# Autotransporters drive biofilm formation and auto-aggregation in the diderm Firmicute Veillonella parvula

Nathalie Bechon, Alicia Jiménez-Fernández, Jerzy Witwinowski, Emilie Bierque, Najwa Taïb, Thomas Cokelaer, Laurence Ma, Jean-Marc Ghigo, Simonetta Gribaldo, Christophe Beloin

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Nathalie Bechon, Alicia Jiménez-Fernández, Jerzy Witwinowski, Emilie Bierque, Najwa Taïb, et al. Autotransporters drive biofilm formation and auto-aggregation in the diderm Firmicute Veillonella parvula. Journal of Bacteriology, 2020, 10.1128/JB.00461-20. pasteur-02924221

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1 **Autotransporters drive biofilm formation and auto-aggregation in the diderm Firmicute *Veillonella parvula*.

3 Nathalie Béchon¹,²,#, Alicia Jiménez-Fernández¹,#, Jerzy Witwinowski³, Emilie Bierque¹,⁴,⁷,

4 Najwa Taib³,⁵, Thomas Cokelaer⁵,⁶, Laurence Ma⁶, Jean-Marc Ghigo¹, Simonetta Gribaldo³ and

5 Christophe Beloin¹, *

6 ¹Genetics of Biofilm Laboratory, Institut Pasteur, UMR CNRS2001, Paris, 75015, France

7 ²Université de Paris, Sorbonne Paris Cité, Paris, France

8 ³Unit Evolutionary Biology of the Microbial Cell, Institut Pasteur, UMR CNRS2001, Paris,

9 France

10 ⁴Sorbonne Université, Collège doctoral, F-75005 Paris, France

11 ⁵Hub de Bioinformatique et Biostatistique – Département Biologie Computationnelle, Institut

12 Pasteur, USR 3756 CNRS, Paris, France

13 ⁶Plate-forme Technologique Biomics – Centre de Ressources et Recherches Technologiques

14 (C2RT), Institut Pasteur, Paris, France

15 ⁷Present address: Leptospirosis Research and Expertise Unit, Institut Pasteur in New

16 Caledonia, Institut Pasteur International Network, Noumea, New Caledonia.

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18 #The authors equally contributed to the work. Author order was determined alphabetically.

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20 Running Title: Autotransporters drive *V. parvula* biofilm.

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22 *Corresponding author: Christophe Beloin (christophe.beloin@pasteur.fr)
23 **ABSTRACT**

The Negativicutes are a clade of Firmicutes that have retained the ancestral diderm character
and possess an outer membrane. One of the best studied Negativicute, *Veillonella parvula*, is
an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial
communities, including the gut and the dental plaque microbiota. Whereas adhesion and biofilm
capacity of *V. parvula* is expected to be crucial for its maintenance and development in these
environments, studies of *V. parvula* adhesion have been hindered by the lack of efficient genetic
tools to perform functional analyses in this bacterium. Here, we took advantage of a recently
described naturally transformable *V. parvula* isolate, SKV38, and adapted tools developed for
the closely related *Clostridia* spp. to perform random transposon and targeted mutagenesis to
identify *V. parvula* genes involved in biofilm formation. We show that type V secreted
autotransporters -typically found in diderm bacteria- are the main determinants of *V. parvula*
auto-aggregation and biofilm formation, and compete with each other for binding either to cells
or to surfaces, with strong consequences on *V. parvula* biofilm formation capacity. The
identified trimeric autotransporters have an original structure compared to classical
autotransporters identified in Proteobacteria with an additional C-terminal domain. We also
show that inactivation of the gene coding for a poorly characterized metal-dependent
phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related
diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for
further molecular characterization of *V. parvula* interactions with other bacteria and the host
within complex microbiota environments.

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45 **IMPORTANCE**

46 Veillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to 47 adhere to surfaces or other bacteria and form biofilms is critical to inhabit complex human 48 microbial communities such as the gut and oral microbiota. Although the adhesive capacity of 49 V. parvula has been previously described, very little is known about the underlying molecular 50 mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took 51 advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to 52 identify surface exposed adhesin called autotransporters as the main molecular determinants of 53 adhesion in this bacterium. This work therefore provides new insights on an important aspect 54 of V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of 55 biofilm formation to the biology of this major commensal of the oral-digestive tract.

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INTRODUCTION

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Negativicutes are atypical and poorly studied Firmicute lineages displaying an outer envelope with lipopolysaccharide (1). Among the Negativicutes, *Veillonella* spp. are anaerobic diderm cocci that commonly inhabit the human and animal microbiota. One of their best studied species, *Veillonella parvula* (2), is a natural inhabitant of multiple different microbiota, including the human gut (3, 4). *V. parvula* is considered a commensal organism, and proposed to play a role in the development of immunity through its capacity to colonize the infant gut (5, 6). It is a key early colonizer of the dental plaque during the establishment of sessile microbial communities called biofilms (7), promoting multi-species growth and playing a central role in the metabolism of community members through lactic acid consumption (8). However, *V. parvula* is also described as an opportunistic pathogen and has been associated with diverse infections, including osteomyelitis, endocarditis, spondylodiscitis, abscesses as well as systemic infections (9–13).

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The importance of *V. parvula* in the development of microbial community spurred our interest in identifying the determinants of its adhesion and biofilm formation capacities. Moreover, considering the presence of an outer membrane in this atypical Firmicute, we wondered whether *V. parvula* uses known diderm or monoderm biofilm determinants, or currently undescribed adhesion factors. We recently studied *V. parvula* DSM2008 as a model diderm Firmicute strain (14) to investigate its outer membrane (OM) protein composition and detected 78 OM proteins, thirteen of which being potential adhesins belonging to the type V family of secreted autotransporter proteins (T5SS) (15). Autotransporter proteins are specifically found in diderms and all share common structural and functional features: a Sec-dependent signal peptide, a passenger domain providing the protein function, and an outer-membrane β-barrel domain that allows secretion of the passenger domain (16). However, the challenge of genetic manipulation in *V. parvula* DSM2008 severely limited the study of these adhesins in this strain.

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Here, we have sequenced and annotated the genome of *V. parvula* SKV38, a recently isolated, naturally transformable and genetically amenable strain (17). We adapted and developed genetic tools for this organism, permitting random and site directed mutagenesis, plasmid complementation and controlled expression using an inducible promoter. This enabled us to identify and characterize factors involved in *V. parvula* biofilm formation. We find that the main *V. parvula* biofilm modulating determinants are T5SS adhesins, i.e. typical diderm
89 determinants. Interestingly, the identified adhesins possess an additional C-terminal domain
90 compared to the known domain architecture of classical autotransporters. We also show that a
91 locus encoding a metal-dependent phosphohydrolase HD domain protein is involved in biofilm
92 formation, similarly to what was shown in the prototypical monoderm *Bacillus subtilis* (18).
93 Therefore, our results demonstrate that diderm Firmicutes use a mixture of diderm and
94 monoderm factors to modulate their ability to engage into biofilm lifestyle, supporting the idea
95 that monoderm and diderm molecular systems could have co-evolved in these atypical
96 Firmicutes.

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## 98 RESULTS

### 99 Random transposon mutagenesis reveals two *V. parvula* SKV38 genes involved in biofilm formation

In order to obtain a framework for genetic work in the recently described naturally competent *V. parvula* SKV38 isolate, we sequenced it using PacBio technology. We obtained a completely assembled genome of 2.146Mbp, encoding 1,912 predicted protein-encoding open reading frame (ORF), 12 rRNA, 49 tRNA and one tmRNA (see Material and Methods). We performed random transposon mutagenesis in *V. parvula* SKV38 using the pRPF215 plasmid carrying an inducible transposase and a mariner-based transposon previously used to mutagenize *Clostridioides difficile* (19), a close relative of the Negativicutes. We screened 940 individual transposon mutants for biofilm formation using crystal violet staining (CV) static biofilm assay in 96-well microtiter plates and identified eight independent mutants with significant reduction in biofilm formation (Figure 1A). Whole genome sequencing localized the transposons in two loci putatively implicated in biofilm formation (Figure 1B). The most affected mutants correspond to insertions in *FNLLGLLA_00516* (seven mutants), encoding a T5SS type Vc trimeric autotransporter. One transposon mutant was inserted in *FNLLGLLA_01127*, encoding a putative HD phosphatase (Figure 1B).

### 116 FNLLGLLA_00516 encodes a trimeric autotransporter involved in auto-aggregation

*FNLLGLLA_00516* encodes a protein containing several domains usually identified in the T5SS type Vc trimeric autotransporters. Trimeric autotransporters are OM proteins specific of diderm bacteria that have been widely studied for their ability to bind to different surfaces or to other bacteria (20). *FNLLGLLA_00516* is a homolog of *V. parvula* DSM2008 *vpar_0464*, which encodes a protein that was detected in the OM (15). *FNLLGLLA_00516* was annotated by PROKKA as BtaF, a trimeric autotransporter identified in *Brucella suis* involved in adhesion to extracellular matrix and abiotic surfaces (21). Here, we renamed it *Veillonella* trimeric autotransporter A (VtaA), as the first trimeric autotransporter involved in biofilm formation identified in *V. parvula* SKV38. We deleted the *vtaA* coding sequence and showed that Δ*vtaA* had no growth defect (Figure S1A) but displayed a marked reduction of biofilm formation in 96-well polystyrene microtiter plate (Figure 2A). Moreover, while *V. parvula* SKV38 cultures strongly aggregated, Δ*vtaA* did not (Figure 2B and S2). We constructed the strain *pTet-vtaA*, where the chromosomal *vtaA* gene is placed under the control of a functional tetracycline/anhydrotetracycline (aTc) inducible promoter (Figure S3) and showed that its
131 aggregation capacity and biofilm formation in 96-well polystyrene microtiter plate directly
132 correlated with aTc concentration (Figure 2C-D), demonstrating that VtaA-mediated cell-to-
133 cell interactions are critical for biofilm formation in these conditions. Whereas the microtiter
134 plate assay corresponds to a static biofilm assay, we also used continuous flow glass
135 microfermentors to investigate the contribution of VtaA to biofilm formation in dynamic
136 conditions. Surprisingly, ΔvtaA formed almost six times more biofilm than the WT strain in
137 these conditions (Figure 2E). Accordingly, scanning electronic microscopy (SEM) images of
138 mature biofilms on microscopic plastic slides in microfermentor showed that ΔvtaA formed a
139 much thicker biofilm when compared to WT (Figure S4). Altogether, these results suggest that
140 auto-aggregation differentially contributes to biofilm formation in static conditions on
141 hydrophobic surfaces versus continuous flow conditions on hydrophilic surfaces.

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143 V. parvula SKV38 encodes sixteen putative autotransporters in addition to VtaA

144 The strong biofilm phenotype displayed by the ΔvtaA mutant in microfermentor led us
to suspect that additional adhesins could modulate V. parvula biofilm formation capacity.
145 Indeed, searching the V. parvula SKV38 genome revealed multiple genes encoding
146 autotransporters (Table 1): three Va classical monomeric autotransporters with a characteristic
147 PFAMPF03797 autotransporter β-domain (renamed Veillonella monomeric autotransporter A
148 to C : VmA to C), and eight other putative Vc trimeric autotransporters with a characteristic
149 PFAM PF03895 YadA _anchor_domain (renamed Veillonella trimeric autotransporter B to I:
150 VtaB to I). We also identified several partial autotransporters: FNLLGLLA_00035, that only
151 contains a PFAM PF11924 Ve inverse autotransporter β-domain but no putative α-domain that
152 normally carries the function of the protein, and FNLLGLLA_00036-37 and
153 FNLLGLLA_00040-41, which are homologs of V. parvula DSM2008 Vpar_0041 and
154 Vpar_0048, respectively, and that appear to be split in SKV38 (Table 1). Interestingly, domain
155 analysis of all trimeric ATs of V. parvula SKV38 showed that they possess an extra C-terminal-
156 domain (SLH or coiled-coil domain) after the YadA anchor domain that is not found in classical
157 trimeric ATs. Among those, six autotransporters plus FNLLGLLA_00035, FNLLGLLA_00036-
158 FNLLGLLA_00040-41 form a potential genomic cluster coding for adhesins (Figure
159 3A), whereas the six others are located in different areas of the genome (Figure 3B).
160

161 We selected eight Veillonella strains to study more precisely the evolution of the adhesin
162 cluster, including SKV38 and DSM2008. The trimeric autotransporter adhesins seem to evolve
163 dynamically with numerous domain swaps, duplications and reductions of gene copies, likely
through homologous recombination suggesting rapid evolutionary changes in the repertoire of *Veillonella* adhesins (Figure 4). Duplications and deletions could be eased by the presence of short ORFs annotated as hypothetical proteins presenting a high degree of sequence identity. The most basal strain in the *Veillonella* phylogeny has a minimal cluster of only three adhesins genes. Throughout the *Veillonella* genus, the size of the cluster is very variable with a minimal form in *V. atypica*, with only two adhesins. This specific adhesin locus, immediately upstream of rRNA coding genes, is to our knowledge a peculiar genomic character of the *Veillonella* genus and is not found in other genera of the Veillonellaceae, suggesting it originated in the common ancestor of all *Veillonella* species.

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174 The cluster of trimeric autotransporters is involved in surface binding and not
175 aggregation.

176 To assess the function of the potential adhesins identified in the *V. parvula* SKV38
177 genome, we constructed -within the cluster of adhesin genes- independent deletion mutants for
178 the two first autotransporters (*vmaA* and *vtaB*) and a large deletion for the eight adjacent genes
179 encoding trimeric autotransporters or partial trimeric autotransporters, hereafter called Δ8
180 (*ΔFNLLGLLA_00036* to *vtaF*). We also generated independent individual mutants for each of
181 the six additional autotransporters located outside of the cluster. These mutants were all tested
182 for biofilm formation in 96-well polystyrene plate and aggregation capacities. None of the
183 mutants, with the exception of the previously mentioned Δ*vtaA* strain, was affected for
184 aggregation capacities (Figure 5A). The Δ8 mutant was the sole mutant, in addition to the Δ*vtaA*
185 mutant, to display lower biofilm formation in 96-well polystyrene microtiter plate (Figure 5B
186 and C), suggesting that the adhesins of this cluster could be involved in biofilm formation
187 independently of cell-to-cell interactions. When tested in microfermentor, Δ8 displayed a
188 slightly reduced ability to form mature biofilm, however, not statistically different from WT
189 (Figure 5D). This reduced ability to form mature biofilms was actually more visible when
190 observing SEM images, since the Δ8 mutant only poorly covered the coverslip with sporadic
191 aggregates of cells producing extracellular matrix (Figure S4). Initial adhesion assay to glass
192 spatula showed that both Δ*vtaA* and Δ8 displayed a lower percentage of initial adhesion than
193 WT, suggesting that VtaA-mediated auto-aggregation contributed to initial adhesion of the WT
194 strain while the adhesin cluster is probably directly involved in surface binding (Figure 5E).
195 This also indicates that Δ*vtaA* does not adhere to glass better than WT, and so the increased
196 biofilm formation of Δ*vta* in microfermenter arises during the continuous flow culture step. The
197 effect of deleting *vtaA* and the 8 adhesin genes on initial adhesion was additive since a Δ*vtaAΔ8*
198 double mutant showed a reduced initial adhesion on microfermentor spatula compared to either
199 WT, Δ*vtaA* or Δ8 (Figure 5E). In addition, Δ*vtaAΔ8* formed 17 times less biofilm than Δ*vtaA*
200 in microfermentor, indicating that in the absence of VtaA, the adhesins encoded by some of
201 these eight genes strongly promote mature biofilm formation in microfermentor (Figure 5D).

202 Taken together, these results demonstrate the differential contribution of VtaA and part
203 of the cluster of adhesin to V. parvula SKV38 adhesion and highlight the existence of potential
204 interference mechanisms between them.

205
206 FNLLGLLA_01127 encodes an HD phosphatase that inhibits biofilm formation

207 In addition to genes encoding potential T5SS proteins, we also identified a transposon
208 mutant in FNLLGLLA_01127, encoding a protein of the HD phosphatase superfamily (Figure
209 1B). The FNLLGLLA_01127 gene is homologous to YqeK, a putative phosphatase required for
210 pellicle formation and the development of biofilm in B. subtilis (18). FNLLGLLA_01127/yqeK
211 is found in a cluster of genes (obg, yhbY, proB, proA, nadD, yqeK, lytR, and rsfS), whose
212 synteny is very well conserved among Negativicutes. This cluster, or part of it, is also well
213 conserved in almost all Firmicutes genomes we analyzed, both monoderm and diderm, as well
214 as in members of other diderm phyla phylogenetically close to the Firmicutes, notably
215 Deinococcus-Thermus (Figure 6 and S5, DataSet 2). A FNLLGLLA_01127 deletion mutant
216 (Δ1127) had a lower carrying capacity compared to WT, maybe due to higher mortality during
217 the stationary phase (Figure S1) and a moderate 1.5-fold decrease in biofilm formation in
218 microtiter plate after correcting for the growth defect (Figure 7A). This mutant also displayed
219 a slightly faster aggregation rate than the WT during early time points (Figure 7B). The
220 strongest phenotype of this mutant was detected in microfermentor with a 9-fold increase in
221 biofilm formation compared to WT (Figure 7C). Expression of FNLLGLLA_01127 gene in
222 trans (plasmid p1127) did not complement the observed growth defect (Figure S1B) but it did
223 complement the increased biofilm formation in microfermentor (Figure 7D), showing that
224 deletion of FNLLGLLA_01127 might have had polar effects on downstream genes of the operon
225 causing a growth defect, but that FNLLGLLA_01127 alone was responsible for the observed
226 inhibition of biofilm formation. Scanning electronic microscopy showed that Δ1127, similarly
227 to Δ*vtaA*, formed a thick layered biofilm, although with fewer filaments and protein deposits
228 compared to WT (Figure 7E). However, contrary to Δ*vtaA* or Δ8 mutants, Δ1127 showed no
229 defect in initial adhesion to a glass spatula (Figure 7F). Interestingly, a Δ1127Δ8 double mutant
230 formed almost 20 times less biofilm than Δ1127 in microfermentor (Figure 7C), suggesting that

231 at least some of the autotransporters of the cluster were necessary for Δ1127 observed strong

232 biofilm formation in microfermentor.

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234
235

236 DISCUSSION

237

238 Originally described as a social organism mostly living in biofilm communities (8), Veillonella
239 is a known bacterial member of multiple human microbiota. Biofilm formation and adhesion
240 are important in these niches, but their study in Veillonella has been hindered by the lack of
241 efficient genetic tools. Here, we used genetics tools adapted from Clostridia to characterize
242 factors promoting biofilm formation in a naturally competent Veillonella parvula isolate.

243 We identified a T5SS type Vc trimeric autotransporter, FNLLGLLA_0516 (VtaA), as an
244 important biofilm factor promoting V. parvula SKV38 auto-aggregation. In addition to Hag1,
245 a YadA-like autotransporter identified from the related species V. atypica involved in
246 interspecies interactions (22), VtaA represents the second Veillonella protein described which
247 is involved in adhesion, and the first involved in abiotic surface adhesion and auto-aggregation
248 in diderm Firmicutes. Beyond the potential impact on Veillonella niche colonization,
249 aggregation capacity is known to contribute to bacterial protection from environmental stresses
250 or host responses (23), promotion of host colonization (24), or pathogenesis (25) in various
251 bacterial species. VtaA is homologous to Brucella suis trimeric autotransporter BtaF. However,
252 while B. suis BtaF promotes biofilm formation in vitro, it was not shown to promote aggregation
253 (21), suggesting that these two proteins have different functions.

254 In diderm bacteria such as E. coli, self-associating autotransporters (SAATs) from the type Va
255 family and type Vc trimeric autotransporters were shown to contribute to biofilm formation
256 through their self-recognition properties (26–32). However, in V. parvula VtaA-mediated auto-
257 aggregation either promoted (plastic surface and static conditions) or strongly impaired (glass
258 surface and continuous-flow conditions) biofilm formation depending on the model used. ΔvtaA
259 initially adhered less to glass spatula compared to WT, even though later on it formed much
260 more biofilm, thus we suspect that the material (glass vs. plastic) is not responsible for the
261 observed difference between our two systems. We hypothesize instead that in the WT, VtaA-
262 mediated aggregates are more sensitive to flow than individual cells, and are thus washed out
263 faster of the microfermentor, and that adhesion to surfaces or to the biofilm extracellular matrix
264 is more important than cell-to-cell interactions when the culture is performed under continuous
265 flow.

266 Interference between cell surface structures is a well-described mechanism by which bacteria
267 modulate their adhesion properties. In E. coli, multiple structures, such as chaperone-usher
268 fimbriae, LPS O-antigen or capsules, interfere with the self-recognizing autotransporter Ag43
269 though unknown mechanisms (33–36). Therefore, it is possible that in *V. parvula*, VtaA could
270 compete with other adhesins through steric hindrance or competition for membrane export and
271 thus limit biofilm formation under continuous-flow conditions. Consistently, Δ*vtaA* enhanced
272 biofilm formation in microfermentor was dependent on the presence of eight genes of the
273 cluster of trimeric autotransporters, suggesting a competition between VtaA and adhesin(s) of
274 this cluster. Moreover, we noticed that both VtaA and the 8-gene cluster are necessary for full
275 initial adhesion to glass spatula in an independent manner, suggesting that any competition
276 between them only arises later on, during continuous-flow cultures. The exact contribution of
277 these different trimeric autotransporters to biofilm formation and their interplay with VtaA will
278 require further characterization.

279 Analysis of *V. parvula* SKV38 genome revealed the presence of seven other potential full-
280 length autotransporters, but no other types of classical diderm adhesins. None of them appeared
281 to be involved in cell-to-cell interactions or biofilm formation on abiotic surfaces, and their
282 function remains to be fully elucidated. As *V. parvula* is present in different microbiota, it is
283 expected that a large arsenal of adhesion factors is necessary to adhere under different
284 mechanical constraints and on different surfaces, such as tooth enamel or various epithelia.
285 Moreover, *Veillonella* is known to co-aggregate with *Streptococci* (37–39), that produce
286 *Veillonella* favored carbon source, lactate (40), and they were shown to specifically co-
287 aggregate with *Streptococci* and *Actinomyces* strains from the same microbiota, showing that
288 co-aggregation could have strong implication in niche colonization of these bacteria (41). *V.
289 parvula* and other *Veillonella* are also associated to different opportunistic infections and the
290 contribution of their adhesins to pathogenicity remains to be addressed. Finally, some
291 autotransporters have been shown to carry non-adhesive functions, including protease activity
292 (42), but we detected no classical protease domain in the *Veillonella* autotransporters.
293

294 Trimeric autotransporters possess a characteristic YadA anchor domain (PF03895) that is
295 found mainly in Proteobacteria, but also in Cyanobacteria, Verrumicrobia, Planctomycetes,
296 Kiritimatiellaeota, Chlorobi, Synergistetes, Fusobacteria and Negativicutes
297 (https://pfam.xfam.org/family/PF03895, Dec 2019 (43)). Interestingly, the YadA anchor of V.
298 parvula SKV38 and all Veillonella trimeric autotransporters is not at the very end of the C-
299 terminus, where it is usually found in Proteobacteria, but is pre-C-terminal, followed by either
300 a coiled domain or a S-layer homology (SLH) domain (Figure 3, DataSet 1). While the function
301 of the coiled domain is unknown, in some bacteria the periplasmic SLH domain binds to
302 peptidoglycan (44), suggesting that Veillonella trimeric autotransporters could be non-
303 covalently attached to the peptidoglycan. These extra-domains after the YadA anchor are also
304 found in other Negativicutes (notably the extra SLH domain) and in some other diderm phyla
305 phylogenetically related to the Firmicutes such as Synergistetes and Fusobacteria (DataSet 1).
306 In addition to possessing trimeric autotransporters with an extra coiled C-terminus domain, the
307 Fusobacterium Streptobacillus moniliformis ATCC14647 carries eight genes encoding unique
308 trimeric autotransporters with an extra OmpA_family domain (PF00691) at their extreme C-
309 terminus, a domain known to display affinity to peptidoglycan (45) (DataSet 1). These data
310 suggest that a subset of phylogenetically close diderm bacteria have evolved trimeric
311 autotransporters integrating different peptidoglycan binding domains. Whether these domains
312 have an impact on trimeric autotransporters function or exposure to the surface, or more
313 generally on outer membrane stabilization is presently unknown.

314 Our screening also led to the identification of FNLLGLLA_01127, the homolog of B. subtilis
315 YqeK, a putative phosphatase required for pellicle formation and the development of biofilm
316 (18). Staphylococcus aureus YqeK was recently shown to be a nucleosidase hydrolyzing
317 diadenosine-tetraphosphate (Ap4A) into ADP (46). In Pseudomonas fluorescens, an increased
318 level of Ap4A increases cyclic-di-GMP concentration and enhances cell-surface exposure of a
319 large adhesin LapA, thus inducing biofilm formation (47). c-di-GMP regulates biofilm
320 formation by modulating production of a variety cell-surface appendages or exopolysaccharides
321 in both monoderm and diderm bacteria (48–52). Interestingly, B. subtilis YqeK induces the
322 epsA-O operon, involved in the production of biofilm matrix-forming polysaccharides (53).
323 Deletion of V. parvula FNLLGLLA_01127 only led to a minor decrease in biofilm formation in
324 96-well plate, but to a strong increase in continuous-flow biofilm formation that was dependent
325 on the presence of the cluster of trimeric autotransporters. Further work is needed to determine
326 whether FNLLGLLA_01127 directly impacts production of the adhesins of the cluster or
327 participates to the production/regulation of an unknown exopolysaccharide, which, contrary to
328 B. subtilis, would interfere with the function or exposure of the adhesins of the cluster rather
329 than favor community development.

330
331 In this study we have shown that classical diderm trimeric autotransporters and a potential
332 nucleotidase, conserved both in monoderms and diderms are crucial for adhesion both between
333 cells and/or to surfaces in the diderm Firmicute V. parvula. Our work also underscores the rapid
334 evolution of a diverse arsenal of trimeric autotransporters in the *Veillonella* genus, both in
335 numbers and size, probably by efficient recombination favored by gene clustering, allowing
336 rapid adaptation to changing environments. Taken together, our results suggest a complex
337 interplay at the surface of *V. parvula* between different cell surface structures that may have
338 co-evolved for a long time in these atypical Firmicutes. Much remains to be discovered on the
339 regulatory circuits controlling these adhesion factors and their role in diderm Firmicutes
340 biology.

341
342

343 MATERIALS AND METHODS

344 Genome preparation and sequencing

345 V. parvula SKV38 genomic DNA was extracted using Qiagen genomic tip 20G kit. It was
346 sequenced to 1,500X coverage using PacBio sequencing of one single molecule real time
347 (SMRT) cell with no multiplexing using the V2.1 chemistry. Only one SMRT cell was used but
348 with no multiplexing, leading to an unusually large amount of subreads: 3 Gbp, meaning about
349 1,500X coverage assuming a 2.1 Mbp genome. This yielded 338,310 reads with a mean subread
350 length of 9,080 bp and N50 read length of 13,500 bp. The longest subread length is above 70
351 kbp. We randomly subsampled the data to avoid misassemblies keeping only 100,000 subreads,
352 which resulted in a 430X coverage. The genome was then assembled using Canu version 1.8
353 (54) keeping the default parameters. In particular, subread below 1,000 bp were dropped. The
354 error correction steps of the Canu algorithm were not tuned, keeping the parameters that control
355 alignment seed length, read length, overlap length and error rates to their default values. We
356 obtained one contig of 2.146 Mbp and an additional contig of only 1,972 bp that was abandoned
357 due to lack of supporting data and was removed by the circularization process. The obtained
358 assembled genome closely matched the genome size (2.1422 Mbp) and GC content (38.7%,
359 expected 38.6%) of the reference V. parvula DSM2008 strain. The resulting assembled genome
360 was polished using Pilon (55) but no correction was required. No gaps or drops of coverage
361 was detected based on sequanaCoverage output (56, 57). The completeness of the candidate
362 assembly was assessed to be 98% using the bacteria mode and the bacteria_db9 lineage-specific
363 profile library of BUSCO software (58), while the number of complete duplicated or
364 fragmented BUSCOs remains at 0, indicative of complete assembly. Alignment of all reads
365 show that only 4% (13,028) remained unmapped and 80% of their length were below 2 kbp.
366 The remaining reads (2000 reads) map on various species and could not be further assembled.
367 Overall, these analyses indicate that the final genome assembly is complete and of good quality.

368

369 Bioinformatic analyses

370 The V. parvula SKV38 genome was annotated using PROKKA (59). The SKV38 annotated
371 genome sequence was deposited in the ENA (European Nucleotide Archive) under the
372 accession number ERZ1303164.
For protein domain visualization, PFAM domains (pfam.xfam.org, Pfam 32.0. (43)) were detected using HMMER (60). Domains with an e-value lower than 10⁻³ were kept and, in case of overlapping domains, the domain having the best e-value was kept. Presence of C-terminal coils structure was determined using the COILS program (https://embnet.vital-it.ch/software/COILS_form.html) (61).

The search for HD phosphatase (YqeK) cluster homologs was conducted as follows: a local databank containing 390 genomes representative of bacterial diversity was mined for the presence of a phosphatase containing HD domain (PF01966) using HMMSEARCH and the --cut_ga option. Protein sequences were then filtered using alignment, functional annotation, protein domains presence and phylogeny. Synteny was investigated in the locus around yqeK by looking, using MacSyFinder (62), for the presence of at least one of the 7 genes surrounding yqeK in V. parvula SKV38, namely obg (containing GTP1_OBG domain, PF01018), yhbY (containing CRS1_YhbY domain, PF01985), proB (containing AA_kinase domain, PF00696), proA (containing Aldedh domain, PF00171), nadD (containing CTP_transf_like domain, PF01467), lytR (containing LytR_cpsA_psr domain, PF03816) and rsfS (containing RsfS domain, PF02410), with no more than eight other genes separating them. All HMM profiles were downloaded from the PFAM site (pfam.xfam.org). As YqeK homologs are widespread in the Firmicutes, another local databank containing 230 representative Firmicutes genomes was queried by the MacSyFinder approach as described above. All trees were visualized with ITOL (63). Details of the results are presented in Dataset S2.

## Strains and growth conditions

Bacterial strains and plasmids are listed in Table 2. V. parvula was grown in either Brain Heart infusion medium (Bacto Brain Heart infusion, Difco) supplemented with 0.1 % L-cysteine and 0.6 % sodium DL-lactate (BHILC) or SK medium (10 g L⁻¹ tryptone (Difco), 10 g L⁻¹ yeast extract (Difco), 0.4 g L⁻¹ disodium phosphate, 2 g L⁻¹ sodium chloride, and 10 mL L⁻¹ 60 % w/v sodium DL-lactate, described in (17)) and incubated at 37°C in anaerobic conditions in anaerobic bags (GENbag anaero, Biomerieux, ref. 45534) or in a C400M Ruskinn anaerobic-microaerophilic station. Escherichia coli was grown in Lysogeny Broth (LB) (Corning) medium under aerobic conditions at 37°C. 20 mg L⁻¹ chloramphenicol (Cm), 200 mg L⁻¹ erythromycin (Ery) or 2.5 mg L⁻¹ tetracycline (Tc) were added to V. parvula cultures, 100 mg L⁻¹ carbenicillin (Cb) or 5 mg L⁻¹ tetracycline (Tc) were added to E. coli cultures when needed.
100 µg L⁻¹ anhydrotetracycline (aTc) was added to induce the pTet promoter unless stated otherwise. All chemicals were purchased from Sigma-Aldrich unless stated otherwise.

407

**Natural transformation**

Cells were resuspended in 1 mL SK media adjusted to 0.4-0.8 OD₆₀₀ and 10 µL were dotted on SK-agar petri dishes. On each drop, 0.5-1 µg plasmid or 75-200 ng µL⁻¹ linear dsDNA PCR product was added, or water for the negative control. The plates were incubated 48 hours. The biomass was resuspended in 500 µL SK medium and plated on SK-agar supplemented with the corresponding antibiotic and incubated for another 48 hours. Colonies were streaked on fresh selective plates and the correct integration of the construct was confirmed by PCR and sequencing.

416

**Random mariner transposon mutagenesis**

Plasmid pRPF215, described for *Clostridium* mutagenesis (Addgene 106377) (19) was transformed into *V. parvula* SKV38 by natural transformation and selected on Cm supplemented SK-agar medium. An overnight culture of *V. parvula* SKV38-pRPF215 in BHILC was then diluted to 0.1 OD₆₀₀ in the same media, supplemented with aTc and grown 5 hours to induce the transposase. After induction, the culture was diluted and plated on BHILC supplemented with Ery and aTc for selection and incubated for 48 hours. From the resulting colonies, 940 were inoculated in Greiner Bio-one polystyrene flat-bottom 96-well plates (655101) and grown in BHILC supplemented with either Ery and aTc, or Cm, to confirm both the presence of the transposon and the loss of pRPF215 and then kept in 15 % glycerol at -80°C. Selected transposon mutants were grown overnight and the genomic DNA was harvested using DNeasy blood and tissue kit (Qiagen). The genomic DNA was then sent for whole genome sequencing at the Mutualized platform for Microbiology of Institut Pasteur.

430

**Cloning-independent allelic exchange mutagenesis**

Site-directed mutagenesis of *V. parvula* SK38 strain was performed as described by Knapp and colleagues (17). Briefly, 1-Kb regions upstream and downstream the target sequence and *V. atypica* tetracycline resistance cassette (*tetM* in pBSJL2) or catP resistance cassette from *C. difficile* (*catP* in pRPF185, Addgene 106367, from (64)) were PCR-amplified with overlapping
primers using Phusion Flash High-Fidelity PCR Master-Mix (Thermo Scientific, F548). PCR products were used as templates in a second PCR round using only the external primers that generated a linear dsDNA with the tetracycline resistance cassette flanked by the upstream and downstream sequences. This construct was transformed into *V. parvula* by natural transformation and its integration into the genome was selected by plating on Tc or Cm supplemented medium. Positive candidates were further confirmed by a set of PCRs and sequencing around the site. Primers used in this study are listed in Table S1.

## Complementation

We replaced the tetracycline resistance gene and its gyrA promoter of the shuttle vector pBSJL2 by a chloramphenicol resistance gene, Pcat:cat from pRPF185 by Gibson assembly. Briefly, the inserts and the plasmids were PCR amplified and then mixed with Gibson master mix 2x (100µL 5X ISO Buffer, 0.2 µL 10,000 U/mL T5 exonuclease (NEB #M0363S), 6.25 µL 2,000 U/mL Phusion HF polymerase (NEB #M0530S), 50 µL 40,000 U/mL Taq DNA ligase (NEB #M0208S), 87 µL dH2O for 24 reactions) and incubated at 50°C for 30-60 min.

The resulting plasmid pBSJL2-cat was digested by Fastdigest BamHI (Thermo scientific) and the band obtained was purified from agarose gel using QIAquick gel extraction kit (Qiagen) to be used as linear plasmid in a second Gibson assembly. The genes and the Pmdh promoter of *V. parvula* SKV38 were amplified by PCR using PhusionFlash Master-mix and cloned in pBSJL2-cat using Gibson assembly. The mix was then transformed in *E. coli* DH5α and plated on LB with carbenicillin. The plasmid was harvested by miniprep using QIAprep spin miniprep kit (Qiagen) and transformed in *V. parvula* as described above.

Alternatively, the anhydrotetracycline inducible expression cassette of pRPF185, hereafter referred to as pTet, (Addgene 106367, (64)) was inserted along with a chloramphenicol marker right before the ATG of the target gene, following the procedure described above for cloning-independent allelic exchange mutagenesis. The functionality of pTet in *V. parvula* was previously verified using measurement of the aTc dependent β-glucuronidase activity generated by the presence of pRPF185 transformed in *V. parvula* SKV38 (Figure S3).

## Biofilm formation in 96-well microtiter plates

Overnight cultures in BHILC medium were diluted to 0.05 OD600 and transferred to three Greiner Bio-one polystyrene flat bottom 96-well plates adding 150 µL per well. After 24 hours of static incubation, one of the three plates was resuspended by pipetting to measure OD600
using a TECAN Infinite-M200-Pro spectrophotometer. The two other plates were used for coloration: cultures were removed by pipetting carefully the supernatant out and biofilms fixed with 150 µL Bouin solution (HT10132, Sigma-Aldrich) for 15 minutes. Bouin solution was removed by inversion and the biofilms were washed once in water. The biofilms were stained with 150 µL crystal violet 1 % (V5265, Sigma-Aldrich) for 15 minutes without shaking, then washed in water twice and left to dry. All washes were made by flicking of the plate. After drying the plate, crystal violet was dissolved with 200 µL absolute ethanol and transferred to a clean 96-well plate for OD₆₂₀ measurement (TECAN Infinite-M200-Pro spectrophotometer).

477

## Biofilm formation in microfermentor

Continuous flow non-bubbled microfermentor containing a removable spatula were used as described in (65, 66) (see https://research.pasteur.fr/en/tool/biofilm-microfermenters/). Briefly, a glass spatula was dipped in an overnight culture diluted to 0.5 OD₆₀₀ in 15 mL BHILC for 15 minutes and returned to the fermentor. Biofilm was grown on the spatula for 48 hours at 37°C. BHILC was constantly supplied through a peristaltic pump at 4 rpm. During the last hour, the speed was increased to 10 rpm to remove planktonic bacteria. A mix of filtered 90% nitrogen/5% hydrogen/5% carbon dioxide was also constantly supplied to maintain anaerobic condition. After 48 hours of growth, the spatula was removed, and the biofilm was resuspended by vortexing in 15 mL BHILC. We measured OD₆₀₀ of the resuspended biofilms with Smart Spec Plus spectrophotometer (BioRad).

489

## Aggregation curve

Overnight cultures were diluted to 0.8 OD₆₀₀ in Brain-heart infusion (BHI) media in semi-micro spectrophotometry cuvette (Fisherbrand) and left to sediment on the bench in presence of oxygen, so no growth should occur. OD₆₀₀ was measured every hour in a single point of the cuvette using SmartSpec spectrophotometer (BioRad).

495

## Initial adhesion on glass

Glass spatula from microfermentor (described above) were dipped in overnight cultures diluted to 0.5 OD₆₀₀ in 15 mL Brain-Heart Infusion (BHI) media for 30 minutes to let bacteria adhere. The spatulas were washed once in 15 mL BHI by submersion and the adhering bacteria were resuspended in 15 mL clean BHI by vortexing. The culture used for inoculation, as well as the resuspended bacteria were serially diluted and plated on SK-agar plate for colony forming unit (CFU) counting.

502
503

504 Statistical analysis

505 Statistical analysis was performed using either R and Rstudio software or GraphPad Prism8

506 (GraphPad software, Inc.). We used only non-parametric test, and when applicable corrected

507 for multiple testing. For microfermentor experiments, 4 replicates of each condition were used.

508 For all the other experiments, at least 6 biological replicates in at least 2 independent experiment
509 were used. A cut-off of p-value of 5% was used for all tests. * p<0.05; ** p<0.05; *** p<0.005.

510 For growth curve analyses, we computed the growth rate and carrying capacity of each

511 biological replicates using Growthcurver 0.3.0 package in R and we performed a Mann-

512 Whitney test comparing both parameters for each mutant to the corresponding WT.

513
514

515 COMPETING FINANCIAL INTERESTS

516 The authors declare no competing financial interests.

517

518

519 ACKNOWLEDGEMENTS

520 We thank Justin Merritt for providing *V. parvula* SKV38 strain, Bruno Dupuy and Robert P.

521 Fagan for providing the different *Clostridium* plasmids, Pierre Simon Garcia for help with the

522 Figure 3 preparation, Daniela Megrian Nuñez and Panagiotis Adam for the genome databank

523 preparation and the platforms France Génomique and IBISA. We wish to acknowledge funding

524 from the French National Research Agency (ANR) (Fir-OM ANR-16-CE12-0010), from the

525 Institut Pasteur "Programmes Transversaux de Recherche" (PTR 39-16), from the French

526 government's Investissement d'Avenir Program, Laboratoire d'Excellence "Integrative Biology

527 of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID) and from the Fondation

528 pour la Recherche Médicale (grant DEQ20180339185). N.B. was supported by a MENESR

529 (Ministère Français de l'Éducation Nationale, de l'Enseignement Supérieur et de la Recherche)

530 fellowship. A.J.F. was supported by a PRESTIGE program from Campus France.

531

532 AUTHORS CONTRIBUTIONS

533 C.B., N.B. and A.J.F. designed the experiments. N.B., A.J.F., E.B. and L.M. performed the

534 experiments. J.W., N.T., and T.C. carried out all genomics and phylogeny analyses under the

535 supervision of SG. N.B., C.B. and A.J.F. wrote the paper, with contribution from S.G., J.W.,

536 T.C. and JM.G. All authors have read and approved the manuscript.

537

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733 66. Beloin C, Valle J, Latour-Lambert P, Faure P, Kzreminski M, Balestrino D, Haagensen JAJ, Molin S, Prensier G, Arbeille B, Ghigo J-M. 2003. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Mol Microbiol 51:659–674.

736 67. Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B, Martin-Verstraete I. 2013. Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile. PLoS Genet 9.

740 68. Liu J, Xie Z, Merritt J, Qi F. 2012. Establishment of a tractable genetic transformation system in Veillonella spp. Appl Environ Microbiol 78:3488–91.

742 69. Sullivan MJ, Petty NK, Beatson SA. 2011. Easyfig: a genome comparison visualizer. Bioinformatics 27:1009–1010.

744

745
746 TABLES

747

748
<table><caption>Table 1: *V. parvula* SKV38 autotransporters</caption><thead><tr><th>Locus tag</th><th>PROKKA Gene name</th><th>Genome position</th><th>Gene size (kb)</th><th>Strand</th><th>Description</th><th>DSM2008 homolog</th><th>Name</th><th>Class</th></tr></thead><tbody><tr><td>FNLLGLLA_00032</td><td>prn 1</td><td>39,354</td><td>41,723</td><td>2,37</td><td>forward</td><td>Autotransporter</td><td>Vpar_0036-0037</td><td>VmA</td><td>Va</td></tr><tr><td>FNLLGLLA_00034</td><td>btaE 1</td><td>42,345</td><td>43,754</td><td>1,41</td><td>reverse</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0039</td><td>VtaB</td><td>Vc</td></tr><tr><td>FNLLGLLA_00035</td><td>hypothetical protein</td><td>44,146</td><td>45,189</td><td>1,04</td><td>forward</td><td>Autotransporter (partial)</td><td>Vpar_0040</td><td></td><td>Vc</td></tr><tr><td>FNLLGLLA_00036</td><td>hypothetical protein</td><td>45,453</td><td>46,883</td><td>1,431</td><td>forward</td><td>none</td><td>split Vpar_0041</td><td></td><td>?</td></tr><tr><td>FNLLGLLA_00037</td><td>omp-alpha</td><td>46,91</td><td>47,878</td><td>969</td><td>forward</td><td>Trimeric Autotransporter/ S-layer homology domain</td><td>split Vpar_0041</td><td></td><td>Vc?</td></tr><tr><td>FNLLGLLA_00038</td><td>upaG 1</td><td>48,397</td><td>56,829</td><td>8,433</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0042</td><td>VtaC</td><td>Vc</td></tr><tr><td>FNLLGLLA_00040</td><td>btaE 2</td><td>57,966</td><td>59,84</td><td>1,875</td><td>forward</td><td>Trimeric Autotransporter: YadA like (Partial)</td><td>split Vpar_0048</td><td></td><td>?</td></tr><tr><td>FNLLGLLA_00041</td><td>ata 1</td><td>59,837</td><td>63,463</td><td>3,627</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>split Vpar_0048</td><td></td><td>Vc?</td></tr><tr><td>FNLLGLLA_00044</td><td>ehaG 1</td><td>65,3</td><td>71,515</td><td>6,216</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0051</td><td>VtaD</td><td>Vc</td></tr><tr><td>FNLLGLLA_00045</td><td>upaG 2</td><td>71,995</td><td>81,42</td><td>9,426</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0052</td><td>VtaE</td><td>Vc</td></tr><tr><td>FNLLGLLA_00046</td><td>ata 2</td><td>81,941</td><td>91,519</td><td>9,579</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0053</td><td>VtaF</td><td>Vc</td></tr><tr><td>FNLLGLLA_00098</td><td>btaE 3</td><td>151,792</td><td>153,522</td><td>1,731</td><td>forward</td><td>Trimeric Autotransporter/ S-layer homology domain</td><td>Vpar_0100</td><td>VtaG</td><td>Vc</td></tr><tr><td>FNLLGLLA_00099</td><td>ata 3</td><td>154,024</td><td>158,982</td><td>4,959</td><td>forward</td><td>Trimeric Autotransporter/ S-layer homology domain</td><td>absent</td><td>VtaH</td><td>Vc</td></tr><tr><td>FNLLGLLA_00335</td><td>prn 2</td><td>414,666</td><td>416,888</td><td>2,223</td><td>forward</td><td>Autotransporter</td><td>Vpar_0330</td><td>VmaB</td><td>Va</td></tr><tr><td>FNLLGLLA_00516</td><td>btaF</td><td>581,236</td><td>590,358</td><td>9,123</td><td>forward</td><td>Trimeric Autotransporter: YadA like</td><td>Vpar_0464</td><td>VtaA</td><td>Vc</td></tr><tr><td>FNLLGLLA_00581</td><td>brkA</td><td>668,34</td><td>670,583</td><td>2,244</td><td>forward</td><td>Autotransporter</td><td>Vpar_1322</td><td>VmaC</td><td>Va</td></tr><tr><td>FNLLGLLA_01790</td><td>ehaG 2</td><td>1,943,661</td><td>1,946,159</td><td>2,499</td><td>reverse</td><td>Trimeric Autotransporter/ S-layer homology domain</td><td>Vpar_1664</td><td>VtaI</td><td>Vc</td></tr></tbody></table>
<table><thead><tr><th>Strain name</th><th>Description</th><th>Reference</th></tr></thead><tbody><tr><td>WT</td><td>Veillonella parvula SKV38</td><td>(17)</td></tr><tr><td>9G5</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>5C5</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>5H1</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>3D6</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>7B11</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>2F7</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>3F7</td><td>Veillonella parvula SKV38 FNLLGLLA_00516::Transposon</td><td>This study</td></tr><tr><td>5E11</td><td>Veillonella parvula SKV38 FNLLGLLA_01127::Transposon</td><td>This study</td></tr><tr><td>ΔvtaA</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00516::tetM</td><td>This study</td></tr><tr><td>pTet-vtaA</td><td>Veillonella parvula SKV38 catP-Term(fdx)-Ptet-FNLLGLLA_00516</td><td>This study</td></tr><tr><td>Δ8</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00036-46::tetM</td><td>This study</td></tr><tr><td>ΔvmaA</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00032::tetM</td><td>This study</td></tr><tr><td>ΔvtaB</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00034::tetM</td><td>This study</td></tr><tr><td>ΔvtaG</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00098::tetM</td><td>This study</td></tr><tr><td>ΔvtaH</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00099::tetM</td><td>This study</td></tr><tr><td>ΔvmaB</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00335::tetM</td><td>This study</td></tr><tr><td>ΔvmaC</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00581::tetM</td><td>This study</td></tr><tr><td>ΔvtaI</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_01790::tetM</td><td>This study</td></tr><tr><td>ΔvtaAΔ8</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_00516::catP ΔFNLLGLLA_00036-46::tetM</td><td>This study</td></tr><tr><td>Δ1127</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_01127::tetM</td><td>This study</td></tr><tr><td>Δ1127Δ8</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_01127::tetM ΔFNLLGLLA_00036-46::catP</td><td>This study</td></tr><tr><td>WT+pEmpty</td><td>Veillonella parvula SKV38-pBSJL2-catP-pmdh</td><td>This study</td></tr><tr><td>Δ1127+pEmpty</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_01127::tetM-pBSJL2-catP-pmdh</td><td>This study</td></tr><tr><td>Δ1127+p1127</td><td>Veillonella parvula SKV38 ΔFNLLGLLA_01127::tetM-pBSJL2-catP-pmdh-FNLLGLLA_01127</td><td>This study</td></tr><tr><td>P<sub>ter-φ</sub></td><td>SKV38- pRPF185ΔgusA</td><td>This study</td></tr><tr><td>P<sub>ter-gusA</sub></td><td>SKV38- pRPF185</td><td>This study</td></tr><tr><td>P<sub>Cwp2-gusA</sub></td><td>SKV38- pRPF144</td><td>This study</td></tr><tr><th>Plasmid</th><th>Description</th><th>Reference</th></tr></tbody></table>
<table><caption>Table 2: Strains and plasmids used in this study</caption><tr><td>pRPF215</td><td>mariner Tn delivery plasmid, P<sub>tet</sub>::Himar1, ITR-ermB-ITR, catP, tetR</td><td>(19)</td></tr><tr><td>pRPF185</td><td>tetracycline inducible expression system fused with β-glucuronidase gusA Term(fdx)- P<sub>tet</sub>-gusA-Term(slpA), catP</td><td>(64)</td></tr><tr><td>pRPF185ΔgusA</td><td>pDIA6103, tetracycline inducible expression system Term(fdx)-P<sub>tet</sub>-Term(slpA), catP</td><td>(67)</td></tr><tr><td>pRPF144</td><td>carries a Clostridium constitutive promoter fused with gusA P<sub>Cwp2-gusA</sub></td><td>(64)</td></tr><tr><td>pBSJL2</td><td>E. coli-Veillonella shuttle plasmid, P<sub>gyrA::tetM</sub></td><td>(68)</td></tr><tr><td>pBSJL2-cat</td><td>E. coli-Veillonella shuttle plasmid, P<sub>cat::catP, pmdh</sub> promoter</td><td>This study</td></tr><tr><td>p1127</td><td>pBSJL2-catP-pmdh-FNLLGLLA_01127</td><td>This study</td></tr></table>

751
752
753
754
755 FIGURES LEGEND

756

757 Figure 1: Random transposon mutagenesis in Veillonella parvula SKV38 led to identification of mutants with reduced biofilm formation. A. 96-well polystyrene plate 758 biofilm assay after CV staining of nine transposon mutants identified by random mutagenesis 759 grown 24h in BHILC. Mean of WT is adjusted to 100 %. Min-max boxplot of 6-15 biological 760 replicates for each strain are represented, each replicate is the mean of two technical replicates. 761 * p-value<0.05, ** p-value <0.005, Mann-Whitney test. B. Schematic representation of the 762 transposon insertion point identified (red arrow) for the 8 transposon mutants. Blue bar 763 represents the size of the gene in nucleotides. 764

765

766 Figure 2: VtaA is an adhesin involved in auto-aggregation and biofilm formation. A. 96-767 well plate biofilm assay after 24h growth in BHILC. Mean of WT is adjusted to 100 %. Min-768 max boxplot of 6 biological replicates for each strain. * p-value<0.05, ** p-value <0.005, 769 Mann-Whitney test between strains. B. and C. Aggregation curve in spectrophotometry cuvette 770 of WT and ΔvtaA (B) and of an inducible vtaA with 0, 0.025 or 0.1 µg/mL of the inducer aTc 771 (C). 100 % represent lack of aggregation, 0 % complete sedimentation of the culture. Median 772 of 6 biological replicates, error bars represent 95% confidence interval. At each time points we 773 computed the Mann-Whitney test between conditions. We applied Bonferroni correction for 774 multiple testing: p-value are only considered significant if *p-value<0.004, **p-value<0.0004, 775 ***p-value<0.00004. Indicated p-values were calculated by comparing in B, WT and ΔvtaA, 776 and in C, pTet-vtaA without aTc and pTet-vtaA with different aTc concentrations. D. 96-well 777 plate biofilm assay after 24h growth of an inducible vtaA in BHILC with different 778 concentrations of aTc. WT without aTc is adjusted to 100 %. Median of 6 biological replicates, 779 each replicate corresponds to the mean of two technical replicates, error bars represent 95% 780 confidence interval. * p-value<0.05, ** p-value <0.005, Mann-Whitney test. E. Biofilm 781 formation in continuous flow microfermentor on glass spatula during 48h in BHILC. WT was 782 adjusted to 100 %. Min-max boxplot of 4 biological replicates for each strain. A picture of the 783 spatula before resuspension is shown below each boxplot bar. * p-value<0.05, Mann-Whitney 784 test. 785

785

786 Figure 3: Veillonella parvula autotransporters domain organization. A. Genetic 787 organization of the V. parvula SKV38 autotransporter adhesin gene cluster and the 788 corresponding adhesin domain organization. B. Domain organization of the six remaining V. 789 parvula SKV38 autotransporter adhesins encoded by genes located outside of the cluster. 790 Domains were detected with the HMMER package (60), only the domains with e-values lower 791 than 10⁻³ are shown. Presence of C-terminal coils structure was determined using the COILS 792 program (https://embnet.vital-it.ch/software/COILS_form.html). All V. parvula trimeric ATs 793 display an additional C-terminal domain (a SLH or a coiled coil domain) following the YadA 794 anchor domain as compared to classical trimeric autotransporters. 795

795

796 Figure 4: Synteny of the adhesin gene cluster in a selection of Veillonella species. The 797 synteny of the proteins of the cluster between the closest relatives was assessed using EasyFig 798 (69). Oblique lines between genes represent tblastx identities (program parameters: maximum 799 e-value of 10¹², minimum length of 30, minimum identity of 30). The V. parvula SKV38 strain
800 used in this study is presented in red. The annotation of the genes of the cluster is indicated on
801 the right.

802

803 **Figure 5: A cluster of eight trimeric autotransporters is involved in surface binding. A.**
804 Aggregation curve in spectrophotometry cuvette. 100 % represent lack of aggregation, 0 %
805 complete sedimentation of the culture. Median of 6 biological replicates, error bars represent
806 95% confidence interval. * Mann-Whitney test, corrected for multiple testing with Bonferroni
807 correction: significance is achieved if p-value < 0.007. **B.** and **C.** 96-well plate biofilm assay
808 after 24 h growth in BHILC. Mean of WT is adjusted to 100 %. Min-max boxplot of 6 biological
809 replicates for each strain, each replicate is the mean of two technical replicates. In **B.** we applied
810 a Mann-Whitney; * p-value<0.05, ** p-value <0.005. In **C.** we applied Bonferroni correction
811 for multiple testing: tests were called significant only if p-value<0.01: * p-value<0.01, ** p-
812 value <0.001, *** p-value <0.0001. **D.** Biofilm formation in continuous flow microfermentor
813 on glass spatula during 48h in BHILC. WT was adjusted to 100 %. Min-max boxplot of 4
814 biological replicates for each strain. * p-value<0.05, Mann-Whitney test. A picture of spatula
815 before resuspension is shown for each mutant below the boxplot. **E.** Initial adhesion on glass
816 spatula. Percentage of CFU that adhered to the spatula controlled by the number of CFU of the
817 inoculation solution. Min-max boxplot of 6-9 replicates per strain is represented. * p-
818 value<0.05, ** p-value <0.005, *** p-value <0.0005, Mann-Whitney test.

819

820 **Figure 6: Occurrence and synteny of HD Phosphatase (YqeK) in diderm and monoderm**
821 **bacteria.** A. The presence of the cluster was investigated using MacSyFinder (62) and the
822 results were plotted onto a schematic reference tree of 187 cultivable bacteria among the 390
823 of the analyzed databank. The cell wall status of each phylum is indicated as: (-) diderm with
824 LPS, (+) monoderm, (atyp.) diderm without LPS, (?) unclear. For the Firmicutes, the diderm
825 lineages are indicated in red (Negativicutes), blue (Halanaerobiales) and purple
826 (Limnochordales).

827

828 **Figure 7: FNLLGLLA_01127 represses biofilm formation in microfermentor. A.** 96-well
829 plate biofilm assay after 24 h growth in BHLC corrected by OD600 after 24 h growth in plate.
830 Mean of WT is adjusted to 100 %. Min-max boxplot of 6 biological replicates for each strain,
831 each replicate is the mean of two technical replicates. * p-value < 0.05, Mann-Whitney test. **B.**
832 Aggregation curve in spectrophotometry cuvette. 100 % represent lack of aggregation, 0 %
833 complete sedimentation of the culture. Median of 6 biological replicates, error bars represent
834 95% confidence interval. * Mann-Whitney test, corrected for multiple testing with Bonferroni
835 correction: significance is achieved if p-value<0.007. **C.** Biofilm formation in continuous flow
836 microfermentor on glass spatula during 48h in BHILC. Mean of WT is adjusted to 100 %. Min-
837 max boxplot of 4 biological replicates for each strain. * p-value < 0.05, ** p-value<0.005,
838 Mann-Whitney test. A picture of a spatula before resuspension is shown for each strain below
839 the histogram. **D.** Biofilm formation in continuous flow microfermentor on glass spatula during
840 48h in BHILC+chloramphenicol. Mean of WT+pEmpty is adjusted to 100 %. Min-max boxplot
841 of 4 biological replicates for each strain. * p-value < 0.05, Mann-Whitney test. A picture of a
842 spatula before resuspension is shown for each strain below the boxplot. **E.** Scanning electronic
843 microscopy of Δ1127 biofilm grown under continuous flow of BHILC in microfermentor on a
844 plastic microscopy slide. Magnification 2K and 5K. **F.** Initial adhesion on glass spatula.
845 Percentage of CFU that adhered to the spatula in 30 min controlled by the number of CFU of
846 the inoculation solution. Min-max boxplot of 6-9 replicates per strain. * p-value<0.05, Mann-

847 Whitney test.

848
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>A</figcaption></figure>

<table><caption>B</caption><thead><tr><th>Gene locus</th><th>Protein name</th><th>Domain organization</th></tr></thead><tbody><tr><td>FNLLGLLA_00032</td><td>Prn_1 / VmaA</td><td>790 aa</td></tr><tr><td>FNLLGLLA_00034</td><td>BtaE_1 / VtaB</td><td>470 aa</td></tr><tr><td>FNLLGLLA_00035</td><td></td><td>348 aa</td></tr><tr><td>FNLLGLLA_00036</td><td></td><td>477 aa</td></tr><tr><td>FNLLGLLA_00037</td><td></td><td>323 aa</td></tr><tr><td>FNLLGLLA_00038</td><td>UpaG_1 / VtaC</td><td>2811 aa</td></tr><tr><td>FNLLGLLA_00040</td><td>BtaE_2</td><td>625 aa</td></tr><tr><td>FNLLGLLA_00041</td><td>Ata_1</td><td>1209 aa</td></tr><tr><td>FNLLGLLA_00044</td><td>EhaG_1 / VtaD</td><td>2072 aa</td></tr><tr><td>FNLLGLLA_00045</td><td>UpaG_2 / VtaE</td><td>3142 aa</td></tr><tr><td>FNLLGLLA_00046</td><td>Ata_2 / VtaF</td><td>3193 aa</td></tr></tbody></table>

<table><thead><tr><th>Gene locus</th><th>Protein name</th><th>Domain organization</th></tr></thead><tbody><tr><td>FNLLGLLA_00098</td><td>BtaE_3 / VtaG</td><td>577 aa</td></tr><tr><td>FNLLGLLA_00099</td><td>Ata_3 / VtaH</td><td>1653 aa</td></tr><tr><td>FNLLGLLA_00335</td><td>Prn_2 / VmaB</td><td>741 aa</td></tr><tr><td>FNLLGLLA_00516</td><td>BtaF / VtaA</td><td>3041 aa</td></tr><tr><td>FNLLGLLA_00581</td><td>BrkA / VmaC</td><td>748 aa</td></tr><tr><td>FNLLGLLA_01790</td><td>EhaG_2 / VtaF</td><td>833 aa</td></tr></tbody></table>

■ extended signal peptide region (PF13018)

■ procyclic acidic repetitive protein (PF05887)

■ DUF1664 (PF07889)

■ YadA head (PF05658)

■ YadA stalk (PF05662)

■ autotransporter (PF03797)

■ inverse autotransporter beta domain (PF11924)

■ YadA anchor (PF03895)

■ S-layer homology (PF00395)

■ Coil (Coils prediction)
<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_15.png" /></figure>
<figure><img src="image_16.png" /></figure>
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /></figure>

D

<figure><img src="image_20.png" /></figure>

E

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

F

<figure><img src="image_23.png" /></figure>