bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Clostridioides difficile LuxS mediates inter-bacterial interactions within

Ross Slater1, Lucy Frost1, Sian Jossi1, Andrew Millard2 and Meera Unnikrishnan1

1University of Warwick, Gibbet Hill Road, Coventry, United Kingdom CV4 7AL

2University of Leicester, University Road, Leicester LE1 7RH, UK

Running title: Role of C. difficile LuxS in biofilms

*Correspondence: Meera Unnikrishnan, Microbiology and Group, Division of

Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry,

CV4 7AL, UK, Email: [email protected]

KEYWORDS: C. difficile, B. fragilis, biofilms, luxS,

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Abstract

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms

that may play an important role in recurrent C. difficile . The mechanisms

underlying C. difficile community formation and inter-bacterial interactions are

nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule

that modulates formation across many bacterial species. We found that a strain

defective in LuxS, the that mediates AI-2 production, is defective in biofilm

development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT)

and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS

mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms

may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile

biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with

other gut species, C. difficile interactions with a common gut bacterium, Bacteroides

fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced

when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C.

difficile LuxS alleviates the B. fragilis mediated growth inhibition. Dual species RNA-

sequencing analyses from single and mixed biofilms revealed differential modulation

of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture,

indicating that AI-2 may be involved in induction of selective metabolic responses in

B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different

mechanisms to mediate formation of single and mixed species communities.

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Introduction

Clostridiodes difficile ( difficile), an anaerobic, opportunistic pathogen, is

the causative agent of C. difficile infection (CDI), a debilitating condition with

symptoms ranging from mild diarrhoea to severe pseudomembranous colitis.

~453,000 cases of CDI were reported in the United States in 2011 [1] and there have

been increasing reports of CDI from different parts of the world [2-4]. Treatment of

CDI is complicated by the fact that 20-36% of cases experience recurrence,

relapsing after completion of initial treatment [5]. CDI is primarily a hospital-acquired

infection with the elderly being at highest risk [6] and has been associated with the

disruption of the gut microbiota as a result of the use of broad-spectrum antibiotics.

However, more recently, there has been a reported increase in community-acquired

cases where patients do not have the typical risk factors such as antibiotic exposure

or recent hospitalisation [7].

Colonisation of C. difficile and development of CDI is influenced by composition of

the native gut microbiota. Broadly, Bacteroides, Prevotella, Difidobacterium,

Enterococcaceae and Leuconostocaceae spp. correlate negatively [8-10], and

Lactobacilli, Aerococcaceae, Enterobacteriaceae, and Clostridium correlate

positively to C. difficile colonisation and disease [8, 10-13]. While mechanisms

underlying colonisation resistance are not entirely clear, some pathways have been

described recently. Secondary bile acids produced by like Clostridium

scindens can inhibit C. difficile growth, while other bile acids such as

chenodeoxycholate can inhibit spore germination [14-16]. Studies have shown that

the ability of C. difficile to utilise metabolites produced by the gut microbiota or

mucosal sugars such as sialic acid promote C. difficile expansion in the gut [17, 18].

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However, gaps still remain in the understanding C. difficile interactions with members

of the gut microbiota.

Research into CDI has primarily focused on the action of two large toxins [19, 20]

that cause tissue damage, neutrophil recruitment and a severe inflammatory

response [21]. More recently, a number of factors have been shown to influence

adhesion of C. difficile to host cells and early colonisation, including cell wall

proteins, adhesins and flagella [22-26]. C. difficile also produces biofilms that confer

increased resistance to antibiotics [27-29] and have recently shown to be associated

with C. difficile infection in vivo, in close association with other commensal gut

species [30, 31].

Formation of adherent communities within the gut requires communication between

bacteria. For many species, quorum sensing (QS) is important for the construction

and/or dispersal of biofilm communities [32], with bacteria utilising diverse QS

systems [32, 33]. Many bacteria possess the metabolic enzyme LuxS, which is

involved in the detoxification of S-adenoslylhomocysteine during the activated methyl

cycle. Whilst catalysing the reaction of S-ribosylhomocysteine to homocysteine,

LuxS produces the bi- 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is an

unstable compound that spontaneously cyclises into several different forms. These

ligands are collectively known as autoinducer-2 (AI-2), a group of potent, cross-

species QS signalling molecules [34]. In many bacteria, including C. difficile, AI-2

plays a role in biofilm formation, with luxS mutants showing a defect during biofilm

formation and development [27, 35-40]. The precise mode of action for LuxS in C.

difficile has remained elusive as a result of conflicting studies and the lack of a clear

receptor for AI-2 [35, 41, 42].

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Here we investigate the role of LuxS within C. difficile and mixed biofilm

communities. Interestingly, we find that C. difficile LuxS/AI-2 mediates the induction

of two putative C. difficile R20291 prophages within C. difficile biofilms. In mixed

biofilms, we show that in the presence of B. fragilis, a gut bacterium, C. difficile

growth is inhibited and this inhibition is alleviated in the absence of LuxS. Dual

species transcriptomics show that distinct metabolic pathways are triggered in mixed

cultures with the wild type (WT) and luxS mutant C. difficile strains.

Results

LuxS mediates biofilm formation in vitro

We previously reported that a R20291 C. difficile luxS mutant (luxS) was defective in

biofilm formation as measured by crystal violet (CV) staining [27]. However, in our

subsequent studies, although we see a reduction in biofilms at 24 h, we observe a

high variability in the WT biofilms formed at 24 h and in the reduction in the luxS

mutant between experiments (Figure 1A). Nevertheless, the biofilm defect for luxS

was very consistent at later time points (72 h) (Figure 1A, B). In spite of differences

in total biofilm content, colony counts from the WT and luxS biofilms were similar at

both times (Figure 1C).

To determine if AI-2 signalling is involved in biofilm formation, we first performed an

AI-2 assay from both planktonic and biofilm supernatants as described by Carter et

al. 2005 (Figure S1A). AI-2 is produced maximally in mid-log and stationary phases

as previously reported [44]. The WT strain produced less AI-2 in 24 h biofilms

compared to log phase culture, while the luxS strain did not produce AI-2 as

expected (Figure S1B). To study if the biofilm defect could be complemented by

chemically synthesised 4,5-dihydroxy-2, 3-pentanedione (DPD), the precursor of AI-

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2, was supplemented in the culture medium. Whilst high concentrations (>1000 nM)

appeared to have no effect on biofilm formation (data not shown), a concentration of

100 nM was capable of restoring the WT phenotype (Figure 1D), indicating that AI-2

may be involved in signalling within C. difficile biofilms.

RNA-seq analysis reveals LuxS-mediated prophage induction

To investigate mechanism of action of LuxS/AI-2 in C. difficile, transcriptional profiles

of planktonically cultured C. difficile WT and luxS strains were first compared using

RNA-seq. However, surprisingly, no differential transcriptional changes were

observed (Accession number E-MTAB-7486). Following this, an RNA-seq analysis

was performed with total RNA isolated from C. difficile WT and luxS biofilms grown in

BHIS + 0.1 M for 18 h (Accession number E-MTAB-7523).

The DESeq2 variance analysis package [45] was used to identify genes that were

differentially expressed in luxS ≥ 1.6-fold relative to the WT strain, with a p -adjusted

value ≤ 0.05. This pairwise analysis identified 21 differentially expressed genes

(Figure 2A & C) (Table 1). Interestingly, all 18 down-regulated genes correspond to

two prophage regions located within the C. difficile R20291 genome (Figure 2B), as

identified using the online phage search tool, Phaster [46, 47]. A Fisher’s exact test

(p-value < 0.001 for both prophage regions) further confirmed an enrichment of

differently regulated genes in prophage regions. There were only three genes

upregulated in the luxS biofilms compared to the WT; two of these were involved in

trehalose utilisation.

To demonstrate the presence of phage in the biofilm, cell-free supernatants were

treated with DNase, before a subsequent DNA extraction was performed. As the

bacterial cells were already removed, only DNA within intact bacteriophages would

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be protected from DNase. A 16S rRNA gene PCR was performed to confirm

digestion of all free extracellular genomic DNA from the biofilm (Figure 3A). PCRs

with primers corresponding to genes specific to each prophage, confirmed that the

DNA extracted had come from the phage (Figure 3 B). Since cell lysis is linked to

phage release, we quantified and compared the total extracellular DNA (eDNA)

content of luxS mutant and WT biofilms. The WT biofilms contained more eDNA

compared to the luxS mutant (Figure 3C). Overall, these data suggest that AI-2 may

play a role in inducing prophages in C. difficile biofilms, which leads to phage-

mediated host cell lysis and may contribute to subsequent biofilm accumulation.

C. difficile is inhibited when cultured with B. fragilis in mixed biofilms

Given the high microbial density within the gut, C. difficile likely needs to interact with

other members of the gut microbiota to establish itself within this niche. As an inter-

species signalling function has been previously proposed for AI-2 [48, 49], we sought

to investigate the interactions between a gut-associated Bacteroides spp and C.

difficile.

C. difficile formed significantly more biofilms in vitro compared with Bacteroides

fragilis in monocultures, as measured by CV staining (Figure 4A). When both

organisms were co-cultured, less biofilm was formed compared to C. difficile

monoculture (Figure 4A). To investigate the impact of co-culturing on both C. difficile

and B. fragilis, bacterial numbers (CFU/ml) were determined from monoculture and

mixed biofilms (Figure 4B). Colony counts obtained from the mono and co-culture

biofilms, confirmed that B. fragilis was a poor biofilm producer when cultured alone.

Interestingly, when both species were co-cultured, the CFU/ml for C. difficile was

significantly reduced, and the CFU/ml of B. fragilis significantly higher. This reduction

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of colony counts of C. difficile was observed at both 24 h (Figure 4B) and 72 h

(Figure S2). AI-2 production from single and mixed biofilms was also quantitated. We

observed no production of AI-2 by B. fragilis, and a reduction of AI-2 production by

the mixed biofilms compared with the WT C. difficile biofilms (Figure S3). Confocal

microscopy analysis of mono and co-culture biofilms showed that the single species

biofilms contained more live bacteria compared to co-culture biofilms, which had

higher numbers of dead cells (Figure 4C). These data suggest that the presence of

B. fragilis in biofilms results in inhibition of C. difficile growth.

To understand if the inhibitory effect was due to a factor secreted by B. fragilis, C.

difficile and B. fragilis were co-cultured under planktonic conditions for 6 h and 10 h

(Figure 4D). However, there were no significant differences in C. difficile bacterial

numbers between mono and co-culture. Additionally, supplementing biofilms with B.

fragilis planktonic or biofilm culture supernatants did not cause C. difficile growth

inhibition (Figure S4), indicating that the observed inhibitory effects were specific to

adherent biofilms i.e. when they are in close proximity to each other.

LuxS is involved in the B. fragilis inhibition of C. difficile

To study the role of LuxS in C. difficile-B. fragilis interactions, WT C. difficile and luxS

strains were co-cultured with B. fragilis in mixed biofilms. CV staining of biofilms

showed that there was less luxS biofilm formed compared to the WT when co-

cultured with B. fragilis (Figure 5A). While colony counts of C. difficile in

monocultures were similar for both WT and luxS strains (Figure 1B) when co-

cultured with B. fragilis the bacterial counts for both C. difficile strains were

significantly reduced, although the reduction was significantly higher for the WT than

luxS (Figure 5B). Colony counts for B. fragilis increased significantly during co-

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culture, with similar levels observed in both co-culture conditions (Figure 5B). These

data suggest that AI2/LuxS is involved in mediating the B. fragilis-induced inhibition

of C. difficile, when they are within adherent communities.

Dual species RNA-seq analysis shows altered in C. difficile and B.

fragilis in the absence of LuxS

To investigate mechanisms underlying the C. difficile inhibition mediated by B.

fragilis, we performed an RNA-seq analysis to compare biofilm monocultures of C.

difficile WT, luxS or B. fragilis with mixed biofilm co-cultures of C. difficile WT or luxS

with B. fragilis (Accession number E-MTAB-7523). Differentially expressed genes

were defined as having ≥ 1.6-fold relative to their respective control (mono-cultures

of either B. fragilis or C. difficile WT), with an adjusted p-adjusted value ≤ 0.05.

A total of 45 genes were up-regulated (21) or down-regulated (24) in C. difficile WT

(Figure 6, Table 2), while 69 genes were differentially expressed in C. difficile luxS of

which 34 were down-regulated and 35 up-regulated, during co-culture with B. fragilis

(Figure 6A, Table 3).

Eight up-regulated genes were specific to C. difficile WT co-culture (Table 2). Of

these, four genes involved in fatty acid biosynthesis and metabolism: fabH encoding

3-oxoacyl-[acyl-carrier protein] synthase III, fabK encoding trans-2-enoyl-ACP

reductase, accC encoding a biotin carboxylase (acetyl-CoA carboxylase subunit A),

and accB encoding a biotin carboxyl carrier protein of acetyl-CoA carboxylase (Table

2). 11 genes were down-regulated exclusively in WT however, these genes do not

coincide with a specific metabolic pathway.

18 up-regulated genes were specific to luxS in co-culture (Table 3). These include a

putative homocysteine S-methyltransferase, a putative osmoprotectant ABC

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transporter, binding/ permease protein, ribonucleoside-diphosphate

reductase alpha chain (nrdE) and two genes from the trehalose operon: a PTS

system II ABC transporter, and trehalose-6-phosphate (treA). 24 genes

were down-regulated (Table 3), which include 3 genes involved in thiamine

metabolism thiD, thiK and thiE1, (CDR20291_1497, CDR20291_1498 and

CDR20291_1499 respectively) which encode a putative phosphomethylpyrimidine

, 4-methyl-5-beta-hydroxyethylthiazole kinase and thiamine-phosphate

pyrophosphorylase respectively.

A total of 26 genes were differentially expressed in both C. difficile WT and C. difficile

luxS when co-cultured with B. fragilis (Figure 6A, Table 4). These include six up-

regulated genes (accB, abfH, abfT, abfD, sucD and cat1) involved in carbon and

butanoate metabolism, with cat1, which encodes succinyl-CoA:coenzyme A

, being the highest up-regulated gene for both C. difficile strains.

In contrast, a higher number of genes (266) were differentially expressed in B.

fragilis when co-cultured with C. difficile (WT and luxS) (Table S3, Figure 6B). A total

of 114 B. fragilis genes were found to be specific to C. difficile WT co-culture, with 56

of these up-regulated and 58 down-regulated (Table 5). Similarly, 91 genes were

found to be specific to C. difficile luxS co-culture, with 56 of these up-regulated and

35 down-regulated (Table 6). Although distinct B. fragilis expression profiles were

observed with the WT and luxS (Figure 6B), there were no clear pathways identified

in the datasets.

Whilst the highest up-regulated gene in both WT and LuxS co-cultures encodes a

putative virus attachment protein, no other viral genes were shown to be up-

regulated (Table S3). Genes encoding iron containing proteins desulfoferrodoxin and

rubrerythrin were highly up-regulated in both conditions. Interestingly, multiple copies

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of fecR, a key regulator for the ferric citrate transport system [50], and ferrous iron

transport protein B were also up-regulated. Additionally, a number of other metabolic

pathways were up-regulated, including four genes (encoding 3-isopropylmalate

dehydratase small subunit, 3-isopropylmalate dehydrogenase, and 3-

isopropylmalate dehydratase large subunit) involved in valine, leucine and isoleucine

biosynthesis and C5-branched dibasic acid metabolism. It should be noted that many

of the up-regulated genes were hypothetical proteins of unknown function. Similarly,

several metabolic pathways were down-regulated in both co-culture conditions.

These include six genes involved in carbon metabolism, four genes involved in

alanine, aspartate and glutamate metabolism, and four genes involved in the

biosynthesis of amino acids, although these appear to be single genes rather than

specific pathways.

Discussion

Inter-bacterial interactions within gut communities are critical in controlling invasion

by intestinal pathogens. Quorum sensing molecules such as AI-2 are instrumental in

bacterial communication, especially during formation of bacterial communities [32,

37, 38, 51-54]. C. difficile produces AI-2, although the mechanism of action of

LuxS/AI-2 in C. difficile, particularly within a biofilm community is unclear. We report

here that the C. difficile LuxS/AI-2 plays an important role in the formation of single

and multi-species communities. In C. difficile biofilms, LuxS mediates the induction of

prophages, which likely contributes to the biofilm structure. Whereas, in a mixed

biofilm of C. difficile and the intestinal commensal and pathogen, B. fragilis, LuxS

likely triggers the induction of differential metabolic responses in B. fragilis, that leads

to growth inhibition of C. difficile. To our knowledge, this is the first time dual species

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RNA-seq [55] has been applied to analyse interactions between anaerobic gut

bacteria in an adherent biofilm community.

Bacterial biofilms contain a number of extracellular components that make up their

complex structure including extracellular DNA (eDNA), a key component that binds

together bacteria within a community. Autolysis is a common mechanism by which

eDNA is released from bacterial cells [56]. In bacteria such as Staphylococcus

aureus and Pseudomonas aeruginosa, eDNA is generated through the lysis of

subpopulations within a biofilm, under the control of quorum sensing [56-59]. In C.

difficile luxS mutant (luxS) biofilms, we observe reduced induction of two C. difficile

prophages compared with the WT. These phage loci were conserved in several C.

difficile strains with the Region 2 encoding a phiC2-like phage [60]. Given that it has

previously been shown that eDNA is a major component of C. difficile biofilms [27,

61], it is likely that phage-mediated bacterial cell lysis and subsequent DNA release

help build a biofilm. However, phages are also known to control biofilm structure in

some organisms: a filamentous phage of P. aeruginosa was reported to be a

structural component of the biofilm [62] and an AI-2 induced phage mediated the

dispersal of faecalis biofilms [43]. Although attempts to visualise

phages from C. difficile biofilms with transmission electron microscopy (data not

shown) were unsuccessful, we cannot rule out the possibility that C. difficile phages

may directly influence the biofilm structure. While the precise mechanisms by which

LuxS/AI-2 controls phage induction are yet to be elucidated, AI-2 appears to be

signalling through a yet unidentified AI-2 receptor in C. difficile. Phage-mediated

control of biofilms may in part explain the variation observed in biofilm formation

between different C. difficile strains [63].

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The gut hosts a variety of bacterial species, which compete or coexist with

each other. It is likely that the bacteria occupying this niche form multi-species

bacterial communities in association with the mucus layer. Interactions within such

communities are important in gaining a better understanding of phenomena such as

‘colonisation resistance’ which prevents pathogens such as C. difficile from

establishing an infection [64]. Whilst sequencing studies have identified members of

the Bacteroides genus as being associated with gut colonisation resistance to C.

difficile, the mechanisms have remained elusive [10]. A recent study demonstrated

that production of the enzyme: bile salt hydrolase, is responsible for the inhibitory

effect of B. ovatus on C. difficile [65]. This study reported that in the presence of bile

acids, cell free supernatants for B. ovatus were capable of inhibiting the growth of C.

difficile whereas in the absence of bile acids, C. difficile growth was promoted [65].

Since bile acids are not supplemented into our media, a different mechanism is likely

responsible for B. fragilis mediated inhibition of C. difficile. Also, the growth

restraining effects of B. fragilis on C. difficile were evident only within mixed biofilms,

not in planktonic culture or with culture supernatants. While it is likely that cell-cell

contact is essential for the inhibitory effect, we cannot exclude involvement of an

inhibitory secreted molecule that accumulates to a higher concentration within a

biofilm environment, or that B. fragilis has a competitive growth advantage in a

biofilm environment.

A dual species RNA-seq analysis performed to understand the interactions between

the two bacterial species, showed that largely all the differentially expressed genes

mapped to distinct metabolic pathways. Overall, a higher number of genes were

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modulated in B. fragilis as compared to C. difficile strain during co-culture, which is in

line with the growth characteristics observed. Carbon and butanoate metabolism

pathways were induced in C. difficile strains in response to co-culture (accB, abfH,

abfT, abfD, sucD and cat1). As B. fragilis is known to produce succinate [66], it is

likely that the upregulation in these pathways results from the increased levels of

succinate in the culture medium. However, since gut microbiota-produced succinate

promotes C. difficile growth in vivo [17], it is unlikely that these changes are directly

responsible for the observed inhibition of C. difficile. However, bacteria utilise

carbohydrates in a sequential manner [67]. Consistent with this, we observed a

down-regulation of genes important for the utilisation of pyruvate such as bcd2 and

idhA encoding for butyryl-CoA dehydrogenase and (r)-2-hydroxyisocaproate

dehydrogenase respectively. Such a shift in metabolism could allow B. fragilis to fully

consume other metabolites, and thus enabling it to outcompete C. difficile.

Additionally, it is interesting to note that a number of copies of the ferric citrate

transport system regulator, fecR, are up-regulated in B. fragilis during C. difficile co-

culture. The ferric citrate transport system is an iron uptake system that responds to

the presence of citrate [50, 68, 69]. Analysis of the C. difficile genome using BLAST

(NCBI) showed that C. difficile does not possess this iron uptake system. Given the

evidence that ferric citrate is an iron source in the gut [50, 70], B. fragilis may have

an advantage over C. difficile in sequestering iron, and thus preventing C. difficile

colonisation. Although the clear modulation of metabolic pathways strongly suggest

a competitive advantage of B. fragilis over C. difficile, it is possible that the genes

with unknown functions that are differentially expressed in B. fragilis (Table S1),

encode pathways for the production of yet to be identified small inhibitory molecules.

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The LuxS/AI-2 quorum sensing system is known to have a cross-species signalling

role in many bacteria [48, 71, 72]. While all sequenced C. difficile strains produce AI-

2, only selected strains of B. fragilis have the ability to produce AI-2 [73]. A recent

study showed that Ruminococcus obeuem inhibited Vibrio cholerae in the gut via

LuxS/AI-2 mediated downregulation of V. cholerae colonisation factors [74]. Also, AI-

2 produced by engineered E. coli was reported to influence /bacteroidetes

ratios in microbiota treated with streptomycin [75]. Our data show the involvement of

LuxS/AI-2 in the B. fragilis-mediated C. difficile growth inhibition. As the B. fragilis

strain used in this study does not produce AI-2, it is likely that B. fragilis responds

differentially to AI-2 produced by C. difficile. Similar to the WT, most transcriptional

changes were in metabolic pathways, although specific sets of genes were

modulated in the luxS mutant. Modulation of prophage genes was not seen, unlike

single biofilm cultures, indicating different dominant mechanisms at play in a multi-

species environment.

It was interesting to note that the trehalose utilisation operon, which provides a

growth advantage to C. difficile against other gut bacteria [76], was upregulated in

both the single luxS biofilms (Table 1) and luxS co-cultured with B. fragilis (Table 3).

The upregulation of a system component and treA (both in the

same operon), likely enables increased utilisation of trehalose, providing an

additional carbon source. Like glucose, trehalose acts as an osmoprotectant [77] and

its presence within the cell may help maintain protein conformation during cellular

dehydration. It is possible that trehalose plays a role in building C. difficile biofilms,

as reported for Candida biofilms [78]. Our preliminary studies with exogenous

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trehalose levels similar to those used by Collins et al. (2018) showed an inhibitory

effect on biofilm formation by both WT and luxS, although no differential effects were

observed between luxS and WT (data not shown). However, further investigations

into accumulation of trehalose within biofilms are required to clarify the role of

trehalose in luxS mediated biofilm formation.

In conclusion, we report that C. difficile LuxS/AI-2 may play a key role in building C.

difficile communities through mediating prophage induction, and subsequent

accumulation of eDNA. In mixed communities, C. difficile AI-2 likely signals to B.

fragilis to induce an altered metabolic response, enabling it to outgrow C. difficile.

Further studies are required to understand the precise AI-2 sensing pathways

involved.

Materials and Methods

Bacterial strains and media: Two bacterial species were used in this study – C.

difficile strain: B1/NAP1/027 R20291 (isolated from the Stoke Mandeville outbreak in

2004 and 2005), and Bacteroides fragilis (kindly provided by Dr Gianfranco Donelli,

Rome). A luxS Clostron R20291 mutant described previously in Dapa et al, 2013

was used in this study. Both species were cultured under anaerobic conditions (80%

N2, 10% CO2, 10% H2) at 37°C in an anaerobic workstation (Don Whitley, United

Kingdom) in BHIS, supplemented with L-Cysteine (0.1% w/v; Sigma, United

Kingdom), yeast extract (5 g/l; Oxoid) and glucose (0.1 M).

Vibrio harveyi strain: BB170 was used to measure AI-2. V. harveyi strains were

cultured in aerobic conditions at 30°C in Lysogeny broth (LB) supplemented with

kanamycin (50 µg/ml).

16 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Biofilm formation assay: Biofilms were grown as per the previously published

protocol [27]. Overnight cultures of C. difficile were diluted 1:100 in fresh BHIS with

0.1 M glucose. 1 ml aliquots were pipetted into 24-well tissue culture treated

polystyrene plates (Costar), and incubated under anaerobic condition at 37°C, for 6 –

120 h. Tissue culture plates were pre-incubated for 48h prior to use. The plates were

wrapped with parafilm to prevent liquid evaporation.

Measurement of biofilm biomass: Biofilm biomass was measured using crystal

violet (CV) [27, 79]. After the required incubation, each well of the 24-well plate was

washed with sterile phosphate buffer saline (PBS) and allowed to dry for a minimum

of 10 mins. The biofilm was stained using 1 ml of filter-sterilised 0.2% CV and

incubated for 30 mins at 37°C, in anaerobic conditions. The CV was removed from

each well, and wells were subsequently washed twice with sterile PBS. The dye was

extracted by incubated with 1 ml methanol for 30 mins at room temperature (RT) in

aerobic conditions. The methanol-extracted dye was diluted 1:1, 1:10 or 1:100 and

OD570 was measured with a spectrophotometer (Biochrom, UK).

For bacterial cell counts from the biofilm, the planktonic phase was removed and

wells were washed once using sterile PBS. The adherent biofilms were then

detached by scrapping with a sterile pipette tip and re-suspended into 1 ml PBS.

Serial dilutions were made and plated onto BHIS plates to determine the CFU

present in the biofilm.

Co-culture biofilm assay: For generation of co-culture biofilms, both C. difficile and

B. fragilis were diluted to an OD600 of 1. Both species were diluted 1:100 into fresh

BHIS with 0.1 M glucose. Biofilms assays were performed as described above and

measured by a combination of CV staining and CFU. To distinguish between C.

difficile and B. fragilis, serial dilutions used for determining CFU were plated on BHIS

17 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

plates additionally supplemented with C. difficile selective supplement (Oxoid, UK).

Colonies can be differentiated by size and colony morphology as B. fragilis form very

small colonies.

Exogenous addition of DPD: To analyse the potential signalling role of AI-2, biofilm

assays were performed as described above in BHIS with 0.1 M glucose containing 1

nM, 10 nM, 100 nM, or 1 µM of chemically synthesised, exogenous 4,5-Dihydroxy-

2,3-pentanedione (Omm Scientific, Texas USA) for both C. difficile WT and LuxS.

BHIS with 0.1 M glucose was used as a control. Samples were washed and stained

with 0.2% CV at either 24 h or 72 h.

AI-2 Assay: The AI-2 bioluminescence assay was carried out essentially as

described by Bassler et al. 1993 [80]. The V. harveyi reporter strain BB170 was

grown overnight in LB medium before being diluted 1: 5000 in Autoinducer Bioassay

(AB) medium containing 10 % (v/v) cell-free conditioned medium collected from

either planktonic or biofilm C. difficile cultures (in BHI) and allowed to grow at 30°C

with shaking. AB medium containing 10 % (v/v) from V. harveyi BB120 was used as

a positive control, and 10 % (v/v) sterile BHI medium as a blank. Luminescence was

measured every hour using a SPECTROstar Omega plate reader. Induction of

luminescence was taken at the time when there was maximal difference between the

positive and negative controls (usually 2–5 h) and is expressed as a percentage of

the induction observed in the positive control.

Confocal Microscopy: C. difficile strains were grown in 4-well glass chamber slides

(BD Falcon, USA) in BHIS + 0.1 M glucose. Chamber slides were incubated at 37°C

in anaerobic conditions for 24 hrs – 72 hrs and stained with BacLight live/dead stain

mixture (Molecular Probes, Invitrogen), which contains the nucleic acid stains Syto 9

and propidium iodide (staining live bacteria green and dead bacteria red

18 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

respectively). Following incubation, wells were gently washed twice with PBS 0.1%

w/v saponin to remove unattached cells and permeabilise the biofilm. Biofilm

samples were incubated with the dye for 15 minutes at 37°C after which the samples

were washed twice with PBS. Cells were fixed with 4% paraformaldeyhyde (PFA) for

15 mins. All samples were visualised with a Zeiss Observer LSM 880 confocal

scanning microscope at 60x – 100x magnification.

RNA-seq Biofilms were grown for 18 h in BHIS + glucose, supernatants were

removed and attached biofilms were washed with 1 ml PBS. Biofilms were disrupted

and RNA was extracted using Trizol (Invitrogen, UK).5 µg of extracted RNA was

treated with RiboZEROTM (Illumina, UK) according to the manufacturer’s protocol to

deplete rRNA. cDNA libraries were prepared using TruSEQ LT (Illumina, UK)

according to the manufacturer’s instructions. Briefly, samples were end-repaired,

mono-adenylated, ligated to index/adaptors. Libraries were quantified by bioanalyzer

and fluorometer assay. The final cDNA library was prepared to a concentration of 10

-12 pM and sequenced using paired end technology using a version-3 150-cycle kit

on an Illumina MiSeqTM (Illumina, UK).

RNA-seq Analysis: The paired-end sequencing reads from RNA-seq experiments

were mapped against the appropriate reference genome (NC_013316 for C. difficile

and a de novo assembly using RNA SPAdes v3.9 with default settings [81] from the

RNA-sequence reads for B. fragilis [Accession number PRJEB29695). The first read

was flipped into the correct orientation using seqtk v1.3 (https://github.com/lh3/seqtk)

and the reads were mapped against the reference genome using BWA v0.7.5 with

the ‘mem’ alignment algorithm [82]. BAM files were manipulated with Samtools

v0.1.18 using the ‘view’ and ‘sort’ settings [82]. Sorted BAM and GFF (general

feature format) files were inputted into the coverageBed tool v2.27.0 with default

19 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

settings [83] to gain abundance of each genomic feature. The R package DESeq2

was used with default settings to calculate differential gene expression using a

negative binomial distribution model [45]. The data was filtered by applying a cut-off

of 1.6 for the fold change and 0.05 for the adjusted p-value. All sequencing reads

were submitted to the European Bioinformatics Institute (Accession numbers E-

MTAB-7486, E-MTAB-7523 and PRJEB29695).

As analysis by BLAST (NCBI) demonstrated species specificity for mapping, co-

culture samples were mapped to each species reference separately. Initial mapping

of the B. fragilis strain to a published reference proved unsuccessful, offering a poor

rate of alignment of 60%. As the B. fragilis strain has not been previously

sequenced, and because we were not successful in generating high quality genome

sequence, a reference was generated from RNA library of B. fragilis using the

software rnaSPAdes v3.9 [84] and annotated using Prokka v1.11 (default settings)

[85]. The reads from each condition were mapped to their respective reference

sequence using BWA v0.7.5 (‘mem’ algorithm) [82, 86] and counted using

coverageBed v2.27.0 [83].

Metabolic pathways in C. difficile were identified using the KEGG mapper [87], a tool

that identifies the function of genes in a published genome. As the B. fragilis strain

used in this study does not have a published reference genome, blastKOALA [88]

was used to search for gene homology within metabolic pathways. Heatmaps were

generated from normalised gene expression data outputted from DESeq2, using the

online tool Heatmapper [89] using the default settings.

PCR analysis

16S PCRs were performed using the universal 16S rRNA bacterial primers 27F and

1392R (Table S2). Primers were constructed for prophage genes CDR20219_1208

20 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

and CDR20291_1436 (Table S2) to confirm the presence of prophage within C.

difficile biofilms. PCR was carried out using Fusion High-Fidelity DNA

(NEB, USA) following the manufacturer’s protocol. Samples were heated to 95°C for

5 mins followed by 35 cycles of: 95°C for 30 seconds, 51°C for 30 seconds and 72°C

for 30 seconds, after which samples were heated to 72°C for 10 mins.

eDNA quantification

eDNA was extracted from C. difficile biofilms grown in a 24-well plate as described

above, using a protocol described in Rice et al. [57]. Briefly, the plate was sealed

with parafilm and chilled at 4oC for 1 hour. 1 μl 0.5 M EDTA was added to each well

and incubated at 4oC for 5 mins. The medium was removed and biofilms were

resuspended in 300 μl 50 mM TES buffer (50 mM Tris HCl /10 mM EDTA/500 mM

NaCl). The OD600 was measured to determine biofilm biomass and the tubes were

centrifuged at 4oC at 18,000 g for 5 mins. 100 μl of supernatant was transferred to a

tube of chilled TE buffer (10 mM Tris HCl/ 1 mM EDTA) on ice. DNA was extracted

using an equal volume of phenol/chloroform/isoamyl alcohol three times. 3 volumes

of ice-cold 100% ethanol and 1/10 volumes 3 M sodium acetate were added to the

aqueous phase to precipitate the DNA. The DNA pellet was washed with 1 ml ice-

cold 70% ethanol, dissolved in 20 μl TE buffer, quantified by Qubit fluorometer

(Thermo Fisher).

Statistical analysis

All experiments were performed in triplicate, with at least three independent

experiments performed. Paired student’s t-test was used to determine if differences

between two groups were significant, and one way-ANOVA was used to compare

multiple groups. Mann-Whitney U tests were used to compare non-parametric data.

21 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fisher’s exact t-test was used to confirm the enrichment of differently regulated

genes in prophage regions.

Acknowledgments

We thank Gianfranco Donelli, Director, Microbial Biofilm Laboratory, IRCCS

Fondazione Santa Lucia, Italy for providing the Bacteroides fragilis isolate and Prof

Paul Williams, University of Nottingham for providing the Vibrio harveyi strain BB170.

This was in part supported by a Seed grant from the Wellcome Warwick Quantitative

Biomedicine Programme (Institutional Strategic Support Fund: 105627/Z/14/Z).

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Figure legends

Figure 1: LuxS biofilm defect is reversed by addition of DPD

(A) WT and LuxS biofilms were grown for 24 h or 72 h and stained with 0.2% CV,

followed by measuring OD570, N=5. (B) Colony counts from biofilms (N=7) after 24 h

and 72 h. (C) Representative pictures of crystal violet stained C. difficile WT and

luxS biofilms after 72 h. (D) The AI-2 precursor, DPD, was exogenously

supplemented to LuxS at a concentration of 100 nM, followed by biofilm staining and

quantitation with 0.2% CV after 72 h. Error bars indicate SD, *p < 0.05, ***p<0.001

as determined by students t-test or by Mann-Whitney U test.

36 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2: Down-regulation of prophage genes in C. difficile luxS mutant

(A) Pairwise analysis identified 21 differentially expressed genes in luxS (red points).

All 18 down-regulated genes clustered into two regions. (B) Three prophage regions

are identified in the C. difficile genome using Phaster. Regions 2 and 3 were down

regulated in luxS. (C) Heat map representation of the genes that were differentially

expressed in luxS red and green indicate down- and up-regulation respectively when

compared to WT. 18 prophage genes were found to be down-regulated in luxS

relative to WT, whilst three metabolic genes involved in trehalose metabolism were

up-regulated in luxS relative to WT. Data shown is the mean of 3 independent

experiments in triplicates. Differential expression was defined as ≥ 1.6-fold change

relative to WT with an adjusted p-value ≤ 0.05.

Figure 3: Presence of phage and eDNA in C. difficile biofilms

The phage origin of DNA isolated from WT biofilms was confirmed by PCR, using

primers for 16S (A) and two phage genes (CDR20291_1436 and CDR20291_1208

(B). WT-1-3 are three biological replicates. (C) Total eDNA extracted from the WT

and luxS mutant biofilms after 24 h, normalised to the biofilm biomass. N=3,

****p<0.0001 as determined by Mann-Whitney U test.

Figure 4: B. fragilis mediated inhibition of C. difficile in mixed biofilms.

(A) Biofilm of C. difficile, B. fragilis and both species co-cultured (mixed) were grown

for 24 h and stained with 0.2% CV, followed by measuring OD570. (B) Colony counts

for both C. difficile and B. fragilis from mono and co-culture biofilms after 24 h. (C)

Mono and co-culture biofilms were visualised by confocal microscopy after 24 h

37 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

culture using a live/dead stain. Live bacteria are stained green and dead bacteria

stained red using Syto 9 and propidium iodide dye respectively. (D) Colony counts

for both C. difficile and B. fragilis from mono and co-culture during planktonic growth.

Data shown is the mean of 3 independent experiments in triplicates and error bars

indicate SD, **p < 0.005, ****p<0.0001 as determined by one way ANOVA.

Figure 5: B. fragilis-mediated inhibition of C. difficile is more prominent for WT

than LuxS

(A) Biofilms for mono and co-cultures of C. difficile WT and luxS with B. fragilis were

grown for 24 h and stained with 0.2% CV and were quantified using a

spectrophotometer OD570. (B) Colony counts for C. difficile WT, C. difficile LuxS

during co-culture with B. fragilis were performed at 24 h. Data shown is the mean of

3 independent experiments in triplicates and error bars indicate SD, **p < 0.01,

***p<0.001 as determined by one way ANOVA.

Figure 6: Dual species RNA-seq shows modulation of metabolic pathways in

C. difficile WT, luxS and B. fragilis

Heat maps showing clustering of up- and down-regulated genes in (A) C. difficile WT

and luxS co-cultured with B. fragilis compared to C. difficile WT mono-culture, and in

(B) B. fragilis co-cultured with C. difficile WT and luxS compared to B. fragilis mono-

culture. Red indicates genes that are down-regulated, whilst green indicates genes

that are up-regulated.

Figure S1: Analysis of AI-2 production by WT and LuxS in planktonic and

biofilm growth conditions.

38 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Strains were grown anaerobically in BHI medium. (A) Planktonic aliquots were

removed for OD600 readings (line) and cell-free supernatants were collected at each

time-point (0 – 12 h) and assayed for AI-2 activity using V. harveyi BB170 reporter

assay (bars). Data shown is the mean of 3 independent experiments in triplicates

and error bars indicate SD (B) Cell free supernatants were collected from WT and

luxS biofilms cultured for 24 h and WT planktonic cultures at 8 h. AI-2 activity was

measured using V. harveyi BB170 reporter assay. Bioluminescence is shown as a

percentage of wildtype V. harveyi BB120 bioluminescence, which was assumed to

be 100 %. Data is representative of 2 independent experiments.

Figure S2 C. difficile inhibition in mixed biofilms

Colony counts from monoculture C. difficile and co-culture biofilms with B. fragilis

after 72 h. Data is representative of 3 independent experiments done in triplicates.

Figure S3: AI-2 production in mixed biofilms.

Late log-phase C. difficile WT is displayed as a control. Cell-free supernatants were

taken from both mono and co-culture biofilms of C. difficile (WT and luxS) and B.

fragilis. These were assayed for AI-2 activity using V. harveyi. Bioluminescence is

shown as a percentage of wild-type V. harveyi BB120 bioluminescence, which was

assumed to be 100%. Data is representative of 2 independent experiments.

Figure S4 Cell free B. fragilis supernatants do not inhibit C. difficile

(A) Colony counts were performed at 24 h for WT and WT resuspended in cell-free

B. fragilis supernatant. (B) WT biofilms were grown for 24 h after which the

supernatant was replaced with either fresh BHIS + 0.1M glucose, cell-free B. fragilis

39 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

biofilm supernatant or cell-free co-culture biofilm supernatant. Samples were

incubated for a further 24 h before colony counts were performed. Data shown is the

mean of 3 independent experiments in triplicates and error bars indicate SD, *p <

0.05 as determined by student’s t-test.

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Table 1: Genes up- and down-regulated in luxS relative to the WT C. difficile

No Gene ID log2 FoldChange Gene annotation 1 CDR20291_1206 -0.994213972 hypothetical protein 2 CDR20291_1207 -1.059645046 hypothetical protein 3 CDR20291_1208 -1.227114763 hypothetical protein 4 CDR20291_1210 -1.08584066 hypothetical protein 5 CDR20291_1211 -1.0763802 hypothetical protein 6 CDR20291_1212 -1.009946545 phage cell wall hydrolase 7 CDR20291_1214 -1.09972952 phage protein 8 CDR20291_1215 -1.038498251 phage protein 9 CDR20291_1216 -1.047731727 phage protein 10 CDR20291_1217 -0.999294901 phage tail fiber protein 11 CDR20291_1218 -0.898724949 hypothetical protein 12 CDR20291_1425 -0.928686375 virulence-associated protein e 13 CDR20291_1432 -1.243533992 phage terminase large subunit 14 CDR20291_1433 -1.154529376 phage portal protein 15 CDR20291_1436 -1.156306053 phage major capsid protein 16 CDR20291_1442 -1.085573967 phage protein 17 CDR20291_1444 -1.257181603 phage protein 18 CDR20291_1449 -1.008627768 phage tail tape measure protein 19 PTS system glucose-specific CDR20291_2554 0.895993497 transporter subunit IIA 20 cellobiose-phosphate degrading CDR20291_2927 1.587075038 protein 21 CDR20291_2930 1.444846424 trehalose-6-phosphate hydrolase

Note: P-adjusted value ≤ 0.05

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Table 2: Genes up and down-regulated in WT during co-culture with B. fragilis

relative to the WT C. difficile monoculture.

No Gene ID log2FoldChange Gene Annotation 1 CDR20291_0194 1.3822081 10 kDa chaperonin glycerol-3-phosphate CDR20291_1016 1.7087847 2 acyltransferase PlsX 3 CDR20291_1017 1.7011992 3-oxoacyl-ACP synthase III 4 CDR20291_1018 1.2469326 trans-2-enoyl-ACP reductase 5 CDR20291_1337 1.3290499 transcriptional regulator biotin carboxylase acetyl-CoA CDR20291_1861 1.412304 6 carboxylase subunit A 7 CDR20291_2027 1.6309458 2-nitropropane dioxygenase 8 CDR20291_3225 1.0229025 formate/nitrite transporter 9 CDR20291_0363 -1.377088 radical SAM protein 10 CDR20291_0364 -1.003595 hypothetical protein (R)-2-hydroxyisocaproate CDR20291_0365 -1.461207 11 dehydrogenase 12 CDR20291_0659 -1.248536 radical SAM protein 13 CDR20291_1271 -1.15672 hypothetical protein 14 CDR20291_1309 -1.208811 phosphohydrolase imidazole glycerol phosphate CDR20291_1400 -1.833327 15 synthase subunit HisH ethanolamine/propanediol CDR20291_1834 -1.411634 16 ammonia- heavy chain 17 CDR20291_2416 -1.231651 hypothetical protein 18 CDR20291_2417 -1.574982 hypothetical protein two-component sensor histidine CDR20291_2610 -1.046807 19 kinase

Note: P-adjusted value ≤ 0.05

42 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 3: Genes up and down-regulated in luxS during co-culture with B. fragilis

relative to the WT C. difficile monoculture.

No Gene ID log2FoldChange Gene Annotation 1 CDR20291_0491 1.0964355 RNA methylase 2 CDR20291_0492 1.1802034 hypothetical protein 3 CDR20291_0493 1.0076105 outer membrane lipoprotein 4 CDR20291_0715 1.6245725 N-acetylmuramoyl-L-alanine amidase 5 CDR20291_1366 0.9274027 ferrous ion transport protein 6 CDR20291_1374 1.1651176 iron-sulfur protein 7 CDR20291_1691 1.4187304 nitrite and sulfite reductase subunit 8 CDR20291_1716 1.3143654 thiol peroxidase 9 CDR20291_1717 1.110872 hypothetical protein 10 CDR20291_1934 1.5456229 hypothetical protein 11 CDR20291_1936 1.3368874 GntR family transcriptional regulator 12 CDR20291_1937 1.3736945 ABC transporter ATP-binding protein 13 CDR20291_2389 1.2619709 competence protein ribonucleoside-diphosphate reductase CDR20291_2830 1.11333 14 subunit alpha 15 CDR20291_2928 1.7538156 PTS system transporter subunit IIABC 16 CDR20291_2930 1.5713168 trehalose-6-phosphate hydrolase osmoprotectant ABC transporter CDR20291_3075 1.3649687 17 substrate-binding/permease sigma-54-dependent transcriptional CDR20291_3104 0.8099098 18 activator 19 CDR20291_3434 1.4402419 homocysteine S-methyltransferase acetoin:2%2C6- CDR20291_0025 -1.32053 dichlorophenolindophenol 20 subunit alpha 21 CDR20291_0615 -0.86915 nucleotide phosphodiesterase ABC transporter substrate-binding CDR20291_0802 -2.260047 22 protein electron transfer subunit CDR20291_0911 -1.24181 23 beta 24 CDR20291_1359 -0.850952 hypothetical protein 25 CDR20291_1370 -1.124726 tyrosyl-tRNA synthetase 26 CDR20291_1497 -1.456296 phosphomethylpyrimidine kinase 27 CDR20291_1498 -1.727285 hydroxyethylthiazole kinase thiamine-phosphate CDR20291_1499 -1.276387 28 pyrophosphorylase dinitrogenase iron-molybdenum CDR20291_1591 -1.51799 29 30 CDR20291_1901 -1.832663 ABC transporter ATP-binding protein

43 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ABC transporter substrate-binding CDR20291_1902 -1.875507 31 protein 32 CDR20291_1903 -1.659329 ABC transporter permease 33 CDR20291_1904 -1.638609 hypothetical protein 34 CDR20291_1925 -1.54806 flavodoxin DNA-directed RNA polymerase CDR20291_2474 -1.434272 35 subunit omega 36 CDR20291_2515 -1.603951 amino acid permease family protein 37 CDR20291_2516 -1.176482 cobalt dependent x-pro dipeptidase teichuronic acid biosynthesis glycosyl CDR20291_2660 -0.958814 38 transferase 39 CDR20291_2870 -2.335518 hypothetical protein 40 CDR20291_3142 -1.665916 pyrroline-5-carboxylate reductase 41 CDR20291_3143 -1.830481 formate acetyltransferase pyruvate formate-lyase 3 activating CDR20291_3144 -1.643573 42 enzyme

Note: P-adjusted value ≤ 0.05

44 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 4: Genes up- and down-regulated in both WT and luxS during co-culture with

B. fragilis relative to the WT C. difficile monoculture.

No Gene ID Log2 Foldchange Gene Annotation 1 CDR20291_0511 -1.947939847 hypothetical protein 2 CDR20291_0512 -1.790659218 hypothetical protein 3 CDR20291_0910 -1.118039675 butyryl-CoA dehydrogenase 4 CDR20291_1799 -1.559955287 hypothetical protein 5 CDR20291_1878 -1.084562908 ABC transporter ATP-binding protein 6 CDR20291_2418 -1.624152909 hypothetical protein 7 CDR20291_2419 -1.384788131 aminotransferase 8 CDR20291_2611 -1.45763049 two-component response regulator 9 CDR20291_2612 -1.940640211 hypothetical protein 10 CDR20291_2613 -1.569696353 deacetylase 11 CDR20291_0196 1.242760946 hypothetical protein 12 CDR20291_0197 1.024719474 hypothetical protein fatty acid biosynthesis transcriptional CDR20291_1015 1.855396639 13 regulator biotin carboxyl carrier protein of acetyl- CDR20291_1862 1.571375262 14 CoA carboxylase 15 CDR20291_1931 1.501260024 AraC family transcriptional regulator 16 CDR20291_1935 1.754871647 hypothetical protein 17 CDR20291_2077 1.310107407 Sodium:dicarboxylate symporter N-acetylmannosamine-6-phosphate 2- CDR20291_2140 2.555234046 18 epimerase NAD-dependent 4-hydroxybutyrate CDR20291_2227 3.630122723 19 dehydrogenase 20 CDR20291_2228 3.789825947 4-hydroxybutyrate CoA transferase 21 CDR20291_2229 3.695347944 hypothetical protein gamma-aminobutyrate metabolism CDR20291_2230 3.544168193 22 dehydratase/ succinate-semialdehyde CDR20291_2231 3.864728796 23 dehydrogenase [NAD(P)+] 24 CDR20291_2232 4.2239099 succinyl-CoA:coenzyme A transferase 25 CDR20291_2233 4.339535053 hypothetical protein 26 CDR20291_3110 1.176093028 hypothetical protein

Note: P-adjusted value ≤ 0.05

45 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 5: Genes up- and down-regulated in B. fragilis specific to co-culture with WT

C. difficile.

No Gene ID log2FoldChange Gene Annotation 1 BF1917_03152 1.311516182 RNA polymerase sigma factor 2 BF1917_01567 1.305829947 N-acetylmuramoyl-L-alanine amidase 3 BF1917_02264 1.296892458 tRNA-Arg(ccg) 4 BF1917_01254 1.287474673 Phytochrome-like protein cph1 5 BF1917_01030 1.273862488 L-fucose-proton symporter 6 BF1917_01257 1.192975873 SusD family protein 7 BF1917_01509 1.125756293 putative N-acetyltransferase YvbK 8 BF1917_01907 1.046014221 hypothetical protein 9 BF1917_02466 0.929316202 hypothetical protein 10 BF1917_00894 0.918517125 7-cyano-7-deazaguanine synthase 3-deoxy-D-manno-octulosonic acid 11 BF1917_02556 0.895506005 transferase 12 BF1917_00770 0.894200966 hypothetical protein Putative multidrug export ATP- 13 BF1917_01245 0.892290589 binding/permease protein 14 BF1917_01085 0.880992869 hypothetical protein NADPH-dependent 7-cyano-7- 15 BF1917_00895 0.880607691 deazaguanine reductase Nicotinamide-nucleotide amidohydrolase 16 BF1917_01448 0.87638903 PncC 17 BF1917_01836 0.862806532 hypothetical protein 18 BF1917_01057 0.845284167 50S ribosomal protein L35 19 BF1917_00537 0.844678344 Peptide-N-glycosidase F%2C N terminal 20 BF1917_01075 0.843492885 Abhydrolase family protein Phospho-N-acetylmuramoyl-pentapeptide- 21 BF1917_00189 0.839780605 transferase 22 BF1917_03080 0.829317439 hypothetical protein Beta-1%2C4-mannooligosaccharide 23 BF1917_00834 0.825832969 phosphorylase 24 BF1917_02026 0.811321047 lipid-A-disaccharide synthase 25 BF1917_01653 0.81109879 hypothetical protein 26 BF1917_02254 0.810810413 Aspartate/alanine antiporter 27 BF1917_02339 0.807753339 Arylsulfatase 28 BF1917_03030 0.802685207 DNA-invertase hin 29 BF1917_01201 0.801621087 D-3-phosphoglycerate dehydrogenase 30 BF1917_02441 0.78894384 PSP1 C-terminal conserved region 31 BF1917_01453 0.778896942 Proline--tRNA 32 BF1917_02227 0.762789235 Ketol-acid reductoisomerase 33 BF1917_00375 0.752440474 Riboflavin biosynthesis protein RibBA

46 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

34 BF1917_01587 0.748273315 Tetratricopeptide repeat protein 35 BF1917_01056 0.739389804 Translation initiation factor IF-3 36 BF1917_01269 0.732209067 hypothetical protein 37 BF1917_00105 0.715996603 Efflux pump membrane transporter BepE 38 BF1917_02136 0.709932491 Putative glycoside hydrolase 39 BF1917_01340 0.685536905 photosystem I assembly protein Ycf3 40 BF1917_01974 0.684836774 Acyl-CoA dehydrogenase 41 BF1917_01403 0.669395421 Aspartate--tRNA ligase 42 BF1917_00449 0.666199374 Beta-hexosaminidase Putative acetolactate synthase small 43 BF1917_02229 0.663450332 subunit 44 BF1917_01837 0.655283138 hypothetical protein 45 BF1917_01102 0.620796646 Multidrug export protein MepA Malonyl CoA-acyl carrier protein 46 BF1917_01352 0.612741808 transacylase Acetylornithine/acetyl-lysine 47 BF1917_00323 0.609404167 aminotransferase 48 BF1917_02919 0.604261412 SusD family protein 49 BF1917_01071 0.5992361 N-acetylneuraminate lyase Aspartate carbamoyltransferase regulatory 50 BF1917_01311 0.577463631 chain 51 BF1917_02617 0.576894078 Na+/Pi-cotransporter Single-stranded-DNA-specific exonuclease 52 BF1917_02330 0.57236035 RecJ 53 BF1917_02512 0.543247595 50S ribosomal protein L30 54 BF1917_01877 0.535723764 Tetracycline resistance protein%2C class B putative propionyl-CoA carboxylase beta 55 BF1917_01897 0.534003138 chain 5 56 BF1917_01328 0.491588359 Penicillin-binding protein 1A 57 BF1917_01113 -0.524719502 hypothetical protein FKBP-type peptidyl-prolyl cis-trans 58 BF1917_02232 -0.572743133 isomerase SlyD 59 BF1917_03010 -0.582199556 Fic/DOC family protein 60 BF1917_01873 -0.635535861 hypothetical protein 61 BF1917_02202 -0.660835242 hypothetical protein 62 BF1917_02685 -0.666560462 Putative O-methyltransferase/MSMEI_4947 63 BF1917_00501 -0.667060521 hypothetical protein 64 BF1917_00109 -0.668328202 hypothetical protein 65 BF1917_02862 -0.669531514 hypothetical protein 66 BF1917_00475 -0.67568016 Sporulation initiation inhibitor protein Soj 67 BF1917_00935 -0.679922007 hypothetical protein 68 BF1917_00669 -0.703597245 hypothetical protein 69 BF1917_00495 -0.735635295 recombination protein F

47 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

70 BF1917_01685 -0.743225598 LL-diaminopimelate aminotransferase Crossover junction endodeoxyribonuclease 71 BF1917_01872 -0.743919917 RuvC 72 BF1917_03069 -0.749607402 Chromosome-partitioning protein ParB 73 BF1917_01229 -0.757098091 hypothetical protein Undecaprenyl-phosphate 74 BF1917_01766 -0.77013931 mannosyltransferase 75 BF1917_02744 -0.771369435 Pyridoxamine 5'-phosphate oxidase 76 BF1917_00880 -0.792153171 hypothetical protein 77 BF1917_01736 -0.827475365 Putative zinc ribbon domain protein 78 BF1917_01302 -0.835788738 hypothetical protein 79 BF1917_01112 -0.858879453 GDP-mannose 4%2C6-dehydratase 80 BF1917_01884 -0.860144817 Tyrocidine synthase 1 81 BF1917_02192 -0.862183423 hypothetical protein 82 BF1917_01418 -0.866621344 Helix-turn-helix Putative SOS response-associated 83 BF1917_01339 -0.884258094 peptidase YedK 84 BF1917_02268 -0.896373658 tRNA 2'-O-methylase Carboxymuconolactone decarboxylase 85 BF1917_02955 -0.923177568 family protein Succinyl-CoA ligase [ADP-forming] subunit 86 BF1917_01351 -0.93045494 beta 87 BF1917_01007 -0.95502202 Flavodoxin 88 BF1917_01605 -0.979335966 89 BF1917_00279 -0.986458154 hypothetical protein 90 BF1917_01321 -0.987569407 putative deferrochelatase/peroxidase YfeX 91 BF1917_02874 -0.987697244 hypothetical protein 92 BF1917_01816 -0.993202168 Alpha-amylase precursor Putative pyridoxal phosphate-dependent 93 BF1917_00505 -0.994106622 aminotransferase EpsN 94 BF1917_02297 -1.007217502 hypothetical protein Dihydrolipoyllysine-residue acetyltransferase component of pyruvate 95 BF1917_01005 -1.012280592 dehydrogenase complex 96 BF1917_00888 -1.051392536 Chagasin family peptidase inhibitor I42 97 BF1917_00419 -1.072148557 Multidrug resistance protein MdtA precursor 98 BF1917_01154 -1.072861576 ECF RNA polymerase sigma factor SigH 99 BF1917_01264 -1.074778365 Hydroxylamine reductase 100 BF1917_00886 -1.076904738 Quercetin 2%2C3-dioxygenase 101 BF1917_01818 -1.090626971 hypothetical protein 102 BF1917_00666 -1.113835599 hypothetical protein 103 BF1917_01014 -1.119101941 hypothetical protein 104 BF1917_00957 -1.12987456 hypothetical protein 105 BF1917_02248 -1.151858985 hypothetical protein

48 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

106 BF1917_00131 -1.190748639 Nitroreductase family protein 107 BF1917_03151 -1.194336742 hypothetical protein 108 BF1917_00525 -1.196694488 hypothetical protein 109 BF1917_02975 -1.225526137 hypothetical protein Fimbrillin-A associated anchor proteins 110 BF1917_01315 -1.288861381 Mfa1 and Mfa2 111 BF1917_02081 -1.362552672 hypothetical protein 112 BF1917_01958 -1.371084162 hypothetical protein 113 BF1917_03077 -1.377462013 hypothetical protein 114 BF1917_03003 -1.403406871 hypothetical protein

Note: P-adjusted value ≤ 0.05

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Table 6: Genes up- and down-regulated in B. fragilis specific to co-culture with luxS

C. difficile.

log2FoldCha No Gene ID Gene Annotation nge 1 BF1917_03116 1.50249 hypothetical protein 2 BF1917_00412 1.48934 Tyrosine recombinase XerD 3 BF1917_02127 1.295481 fec operon regulator FecR 4 BF1917_02593 1.280656 tRNA-Leu(caa) 5 BF1917_01792 1.235375 Reverse rubrerythrin-1 6 BF1917_02909 1.220847 hypothetical protein ATP-dependent Clp protease ATP-binding 7 BF1917_01427 1.200963 subunit ClpC 8 BF1917_02997 1.188287 hypothetical protein 9 BF1917_01065 1.176729 tRNA-Thr(cgt) 10 BF1917_00574 1.133713 hypothetical protein 11 BF1917_01993 1.109684 60 kDa chaperonin 12 BF1917_01994 1.09403 10 kDa chaperonin 13 BF1917_02911 1.08306 Tyrosine recombinase XerC 14 BF1917_02143 1.066955 NAD-specific glutamate dehydrogenase 15 BF1917_02972 1.063195 fec operon regulator FecR 16 BF1917_00620 1.03149 fec operon regulator FecR 17 BF1917_01491 1.021451 hypothetical protein 18 BF1917_02039 1.015769 tRNA-Phe(gaa) 19 BF1917_03113 0.990297 hypothetical protein 20 BF1917_03172 0.98787 tRNA-Arg(gcg) 21 BF1917_00358 0.980119 MarR family protein 22 BF1917_00832 0.956639 hypothetical protein 23 BF1917_00767 0.951389 hypothetical protein 24 BF1917_01889 0.938171 Septum formation protein Maf 25 BF1917_01282 0.932255 ATP synthase subunit beta 26 BF1917_03056 0.92593 hypothetical protein 27 BF1917_02999 0.921887 hypothetical protein 28 BF1917_00178 0.905375 30S ribosomal protein S20 29 BF1917_02814 0.89716 hypothetical protein 30 BF1917_02866 0.885735 hypothetical protein 31 BF1917_02896 0.871156 hypothetical protein 32 BF1917_00295 0.865619 50S ribosomal protein L34 33 BF1917_03169 0.843942 tRNA-Asp(gtc) 34 BF1917_01072 0.843727 Cellobiose 2-epimerase 35 BF1917_02507 0.835563 50S ribosomal protein L36 36 BF1917_02553 0.817012 30S ribosomal protein S21

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37 BF1917_03177 0.802377 tRNA-Lys(ttt) 38 BF1917_01910 0.800503 50S ribosomal protein L31 type B putative ABC transporter ATP-binding 39 BF1917_01181 0.795826 protein YjjK 40 BF1917_03168 0.776641 tRNA-Pro(tgg) 41 BF1917_02950 0.740702 transcriptional regulator BetI 42 BF1917_02033 0.735701 hypothetical protein 43 BF1917_01372 0.732434 hypothetical protein 44 BF1917_00179 0.697125 tRNA-Glu(ttc) 45 BF1917_01819 0.691297 4Fe-4S binding domain protein 46 BF1917_01081 0.670376 F5/8 type C domain protein 47 BF1917_01847 0.651964 hypothetical protein 48 BF1917_02253 0.635549 Methylmalonyl-CoA mutase 49 BF1917_01261 0.623302 hypothetical protein 50 BF1917_02778 0.561439 enterobactin exporter EntS 51 BF1917_02391 0.556052 50S ribosomal protein L13 52 BF1917_02815 0.541997 Phosphoenolpyruvate carboxykinase [ATP] 53 BF1917_02820 0.538608 Phosphoenolpyruvate carboxykinase [ATP] 54 BF1917_02819 0.535546 Phosphoenolpyruvate carboxykinase [ATP] 55 BF1917_02049 0.508167 putative enoyl-CoA hydratase 1 56 BF1917_00123 0.446281 Acyl carrier protein 57 BF1917_02261 -0.51363 Glucose-6-phosphate isomerase 58 BF1917_00321 -0.53822 DNA-binding transcriptional repressor PuuR Bifunctional transcriptional activator/DNA 59 BF1917_00914 -0.56999 repair enzyme AdaA 60 BF1917_00594 -0.60044 DNA replication and repair protein RecF 61 BF1917_01306 -0.60418 Formate--tetrahydrofolate ligase 62 BF1917_00287 -0.64751 Phosphate acetyltransferase 63 BF1917_00206 -0.68829 tetratricopeptide repeat protein 64 BF1917_00107 -0.72261 Beta-galactosidase 65 BF1917_01926 -0.72606 Outer membrane efflux protein 66 BF1917_01224 -0.7469 putative GTP-binding protein EngB 67 BF1917_01293 -0.75882 Transcriptional regulatory protein CusR 68 BF1917_01212 -0.76206 hypothetical protein 69 BF1917_01767 -0.77069 Dihydroorotase 70 BF1917_01707 -0.78791 hypothetical protein 71 BF1917_00291 -0.79844 UDP-2%2C3-diacylglucosamine hydrolase 72 BF1917_01301 -0.80259 dTDP-4-dehydrorhamnose reductase 73 BF1917_02606 -0.8053 Aminopeptidase YwaD precursor 74 BF1917_01852 -0.81525 Transaldolase 75 BF1917_01735 -0.83554 putative oxidoreductase 76 BF1917_02275 -0.8445 FMN reductase [NAD(P)H]

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Lipoprotein-releasing system 77 BF1917_02683 -0.86216 transmembrane protein LolE putative A/G-specific adenine glycosylase 78 BF1917_00953 -0.87106 YfhQ 79 BF1917_00810 -0.9142 hypothetical protein 80 BF1917_02167 -0.91437 Pyruvate formate-lyase 1-activating enzyme Bifunctional transcriptional activator/DNA 81 BF1917_02588 -0.9256 repair enzyme Ada Cadmium%2C zinc and cobalt-transporting 82 BF1917_00809 -0.98589 ATPase 83 BF1917_01476 -1.01713 Colicin I receptor precursor 84 BF1917_01210 -1.01929 putative metallo-hydrolase 85 BF1917_00766 -1.03246 hypothetical protein Molecular chaperone Hsp31 and glyoxalase 86 BF1917_01961 -1.06955 3 87 BF1917_02112 -1.11145 Lactate utilization protein C 88 BF1917_00727 -1.19678 hypothetical protein 89 BF1917_03146 -1.33023 hypothetical protein 90 BF1917_01478 -1.33473 hypothetical protein 91 BF1917_02716 -1.4039 hypothetical protein

Note: P-adjusted value ≤ 0.05

52 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S1: Genes up- and down-regulated in B. fragilis co-cultured with both WT and

luxS C. difficile.

log2FoldChan Gene identifier Product No ge 1 BF1917_00021 0.8222 hypothetical protein 2 BF1917_00043 1.67588 tRNA-Gln(ttg) 3 BF1917_00113 0.95251 Transcriptional regulatory protein CssR 4 BF1917_00148 1.47898 L-fucose isomerase 5 BF1917_00150 0.84558 Lactaldehyde reductase DNA-binding transcriptional repressor BF1917_00221 2.16886 6 MngR 7 BF1917_00222 2.55126 hypothetical protein 8 BF1917_00365 1.42774 fec operon regulator FecR 9 BF1917_00371 1.24232 tRNA-Met(cat) 10 BF1917_00577 1.0794 HTH-type transcriptional activator Btr 11 BF1917_00591 1.24631 tRNA-Tyr(gta) 12 BF1917_00627 0.98501 Glycosyl hydrolase family 57 13 BF1917_00674 1.31953 fec operon regulator FecR 14 BF1917_00677 2.94941 hypothetical protein 15 BF1917_00753 0.99535 hypothetical protein 16 BF1917_00756 0.86105 50S ribosomal protein L25 17 BF1917_00791 1.45917 Alkyl hydroperoxide reductase subunit F 18 BF1917_00793 0.93169 Alkyl hydroperoxide reductase subunit C 19 BF1917_00811 0.95941 Outer membrane protein 41 precursor 20 BF1917_00831 0.99547 fec operon regulator FecR 21 BF1917_00843 0.85771 Alpha-xylosidase 22 BF1917_00846 1.31112 hypothetical protein 23 BF1917_00863 1.01842 Ferrous iron transport protein B 24 BF1917_00864 1.21647 hypothetical protein 25 BF1917_00865 1.03526 tRNA-Cys(gca) 26 BF1917_00893 0.82647 hypothetical protein 27 BF1917_01029 0.94956 Galactokinase 28 BF1917_01061 1.35058 indolepyruvate oxidoreductase subunit beta 29 BF1917_01086 1.16541 hypothetical protein 30 BF1917_01126 1.12128 hypothetical protein 31 BF1917_01202 1.16407 hypothetical protein 32 BF1917_01239 1.06702 Leucine Rich repeats (2 copies) 33 BF1917_01253 1.2035 UDP-glucose 4-epimerase 34 BF1917_01277 0.9888 Undecaprenol kinase 35 BF1917_01283 1.00392 ATP synthase epsilon chain 36 BF1917_01284 1.16107 hypothetical protein

53 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

37 BF1917_01305 1.61776 Sensor RcsC 7%2C8-dihydro-6-hydroxymethylpterin- BF1917_01329 0.88732 38 pyrophosphokinase (HPPK) 39 BF1917_01338 1.66918 tRNA-Arg(tct) 40 BF1917_01379 1.5168 tRNA-Lys(ctt) 41 BF1917_01426 1.40284 Chaperone protein HtpG 42 BF1917_01440 0.80392 LysM domain/BON superfamily protein 43 BF1917_01445 0.98821 hypothetical protein 44 BF1917_01565 1.45037 hypothetical protein 45 BF1917_01566 1.3672 transcriptional repressor DicA 46 BF1917_01568 1.24792 integration host factor subunit alpha 47 BF1917_01586 0.97287 Enamine/imine deaminase 48 BF1917_01618 2.07361 hypothetical protein 49 BF1917_01668 1.0958 ACT domain protein 50 BF1917_01699 1.01266 hypothetical protein 51 BF1917_01704 0.93246 Oxygen regulatory protein NreC 52 BF1917_01705 1.11144 hypothetical protein 53 BF1917_01783 1.19659 hypothetical protein 54 BF1917_01784 1.06401 hypothetical protein 55 BF1917_01794 1.03333 Inner membrane protein YhaI 56 BF1917_01838 0.92354 hypothetical protein 57 BF1917_01900 0.80784 Glutaconyl-CoA decarboxylase subunit beta 58 BF1917_01987 1.62881 hypothetical protein 59 BF1917_02028 1.2856 3-isopropylmalate dehydrogenase 60 BF1917_02029 1.43244 2-isopropylmalate synthase 3-isopropylmalate dehydratase small BF1917_02030 1.22383 61 subunit 3-isopropylmalate dehydratase large BF1917_02031 1.22256 62 subunit NADP-dependent 7-alpha-hydroxysteroid BF1917_02052 1.77273 63 dehydrogenase multifunctional tRNA nucleotidyl transferase/2'3'-cyclic BF1917_02145 0.80664 phosphodiesterase/2'nucleotidase/phosphat 64 ase 65 BF1917_02150 1.24574 hypothetical protein Acetolactate synthase isozyme 2 large BF1917_02230 0.86406 66 subunit 67 BF1917_02241 1.6209 fec operon regulator FecR 68 BF1917_02252 1.04906 Methylmalonyl-CoA mutase large subunit 69 BF1917_02364 0.99688 Outer membrane protein TolC precursor 70 BF1917_02388 0.80476 Elongation factor Ts 71 BF1917_02389 1.00102 30S ribosomal protein S2 72 BF1917_02390 0.88503 30S ribosomal protein S9

54 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

73 BF1917_02399 1.54669 hypothetical protein Fimbrillin-A associated anchor proteins BF1917_02408 1.20627 74 Mfa1 and Mfa2 75 BF1917_02411 1.43474 Carbamoyl-phosphate synthase large chain 76 BF1917_02433 1.18261 Transcriptional regulatory protein DegU 77 BF1917_02533 1.01466 30S ribosomal protein S7 78 BF1917_02534 0.85195 30S ribosomal protein S12 79 BF1917_02590 1.09676 site-specific tyrosine recombinase XerC 80 BF1917_02599 1.13134 fec operon regulator FecR Autoinducer 2 sensor kinase/phosphatase BF1917_02675 1.09631 81 LuxQ 82 BF1917_02743 1.69888 RNA polymerase sigma factor SigV 83 BF1917_02779 1.00209 tRNA-Ser(tga) 84 BF1917_02781 0.80239 Type-1 restriction enzyme R protein 85 BF1917_02832 1.0499 23S ribosomal RNA 86 BF1917_02841 1.26941 hypothetical protein 87 BF1917_02895 1.17407 hypothetical protein putative type I restriction enzymeP M BF1917_02908 0.85299 88 protein 89 BF1917_02926 1.35419 hypothetical protein 90 BF1917_02940 1.72848 fec operon regulator FecR 91 BF1917_02946 1.05842 Tyrosine recombinase XerC 92 BF1917_03008 1.45389 mRNA interferase YafQ 93 BF1917_03009 1.16616 hypothetical protein 94 BF1917_03031 1.47571 hypothetical protein 95 BF1917_03037 1.20593 hypothetical protein 96 BF1917_03072 5.99042 Rubrerythrin 97 BF1917_03086 7.42484 Desulfoferrodoxin 98 BF1917_03094 1.64632 hypothetical protein 99 BF1917_03132 1.26258 hypothetical protein 100 BF1917_03138 1.21443 hypothetical protein 101 BF1917_03154 1.10663 tRNA-Leu(taa) 102 BF1917_03155 9.49728 Virus attachment protein p12 family protein 103 BF1917_03166 5.22671 hypothetical protein 104 BF1917_03176 1.19636 tRNA-Cys(gca) 105 BF1917_00094 -1.0009 putative oxidoreductase UxuB 106 BF1917_00133 -1.0426 hypothetical protein 107 BF1917_00198 -1.0327 ECF RNA polymerase sigma factor SigH 108 BF1917_00199 -1.4827 hypothetical protein putative TonB-dependent receptor BF1917_00200 -1.672 109 precursor 110 BF1917_00201 -2.2887 hypothetical protein 111 BF1917_00233 -1.5951 hypothetical protein

55 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

112 BF1917_00234 -1.7168 hypothetical protein 113 BF1917_00235 -1.6642 Periplasmic beta-glucosidase precursor 114 BF1917_00236 -1.4454 SusD family protein TonB-dependent Receptor Plug Domain BF1917_00237 -1.7551 115 protein 116 BF1917_00238 -1.1093 hypothetical protein 117 BF1917_00253 -1.591 Cytochrome c-type protein NrfH 118 BF1917_00254 -1.4394 hypothetical protein 119 BF1917_00272 -1.2798 Glutamate decarboxylase 120 BF1917_00273 -1.0404 Glutaminase 1 121 BF1917_00311 -1.2125 Chaperone protein Skp precursor 122 BF1917_00329 -1.7304 Glutamate/gamma-aminobutyrate antiporter 123 BF1917_00330 -1.3858 hypothetical protein 124 BF1917_00331 -1.2441 hypothetical protein 125 BF1917_00332 -1.6386 Malate dehydrogenase 126 BF1917_00355 -1.4376 hypothetical protein 127 BF1917_00356 -1.3021 hypothetical protein 128 BF1917_00417 -1.0045 Outer membrane protein TolC precursor 129 BF1917_00418 -0.9189 Efflux pump membrane transporter BepE 130 BF1917_00452 -1.2843 hypothetical protein 131 BF1917_00556 -1.5284 LemA family protein 132 BF1917_00557 -1.0771 Alpha/beta hydrolase family protein 133 BF1917_00619 -1.1745 RNA polymerase sigma factor SigM 134 BF1917_00656 -1.1581 Thioredoxin reductase 135 BF1917_00665 -1.1051 ECF RNA polymerase sigma factor SigW 136 BF1917_00667 -0.8588 hypothetical protein 137 BF1917_00695 -1.033 hypothetical protein 138 BF1917_00721 -1.3608 hypothetical protein 139 BF1917_00779 -1.1636 H(+)/Cl(-) exchange transporter ClcA 140 BF1917_00796 -0.9578 hypothetical protein 141 BF1917_00797 -0.9605 hypothetical protein 5-amino-6-(5-phosphoribosylamino)uracil BF1917_00804 -0.9906 142 reductase lipoprotein involved with copper BF1917_00844 -1.1187 143 homeostasis and adhesion 144 BF1917_00854 -1.4374 hypothetical protein 145 BF1917_00858 -1.0785 VIT family protein Sodium-dependent dicarboxylate BF1917_00878 -1.1337 146 transporter SdcS 147 BF1917_00891 -2.0183 Porin subfamily protein Histidine decarboxylase proenzyme BF1917_00908 -1.9735 148 precursor

56 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Bifunctional aspartate aminotransferase and BF1917_00930 -1.2932 149 L-aspartate beta-decarboxylase 150 BF1917_00931 -1.4384 Aspartate/alanine antiporter 151 BF1917_00956 -1.1286 hypothetical protein 152 BF1917_00958 -1.3981 hypothetical protein 153 BF1917_00960 -1.4226 hypothetical protein 154 BF1917_00961 -1.862 hypothetical protein 155 BF1917_00980 -1.3254 putative inner membrane protein 156 BF1917_00989 -1.5085 DUF based on B. Theta Gene description 157 BF1917_00990 -1.542 hypothetical protein 158 BF1917_00996 -1.0481 metal-dependent hydrolase 159 BF1917_00998 -1.7058 hypothetical protein 160 BF1917_00999 -1.4252 hypothetical protein 161 BF1917_01000 -1.9487 hypothetical protein 2-oxoisovalerate dehydrogenase subunit BF1917_01006 -0.951 162 beta 163 BF1917_01090 -1.5908 ECF RNA polymerase sigma factor SigW 164 BF1917_01091 -1.9091 hypothetical protein 165 BF1917_01092 -1.6308 hypothetical protein 166 BF1917_01094 -1.1489 site-specific tyrosine recombinase XerD 167 BF1917_01143 -0.862 Glycerate dehydrogenase 168 BF1917_01153 -1.0557 LemA family protein 169 BF1917_01167 -1.2786 Collagen triple helix repeat (20 copies) 170 BF1917_01240 -1.1545 hypothetical protein 171 BF1917_01266 -1.2582 Transcriptional activator protein Anr 172 BF1917_01273 -1.0268 Dihydroorotate dehydrogenase (quinone) 173 BF1917_01274 -2.1585 Acetylxylan esterase precursor 174 BF1917_01295 -1.3227 inner membrane protein 175 BF1917_01307 -1.8784 hypothetical protein 176 BF1917_01325 -1.1429 hypothetical protein 177 BF1917_01357 -1.3565 putative peroxiredoxin 178 BF1917_01363 -1.8439 Inner membrane protein YhiM 179 BF1917_01395 -1.288 hypothetical protein 180 BF1917_01396 -1.2754 hypothetical protein 181 BF1917_01397 -0.9999 hypothetical protein 182 BF1917_01412 -1.7894 Outer membrane protein OprM precursor 183 BF1917_01413 -2.3962 Efflux pump periplasmic linker BepF 184 BF1917_01414 -1.7576 Efflux pump membrane transporter BepE 185 BF1917_01465 -0.8542 Outer membrane efflux protein 186 BF1917_01477 -1.3277 Sirohydrochlorin cobaltochelatase 187 BF1917_01486 -0.9303 Multidrug export protein EmrB 188 BF1917_01487 -1.1674 putative multidrug resistance protein EmrK

57 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Ribonucleoside-diphosphate reductase BF1917_01503 -1.2942 189 subunit beta 190 BF1917_01543 -1.4322 hypothetical protein 191 BF1917_01544 -1.5553 hypothetical protein 192 BF1917_01562 -1.1674 acid-resistance membrane protein 193 BF1917_01610 -0.8239 Superoxide dismutase [Mn/Fe] 194 BF1917_01629 -0.8023 hypothetical protein 195 BF1917_01684 -1.2908 Diaminopimelate epimerase Guanosine-5'-triphosphate%2C3'- BF1917_01690 -1.3927 196 diphosphate pyrophosphatase 197 BF1917_01709 -0.9715 Thioredoxin-1 5-methyltetrahydropteroyltriglutamate-- BF1917_01711 -2.0211 198 homocysteine methyltransferase 199 BF1917_01757 -1.3548 hypothetical protein 200 BF1917_01771 -0.8441 ECF RNA polymerase sigma factor SigG 201 BF1917_01800 -0.9265 Quercetin 2%2C3-dioxygenase Toluene efflux pump periplasmic linker BF1917_01920 -1.2429 202 protein TtgD precursor 203 BF1917_01957 -1.2235 putative oxidoreductase/MSMEI_2347 204 BF1917_01960 -1.3016 Oxygen-insensitive NAD(P)H nitroreductase 205 BF1917_01989 -1.1508 Pyruvate dehydrogenase [ubiquinone] 206 BF1917_02034 -1.2158 bacterioferritin 207 BF1917_02045 -1.0975 Cupin domain protein 208 BF1917_02050 -1.0708 hypothetical protein 209 BF1917_02051 -1.4625 hypothetical protein 210 BF1917_02058 -1.2098 2-oxoglutarate carboxylase small subunit Methylmalonyl-CoA carboxyltransferase BF1917_02059 -0.9484 211 1.3S subunit 212 BF1917_02075 -1.4466 hypothetical protein 213 BF1917_02077 -1.0832 Beta-lactamase type II precursor 214 BF1917_02080 -1.5189 hypothetical protein 215 BF1917_02082 -1.3791 hypothetical protein 216 BF1917_02083 -1.3787 Phosphotransferase RcsD Membrane-bound lytic murein BF1917_02086 -1.0143 217 transglycosylase D precursor Peptide methionine sulfoxide reductase BF1917_02095 -1.143 218 MsrA/MsrB 219 BF1917_02111 -1.0889 Pyridoxine kinase 220 BF1917_02113 -1.6011 Lactate utilization protein B 221 BF1917_02114 -1.2751 Lactate utilization protein A 222 BF1917_02120 -0.9187 hypothetical protein Anaerobic ribonucleoside-triphosphate BF1917_02166 -1.5855 223 reductase 224 BF1917_02197 -1.8031 Outer membrane efflux protein

58 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

4-hydroxy-3-methylbut-2-enyl diphosphate BF1917_02219 -1.4523 225 reductase 226 BF1917_02244 -1.662 Choloylglycine hydrolase Ribonucleoside-diphosphate reductase BF1917_02276 -1.4009 227 NrdZ 228 BF1917_02308 -0.9336 Glutathione peroxidase homolog BsaA 229 BF1917_02334 -1.3017 P-protein 230 BF1917_02414 -1.5256 Outer membrane protein 40 precursor 231 BF1917_02469 -2.1202 tetratricopeptide repeat protein 232 BF1917_02470 -1.2335 PBP superfamily domain protein 233 BF1917_02471 -2.365 Gram-negative bacterial tonB protein 234 BF1917_02472 -2.9219 Biopolymer transport protein ExbD/TolR 235 BF1917_02473 -2.8621 Biopolymer transport protein ExbD/TolR 236 BF1917_02474 -2.5669 colicin uptake protein TolQ 237 BF1917_02536 -1.0238 hypothetical protein 238 BF1917_02537 -1.1719 hypothetical protein 239 BF1917_02538 -1.1514 hypothetical protein Anaerobic C4-dicarboxylate transporter BF1917_02567 -1.0561 240 DcuA 241 BF1917_02568 -1.6947 L-asparaginase 2 precursor 242 BF1917_02569 -1.7391 hypothetical protein 243 BF1917_02659 -1.1812 Rhomboid family protein FKBP-type 22 kDa peptidyl-prolyl cis-trans BF1917_02707 -1.5013 244 isomerase 245 BF1917_02722 -1.049 hypothetical protein Putative NAD(P)H-dependent FMN- BF1917_02802 -1.4168 246 containing oxidoreductase YwqN NADP-dependent alcohol dehydrogenase C BF1917_02803 -0.8956 247 2 248 BF1917_02905 -1.5008 ISXO2-like domain protein 249 BF1917_02906 -1.3578 hypothetical protein 250 BF1917_02914 -0.8332 hypothetical protein 251 BF1917_02930 -1.5235 fermentation/respiration switch protein 252 BF1917_02976 -1.051 hypothetical protein 253 BF1917_02993 -1.1518 LexA repressor 254 BF1917_03013 -0.819 Iron-binding zinc finger CDGSH type

Note: P-adjusted value ≤ 0.05

59 bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table S2: PCR primers for Phage genes identified by RNA-seq

Gene Name Sequence CDR20291_1208 CDR20291_1208_FW atatatAGGCTCCAACACCAAAAGAA CDR20291_1208_REV cgcgcgACCATAACCCATAACATCTTGAA GT CDR20291_1436 CDR20291_1436_FW atatatACAAAAGTTTCCAGAGATGGATGC CDR20291_1436_REV cgcgcgTGCTGTGTTACCTAATGCGGT Universal 16S 27F AGAGTTTGATCCTGGCTCAG rRNA bacterial GGTTACCTTGTTACGACTT primers 1392R

60

bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1

A 25 *** 1.2 ** * 1 20 0.8 15 0.6 Change 10 OD 570 0.4 Fold 5 0.2

0 0 WT luxS WT luxS WT luxS WT luxS 24 h 72 h 24 h 72 h

B C 1.0E+08

1.0E+06 WT 1.0E+04 CFU / ml

1.0E+02 luxS 1.0E+00 WT luxS WT luxS 24 h 72 h D 1.4 *

1.2 *

1

0.8

0.6

Fold change 0.4

0.2

0 WT luxS luxS + 100 nM DPD bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2

A C. difficile R20291 gene locus B 0 1000 2000 3000 2

0 1.5 4.00 M

1

0.5 1.00 3.00 M M change 0

1.43 M -0.5 2.00 M 1.46 M

Log2 fold Region 1 1.67 M -1 1.73 M Reg io n 2 -1.5

C

CDR20291_1442 phage protein CDR20291_1425 virulence-associated protein e CDR20291_1444 phage protein CDR20291_1211 hypothetical protein CDR20291_1215 phage protein CDR20291_1214 phage protein CDR20291_1217 phage tail fiber protein CDR20291_1216 phage protein CDR20291_1207 hypothetical protein CDR20291_2554 PTS system glucose-specific transporter CDR20291_1433 phage portal protein CDR20291_1432 phage terminase large subunit CDR20291_1208 hypothetical protein CDR20291_1436 phage major capsid protein CDR20291_1449 phage tail tape measure protein CDR20291_1206 hypothetical protein CDR20291_1212 phage cell wall hydrolase CDR20291_2927 cellobiose-phosphate degrading protein CDR20291_1210 hypothetical protein CDR20291_1218 hypothetical protein CDR20291_2930 trehalose-6-phosphate hydrolase bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (1208) (1436) ve ve WT 2 WT (1208) 1 WT (1208) 2 WT (1208) 3 WT - A Ladder DNA Genomic 1 WT 3 WT B Ladder DNA Genomic (1436) 1 WT (1436) 2 WT (1436) 3 WT -

C 1.4 **** 1.2

1

0.8

0.6 / biomass

0.4 eDNA 0.2

0 WT luxS bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4

A B C. difficile ** B. fragilis 30 1.00E+06 ** **** 25 1.00E+05

20 1.00E+04

15 1.00E+03 OD 570 10 CFU / ml 1.00E+02

5 1.00E+01

0 1.00E+00 C. difficile Mixed B. fragilis Single Mixed

C C. difficile C. difficile + B. fragilis

1.0E+10 D C. difficile ns B. fragilis 1.0E+09

1.0E+08 CFU / ml

1.0E+07

1.0E+06 Single Mixed Single Mixed 6 h 10 h bioRxiv preprint doi: https://doi.org/10.1101/494245; this version posted December 13, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5

A B C. difficile ** B. fragilis *** 40 1.0E+06 *** 35 1.0E+05 30 25 *** 1.0E+04 20 1.0E+03 15 OD 570 CFU/ml 1.0E+02 10 1.0E+01 5 0 1.0E+00 WT WT + B. luxS luxS + B. WT+ B. luxS + B. fragilis fragilis fragilis fragilis A B bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. doi: https://doi.org/10.1101/494245 B. fragilis1 WT1

B. fragilis2 WT2 B. fragilis3 WT3

B. fragilis + WT1 WT + B. fragilis1 ; B. fragilis + WT2 WT + B. fragilis2 this versionpostedDecember13,2018.

B. fragilis + WT3 WT + B. fragilis3

B. Fragilis + luxS1 luxS + B. fragilis1

B. fragilis + luxS2 luxS + B. fragilis2

B. fragilis + luxS3 luxS + B. fragilis3 The copyrightholderforthispreprint(which