1 Inactivation of the SecA2 Protein Export Pathway in

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2 Listeria monocytogenes Promotes Cell Aggregation, Impacts

3 Biofilm Architecture and Induces Biofilm Formation in

4 Environmental Condition1

5 Sandra RENIER 1, Caroline CHAGNOT 1, Julien DESCHAMPS 2, Nelly CACCIA 1, Julie

6 SZLAVIK 3, Susan A. JOYCE 4, Magdalena POPOWSKA 5, Colin HILL 4, Susanne

7 KNØCHEL 3, Romain BRIANDET 2, Michel HÉBRAUD 1 and Mickaël DESVAUX 1,*

1
8 INRA, UR454 Microbiologie, F-63122 Saint-Genès-Champanelle, France
2
9 INRA, UMR1319 MicAliS, F-91300 Massy, France
3
10 Department of Food Science, University of Copenhagen, Rolighedsvej 30, 1958

11 Frederiksberg C, Denmark
4
12 Alimentary Pharmabiotic Centre & Microbiology Department, University College Cork,

13 Cork, Ireland
5
14 Department of Applied Microbiology, Institute of Microbiology, University of Warsaw,

15 Warsaw, Poland

16 Running title: SecA2 impacts biofilm formation in L. monocytogenes

17 Keywords: Listeria monocytogenes, Biofilm formation, Bacterial adhesion, SecA2

18 translocase, Protein secretion system, Cell-wall hydrolase.

This article has been accepted for publication and undergone full peer review but has not been through the
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version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12257

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 19 *Corresponding author: INRA Clermont-Ferrand Research Centre, UR454 Microbiology, Site
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of Theix, F-63122 Saint-Genès Champanelle, France. Dr Mickaël Desvaux, Tel.: +33 (0)4 73

62 47 23, Fax: +33 (0)4 73 62 45 81, e-Mail: mickael.desvaux@clermont.inra.fr.

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 22
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SUMMARY

Listeria monocytogenes has a dichotomous lifestyle, existing as an ubiquitous

25 saprophytic species and as an opportunistic intracellular pathogen. Besides its capacity to

26 grow in a wide range of environmental and stressful conditions, L. monocytogenes has the

27 ability to adhere to and colonize surfaces. Morphotype variation to elongated cells forming

28 rough colonies has been reported for different clinical and environmental isolates, including

29 biofilms. This cell differentiation is mainly attributed to the reduced secretion of two SecA2-

30 dependent cell-wall hydrolases, CwhA and MurA. SecA2 is a non-essential SecA paralogue

31 forming an alternative translocase with the primary Sec translocon. Following investigation at

32 temperatures relevant to its ecological niches, i.e. infection (37°C) and environmental (20°C)

33 conditions, inactivation of this SecA2-only protein export pathway led, despite reduced

34 adhesion, to the formation of filamentous biofilm with aerial structures. Compared to the wild

35 type strain, inactivation of the SecA2 pathway promoted extensive cell aggregation and

36 sedimentation. At ambient temperature, this effect was combined with the abrogation of cell

37 motility resulting in elongated sedimented cells, which got knotted and entangled together in

38 the course of filamentous-biofilm development. Such a cell differentiation provides a decisive

39 advantage for listerial surface colonization under environmental condition. As further

40 discussed, this morphotypic conversion has strong implication on listerial physiology and is

41 also of potential significance for asymptomatic human/animal carriage.

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 42
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44
INTRODUCTION

Listeria monocytogenes is an opportunistic pathogen Gram-positive bacterium

45 associated with many foodborne disease outbreaks. Infection mainly occurs after ingestion of

46 contaminated food (Gahan and Collins, 1991) and affects predominantly pregnant women,

47 neonates, the elderly and immunocompromised patients (Farber and Peterkin, 1991). In the

48 food industry, L. monocytogenes represents a major problem as a source of contamination of

49 raw and processed foods. Besides its ability to grow in a wide range of conditions including

50 pH (4.3 to 9.6), temperature (1 to 45°C), salt concentration (up to 10 % NaCl) and water

51 activity (Aw down to 0.93), L. monocytogenes can adhere to and colonizes abiotic surfaces,

52 which contributes to its strong resistance to technological treatments and environmental

53 stresses (Carpentier and Cerf, 2011). While different definitions can be found in the literature

54 (Dunne, 2002), a biofilm can be broadly defined as the sessile development of microbial cells.

55 The bacterial cells adhering to each other and/or to a surface or interface are generally

56 surrounded by a matrix of extracellular polymers (Kolter, 2005). In L. monocytogenes, the

57 existence of such an exopolysaccharide matrix has never been evidenced (Nilsson et al., 2011;

58 Renier et al., 2011). Extracellular DNA (eDNA) could play a role in adhesion and early stages

59 of biofilm formation but only under certain growth conditions (Harmsen et al., 2010). Instead,

60 cell-surface proteins are the major adhesion factors contributing to biofilm formation in

61 L. monocytogenes (Smoot and Pierson, 1998; Longhi et al., 2008; Franciosa et al., 2009),

62 especially the flagella and some yet unidentified proteins (Renier et al., 2011).

63 In monoderm bacteria, cell-surface displayed proteins need to be first translocated

64 across the cytoplasmic membrane (Desvaux et al., 2005a; Desvaux et al., 2005b; Desvaux et

65 al., 2006a; Desvaux et al., 2009). From the most recent proteogenomic analyses and with 714

66 proteins exhibiting an N-terminal signal peptide (Renier et al., 2012), the Sec (Secretion)

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 67 pathway appears as the main route for protein secretion in L. monocytogenes (Desvaux and
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69
Hébraud, 2006; Desvaux, 2012). The Sec translocase, composed of the transmembranar

SecYEG translocon and the peripheral SecA ATPase, is essential for the passage of

70 preproteins through the cytoplasmic membrane (Du Plessis et al., 2011). As in several other

71 pathogenic Gram-positive bacteria, a paralogue of SecA, named SecA2, has been identified in

72 L. monocytogenes. Upon deletion of the secA2 gene, the cell morphotype changes from

73 discrete cells forming smooth colonies in L. monocytogenes wild type (wt) to long-chain cells

74 forming rough colonies and was associated to virulence defect (Lenz and Portnoy, 2002; Lenz

75 et al., 2003). Similar morphotypes have been isolated from clinical patients, food samples and

76 environmental biofilms (Rowan et al., 2000; Monk et al., 2004), and reversible conversion has

77 been observed upon acid-, temperature- and salt-induced stresses (Brzin, 1975; Jørgensen et

78 al., 1995; Bereksi et al., 2002; Geng et al., 2003; Jydegaard-Axelsen et al., 2005; Hazeleger et

79 al., 2006; Giotis et al., 2007). While secretion of several proteins is dependent on SecA2

80 (Lenz et al., 2003; Renier et al., 2013), this phenotypic modification in L. monocytogenes

81 ΔsecA2 is mainly attributed to reduced secretion of two extracellular cell-wall hydrolases,

82 namely CwhA (cell-wall hydrolase A), previously called Iap (invasion associated protein) or

83 P60 (protein of 60 kDa), and MurA (muramidase A), also called NamA (N-acetylmuramidase

84 A) (Lenz et al., 2003; Machata et al., 2005). Simultaneous deletion of cwhA and murA is

85 known to result in the rough colony morphotype (Machata et al., 2005). A recent study

86 showed that strains lacking the divIVA gene also form rough colonies (Halbedel et al., 2012).

87 Actually, DivIVA is involved in the recruitment of CwhA and MurA to the cell poles prior to

88 export in a SecA2-dependent manner.

89 While rough colony variants and/or related mutants have been extensively studied with

90 regards to their virulence level, a few investigations examined their ability to influence

91 biofilm formation. Paradoxically, deletion of divIVA leads to defective sessile development

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 92 (Halbedel et al., 2012) but rough colony variants would enhance biofilm formation (Monk et
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al., 2004). Despite the combined importance of SecA2 and the two associated cell-wall

hydrolases CwhA and MurA in the conversion to the rough colony morphotype, a

95 contribution to biofilm formation has not been established. Here we investigate the role of the

96 SecA2 protein export pathway at different stages of biofilm formation and under different

97 conditions relevant to the physiology of L. monocytogenes, especially the growth temperature.

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RESULTS

Deletion of the secA2 gene leads to the formation of biofilm with a dramatically different

101 architecture at 37°C

102 The involvement of the SecA2 pathway in L. monocytogenes biofilm formation was

103 first investigated at temperatures relevant to its pathogenic lifestyles, i.e. 37°C, the

104 temperature encountered in the course of a human infection or animal carriage. As previously

105 observed (Lenz and Portnoy, 2002; Machata et al., 2005), the isogenic L. monocytogenes

106 secA2 mutant exhibited a rough colony morphotype and elongated cells at 37°C (Fig. 1); the

107 discrete cells and smooth colony phenotypes were restored upon complementation (Fig. S1).

108 To investigate biofilm formation, all stages were considered, i.e. initial adhesion, early

109 (microcolony formation) and later stages (mature sessile biomass) of biofilm development,

110 and evaluated by the BioFilm Ring Test (BRT) (Chavant et al., 2007) and the crystal violet

111 (CV) methods (Borucki et al., 2003).

112 Investigating bacterial cell aggregation at 37°C, it clearly appeared that

113 L. monocytogenes ΔsecA2 aggregated and flocculated more rapidly than the wt strain leading

114 to sedimentation (Fig. 2A, 2B and 2C). Despite the fact that initial bacterial adhesion was

115 significantly reduced for the secA2 mutant compared to the wt in both static and dynamic

116 conditions (Fig. 3A and Fig. S2), no difference in the early stages of biofilm formation could

117 be observed since microcolonies from both the wt and secA2 mutant strains blocked the

118 microbeads (BioFilm Index [BFI] < 2) from 6 hours of incubation onwards (Fig. 3B).

119 Investigating later stages of biofilm formation, no significant difference could be observed up

120 to 48 h sessile growth (Fig. 3C). However, the amount of sessile biomass for the secA2

121 mutant was significantly reduced from 54 h onwards but was not associated with a lower

122 maximum specific growth rate (Fig. S3). These results were biased as it could be visually

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 123 observed that compared to the wt, significant clumps of the sessile biomass from
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L. monocytogenes ΔsecA2 detached at each washing steps of the CV method.

In order to limit artefactual observations and get further access to spatial organization of

126 the biofilm, the sessile development L. monocytogenes wt and secA2 mutant was investigated

127 using confocal laser scanning microscope (CLSM) under static conditions. After 24 h of

128 sessile growth at 37°C, the wt strain formed a biofilm that covered the surface almost entirely

129 and homogeneously with an average thickness of 30 μm (Fig. 3D and 3E). In contrast,

130 L. monocytogenes ΔsecA2 formed a filamentous biofilm with aerial structures (Fig. 3D and

131 S4) but the surface coverage was significantly reduced and heterogeneous (Fig. 3E and S4).

132 While no significant difference was evidenced with regards of the biofilm thickness, the

133 biofilm roughness for the secA2 mutant was significantly higher than that of

134 L. monocytogenes wt (Fig. 3G). Considering that the rough morphotype had previously been

135 reported and isolated from L. monocytogenes biofilms (Monk et al., 2004), its contribution to

136 sessile development was addressed by co-culture experiments of L. monocytogenes wt mixed

137 with ΔsecA2 mutant strain (Fig. S5). While the CV method was still quite inappropriate to

138 appreciate biofilm formation, CLSM observations revealed that mixed biofilms displayed an

139 aerial structure formed by the secA2 mutant in whom the wt strain was embedded (Fig. S5).

140 Under dynamic conditions, L. monocytogenes ΔsecA2 could also undergo sessile development

141 and similar results regarding the architecture, surface coverage and roughness of the biofilm

142 were obtained in comparison the wt strain (Fig. 4).

143 All-in-all, it appears that at 37°C both the L. monocytogenes wt and secA2 mutant form

144 biofilm, which differ extensively in their architecture. Modification of the bacterial

145 morphology in L. monocytogenes ΔsecA2 promoted cell aggregates thereby leading to the

146 formation of a filamentous biofilm with aerial structures in both static and dynamic

147 conditions.

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 148 Simultaneous deletion of murA and cwhA genes exacerbates the ΔsecA2 biofilm phenotype at
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37°C

In order to investigate the contribution of the two major proteins secreted by the SecA2

151 pathway and associated with rough morphotype, adhesion and biofilm formation abilities of

152 L. monocytogenes ΔmurA, ΔcwhA and ΔmurAΔcwhA were compared to the wt. As previously

153 reported at 37°C (Machata et al., 2005), deletion of the murA or cwhA gene also led to

154 elongated cells but to a much lesser extent than L. monocytogenes secA2 mutant (Fig. 1).

155 Regarding colonies, those from L. monocytogenes ΔcwhA retained a smooth morphotype,

156 whereas L. monocytogenes ΔmurA colonies were very slightly rippled (Fig. 1); the wt

157 phenotype for cell and colony morphologies was restored upon complementation (Fig. S1).

158 Contrary to L. monocytogenes ΔmurA, which behaved quite similarly to the wt strain,

159 L. monocytogenes ΔcwhA formed some cell aggregates and sedimented slightly more rapidly

160 (Fig. 2A, 2B and 2C). Regarding bacterial adhesion, no difference with the wt strain was

161 observed for the murA mutant under static or dynamic conditions, whereas it was significantly

162 decreased upon deletion of cwhA at 37°C (Fig. 5A and S2). Nonetheless, the cwhA mutant

163 blocked the microbeads significantly earlier than the wt strain (at 4 h against 6 h for the wt

164 strain), whereas microbead blockage by microcolonies occurred 2 h later with the murA

165 mutant (Fig. 5B). These slight differences were offset at later stages of biofilm formation

166 since no differences could be observed using the CV method when comparing

167 L. monocytogenes ΔcwhA or ΔmurA to the wt (Fig. 5C). CLSM observations confirmed and

168 indicated L. monocytogenes ΔmurA and ΔcwhA formed homogeneous biofilm (Fig. 5D), in

169 which surface coverage, thickness and roughness were similar to the wt (Fig 5E, 5F and 5G).

170 Simultaneous deletion of murA and cwhA, however, had much more drastic effects. As

171 previously shown (Machata et al., 2005), this double mutant led to the formation of rough

172 colonies with even more elongated cells than the secA2 mutant (Fig. 1). L. monocytogenes

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 173 ΔmurAΔcwhA formed intricate and entangled cell aggregates, which flocculated and
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sedimented more rapidly than the secA2 mutant (Fig. 2A, 2B and 2C). In a manner similar to

the cwhA mutant, initial adhesion of L. monocytogenes ΔmurAΔcwhA was significantly

176 reduced and almost undetectable under dynamic conditions (Fig. 5A and S2). Also similar to

177 the murA mutant, L. monocytogenes ΔmurAΔcwhA microcolonies blocked the BRT

178 microbeads earlier than the wt strains (Fig. 5B). However, the results from the CV method

179 were completely biased (Fig. 5C), in that it showed a severely reduced sessile biomass for

180 L. monocytogenes ΔmurAΔcwhA compared to the wt. Indeed, it was not just some significant

181 clumps of the sessile biomass that were removed at each washing steps of the CV method as

182 observed for the secA2 mutant but nearly the entire L. monocytogenes ΔmurAΔcwhA biofilm

183 that detached at once. Clearly, the CV method was not appropriate and biofilm was further

184 investigated by CLSM.

185 As observed with L. monocytogenes ΔsecA2 under static conditions, the simultaneous

186 deletion of murA and cwhA led to the formation of a filamentous biofilm with aerial structures

187 (Fig. 5D). Image analyses confirmed that: (i) surface coverage was significantly reduced to

188 less than 20 % of the wt strain (Fig. 5E); (ii) the average thickness was higher than

189 L. monocytogenes wt, reaching up to 75 μm (Fig. 5F); and (iii) the biofilm roughness was

190 significantly increased (Fig. 5G). Interestingly and despite its tendency to easily detach from

191 the support, L. monocytogenes ΔmurAΔcwhA could be maintained under dynamic conditions

192 (Fig. 4). This biofilm was thicker and rougher than the wt but with a significantly reduced

193 surface coverage. In the end, the total absence of the MurA and CwhA proteins in the double

194 mutant resulted in an exaggerated effect over the L. monocytogenes ΔsecA2 biofilm

195 phenotype, where these proteins were just secreted at lower level. Those especially elongated

196 cells promoted autoaggregation thereby leading to the formation of a filamentous biofilm,

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 197 which was rougher than the wt strain and even thicker than secA2 mutant in both static and
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dynamic conditions.

Inactivation of the SecA2 pathway, or simultaneous deletion of murA and cwhA, enhances the

200 surface colonization at 20°C

201 The involvement of the SecA2 pathway in L. monocytogenes biofilm formation was

202 further investigated at a temperature relevant to its saprophytic lifestyles in the environment.

203 At a standard ambient temperature of 20°C, all mutant strains presented similar cell and

204 colony phenotypes to those described at 37°C (Fig. 1); L. monocytogenes ΔsecA2 and

205 ΔmurAΔcwhA exhibited elongated cells forming a rough colony morphotype, whereas

206 L. monocytogenes ΔmurA and ΔcwhA formed very slightly rippled and smooth colonies

207 respectively.

208 L. monocytogenes ΔsecA2 and ΔmurAΔcwhA clearly formed intricate and entangled cell

209 aggregates and sedimentation was much more rapid than for L. monocytogenes wt, ΔcwhA or

210 ΔmurA (Fig 2D, 2E, and 2F). Initial bacterial adhesion was significantly reduced for

211 L. monocytogenes ΔsecA2, ΔcwhA, ΔmurA and ΔmurAΔcwhA in static conditions (Fig. 6A

212 and 6B); this contrasts with results at 37°C where only the murA mutant was able to adhere

213 similarly to the wt in both static and dynamic conditions. In dynamic conditions, the rate of

214 initial attachment (IAR) was significantly reduced for all isogenic mutants and adhered cells

215 were almost undetectable for L. monocytogenes ΔsecA2 and ΔmurAΔcwhA (Fig. S2).

216 Nonetheless, L. monocytogenes ΔsecA2 and ΔmurAΔcwhA were completely blocked by the

217 microbeads within 24 h contrary to the wt strain or murA and cwhA mutants (Fig. 6C and 6D).

218 Results generated from the CV method for L. monocytogenes ΔsecA2 and ΔmurAΔcwhA were

219 once again not representative since it could be visually observed that the entire biofilms were

220 removed at once from the first washing steps (Fig. S6).

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 221 For L. monocytogenes wt, the sessile biomass formed in the course of biofilm
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development was very low compared to the results obtained at 37°C (Fig. 3, 5 and S6).

Interestingly, microscopic observations under static conditions revealed L. monocytogenes

224 EGD-e wt was highly motile and could not form a biofilm at 20°C (Fig. 7A, 7B and

225 Video S1). In agreement with aggregation and initial adhesion results (Fig. 2, 6 and S2), the

226 very low but still increasing sessile biomass measured over time by the CV method (Fig S6)

227 was related to bacterial adhesion rather than sessile growth. Similarly, the murA and cwhA

228 mutants were also motile at 20°C and did not form a biofilm (Videos S2 and S3).

229 L. monocytogenes ΔsecA2 and ΔmurAΔcwhA, however, were clearly non-motile at 20°C

230 (Videos S4 and S5).

231 While no biofilm could be observed with L. monocytogenes wt at 20°C, CLSM

232 observations confirmed L. monocytogenes ΔsecA2 and ΔmurAΔcwhA formed filamentous

233 biofilms (Fig. 7A); compared to the biofilms observed at 37°C in static conditions (Fig. 3 and

234 5), they were as rough (Fig. 7D) and even thicker in average, i.e. between 60 μm and 77 μm

235 (Fig. 7C) but with a lower surface coverage not exceeding 10 % (Fig. 7B). When co-cultured

236 with L. monocytogenes wt, biofilm at 20°C was exclusively formed by the secA2 mutant cells

237 (Fig. S5); in contrast to 37°C, the wt listerial cells were thus not able to establish themselves

238 within the biofilm. Investigation of the biofilm formation under dynamic conditions

239 confirmed motile L. monocytogenes could not form a biofilm contrary to L. monocytogenes

240 ΔsecA2 and ΔmurAΔcwhA, which could indeed be maintained within flow cells (Fig. 7E).

241 Again, L. monocytogenes ΔsecA2 (or the simultaneous deletion of murA and cwhA) led to the

242 formation of filamentous biofilms with aerial structures. Thus, inactivation of the SecA2

243 pathway allowed L. monocytogenes to colonize a surface at 20°C. Reduced secretion of its

244 two major substrates MurA and CwhA resulted in cell elongation, which abrogated motility

245 and propped up aggregation leading to cell sedimentation then promoting sessile growth.

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 246
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248
DISCUSSION

The architecture of L. monocytogenes biofilms is highly polymorphic ranging from

249 monolayer, honey-comb, mushroom-shaped and most recently knitted-chains network (Chae

250 and Schraft, 2000; Chavant et al., 2002; Borucki et al., 2003; Rieu et al., 2008). The present

251 investigation reveals a new kind of biofilm structural design in L. monocytogenes.

252 Inactivation of SecA2 pathway in L. monocytogenes systematically led to the formation of

253 filamentous biofilms with aerial and fluffy structures. They are rougher and thicker than the

254 wt biofilm and unevenly covered the colonized surface. It clearly appeared the phenotype of

255 filamentous biofilm was associated with the reduced secretion of the two major SecA2-

256 dependent cell hydrolases, MurA and CwhA, which resulted in bacterial cell elongation

257 (Machata et al., 2005). Those cell-wall hydrolases are important enzymes to maintain the cell

258 wall integrity and murein sacculus turnover (Popowska, 2004).

259 Contrary to MurA, the secretion of CwhA is significantly reduced but not completely

260 abolished in L. monocytogenes ΔsecA2 (Lenz and Portnoy, 2002; Dumas et al., 2009;

261 Desvaux et al., 2010; Renier et al., 2013). Actually, the total absence of the MurA and CwhA

262 proteins in L. monocytogenes ΔmurAΔcwhA resulted in an exaggerated effect as compared

263 with inactivation of the SecA2 pathway. Bacterial cells were especially elongated,

264 autoaggregation was quicker and the biofilm was rougher than the wt strain but also even

265 thicker and more fragile than the secA2 mutant. A similar reduction of adhered biomass was

266 also reported in a divIVA mutant, where bacterial cells were elongated as a result of a down-

267 secretion of MurA and CwhA (Halbedel et al., 2012). As for the virulence, it is most certainly

268 an indirect and collateral effect of the change in cell morphology (Desvaux and Hébraud,

269 2006); the loss of bacterial septum formation is accompanied by mislocalization and

270 misassembly of some cell-surface proteins (Carroll et al., 2003; Pilgrim et al., 2003), which

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 271 can be of importance for bacterial adhesion and biofilm formation as evidenced for the
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flagella (Desvaux et al., 2006b; Lemon et al., 2010; Renier et al., 2011) or more recently for

ActA (Travier et al., 2013). This also stressed that much remains to be learned about the

274 biochemistry of the cell-wall hydrolases and their respective implication in cell wall

275 biogenesis in L. monocytogenes (Popowska, 2004).

276 Nonetheless, L. monocytogenes could settle and colonize a surface under both static

277 and dynamic conditions following SecA2 inactivation. The combinatory approach here

278 performed (i.e. cell aggregation, bacterial adhesion in static and dynamic conditions, early and

279 late stages of sessile development using BRT and the crystal violet method, CLSM in static

280 and dynamic conditions) proved a powerful approach to decipher biofilm formation in

281 L. monocytogenes. Also, the defect of biofilm formation previously reported in divIVA mutant

282 (Halbedel et al., 2012) should be regarded with caution considering CV method is not

283 appropriate to observe filamentous biofilm. Besides, temperature has a great influence on

284 listerial cell physiology in terms of genetic expression but it had essentially been understood

285 through the lens of pathogenicity and virulence factors (Toledo-Arana et al., 2009; de las

286 Heras et al., 2011). Filamentous biofilms could form under temperatures relevant to infection

287 condition (37°C) and environmental condition (20°C), albeit with a major difference. It is

288 well known that L. monocytogenes motility is regulated by temperature, where flagella are

289 expressed at 20°C but not at 37°C (Renier et al., 2011). At 37°C, L. monocytogenes wt could

290 form a biofilm and participate to filamentous-biofilm formation when co-cultured with

291 L. monocytogenes ΔsecA2. At 20°C, the wt strain was motile and surface colonization arose

292 through adhesion rather than sessile growth; when co-cultured with L. monocytogenes

293 ΔsecA2, the wt strain could not establish itself within the filamentous biofilm. In

294 environmental condition, SecA2 inactivation clearly gives a competitive advantage for surface

295 colonization over L. monocytogenes wt.

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 296 All-in-all, this investigation provided further significance of morphotypic conversion
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298
to rough colony in L. monocytogenes biofilm formation (Monk et al., 2004). Listerial cell

aggregation was rarely addressed before in the literature but reported only recently in

299 L. monocytogenes (Travier et al., 2013). Compared to the wt strain, however, inactivation of

300 the SecA2 pathway here appeared to have a dramatic effect by promoting extensive

301 sedimentation following cell aggregation and flocculation, which was unlike previously

302 reported cell aggregation in L. monocytogenes. Actually, down-secretion of MurA and CwhA

303 led to elongated cells, which further promoted autoaggregation. At 20°C, this effect is

304 combined with the abrogation of cell motility resulting in elongated sedimented cells, which

305 got knotted and entangled together in the course of filamentous-biofilm development.

306 Considering that the rough colony morphotype was isolated from clinical to environmental

307 samples (Rowan et al., 2000; Monk et al., 2004) and further occur under stressful conditions

308 (Jørgensen et al., 1995; Bereksi et al., 2002; Hazeleger et al., 2006; Giotis et al., 2007), SecA2

309 inactivation could contribute to asymptomatic animal/human carriage at 37°C (Travier et al.,

310 2013) and clearly participate in biofilm settlement in the environment at ambient temperature.

311 Here a parallel can be drawn with the biofilm network of knitted chains, where it was shown

312 to be dependent on RecA and YneA activated by the SOS response, leading to the elongation

313 of cells and allowing a better stress resistance (van der Veen et al., 2010). Therefore, it is

314 tempting to hypothesize about a connection between the SOS response in L. monocytogenes

315 and the SecA2 pathway, which could be implied in cell differentiation in response to external

316 conditions and thus biofilm formation. The molecular mechanisms responsible for SecA2

317 inactivation and the regulation of the reversible cell differentiation/colony morphotype have

318 yet to be clearly established in L. monocytogenes (Lenz and Portnoy, 2002; Rigel and

319 Braunstein, 2008). In this context, phase-variation is a promising research direction that

320 would deserve further in-depth investigations in Gram-positive bacteria in general (Henderson

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 321 et al., 1999; Lenz and Portnoy, 2002). Differential regulation of biofilm formation in response
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to environmental conditions has been previously described in different bacterial species (Hall-

Stoodley and Stoodley, 2002), namely in relation to the expression of different

324 exopolysaccharides (Franklin et al., 2011; Colvin et al., 2012; Young et al., 2012), cell-

325 surface proteins and/or pili (Korea et al., 2010; Heilmann, 2011; Giraud and de Bentzmann,

326 2012). Except for eDNA only expressed in some particular conditions and resulting most

327 certainly from cell lysis (Harmsen et al., 2010), a main singularity of biofilm formation in

328 L. monocytogenes is the absence of a dense exopolymeric matrix as observed in most other

329 microbial biofilms (Renier et al., 2011). Instead, extracytoplasmic proteins are the major

330 determinants contributing to biofilm formation in L. monocytogenes.

331 SecA2 has been previously shown and essentially considered as critical for listerial

332 virulence (Lenz et al., 2003; Rigel and Braunstein, 2008; Halbedel et al., 2012) but its

333 implication in colonization processes has been overlooked (Lenz and Portnoy, 2002; Monk et

334 al., 2004). In this study, we demonstrated that the inactivation of the SecA2 pathway provides

335 a decisive advantage in listerial surface colonization under environmental conditions. Cell

336 differentiation could be involved in the conversion from rough morphotype in environmental

337 conditions to virulent smooth morphotype under infection conditions. The morphotypic

338 conversion in L. monocytogenes could be further considered as a risk factor for contamination

339 of industrial production chain line and food products but also of potential significance for

340 asymptomatic human/animal carriage. Understanding the regulation of the morphotypic

341 conversion would facilitate the eradication of listerial biofilm in food plants and allow

342 controlling listerial infection. Considering that biofilms are generally multispecies rather than

343 monospecies, this cell differentiation could have consequences on L. monocytogenes

344 implantation and interaction with other microbial species in various ecological niches

345 (Sasahara and Zottola, 1993).

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 346
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348
EXPERIMENTAL PROCEDURES

Bacterial strains and culture conditions

349 The bacterial strains used in this study are listed in Table 1. Routinely, cells of

350 L. monocytogenes were cultivated in BHI (brain-heart infusion) broth or BHI agar plates at

351 20°C or 37°C. When necessary, X-Gal (100 μg ml-1) and/or antibiotics were added at the

352 following concentrations: erythromycin (5 μg ml-1), kanamycin (50 μg ml-1). Escherichia coli

353 TOP10 (Invitrogen) was used as the standard plasmid host for all cloning procedures

354 (Sambrook and Russell, 2001). Growth curves were obtained using a Bioscreen C

355 (Labsystems).

356 Construction of in-frame ΔsecA2, ΔmurA and ΔmurAΔcwhA L. monocytogenes mutants and

357 gene complementation

358 The genes encoding SecA2 (lmo0583) and MurA (lmo2691) were deleted by allelic

359 exchange using the pMAD vector as previously described (Arnaud et al., 2004). From

360 L. monocytogenes EGD-e genomic DNA purified with Wizard Genomic DNA Purification

361 Kit (Promega), upstream and downstream DNA fragments flanking the gene of interest were

362 amplified by high-fidelity PCR using TaKaRa LA Taq DNA polymerase with two pairs of

363 primers (Guedon et al., 1999; Guedon et al., 2000), i.e. Fw1/Rv2 and Fw3/Rv4 respectively

364 (Table 1S). The two PCR products then served as a matrix for the SOE-PCR using Fw1/Rv4

365 primers (Desvaux et al., 2006c; Desvaux et al., 2007). Following standard molecular cloning

366 technique (Sambrook and Russell, 2001), the resulting amplicon was cloned into pMAD

367 following DNA restriction digestion with NcoI and MluI, ligation, transformation into E. coli

368 TOP10 (Invitrogen) and selection on LB (lysogeny broth) agar with ampicillin (100 µg ml -1).

369 After purification from E. coli using Nucleospin Plasmid QuickPure (Macherey-Nagel), the

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 370 resulting plasmid pMAD-ΔsecA2 and pMAD-ΔmurA were electroporated into
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L. monocytogenes EGD-e (Monk et al., 2008) and also in the in-frame ΔcwhA mutant with the

selection performed on BHI agar containing erythromycin. As previously described (Arnaud

373 et al., 2004), blue-white screening was applied to select gene knockout events. The isogenic

374 mutants were identified by colony PCR with outFw/outRv primers using GoTaq DNA

375 polymerase (Promega) (Table 1S) and were further confirmed by DNA sequencing (GATC-

376 Biotech) on both strands using primers Fw1 and Rv4, respectively.

377 For gene complementation, the entire CDS (coding sequence) was amplified from

378 genomic DNA by PCR using TaKaRa LA Taq DNA polymerase and the primers

379 SecA2BspHFw/SecA2PstRv and MurANcoFw/MurAPstRv, respectively. The amplicon was

380 cloned into pIMK2 (Monk et al., 2008) following DNA digest with NcoI/PstI restriction

381 enzymes, ligation, electroporation into E. coli TOP10 and selection on LB agar with

382 kanamycin (Rossiter et al., 2011). After plasmid purification, the resulting pIMK2-secA2 and

383 pIMK2-murA were electroporated into L. monocytogenes EGD-e ΔsecA2 and

384 L. monocytogenes EGD-e ΔmurA, respectively. Site-specific integration of the plasmid was

385 confirmed following plating on by kanamycin BHI agar and colony PCR was performed

386 using primers SecA2BspHFw/ SecA2PstRv and MurANcoFw/MurAPstRv respectively. For

387 the complemented L. monocytogenes strains, restoration of the cell and colony morphotype

388 was checked by microscopic observations as detailed below (Fig. 1S).

389 Microsocopic observation

390 Microscopic observations of bacterial colony and individual listerial cells were

391 performed with an inverted contrast phase microscope (Olympus LH50A). For microscopic

392 images of bacterial colonies, the strains were grown on BHI agar plates at 37 and 20°C for

393 24 h and resultant colonies were observed at 150× original magnification. For visualization of

394 bacterial cells, cultures grown in BHI broth, at 37 and 20°C, were sampled during the

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 395 exponential phase and fixed onto glass slides for microscopic analysis at 600× original
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397
magnification.

Bacterial cell aggregation assay

398 Based on a previously described assay (Chagnot et al., 2013), listerial cell suspensions

399 from overnight cultures (early stationary phase) of L. monocytogenes strains previously grown

400 in BHI at 37 or 20°C were adjusted to the same OD600 nm. Briefly, chloramphenicol was added

401 at a final concentration of 90 µg ml-1 and each suspension was placed vertically and

402 statistically in tubes at the relevant temperature up to 24 h. To follow cell sedimentation,

403 samples of 500 µl were taken from the top of the tube at different time points to measure the

404 OD600 nm. To visualize cell aggregates, samples were taken at the bottom of the tubes after

405 24 h of incubation for observations in phase-contrast microscopy as described above.

406 Initial adhesion

407 This assay is based on the crystal violet (CV) method as described below. Briefly, were

408 adjusted at 1.5 (OD600 nm) in sterile BHI medium and loaded into the wells of a 96-wells

409 polystyrene microtiter plate prior to static incubation at 20 or 37°C. After one hour, the

410 supernatant was removed from the wells, which were washed with TS and directly stained

411 with an aqueous solution of crystal violet (0.1 %). After washing, the bound dye was

412 solubilized in acetic acid (33 %), then transferred to a clean microtiter plate where the

413 absorbance was finally measured. At least 5 independent experiments with at least two repeats

414 each were performed for each strain.

415 Adhesion assay under liquid flow

416 Initial adhesion in dynamic conditions was assayed using a recently described standard

417 protocol (Szlavik et al., 2012). Instead of glass, however, plastic coverslips (Agar Scientific,

418 dimension 22 mm×22 mm) made of clear unbreakable polystyrene were used. Briefly, cells

419 were diluted in citric acid-Na2HPO4 buffer (pH 6.6) to a final volume of 50 ml and

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 420 OD600 nm = 0.1 (cell density of 108 CFU ml-1). The tested bacterial solution was connected to a
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422
peristaltic pump (Spectec, Perimax 16/1) with a pumping velocity of 0.76 ml min -1 giving a

wall shear stress of 0.0505 pa. The chamber was mounted on an inverted microscope (Zeiss,

423 Axiovert 25) with an attached camera (QImaging, MicroPublisher v3.3). After activation of

424 the pumps, consecutive pictures were taken in three separate vistas every 5 min for 30 min

425 and the adhered cells were enumerated. The median was selected for each time point and the

426 initial adhesion rate (IAR) was calculated using linear regression from the medians. All

427 adhesion tests were performed at least in triplicates.

428 Biofilm formation assay at early stages of sessile development

429 The assay was conducted using the BioFilm Ring Test (BRT) (Chavant et al., 2007)

430 following BioFilm Control (BFC) supplier recommendations from overnight cultures

431 L. monocytogenes EGD-e wt or mutant strains adjusted at OD600nm = 0.01 (approximately

432 105 CFU ml-1) in sterile BHI medium. Briefly, a suspension of paramagnetic microbeads

433 (Ton5: 2.8 µm in diameter) was added at 10 μl ml−1 final concentration, homogenized by

434 vortexing prior to 200 μl loading into 96-wells BFC Polystyrene Microtiter plates or 8-wells

435 BFC Polystyrene Strips and static incubation at 20°C or 37°C. Control wells were filled with

436 sterile BHI and Ton5. For reading at the different time points, wells of microtiter plates were

437 first covered with 100 μl of BFC Contrast Liquid prior to scanning before and after one-

438 minute magnetization using a BFC Magnetic Rack. Results were expressed as Biofilm

439 Formation Index (BFI) (Chavant et al., 2007; Macé et al., 2008). Basically, in the course of

440 bacterial sessile development, BFI decreases and a BFI ≤ 2 indicates a full immobilization of

441 the paramagnetic microbeads by L. monocytogenes EGD-e microcolonies (Chavant et al.,

442 2007). The BRT is restricted to early stages of biofilm formation since once the microbeads

443 are blocked, the BFI does not change anymore; still, the sessile biomass can continue to grow

444 and later stages of biofilm formation were thus investigated using the crystal violet (CV)

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 445 method described here below. At least 5 independent experiments with at least two repeats
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447
each were performed for each strain and incubation time.

Biofilm formation assay at late stages of sessile development

448 The assay is based on the crystal violet (CV) method (Djordjevic et al., 2002; Borucki et

449 al., 2003). Briefly, overnight cultures of L. monocytogenes strains were adjusted at 0.01

450 (OD600nm) in sterile BHI medium and 200 µl loaded into the wells of a 96-wells polystyrene

451 microtiter plate prior to static incubation at 20 or 37°C (de Luna et al., 2008). At different

452 time points, the supernatant was removed from the wells, which were washed with TS

453 (tryptone salt). Absolute ethanol was then applied for fixation (20 min). After emptying and

454 air-drying the wells, 200 µl of an aqueous solution of crystal violet (0.1 %) was added and left

455 for 10 min. After washing with water, the bound dye was solubilized with 200 µl of an

456 aqueous solution of acetic acid (33 %). Contents of each well (150 µl) were transferred to a

457 clean microtiter plate and absorbance was finally measured using a microtiter plate reader set

458 to 595 nm. At least 5 independent experiments with at least two repeats each were performed

459 for each strain and incubation time.

460 Biofilm growth conditions for CLSM

461 (i) Static biofilm experiments

462 Overnight cultures of L. monocytogenes strains carrying the pNF8 plasmid expressing

463 the green fluorescent protein GFPmut1 (Fortinea et al., 2000), were adjusted at 0.01

464 (OD600 nm) in sterile BHI medium. 200 μl of these cultures were pipetted into the wells of 96

465 wells polystyrene microtiter plate (Greiner Bio-One) which enables high resolution

466 fluorescence imaging. Then, the plates were incubated at 20 or 37°C. After 2 hours, the

467 medium was removed, and 200 μl of fresh BHI was added. Biofilm development was

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 468 evaluated by microscopic observations after 24 h of incubation. At least three independent
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470
experiments were performed for each strain.

(ii) Flowcell biofilm experiments

471 Biofilms under dynamic conditions were performed in flowcells (DTU Systems

472 Biology) with individual channel dimensions of 1 by 4 by 40 mm. Flow chambers were

473 inoculated with overnight cultures of L. monocytogenes EGD-e wt (pNF8), ΔsecA2 (pNF8)

474 and ΔmurAΔcwhA (pNF8) strains adjusted at 0.01 (OD600 nm) in fresh BHI medium. After

475 inoculation (2 ml), the medium flow was stopped for 1 h to allow bacterial adhesion, and

476 thereafter the medium was pumped through the flowcells at 4 ml h-1 by using a peristaltic

477 pump (Watson-Marlow, Model 205S). Two independent experiments with two replicates each

478 were made.

479 (iii) Co-cultured assays

480 Static and flowcell biofilm experiments were also performed in co-cultures. Overnight

481 cultures of L. monocytogenes EGD-e wt and ΔsecA2 (pNF8) or L. monocytogenes EGD-e wt

482 and ΔmurAΔcwhA (pNF8) were mixed in equal quantity and then, adjusted at 0.01 (OD600 nm)

483 before being added in wells or inoculated in the flowcell chamber. Then, sessile cells were

484 stained with the red nucleic acid stain SYTO 61 (0.01 %) (Invitrogen) before microscopic

485 observation.

486 (iv) CLSM and image processing

487 Horizontal plane images of the biofilms were acquired using a Leica SP2 AOBS CLSM

488 (Leica Microsystems) at the MIMA2 microscopy platform

489 (http://www6.jouy.inra.fr/mima2_eng/). When necessary the CLSM allowed simultaneous

490 monitoring of GFP and SYTO 61 dyes. The excitation wavelength used for GFP was 488 nm,

491 and emitted fluorescence was recorded within the range of 500 to 550 nm. The red fluorescent

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 492 nucleic acid stain SYTO 61 was excited at 633 nm, and the emitted fluorescence was
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494
collected in the range of 650 to 700 nm. Images were collected through a 63x Leica oil

immersion objective (numerical aperture, 1.4).

495 3D projections were performed with IMARIS software (Bitplane). The biofilm

496 structural parameters (thickness, roughness and substratum coverage) were evaluated using

497 the PHLIP Matlab program developed by J. Xavier (http://sourceforge.net/projects/phlip/).

498 For each experiment, at least 3 microscopic fields were analyzed. Considering the

499 heterogeneity of the ΔsecA2 and ΔmurAΔcwhA, only images containing a biofilm were

500 considered for the analysis.

501 Statistical analysis

502 In order to test the significance of the differences observed in each assay between the wt

503 and the different mutants, a pair Student’s t-test was performed. Differences were considered

504 significant from P < 0.05.

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 505
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507
ACKNOWLEDGMENTS

This work was supported in part by INRA (French National Institute for Agronomical

508 Research), the European Framework Program 6 (FP6) with the ProSafeBeef (Advancing Beef

509 Safety and Quality through Research and Innovation) research consortium

510 (www.prosafebeef.eu), the COST (European Cooperation in Science and Technology) Action

511 FA1202 BacFoodNet (A European Network For Mitigating Bacterial Colonisation and

512 Persistence On Foods and Food Processing Environments), the EGIDE PHC (Programme

513 Hubert Curien) France-Ireland ULYSSES 2010 from the "Ministère des Affaires Etrangères

514 et Européennes" (n°23755ZD), EGIDE PHC France-Poland POLONIUM 2013 (n°28298ZE)

515 and CSU ("Coopération Scientifique Universitaire") France-Denmark 2012 from the Embassy

516 of France in Denmark IFD ("Institut Français du Danemark") (n°14/2012/CSU.8.2.1). The

517 authors are very grateful to Jens Bo Andersen and Tine Rask Licht (Technical University of

518 Denmark, Soeborg) for kindly providing multiple fluorescence labelling system in

519 L. monocytogenes. The authors thank Chantal Bizet (Institut Pasteur, Paris, France) for

520 providing pMAD under MTA (Material Transfer Agreement). The excellent technical

521 assistance of Marina Bjørklund (Copenhagen University) was highly appreciated as well as

522 Danièle François and Amine Zorgani (INRA Clermont-Ferrand). Caroline Chagnot is a PhD

523 Research Fellow granted by the "Région Auvergne – FEDER (Fonds Européen de

524 Développement Régional)". Dr Sandra Renier had a PhD research fellowship granted by the

525 French "Ministère de l’Enseignement Supérieur et de la Recherche".

526 SUPPORTING INFORMATION

527 Oligonucleotide sequences used in this study are provided in Table S1. Microscopic

528 analysis of colony and cell morphology of complemented L. monocytogenes mutant strains

529 are provided in Figure S1. Initial bacterial adhesion assayed under liquid flow is depicted in

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 530 Figure S2. Growth curves of L. monocytogenes wt and mutant strains performed at 37°C and
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532
20°C are provided in Figure S3. 3D reconstruction of L. monocytogenes ΔsecA2 biofilm at

37°C is provided in Figure S4. CLSM of biofilm from mixed cultures of L. monocytogenes

533 EGD-e wt and ΔsecA2 mutant strains are provided in Figure S5. Biofilm formation assays

534 with the crystal violet method for L. monocytogenes EGD-e wt and the isogenic mutants at

535 20°C are provided in Figure S6. Cell motility of L. monocytogenes EGD-e wt at 20°C was

536 recorded in contrast-phase microscopy (Video S1) after 24 h incubation in static biofilm

537 culture conditions, as well as for L. monocytogenes ΔmurA, ΔcwhA, ΔsecA2 and

538 ΔmurAΔcwhA (Videos S2, S3, S4 and S5 respectively).

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 539
Accepted Article
540 FIGURE LEGENDS

541 Figure 1: Microscopic analysis of colony and cell morphology of L. monocytogenes EGD-

542 e wt and isogenic mutant strains grown in BHI at 37 and 20°C. (On the left side) From

543 phase-contrast microscopy observations of bacterial colonies, the wild type (wt) showed a

544 smooth outline, whereas ΔsecA2 and ΔmurAΔcwhA exhibited a rough colony morphotype at

545 both 37 and 20°C. While L. monocytogenes ΔcwhA colonies remain smooth, colonies of

546 L. monocytogenes ΔmurA were very slightly rippled at both 37 and 20°C. Bars, 100 μm. (On

547 the right side) From phase-contrast microscopy observations of listerial cells at both

548 temperatures, all isogenic mutant strains exhibited elongated bacterial cell forming chains.

549 Listerial cells formed especially long filaments in L. monocytogenes ΔsecA2 and

550 ΔmurAΔcwhA. Bars, 20 μm.

551 Figure 2: Aggregation of L. monocytogenes EGD-e wt and isogenic mutant strains grown

552 in BHI at 37°C and 20°C. The sedimentation assays were performed at 37°C (A) and 20°C

553 (D) with L. monocytogenes wt ( ),ΔsecA2 ( ), ΔmurA ( ), ΔcwhA ( ) and ΔmurAΔcwhA

554 ( ). The listerial cell aggregates were visualized by phase-contrast microscopy following

555 sampling at 24 h incubation time from cultures at 37°C (B) and 20°C (E). Bars, 20 μm.

556 Sedimentation was visualized after 24 h incubation from cultures at 37°C (C) and 20°C (F).

557 Figure 3: Adhesion and biofilm formation of L. monocytogenes EGD-e wt and the

558 isogenic mutant ΔsecA2 at 37°C. (A) Initial adhesion assay based on crystal violet staining.

559 (B) Biofilm formation at early stages of sessile development assayed with the BRT. (C)

560 Biofilm formation at later stages of sessile development assayed with the crystal violet

561 method. (D) CLSM images, (E) surface coverage, (F) thickness and (G) roughness resulted

562 from image analyses of bacterial strains bearing pNF8 after 24 h of sessile development in

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This article is protected by copyright. All rights reserved.
 563 static conditions as described in the Experimental Procedures. Statistical significance of the
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565
results is indicated by an asterisk (P < 0.05). L. monocytogenes wt ( ) and ΔsecA2 ( ).

Figure 4: Biofilm architecture of L. monocytogenes wt, ΔsecA2 and ΔmurAΔcwhA under

566 dynamic conditions at 37°C. (A) CLSM images, (B) surface coverage, (C) thickness and (D)

567 roughness resulted from image analyses of bacterial strains bearing pNF8 after 24 h of sessile

568 development in flow cells as described in the Experimental Procedures. Statistical

569 significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt ( ),

570 ΔsecA2 ( ) and ΔmurAΔcwhA ( ).

571 Figure 5: Adhesion and biofilm formation of L. monocytogenes EGD-e wt and the

572 isogenic mutants ΔmurA, ΔcwhA and ΔmurAΔcwhA at 37°C. (A) Initial adhesion assay

573 based on crystal violet staining. (B) Biofilm formation at early stages of sessile development

574 assayed with the BRT. (C) Biofilm formation at late stages of sessile development assayed

575 with the crystal violet method. (D) CLSM images, (E) surface coverage, (F) thickness and (G)

576 roughness resulted from image analyses of bacterial strains bearing pNF8 after 24 h of sessile

577 development in static conditions as described in the Experimental Procedures. Statistical

578 significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt ( ),

579 ΔmurA ( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ).

580 Figure 6: Adhesion and biofilm formation of L. monocytogenes EGD-e wt and the

581 isogenic mutants at 20°C. (A, B) Initial adhesion assay based on crystal violet staining. (C,

582 D) Biofilm formation at early stages of sessile development assayed with the BRT. Biased

583 results from crystal violet method are provided as supplementary material (Fig S6). Statistical

584 significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt ( ),

585 ΔsecA2 ( ), ΔmurA ( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ).

586

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 587 Figure 7: Biofilm architecture of L. monocytogenes wt and the isogenic mutants ΔsecA2
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589
and ΔmurAΔcwhA strains under static and dynamic conditions at 20°C. (On the left side)

(A) CLSM images, (B) surface coverage, (C) thickness and (D) roughness resulted from

590 image analyses of bacterial strains bearing pNF8 after 24 h of sessile development in static

591 conditions as described in the Experimental Procedures. (On the right side) (E) CLSM

592 images, (F) surface coverage, (G) thickness and (H) roughness resulted from image analyses

593 of bacterial strains bearing pNF8 after 24 h of sessile development in flow cells as described

594 in the Experimental Procedures. Statistical significance of the results is indicated by an

595 asterisk (P < 0.05). L. monocytogenes wt ( ), ΔsecA2 ( ) and ΔmurAΔcwhA ( ).

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Accepted Article
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597
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37
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Accepted Article
TABLES

Table 1. Strains and plasmids used in this study.

Name Relevant characteristics Source/Reference

Plasmids

pMAD AmpR, EmR, bgaB Arnaud et al. (2004)

pIMK2 Site-specific listerial integrative vector, phelp, KanR Monk et al. (2008)

pMAD-ΔsecA2 AmpR, EmR, bgaB, ΔsecA2 construct This work

pMAD-ΔmurA AmpR, EmR, bgaB, ΔmurA construct This work

pIMK2-secA2 Site-specific listerial integrative vector, Phelp- This work

secA2, KanR

pIMK2-murA Site-specific listerial integrative vector, Phelp- This work

murA, KanR

pNF8 EmR, oriR pAMβ1, oriR pUC, Pdlt-gfpmut1 Fortinea et al. (2000)

L. monocytogenes strains

EGD-e L. monocytogenes wt (wild type) Glaser et al. (2001)

ΔsecA2 Isogenic mutant of L. monocytogenes EGD-e Renier et al. (2013)

deleted of secA2 (lmo0583)

ΔmurA Isogenic mutant of L. monocytogenes EGD-e This work

deleted of murA (lmo2691)

ΔcwhA Isogenic mutant of L. monocytogenes EGD-e Monk et al. (2008)

deleted of ΔcwhA (lmo0582)

ΔmurAΔcwhA Isogenic mutant of L. monocytogenes EGD-e This work

deleted of both murA and cwhA genes

ΔsecA2::pIMK2-secA2 ΔsecA2:: pIMK2secA2 integrated at tRNAArg with This work

SecA2 expressed from the Phelp promoter

ΔmurA::pIMK2-murA ΔmurA:: pIMK2murA integrated at tRNAArg with This work

MurA expressed from the Phelp promoter

ΔcwhA::pIMK2-cwhA ΔcwhA:: pIMK2cwhA integrated at tRNAArg with Monk et al. (2008)

CwhA expressed from the Phelp promoter

This article is protected by copyright. All rights reserved.

 EGD-e (pNF8) Green autofluorescent strain This work

Accepted Article
ΔsecA2 (pNF8)

ΔmurA (pNF8)

ΔcwhA (pNF8)
Green autofluorescent strain

Green autofluorescent strain

Green autofluorescent strain

This work

This work

This work

ΔmurAΔcwhA (pNF8) Green autofluorescent strain This work

This article is protected by copyright. All rights reserved.

Figure 1
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Figure 2
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 Accepted Article

Figure 3
 A B
100
90
Accepted Article wt 80
70

Surface coverage (%)

*
60
50
40
30 *
20
10

30 µm 0

C
70
wt ΔsecA2 ΔmurAΔcwhA

∆secA2
*
60

50

Mean thickness (μm)

40

30

20

30 µm 10

D
wt ΔsecA2 ΔmurAΔcwhA

∆murA∆cwhA 0.7

0.6 *
*
0.5
Biofilm roughness

0.4

0.3

0.2

30 µm
0.1
Figure 4
0.0
wt ΔsecA2 ΔmurAΔcwhA
Figure 5
Accepted Article
 A C
Accepted Article 0.30

0.25
24
22
20
18

0.20 16
Abs 595nm

14

BFI
0.15 12
10
*
0.10 8
6

0.05 4 *
* 2

0.00 0
1 2 3 4 5 6 7 12 24
wt ΔsecA2 Time (h)

B D
0.30 24
22
0.25 20
18
0.20 16
Abs 595nm

14
0.15

BFI
12
*
* 10
0.10 8
* 6
*
0.05 4
*
2
0.00 0
Figure 6 wt ΔmurA ΔcwhA ΔmurAΔcwhA 1 2 3 4 5 6 7 12 24
Time (h)
 A B E F
Accepted Article
wt
30

wt
30 *

Surface coverage (%)

Surface coverage (%)
20 20

*
*
*
10 10

30 µm 30 µm
0 0
wt ΔsecA2 ΔmurAΔcwhA wt ΔsecA2 ΔmurAΔcwhA
C G
90 90 *

ΔsecA2 80
*

*
∆secA2 80
70 70

Mean thickness (μm)

Mean thickness (μm)

60 60
50 50
40 40 *

30 30
20 20
30 µm 10 30 µm 10
0 0
wt ΔsecA2 ΔmurAΔcwhA wt ΔsecA2 ΔmurAΔcwhA
D H
0.7 0.7
∆murA∆cwhA
*
∆murAΔcwhA 0.6 *
0.6
* 0.5

Biofilm roughness
0.5 *
Biofilm roughness

0.4 0.4

0.3 0.3

0.2 0.2
30 µm 30 µm
0.1 0.1

0 0
wt ΔsecA2 ΔmurAΔcwhA wt ΔsecA2 ΔmurAΔcwhA

Figure 7