch, and fliC, which encodes flagellin and is regulated by SigD [22,25]. The flgB transcript abundance was higher in MS5 and MS10 than recV flg OFF, though the differences were not significant (Fig 2E, left). Both MS5 and MS10 had significantly increased fliC transcript levels compared to recV flg OFF (Fig 2E,  Together these results indicate that Rho regulates flagellum production and swimming motility in C. difficile by directly or indirectly inhibiting transcription of flagellar genes.

Rho inhibits heterogeneous flagellar gene expression
Analysis of flagellar gene expression by qRT-PCR reflects the transcript abundance averaged across the bacterial population. Yet, phase variation generates a heterogeneous population of

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Rho factor mediated phase variation bacteria, some of which express flagellar genes, and some of which do not. To analyze the effects of Rho on flagellar gene expression at the single cell level, we used fluorescence microscopy with a red fluorescence protein (mCherryOpt) reporter gene under the control of the flgM promoter (P flgM ), which is a SigD-dependent promoter in the late stage flagellar operon [22,23]. Expression of mCherryOpt from the flgM promoter is thus an indirect indication of the flagellar switch orientation. As previously observed, a population of wild-type C. difficile R20291 exhibited heterogeneity in fluorescence, with the majority of cells expressing mCher-ryOpt (Fig 3) [33]. In contrast, virtually no red cells were detectable for recV flg OFF C. difficile.
Heterogeneous fluorescence was restored in MS5 and MS10 carrying P flgM ::mCherryOpt ( Fig  3). Thus, Rho is necessary for suppression of flagellar gene expression in recV flg OFF bacteria.

Rho negatively impacts toxin production
The alternative sigma factor SigD, encoded in the flgB operon, positively regulates the expression of tcdR, tcdA, and tcdB [23,27]. We therefore predicted that inhibition of flgB operon

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Rho factor mediated phase variation transcription by Rho would concomitantly inhibit toxin gene expression. We evaluated TcdA production in MS5 and MS10 carrying vector or pRho by immunoblot. MS5 and MS10 showed a 3-to 4-fold increase in TcdA production, respectively, compared to the recV flg OFF parent ( Fig 4A). Expression of wild-type rho in MS5 and MS10 decreased TcdA levels, resulting in TcdA levels comparable to recV flg OFF bacteria. To determine whether changes in protein levels correlate with changes in transcript abundance, we examined expression of tcdR, tcdA, and tcdB by qRT-PCR. As observed previously, tcdA and tcdB transcript abundance was significantly higher in recV flg ON than recV flg OFF bacteria; tcdR was similarly altered, though the difference was not statistically significant ( Fig 4B) [33]. Consistent with the negative impact of Rho on flagellar gene expression, tcdA, tcdB, and tcdR transcript levels were higher in MS5 and MS10 than in the recV flg OFF parent ( Fig 4B). Providing rho in trans decreased tcdA, tcdB, and tcdR transcript abundances of MS5 and MS10 to the parental recV flg OFF levels. Therefore, in addition to inhibiting motility and growth, Rho negatively affects toxin gene expression and production. This effect is likely mediated through SigD encoded in the flgB operon [23,27].

Mutant rho alleles confer dominant negative motility phenotypes
Rho factor functions as a homohexamer [57]. We hypothesized that overexpression of mutant rho alleles could result in incorporation of aberrant subunits into the hexamer, interfering

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Rho factor mediated phase variation with Rho function. To test for this dominant negative effect, we introduced mutant rho alleles from six different MS into the recV flg OFF strain, with the expectation that incorporation of non-functional Rho monomers would prevent inhibition of swimming motility by the wildtype, chromosomally-encoded Rho. The wild-type rho allele was also introduced as a control. To achieve overexpression of the cloned alleles, the genes were placed under the control of an anhydrotetracycline (ATc)-inducible promoter in the multi-copy plasmid pRPF185. Strains bearing these expression plasmids were assayed for swimming motility in soft agar medium. Expression of five of the six of the mutant rho alleles restored swimming motility to varying extents, while the wild-type allele did not alter motility (Fig 5A and 5B). These results were obtained regardless of ATc induction, suggesting leaky expression from the P tet promoter as previously reported [58,59]. The only mutant allele that did not lead to a dominant negative motility phenotype was N66-FS derived from MS5 (Fig 2A). Because this rho allele contains a nonsense mutation resulting in an early stop codon at residue 73, the truncated gene product may be unstable or unable to incorporate into the Rho hexamer, further justifying the use of the MS5 strain as a rho-null mutant.

Rho preferentially inhibits transcription of flg OFF mRNA
Our prior work suggests that transcription termination of flg OFF bacteria is mediated by a trans-acting factor specific to C. difficile [33]. This role could be fulfilled by Rho. While Rho typically terminates transcription of genes and operons 3' of coding sequences, recent studies have shown that Rho can also terminate transcription in some 5' leader regions in gram-negative bacteria [60][61][62][63]. This mechanism would have a regulatory effect on the downstream gene (s). We therefore hypothesized that Rho is this trans-acting factor affecting transcription of the flgB operon, preferentially inhibiting transcription in flgB transcripts containing the flagellar switch in the OFF orientation. To test this hypothesis, we transcriptionally fused a phoZ reporter gene to the flgB coding sequence and the upstream regulatory region of the flgB operon. This 1045 bp region includes the σA-dependent promoter and the 498 bp 5' untranslated region containing with the flagellar switch in either the ON or OFF orientation: P flgB -UTR ON ::phoZ and P flgB -UTR OFF ::phoZ respectively. Promoterless (::phoZ) and promoter-only

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Rho factor mediated phase variation constructs (P flgB ::phoZ) were included as controls. These plasmid-borne reporters were introduced into recV flg ON and OFF strains, which encode wild-type Rho, and MS5 and MS10, which contain mutant rho alleles, and alkaline phosphatase activity was assayed [64]. As anticipated, the no-promoter control lacked activity in all strains, and no differences were observed for activity of the promoter only reporter suggesting that Rho does not regulate transcription at the level of promoter (S5 Fig). For the P flgB -UTR ON ::phoZ reporter, activity was modestly (~3-fold) higher in MS5 and MS10 compared to the recV flg OFF parent (Fig 6A). In comparison, activity was~15-fold higher in MS5 and MS10 compared to recV flg OFF for the P flgB -UTR OFF ::phoZ reporter. Therefore, mutation of rho had a greater effect on flagellar gene transcription for bacteria with the flagellar switch in the OFF orientation, suggesting that Rho preferentially inhibits transcription of the flg OFF transcript. The results of alkaline phosphatase assays in C. difficile imply that Rho negatively regulates flgB operon transcription, but do not distinguish between a direct and indirect mechanism of regulation. Rho could directly act on the flgB UTR to terminate transcription from flgB OFF mRNA, or Rho could impact the production of another protein involved in flgB regulation. We previously showed that, whereas the P flgB -UTR ON ::phoZ reporter resulted in significantly higher activity than the P flgB -UTR OFF ::phoZ reporter in C. difficile, the difference was lost when these reporters were assayed in B. subtilis [33]. These results indicate that B. subtilis does not encode the factor that mediates regulation. We postulated that if Rho directly terminates transcription within the flgB 5' UTR, introducing C. difficile rho (Cd-rho) into B. subtilis strains carrying the P flgB -UTR ON ::phoZ and P flgB -UTR OFF ::phoZ reporters would restore the regulation seen in C. difficile. To test this idea, the wild-type Cd-rho allele was introduced into the previously constructed B. subtilis reporter strains [33], and alkaline phosphatase activity was assayed. As seen previously, reporter activity was the same in B. subtilis with P flgB -UTR ON ::

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Rho factor mediated phase variation phoZ and P flgB -UTR OFF ::phoZ (Fig 6B). Expression of Cd-rho resulted in decreased activity only in B. subtilis with the P flgB -UTR OFF ::phoZ reporter. Finally, the addition of bicyclomycin, a specific inhibitor of Rho ATPase activity [65], abrogated this effect. These data indicate that Rho, and not another C. difficile protein, is the trans-acting factor which directly inhibits flagellar gene expression, and that Rho selectively prevents transcription in flg OFF bacteria.

Rho is important for early colonization in a mouse model of infection and efficient sporulation
In vitro, Rho affects several phenotypes including growth, motility, and toxin production. Because these characteristics are important during CDI, we analyzed the effect of a rho mutation in a mouse model of infection. MS5 and MS10 were derived from two recV mutant strains that differ in cwpV status-MS5 was derived from RT1694 (cwpV ON) while MS10 was derived from RT1693 (cwpV OFF) [48]. The role of cwpV in vivo has not been previously reported, so both recV flg OFF parental strains were tested. RecV is required for site-specific recombination of multiple invertible sequences, not only the flagellar and cwpV switches [44,45,48]. To ensure appropriate attribution of phenotypes, we confirmed that the parental strains and motile suppressors are isogenic for the other sequences and only differ in the cwpV switch (S6A Fig). Male and female C57BL/6 mice were treated with antibiotics to render them susceptible to C. difficile and then inoculated by oral gavage with 10 5 spores of wild-type R20291, MS5, MS10, and their respective parent strains.
As observed previously, wild-type R20291 colonized the mice within 1 day post-inoculation (reaching more than 10 6 CFU/g feces), maintained this level of colonization for 1-3 days, then was gradually cleared typically between days 3 and 7 post-inoculation (S6B Fig). A similar, but not identical pattern of colonization was seen for both recV flg OFF strains, suggesting that cwpV expression does not consistently impact colonization in this model. For MS5 and MS10, the bacterial burden in feces was significantly lower on day 1 post-inoculation compared to the respective recV flg OFF parents and wild-type R20291 (Fig 7A). About 50% of the animals inoculated with MS5 or MS10 had undetectable levels of C. difficile in their feces; most of the remaining animals showed intermediate or parental levels of colonization. Interestingly, colonization of both MS5 and MS10 recovered to parental levels starting at day 2 post infection and were cleared within a similar time frame (S6B Fig), suggesting that Rho is important for initial colonization in a mouse model of infection. Notably, although the recV flg ON, MS5, and MS10 strains differ in toxin production compared to the recV flg OFF strain in vitro, we did not observe significant differences in weight loss or diarrheal symptoms between the groups of infected animals. These results may be attributable to the subclinical colitis caused by R20291 in this animal model, as previously reported [66][67][68]. It is also possible that other regulators of toxin gene expression, such as CodY and CcpA, unlink co-expression of the toxin and flagellar genes [69][70][71][72].
Sporulation and germination are important for colonization of the mouse model [73]. Because both MS5 and MS10 were attenuated for colonization on day 1 of infection, we considered that this difference is attributable to a germination and/or sporulation defect. We assessed sporulation and spore viability by enumerating ethanol resistant spores as a percentage of total cells (spore plus vegetative) [74]. While the sporulation efficiency for the wild type and both recV flg OFF strains was between 10 and 15%, sporulation efficiency was <1% for both motile suppressors (Fig 7B). Our data implicate Rho as an important factor that positively regulates sporulation, which may contribute to the colonization defect observed in the mouse model. It is possible that additional SNPs in the motile suppressors also contribute to the sporulation defect.

Rho factor mediated phase variation
To examine germination, purified spores of wild-type R20291, both recV flg OFF strains, MS5, and MS10 were assayed in buffer supplemented with the spore germinant 10 mM taurocholic acid (TA) as previously described [75]. In the absence of TA, no germination was detected. In the presence of TA, all strains germinated to the same level, indicating that Rho does not influence germination rate (S7 Fig).

Discussion
In this study, we identified Rho as a trans-acting factor that controls phase variation of flagella and toxins in vitro. The regulation exerted by Rho contributes to the ability of a population of C. difficile to continually maintain motile, toxin-producing flg ON cells as well as non-motile, atoxigenic flg OFF cells. Comparative transcriptional analyses using C. difficile as well as B. subtilis as a heterologous system support that Rho inhibits flagellar gene transcription selectively in flg OFF bacteria. These results implicate Rho as an important regulatory component mediating phase variation of flagella, and by extension toxins, and reveal a new role for Rho-mediated transcription termination in regulation of gene expression.
All of the 14 motile suppressors (MS) contained nucleotide polymorphisms in Rho conferring a missense or nonsense mutation that presumably abrogated Rho function. Five of six rho alleles, which correlated with a range of growth defects in the respective MS, led to a dominant negative effect and relieved inhibition of motility in flg OFF bacteria when over expressed. In E. coli, a dominant negative effect resulted from less efficient binding of mRNA to the Rho secondary binding site and decreased translocation of Rho along the mRNA towards RNA polymerase [76]. In C. difficile, incorporation of mutant subunits into the Rho homohexamer could negatively affect mRNA binding, ATP processing and/or resulting helicase activity. These mechanisms are not mutually exclusive-mutations in different domains of Rho may affect Rho activity by different mechanisms while imparting the same effect on motility. We

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Rho factor mediated phase variation note that we were unable to generate an independent mutation in rho in a wild-type R20291 background, however the MS5 and MS10 strains with truncated Rho represent useful mutants for characterization of rho-null C. difficile. Further studies will determine how the mutant Rho proteins are altered in function and the mechanism by which mutant rho alleles interfere with function of wild-type Rho.
Rho suppressor mutants exhibit restored motility compared to the flg OFF bacteria from which they were derived. The increased motility of motile suppressors corresponds with increased expression of flagellar genes flgB and fliC and higher levels of the major flagellin FliC. Furthermore, Rho contributes to heterogeneity of flagellar expression at the single cell level as evidenced by differences in mCherry signal driven by the SigD-dependent flgM promoter. Unlike the recV flg OFF strain that lacks fluorescence, motile suppressors derived from flg OFF bacteria are mCherry positive and appear similar to a wild-type population. That loss of Rho in the MS resulted in heterogenous fluorescence intensity among individual cells indicates that another factor influences expression. A c-di-GMP riboswitch is encoded between the flgB transcriptional start site and the flagellar switch, and we speculate that fluorescence intensity reflects varying levels of c-di-GMP [77,78].
Experiments with transcriptional phoZ fusions in C. difficile and B. subtilis indicate that Rho strongly inhibits transcription when the flagellar switch is in the OFF orientation. We propose two alternative models for direct control of phase variable expression of the flgB operon by Rho through the selective, premature termination of flg OFF transcripts (Fig 8). In model 1, Rho distinguishes between flg ON and OFF mRNA by preferentially binding to flg OFF mRNA due to the presence of rut sequences that are absent in the flg ON. In model 2, Rho binds flg ON and OFF mRNAs equally, 5' of the flagellar switch. Transcription termination may then be differentially influenced by the presence of an additional sequence required for termination. For example, an RNA polymerase pause site may appear only in flg OFF mRNA [61]. In either model, Rho would selectively terminate flgB operon transcription and inhibit linked phenotypes in bacteria with the flagellar switch in the OFF orientation. More work is needed to distinguish between these two models. Using the RhoTermPredict algorithm [79], we were unable to identify any predicted rut sites within the flgB leader sequence of either flg ON or OFF sequences. RhoTermPredict is based on E. coli, B. subtilis, and S. enterica databases and searches for rut sites with regularly spaced C residues and C>G content followed by a

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Rho factor mediated phase variation putative RNA polymerase pause site [79]. However, the C. difficile genome has low G+C content (<30%) [80], and the flgB leader sequence has 23% G+C content. The rut site characteristics in C. difficile therefore may be different from those previously described in other bacteria, so the RhoTermPredict algorithm may be unsuitable for predicting rut sites in C. difficile.
There are several examples of Rho exerting regulation on 5' leader sequences in gram-negative bacteria, where gene regulation by Rho is achieved by multiple mechanisms involving other proteins, small RNAs, and potentially yet unidentified factors. In E. coli, Rho seems to preferentially regulate expression of >250 genes with long 5' UTRs [81]. In addition, the RNA-binding protein CsrA binds to the 5' UTR of pgaA to prevent the formation of an RNA secondary structure that otherwise sequesters the rut site [82]. In S. enterica serovar Typhimurium, Rho binds within the leader sequences of three genes encoding Mg 2+ transporters to control expression [60,62,83], and the small RNA ChiX inhibits expression of the chiPQ operon by inducing premature Rho-dependent termination [84]. Finally, most known E. coli riboswitches modulate gene expression by either translational regulation or Rho-dependent termination [60,85]. However, riboswitches in C. difficile, including the c-di-GMP riboswitch upstream of the flgB operon, appear to act through Rho-independent mechanisms [77,78]. To our knowledge, this is the first example of Rho-mediated transcription termination within a 5' UTR that results in modulation of downstream gene expression in a gram-positive species.
In E. coli, Rho requires cofactors NusA and NusG to terminate transcription at many sites [86]. NusG and NusA are essential for growth of C. difficile R20291 [53], but the cofactor requirements for C. difficile Rho are currently unknown. Introduction of C. difficile rho into a heterologous host B. subtilis did not alter the ability of C. difficile Rho to terminate transcription of the flg OFF construct. These data suggest that either Rho is able to terminate flagellar transcription without additional cofactors, or it is able to use homologs of NusA, NusG, or other potential cofactors present in B. subtilis. Interestingly, while both C. difficile and B. subtilis encode Rho, only the C. difficile factor terminates flg OFF transcription. This difference could be caused by a difference in structure of these two proteins. Although many of the features of Rho are conserved across bacteria, in~35% of species, including C. difficile, Rho contains an N-terminal insertion domain (NID) whose length and composition are not conserved among species [87]. In other bacterial species with an NID-containing Rho, the NID imparts diverse functions [88][89][90]. It is therefore possible that the insertion domain of C. difficile Rho confers the ability to terminate flg OFF transcription.
Mutations in rho negatively affect initial colonization in a mouse model of infection, resulting in a delay in colonization. We ruled out contributions from other phase-variable loci by ensuring that MS5 and MS10 are isogenic with the parental strains at these sites. The rho mutations likely have pleiotropic effects that impact colonization [91,92], however the delayed colonization may be due in part to the defects in growth and sporulation of the motile suppressor mutants. Interestingly, a high-throughput screen in C. difficile R20291 did not identify rho as a gene required for sporulation [53]. How Rho affects growth and sporulation in C. difficile is unknown, but may arise from pervasive transcription, particularly loss of suppression of antisense transcription, or other potential consequences of loss of Rho [86,[93][94][95]. Further studies are needed to elucidate the effects of Rho on global transcription in C. difficile to determine the cause of the observed growth defects as well as other phenotypes affected by Rho.

Growth and maintenance of bacterial strains
Strains and plasmids used in this study are listed in S3 Table. C. difficile was maintained in an anaerobic chamber (Coy Laboratories) in an atmosphere of 85% N 2 , 5% CO 2 , and 10% H 2 . C. difficile and B. subtilis were routinely cultivated in Brain Heart Infusion medium (Becton Dickinson) supplemented with 5% yeast extract (Becton Dickinson) (BHIS) at 37˚C. Where indicated, bacteria were cultured in Tryptone Yeast (TY) broth. All C. difficile broth cultures were grown statically, with 10 μg/mL thiamphenicol (Tm) for plasmid maintenance as needed. E. coli DH5α and HB101(pRK24) were cultured under aerobic conditions at 37˚C in Luria-Bertani (LB) broth. For selection of plasmids in E. coli, 100 μg/mL ampicillin (Amp) and/or 10 μg/ mL chloramphenicol (Cm) was used, as indicated. Kanamycin (Kan) 100 μg/mL was used to select against E. coli after conjugations with C. difficile. Spectinomycin (Spec) 100 μg/mL was used to select for B. subtilis transformants containing Cd-rho.

Soft agar swimming motility assay
Flagellum-dependent swimming motility was assayed in 0.5X BHIS-0.3% agar as previously described [25]. When appropriate, Tm was added for plasmid maintenance, and 10 ng/mL anhydrotetracycline (ATc) was added to induce gene expression. The diameter of motile growth was measured after 24, 48, and 72 hours. Three independent experiments were performed, each with six technical replicates. Images were taken using the G:BOX Chemi imaging system with the Upper White Light illuminator.

Isolation and sequencing of motile suppressor mutants
The recV flg OFF mutants RT1693 and RT1694 (which contain the cwpV switch in the OFF or ON orientation, respectively) were grown in BHIS broth until OD 600 of 1, then 1.5 μL were inoculated into 0.5X BHIS-0.3% agar motility medium and incubated at 37˚C for 48-96 hours. Each plate included a non-motile sigD negative control (RT1566) and recV flg ON (RT1702) and recV flg OFF controls. Plates were examined for expansion of the recV flg OFF colonies, which appeared in a subset of plates. Bacteria were collected from the outer edge of motile growth and subcultured on BHIS agar.
Genomic DNA was extracted from seven motile isolates (RT1705 to RT1711 (MS 1-7), S3 Table) and the parental recV flg OFF strains (RT1693, RT1694) as previously described [96]. Genomic DNA of MS 1-7 was prepared using the KAPA HyperPrep Kit (Roche) and sequenced using an Illumina HiSeq 2500 Rapid Run platform with paired ends and 100X coverage by the UNC-CH High Throughput Genomic Sequencing Facility. The sequencing data is available on the National Center for Biotechnology Information (NCBI) Sequence Read Archive Database, accession number PRJNA630461. Sequencing reads were mapped to the reference C. difficile R20291 genome (Accession No. FN545816.1) using CLC Genomics Workbench v. 9 software (Qiagen), and nucleotide polymorphisms were identified using the fixed ploidy variant detector function with default parameters. Whole genome sequencing was not performed for MS 8-14. Instead, for MS 8-14 (RT1939-1945, the rho gene (CDR20291_3324) was amplified by PCR with primers R2307 and R2308, and the products were Sanger sequenced using primers R2307, R2308, R2366, and R2367. Primer sequences are provided in S1 Table. Nucleotide polymorphisms were identified by alignment with the wildtype sequence from R20291 using ClustalOmega [97].

Determination of invertible switch orientation by orientation-specific PCR
C. difficile was cultured from glycerol stocks on BHIS agar for 24 hours at 37˚C. A single colony was suspended in 20 μL of dH 2 O and heated at 100˚C for 10 minutes. These lysates served as templates for PCR using primers that discriminate between each flagellar switch sequence orientation in R20291 (S1 Table). Primers R1614 and R857 were used to amplify the ON orientation of the flagellar switch, which corresponds to the published sequence of R20291. Primers R1615 and R857 were used to amplify the OFF orientation of the flagellar switch. Similarly, orientation-specific PCR was used to determine the orientations of the other invertible sequences using primers listed in S1 Table, which follow the naming pattern LOCUS_pubF and LOCUS_R for detection of the orientation in R20291 reference genome, and LOCUS_invF and LOCUS_R for the inverse orientations. Three independent experiments were done.

Detection of FliC and TcdA by immunoblot
Western blots for TcdA and FliC production were performed as previously described [33,34]. Cultures for TcdA immunoblotting were grown in TY broth overnight (~16 hours), diluted 1:50 in fresh TY broth, and grown until late stationary phase (OD 600 of 1.8 to 2.0). Cultures for immunoblotting FliC were grown overnight (~16 hours) in BHIS broth. For complementation experiments, Tm was included in all growth media, and 10 ng/mL ATc was added to induce gene expression. For both FliC and TcdA detection, samples were normalized to an OD 600 1.0, and then cells were collected by centrifugation at 16,000 x g for 5 minutes (TcdA) or 2,000 x g for 10 minutes (FliC). Bacterial pellets were suspended in 1x SDS-PAGE sample buffer. The lysates were separated on a 12% SDS-polyacrylamide gel for FliC detection or on an 8% SDSpolyacrylamide gel for TcdA detection, then transferred to a nitrocellulose membrane (Bio-Rad). Membranes were stained with Ponceau S (Sigma) to assess equal loading and imaged using the G:Box Chemi imaging system. FliC was detected using α-FliC hamster sera (generous gift from Dr. Ghose-Paul) [34,98] followed by goat anti-hamster IgG (H+L) secondary antibody conjugated to DyLight 800 (Novus Biologicals). TcdA was detected using mouse α-TcdA antibody (Novus Biologicals) followed by goat anti-mouse IgG secondary antibody conjugated to DyLight 800 4x PEG (Invitrogen). Blots were imaged using the Odyssey imaging system (LI-COR), and quantification was performed with Image Studio Software. All strains were assayed in at least three independent experiments.

Growth curves
Overnight cultures were diluted 1:50 into BHIS medium including 10 μg/mL Tm and 10 ng/ mL ATc as needed. Optical density (OD 600 ) was measured every 30 minutes for 8 hours. Doubling times were calculated based on the change in optical density during exponential growth. Six biological replicates were assayed in two independent experiments.

Quantitative reverse transcriptase-PCR
Overnight cultures were diluted in BHIS medium containing thiamphenicol as needed. Cells were grown to mid-exponential phase (OD 600 0.8-1) or stationary phase (OD 600~1 .5) for analysis of flagellum (flgB, fliC) and toxin (tcdA, tcdB, tcdR) gene expression, respectively. RNA was isolated as described previously [33,96]. Briefly, cells were collected by centrifugation and stored in ethanol:acetone (1:1) at -80˚C overnight. Cells were lysed by bead beating in cold Tri-SURE (Bioline). Nucleic acids were extracted with chloroform, precipitated from the aqueous phase with isopropanol, washed with ethanol, and suspended in RNase-free water. RNA was treated with TURBO DNase (Thermo Fisher) according to the manufacturer's protocol. Synthesis of cDNA was done using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random hexamers according to the manufacturer's instructions. No-reverse transcriptase controls were included in all experiments. Real-time PCR was performed using 10 ng of cDNA, a final primer concentration of 1 μM, and SYBR Green Real-Time qPCR reagents (Bioline). Relative transcript abundance was calculated using the ΔΔCt method, with rpoC as the control gene and the indicated reference condition/strain. Primers used are listed

PLOS PATHOGENS
Rho factor mediated phase variation in S1 Table, with forward and reverse primers named according to the pattern gene-qF and-qR, respectively.

Visualizing heterogeneity using fluorescent reporters
To visually examine population heterogeneity, we used a previously described protocol [99,100]. Briefly, overnight cultures of strains containing the P flgM ::mCherryOpt reporter were diluted 1:100 into BHIS-Tm. Bacteria were grown anaerobically at 37˚C until OD 600~0 .5, 1 mL of culture was collected by centrifugation, and the remaining steps were performed aerobically. Cell pellets were washed with PBS, then suspended in 500 μl PBS and 120 μl 5x fixative (20 μl NaPO 4 , pH 7.4; 100 μl 16% paraformaldehyde) [99]. The solution was incubated in the dark at room temperature for 30 minutes followed by 30 minutes at 4˚C. After the fixative was removed, cells were washed three times with PBS before suspension in 500 μl PBS and incubation overnight in the dark at 4˚C to allow for fluorophore maturation. Slides for microscopy were prepared by placing 10 μl of concentrated culture onto a thin layer of 1% agarose applied directly to the surface of the slide. Microscopy was performed using a 60x oil immersion Nikon Plan Apo objective on a Keyence BZ-X810 equipped with Chroma 49005-UF1 for RFP detection.

Generation of strains
To generate rho expression plasmids, wild-type and mutant rho alleles were amplified from genomic DNA of recV flg OFF (RT1693) bacteria and 6 selected motile suppressors by PCR using primers R2308 and R2307 [96]. PCR products were cloned via the EcoRV and BamHI sites in pRT1611, a derivative of pRPF185 in which the gusA reporter gene was removed [33,58]. After transformation into E. coli DH5α, Cm-resistant clones were recovered at 30˚C to hinder additional mutations in rho. The presence of the rho insert and its sequence integrity were confirmed using primers R2308, R2307, R2366, and R2367. The expression plasmids and the pRT1611 control were introduced into C. difficile strains RT1693 (recV flg OFF), RT1702 (recV flg ON), RT1709 (MS5), and RT1941 (MS10) via conjugation with E. coli HB101 (pRK24). The presence of the expected plasmid was confirmed by PCR with vector-specific primers R1832 and R1833.
To introduce C. difficile rho (Cd-rho) into B. subtilis BS49, Cd-rho including its native ribosomal binding site (RBS) was amplified from R20291 genomic DNA by PCR using R2656 and R2657, digested with HindIII and SphI, and ligated into similarly digested pDR111, which allows for integration at the amyE site [102]. The resulting plasmid was transformed into B. subtilis BS49 strains bearing previously described transcriptional fusions of phoZ to flgB and its upstream regulatory region, and transformants were selected on LB-Spec agar.

Alkaline phosphatase assays
Overnight (~16 h) cultures of B. subtilis BS49 and C. difficile phoZ reporter strains were diluted 1:50 (C. difficile) or 1:100 (B. subtilis) into BHIS medium. Thiamphenicol was added to C. difficile growth media for plasmid maintenance. To induce expression of Cd-rho in B. subtilis, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the growth medium when cultures reached OD 600~0 .3. Controls without induction were processed in parallel. Where indicated, bicyclomycin (Cayman Chemical) was used at 50 μg/mL. Mid-exponential phase cells (OD 600 0.8-1.3, 1 mL) were collected by centrifugation, the supernatant was discarded, and pellets were stored at -20˚C overnight. Frozen pellets were thawed on ice, and the alkaline phosphatase (AP) assay was performed as previously described [64].

Spore purification
Overnight cultures (100 μL) were plated on ten 70:30 agar plates [103]. After 72 hours of growth at 37˚C, bacterial growth was scraped, suspended in 10 mL DPBS (Gibco) and kept at room temperature overnight. Spores were purified by collection of the growth in DPBS, then washing of the suspension four times with DPBS before purification using a sucrose gradient as described [104]. After discarding supernatant containing cell debris, the spore pellet was washed five more times with DPBS + 1% BSA. Spores were stored at room temperature until use.

Germination assay
Spore germination was analyzed at room temperature (27˚C) by measuring the change in OD 600 [75]. Germination was carried out in clear 96-well flat bottom plates (Corning) in a final volume of 100 μl and final concentration of 30 mM glycine, 50 mM Tris, 100 mM NaCl, pH 7.5. Spores were heat-activated at 65˚C for 30 minutes, cooled on ice and suspended to a final OD 600 of 0.7. At the initiation of the experiment, 10 mM sodium taurocholate (Sigma Aldrich) (TA) was added to induce germination; no-taurocholate controls were done in parallel. Optical density at 600 nm was measured every 2 minutes for 1 hour using a BioTek Synergy plate reader.

Sporulation assay
Sporulation assays were performed as described previously [74]. Briefly, C. difficile cultures were grown overnight in BHIS broth supplemented with 0.1% TA and 0.2% fructose to prevent spore accumulation. Cultures were diluted 1:30 in BHIS-0.1% TA-0.2% fructose, grown to an OD 600 of 0.5 and 250 μl of culture applied to 70:30 agar as a lawn [103]. A control ethanol resistance sporulation assay was performed at this point to ensure no spores were present in exponential phase cultures. After 24 hours of incubation at 37˚C, cells were suspended in BHIS to an OD 600 of 1.0, and an ethanol resistance sporulation assay was performed. A 0.5 ml aliquot was mixed with 0.5 ml of 57% ethanol to achieve a final concentration of 28.5% ethanol, vortexed, and incubated for 15 minutes to eliminate all vegetative cells. Serial dilutions were made in PBS-0.1% TA and plated on BHIS-0.1% TA agar for spore enumeration. Vegetative cells were enumerated by plating serial dilutions of the BHIS cell suspension on BHIS agar. Sporulation efficiency was calculated as the total number of spores divided by the total number of viable cells (spores plus vegetative).

Ethics statement
All animal studies were done in compliance with protocols approved by the UNC-CH Institutional Animal Care and Use Committee.
antibiotic cocktail consisted of kanamycin (400 mg/L), gentamicin (35 mg/L), colistin (850,000 units/L), vancomycin (45 mg/L), and metronidazole (215 mg/L) [105]. Four days prior to inoculation, the mice were switched to regular water for the remainder of the experiment. Clindamycin (10 μg/g body weight) was administered by intraperitoneal injection 48 hours prior to infection [14]. Mice were inoculated with 10 5 spores by oral gavage; control mice received PBS only. Inoculums were quantified by plating serial dilution on BHIS-0.1% TA agar and enumerating CFU. The animals were subsequently monitored for weight loss and diarrheal disease. Fecal samples were collected in pre-weighed tubes every 24 hours for 9 days. Fecal pellets were suspended in 1 mL DPBS and heated at 55˚C for 30 minutes. Serial dilutions were plated on TCCFA to enumerate CFU per gram feces [14]. Two independent experiments were done, each with 3 male and 3 female mice per C. difficile strain tested, for a total of 12 mice inoculated with each strain.  Fig. Supporting data for animal studies. (A) Orientation-specific PCR for the 6 additional invertible sequences found in R20291, in recV flg OFF (RT1693), MS5, and MS10. WT R20291 was included as a control. Orientation is labelled as ON/OFF for the three invertible sequences whose regulation has been studied (cwpV, flg, cmrRST) or as published (pub) or inverse (inv) based on the R20291 reference genome for the Cdi2, Cdi3, and Cdi5 sequences whose effects on gene expression are not known. (B) Antibiotic-treated male and female C57BL6 mice were inoculated with 10 5 spores of the indicated C. difficile strain. CFU in feces collected every 24 hours post inoculation were enumerated as an indication of intestinal burden of C. difficile. Shown are the full courses of infection for two independent experiments that each included 3 male and 3 female mice. The data are separated by motile suppressor and its respective parent strain for clarity with the same data for wildtype R20291 in both upper and lower panels, with means and standard deviation shown. Dotted line represents a limit of detection.