Inactivation of the SecA2 protein export pathway inListeria monocytogenes promotes cell aggregation,impacts biofilm architecture and induces biofilmformation in environmental condition
Sandra Renier,1 Caroline Chagnot,1
Julien Deschamps,2 Nelly Caccia,1 Julie Szlavik,3
Susan A. Joyce,4 Magdalena Popowska,5 Colin Hill,4
Susanne Knøchel,3 Romain Briandet,2
Michel Hébraud1 and Mickaël Desvaux1*1INRA, UR454 Microbiologie, Saint-Genès-ChampanelleF-63122, France.2INRA, UMR1319 MicAliS, Massy F-91300, France.3Department of Food Science, University ofCopenhagen, Rolighedsvej 30, Frederiksberg C 1958,Denmark.4Alimentary Pharmabiotic Centre & MicrobiologyDepartment, University College Cork, Cork, Ireland.5Department of Applied Microbiology, Institute ofMicrobiology, University of Warsaw, Warsaw, Poland.
Summary
Listeria monocytogenes has a dichotomous lifestyle,existing as an ubiquitous saprophytic species and asan opportunistic intracellular pathogen. Besides itscapacity to grow in a wide range of environmental andstressful conditions, L. monocytogenes has the abilityto adhere to and colonize surfaces. Morphotype vari-ation to elongated cells forming rough colonies hasbeen reported for different clinical and environmentalisolates, including biofilms. This cell differentiation ismainly attributed to the reduced secretion of twoSecA2-dependent cell-wall hydrolases, CwhA andMurA. SecA2 is a non-essential SecA paralogueforming an alternative translocase with the primarySec translocon. Following investigation at tempera-tures relevant to its ecological niches, i.e. infection(37°C) and environmental (20°C) conditions, inactiva-tion of this SecA2-only protein export pathway led,despite reduced adhesion, to the formation of filamen-tous biofilm with aerial structures. Compared to thewild type strain, inactivation of the SecA2 pathway
promoted extensive cell aggregation and sedimenta-tion. At ambient temperature, this effect was combinedwith the abrogation of cell motility resulting in elon-gated sedimented cells, which got knotted and entan-gled together in the course of filamentous-biofilmdevelopment. Such a cell differentiation provides adecisive advantage for listerial surface colonizationunder environmental condition. As further discussed,this morphotypic conversion has strong implicationon listerial physiology and is also of potential signifi-cance for asymptomatic human/animal carriage.
Introduction
Listeria monocytogenes is an opportunistic pathogenGram-positive bacterium associated with many foodbornedisease outbreaks. Infection mainly occurs after ingestionof contaminated food (Gahan and Collins, 1991) andaffects predominantly pregnant women, neonates, theelderly and immunocompromised patients (Farber andPeterkin, 1991). In the food industry, L. monocytogenesrepresents a major problem as a source of contaminationof raw and processed foods. Besides its ability to grow in awide range of conditions including pH (4.3 to 9.6), tempera-ture (1 to 45°C), salt concentration (up to 10% NaCl) andwater activity (Aw down to 0.93), L. monocytogenes canadhere to and colonizes abiotic surfaces, which contrib-utes to its strong resistance to technological treatmentsand environmental stresses (Carpentier and Cerf, 2011).While different definitions can be found in the literature(Dunne, 2002), a biofilm can be broadly defined as thesessile development of microbial cells. The bacterial cellsadhering to each other and/or to a surface or interface aregenerally surrounded by a matrix of extracellular polymers(Kolter, 2005). In L. monocytogenes, the existence of suchan exopolysaccharide matrix has never been evidenced(Nilsson et al., 2011; Renier et al., 2011). Extracellulardeoxyribonucleic acid (eDNA) could play a role in adhesionand early stages of biofilm formation but only under certaingrowth conditions (Harmsen et al., 2010). Instead, cell-surface proteins are the major adhesion factors contribut-ing to biofilm formation in L. monocytogenes (Smoot and
Pierson, 1998; Longhi et al., 2008; Franciosa et al., 2009),especially the flagella and some yet unidentified proteins(Renier et al., 2011).
In monoderm bacteria, cell-surface displayed proteinsneed to be first translocated across the cytoplasmic mem-brane (Desvaux et al., 2005a,b; 2006a; 2009). Fromthe most recent proteogenomic analyses and with 714proteins exhibiting an N-terminal signal peptide (Renieret al., 2012), the Sec (Secretion) pathway appears as themain route for protein secretion in L. monocytogenes(Desvaux and Hébraud, 2006; Desvaux, 2012). The Sectranslocase, composed of the transmembranar SecYEGtranslocon and the peripheral SecA ATPase, is essentialfor the passage of pre-proteins through the cytoplasmicmembrane (Du Plessis et al., 2011). As in several otherpathogenic Gram-positive bacteria, a paralogue of SecA,named SecA2, has been identified in L. monocytogenes.Upon deletion of the secA2 gene, the cell morphotypechanges from discrete cells forming smooth colonies inL. monocytogenes wild type (wt) to long-chain cellsforming rough colonies and was associated to virulencedefect (Lenz and Portnoy, 2002; Lenz et al., 2003). Similarmorphotypes have been isolated from clinical patients,food samples and environmental biofilms (Rowan et al.,2000; Monk et al., 2004), and reversible conversion hasbeen observed upon acid, temperature and salt-inducedstresses (Brzin, 1975; Jørgensen et al., 1995; Bereksiet al., 2002; Geng et al., 2003; Jydegaard-Axelsen et al.,2005; Hazeleger et al., 2006; Giotis et al., 2007). Whilesecretion of several proteins is dependent on SecA2(Lenz et al., 2003; Renier et al., 2013), this phenotypicmodification in L. monocytogenes ΔsecA2 is mainly attrib-uted to reduced secretion of two extracellular cell-wallhydrolases, namely CwhA (cell-wall hydrolase A), previ-ously called Iap (invasion associated protein) or P60(protein of 60 kDa) (Pilgrim et al., 2003) and MurA(muramidase A), also called NamA (N-acetylmuramidaseA) (Lenz et al., 2003; Machata et al., 2005). Simultaneousdeletion of cwhA and murA is known to result in the roughcolony morphotype (Machata et al., 2005). A recent studyshowed that strains lacking the divIVA gene also formrough colonies (Halbedel et al., 2012). Actually, DivIVA isinvolved in the recruitment of CwhA and MurA to the cellpoles prior to export in a SecA2-dependent manner.
While rough colony variants and/or related mutantshave been extensively studied with regards to their viru-lence level, a few investigations examined their ability toinfluence biofilm formation. Paradoxically, deletion ofdivIVA leads to defective sessile development (Halbedelet al., 2012) but rough colony variants would enhancebiofilm formation (Monk et al., 2004). Despite the com-bined importance of SecA2 and the two associated cell-wall hydrolases CwhA and MurA in the conversion to therough colony morphotype, a contribution to biofilm forma-
tion has not been established. Here, we investigate therole of the SecA2 protein export pathway at differentstages of biofilm formation and under different conditionsrelevant to the physiology of L. monocytogenes, espe-cially the growth temperature.
Results
Deletion of the SecA2 gene leads to the formation ofbiofilm with a dramatically different architecture at 37°C
The involvement of the SecA2 pathway in L.monocytogenes biofilm formation was first investigated attemperatures relevant to its pathogenic lifestyles, i.e.37°C, the temperature encountered in the course of ahuman infection or animal carriage. As previouslyobserved (Lenz and Portnoy, 2002; Machata et al., 2005),the isogenic L. monocytogenes secA2 mutant exhibited arough colony morphotype and elongated cells at 37°C(Fig. 1); the discrete cells and smooth colony phenotypeswere restored upon complementation (Fig. S1). To inves-tigate biofilm formation, all stages were considered, i.e.initial adhesion, early (microcolony formation) and laterstages (mature sessile biomass) of biofilm development,and evaluated by the BioFilm Ring Test (BRT) (BioFilmControl, Saint-Beauzire, France) (Chavant et al., 2007)and the crystal violet (CV) methods (Borucki et al., 2003).
Investigating bacterial cell aggregation at 37°C, itclearly appeared that L. monocytogenes ΔsecA2 aggre-gated and flocculated more rapidly than the wt strainleading to sedimentation (Fig. 2A–C). Despite the fact thatinitial bacterial adhesion was significantly reduced for thesecA2 mutant compared to the wt in both static anddynamic conditions (Fig. 3A and Fig. S2), no difference inthe early stages of biofilm formation could be observedsince microcolonies from both the wt and secA2 mutantstrains blocked the microbeads [BioFilm Index (BFI) < 2]from 6 h of incubation onwards (Fig. 3B). Investigatinglater stages of biofilm formation, no significant differencecould be observed up to 48 h sessile growth (Fig. 3C).However, the amount of sessile biomass for the secA2mutant was significantly reduced from 54 h onwards butwas not associated with a lower maximum specific growthrate (Fig. S3). These results were biased as it could bevisually observed that compared to the wt, significantclumps of the sessile biomass from L. monocytogenesΔsecA2 detached at each washing steps of the CVmethod.
In order to limit artefactual observations and get furtheraccess to spatial organization of the biofilm, the sessiledevelopment L. monocytogenes wt and secA2 mutantwas investigated using confocal laser scanning micro-scope (CLSM) under static conditions. After 24 h ofsessile growth at 37°C, the wt strain formed a biofilm thatcovered the surface almost entirely and homogeneously
SecA2 impacts biofilm formation in L. monocytogenes 1177
with an average thickness of 30 μm (Fig. 3D and E). Incontrast, L. monocytogenes ΔsecA2 formed a filamentousbiofilm with aerial structures (Figs 3D and S4) but thesurface coverage was significantly reduced and hetero-geneous (Figs 3E and S4). While no significant differencewas evidenced with regards of the biofilm thickness, thebiofilm roughness for the secA2 mutant was significantlyhigher than that of L. monocytogenes wt (Fig. 3G). Con-
sidering that the rough morphotype had previously beenreported and isolated from L. monocytogenes biofilms(Monk et al., 2004), its contribution to sessile develop-ment was addressed by co-culture experiments ofL. monocytogenes wt mixed with ΔsecA2 mutant strain(Fig. S5). While the CV method was still quite inappropri-ate to appreciate biofilm formation, CLSM observationsrevealed that mixed biofilms displayed an aerial structure
Fig. 1. Microscopic analysis of colony and cell morphology of L. monocytogenes EGD-e wt and isogenic mutant strains grown in BHI at 37and 20°C. (On the left side) From phase-contrast microscopy observations of bacterial colonies, the wild type (wt) showed a smooth outline,whereas ΔsecA2 and ΔmurAΔcwhA exhibited a rough colony morphotype at both 37°C and 20°C. While L. monocytogenes ΔcwhA coloniesremain smooth, colonies of L. monocytogenes ΔmurA were very slightly rippled at both 37°C and 20°C. Bars, 100 μm. (On the right side)From phase-contrast microscopy observations of listerial cells at both temperatures, all isogenic mutant strains exhibited elongated bacterialcell forming chains. Listerial cells formed especially long filaments in L. monocytogenes ΔsecA2 and ΔmurAΔcwhA. Bars, 20 μm.
Fig. 2. Aggregation of L. monocytogenes EGD-e wt and isogenic mutant strains grown in BHI at 37°C and 20°C. The sedimentation assayswere performed at 37°C (A) and 20°C (D) with L. monocytogenes wt (□),ΔsecA2 (■), ΔmurA ( ), ΔcwhA ( ), and ΔmurAΔcwhA ( ). Thelisterial cell aggregates were visualized by phase-contrast microscopy following sampling at 24 h incubation time from cultures at 37°C (B) and20°C (E). Bars, 20 μm. Sedimentation was visualized after 24 h incubation from cultures at 37°C (C) and 20°C (F).
SecA2 impacts biofilm formation in L. monocytogenes 1179
formed by the secA2 mutant in whom the wt strainwas embedded (Fig. S5). Under dynamic conditions,L. monocytogenes ΔsecA2 could also undergo sessiledevelopment and similar results regarding the architec-ture, surface coverage and roughness of the biofilm wereobtained in comparison the wt strain (Fig. 4).
All-in-all, it appears that at 37°C both the L.monocytogenes wt and secA2 mutant form biofilm, whichdiffer extensively in their architecture. Modification ofthe bacterial morphology in L. monocytogenes ΔsecA2
promoted cell aggregates thereby leading to the formationof a filamentous biofilm with aerial structures in both staticand dynamic conditions.
Simultaneous deletion of murA and cwhA genesexacerbates the ΔsecA2 biofilm phenotype at 37°C
In order to investigate the contribution of the two majorproteins secreted by the SecA2 pathway and associatedwith rough morphotype, adhesion and biofilm formation
Fig. 3. Adhesion and biofilm formation of L. monocytogenes EGD-e wt and the isogenic mutant ΔsecA2 at 37°C.A. Initial adhesion assay based on crystal violet staining.B. Biofilm formation at early stages of sessile development assayed with the BRT.C. Biofilm formation at later stages of sessile development assayed with the crystal violet method.D. CLSM images of bacterial strains bearing pNF8 after 24 h of sessile development in static conditions as described in the Experimentalprocedures.E. Surface coverage calculated from analyses of CLSM images.F. Thickness calculated from analyses of CLSM images.G. Roughness calculated from analyses of CLSM images.Statistical significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt (□) and ΔsecA2 (■).
abilities of L. monocytogenes ΔmurA, ΔcwhA andΔmurAΔcwhA were compared to the wt. As previouslyreported at 37°C (Machata et al., 2005), deletion of themurA or cwhA gene also led to elongated cells butto a much lesser extent than L. monocytogenes secA2mutant (Fig. 1). Regarding colonies, those from L.monocytogenes ΔcwhA retained a smooth morphotype,whereas L. monocytogenes ΔmurA colonies were veryslightly rippled (Fig. 1); the wt phenotype for cell and colonymorphologies was restored upon complementation(Fig. S1). Contrary to L. monocytogenes ΔmurA, whichbehaved quite similarly to the wt strain, L. monocytogenesΔcwhA formed some cell aggregates and sedimentedslightly more rapidly (Fig. 2A–C). Regarding bacterialadhesion, no difference with the wt strain was observedfor the murA mutant under static or dynamic conditions,whereas it was significantly decreased upon deletion ofcwhA at 37°C (Figs 5A and S2). Nonetheless, the cwhAmutant blocked the microbeads significantly earlier thanthe wt strain (at 4 h against 6 h for the wt strain), whereasmicrobead blockage by microcolonies occurred 2 h later
with the murA mutant (Fig. 5B). These slight differenceswere offset at later stages of biofilm formation since nodifferences could be observed using the CV method whencomparing L. monocytogenes ΔcwhA or ΔmurA to the wt(Fig. 5C). CLSM observations confirmed and indicatedL. monocytogenes ΔmurA and ΔcwhA formed homogene-ous biofilm (Fig. 5D), in which surface coverage, thicknessand roughness were similar to the wt (Fig. 5E–G).
Simultaneous deletion of murA and cwhA, however,had much more drastic effects. As previously shown(Machata et al., 2005), this double mutant led to theformation of rough colonies with even more elongatedcells than the secA2 mutant (Fig. 1). L. monocytogenesΔmurAΔcwhA formed intricate and entangled cell aggre-gates, which flocculated and sedimented more rapidlythan the secA2 mutant (Fig. 2A–C). In a manner similar tothe cwhA mutant, initial adhesion of L. monocytogenesΔmurAΔcwhA was significantly reduced and almost unde-tectable under dynamic conditions (Figs 5A and S2).Also similar to the cwhA mutant, L. monocytogenesΔmurAΔcwhA microcolonies blocked the BRT microbeads
Fig. 4. Biofilm architecture ofL. monocytogenes wt, ΔsecA2 andΔmurAΔcwhA under dynamic conditions at37°C.A. CLSM images of bacterial strains bearingpNF8 after 24 h of sessile development inflow cells as described in the Experimentalprocedures.B. Surface coverage calculated from analysesof CLSM images.C. Thickness calculated from analyses ofCLSM images.D. Roughness calculated from analyses ofCLSM images.Statistical significance of the results isindicated by an asterisk (P < 0.05).L. monocytogenes wt (□), ΔsecA2 (■) andΔmurAΔcwhA ( ).
SecA2 impacts biofilm formation in L. monocytogenes 1181
earlier than the wt strains (Fig. 5B). However, the resultsfrom the CV method were completely biased (Fig. 5C), inthat it showed a severely reduced sessile biomass forL. monocytogenes ΔmurAΔcwhA compared to the wt.Indeed, it was not just some significant clumps of thesessile biomass that were removed at each washingsteps of the CV method as observed for the secA2 mutantbut nearly the entire L. monocytogenes ΔmurAΔcwhAbiofilm that detached at once. Clearly, the CV method wasnot appropriate, and biofilm was further investigated byCLSM.
As observed with L. monocytogenes ΔsecA2 understatic conditions, the simultaneous deletion of murA and
cwhA led to the formation of a filamentous biofilm withaerial structures (Fig. 5D). Image analyses confirmedthat: (i) surface coverage was significantly reduced to lessthan 20% of the wt strain (Fig. 5E); (ii) the average thick-ness was higher than L. monocytogenes wt, reachingup to 75 μm (Fig. 5F); and (iii) the biofilm roughnesswas significantly increased (Fig. 5G). Interestingly anddespite its tendency to easily detach from the support,L. monocytogenes ΔmurAΔcwhA could be maintainedunder dynamic conditions (Fig. 4). This biofilm was thickerand rougher than the wt but with a significantly reducedsurface coverage. In the end, the total absence of theMurA and CwhA proteins in the double mutant resulted
Fig. 5. Adhesion and biofilm formation of L. monocytogenes EGD-e wt and the isogenic mutants ΔmurA, ΔcwhA and ΔmurAΔcwhA at 37°C.A. Initial adhesion assay based on crystal violet staining.B. Biofilm formation at early stages of sessile development assayed with the BRT.C. Biofilm formation at late stages of sessile development assayed with the crystal violet method.D. CLSM images of bacterial strains bearing pNF8 after 24 h of sessile development in static conditions as described in the Experimentalprocedures.E. Surface coverage calculated from analyses of CLSM images.F. Thickness calculated from analyses of CLSM images.G. Roughness calculated from analyses of CLSM images.Statistical significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt (□), ΔmurA ( ), ΔcwhA ( ) andΔmurAΔcwhA ( ).
in an exaggerated effect over the L. monocytogenesΔsecA2 biofilm phenotype, where these proteins were justsecreted at lower level. Those especially elongated cellspromoted autoaggregation thereby leading to the forma-tion of a filamentous biofilm, which was rougher than thewt strain and even thicker than secA2 mutant in both staticand dynamic conditions.
Inactivation of the SecA2 pathway, or simultaneousdeletion of murA and cwhA, enhances the surfacecolonization at 20°C
The involvement of the SecA2 pathway inL. monocytogenes biofilm formation was further investi-gated at a temperature relevant to its saprophyticlifestyles in the environment. At a standard ambient tem-perature of 20°C, all mutant strains presented similar celland colony phenotypes to those described at 37°C(Fig. 1); L. monocytogenes ΔsecA2 and ΔmurAΔcwhAexhibited elongated cells forming a rough colonymorphotype, whereas L. monocytogenes ΔmurA andΔcwhA formed very slightly rippled and smooth coloniesrespectively.
L. monocytogenes ΔsecA2 and ΔmurAΔcwhA clearlyformed intricate and entangled cell aggregates, andsedimentation was much more rapid than for L.monocytogenes wt, ΔcwhA or ΔmurA (Fig. 2D–F). Initialbacterial adhesion was significantly reduced forL. monocytogenes ΔsecA2, ΔcwhA, ΔmurA andΔmurAΔcwhA in static conditions (Fig. 6A and B); thiscontrasts with results at 37°C where only the murA mutantwas able to adhere similarly to the wt in both static and
dynamic conditions. In dynamic conditions, the rate ofinitial attachment (IAR) was significantly reduced for allisogenic mutants, and adhered cells were almost unde-tectable for L. monocytogenes ΔsecA2 and ΔmurAΔcwhA(Fig. S2). Nonetheless, L. monocytogenes ΔsecA2 andΔmurAΔcwhA were completely blocked by themicrobeads within 24 h contrary to the wt strain or murAand cwhA mutants (Fig. 6C and D). Results generatedfrom the CV method for L. monocytogenes ΔsecA2 andΔmurAΔcwhA were once again not representative since itcould be visually observed that the entire biofilms wereremoved at once from the first washing steps (Fig. S6).
For L. monocytogenes wt, the sessile biomass formedin the course of biofilm development was very low com-pared to the results obtained at 37°C (Figs 3, 5, and S6).Interestingly, microscopic observations under static con-ditions revealed L. monocytogenes EGD-e wt was highlymotile and could not form a biofilm at 20°C (Fig. 7A, B andVideo S1). In agreement with aggregation and initialadhesion results (Figs 2, 6, and S2), the very low but stillincreasing sessile biomass measured over time by the CVmethod (Fig. S6) was related to bacterial adhesion ratherthan sessile growth. Similarly, the murA and cwhAmutants were also motile at 20°C and did not form abiofilm (Videos S2 and S3). L. monocytogenes ΔsecA2and ΔmurAΔcwhA, however, were clearly non-motile at20°C (Videos S4 and S5).
While no biofilm could be observed withL. monocytogenes wt at 20°C, CLSM observations con-firmed L. monocytogenes ΔsecA2 and ΔmurAΔcwhAformed filamentous biofilms (Fig. 7A); compared to thebiofilms observed at 37°C in static conditions (Fig. 3 and
Fig. 6. Adhesion and biofilm formation ofL. monocytogenes EGD-e wt and the isogenicmutants at 20°C.A, B. Initial adhesion assay based on crystalviolet staining.C, D. Biofilm formation at early stages ofsessile development assayed with the BRT.Biased results from crystal violet method areprovided as supplementary material (Fig. S6).Statistical significance of the results isindicated by an asterisk (P < 0.05).L. monocytogenes wt (□), ΔsecA2 (■), ΔmurA( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ).
SecA2 impacts biofilm formation in L. monocytogenes 1183
5), they were as rough (Fig. 7D) and even thicker inaverage, i.e. between 60 μm and 77 μm (Fig. 7C) but witha lower surface coverage not exceeding 10% (Fig. 7B).When co-cultured with L. monocytogenes wt, biofilm at20°C was exclusively formed by the secA2 mutant cells(Fig. S5); in contrast to 37°C, the wt listerial cells werethus not able to establish themselves within the biofilm.Investigation of the biofilm formation under dynamic con-ditions confirmed motile L. monocytogenes could not forma biofilm contrary to L. monocytogenes ΔsecA2 andΔmurAΔcwhA, which could indeed be maintained withinflow cells (Fig. 7E). Again, L. monocytogenes ΔsecA2 (orthe simultaneous deletion of murA and cwhA) led to theformation of filamentous biofilms with aerial structures.Thus, inactivation of the SecA2 pathway allowedL. monocytogenes to colonize a surface at 20°C.Reduced secretion of its two major substrates MurA andCwhA resulted in cell elongation, which abrogated motility
and propped up aggregation leading to cell sedimentationthen promoting sessile growth.
Discussion
The architecture of L. monocytogenes biofilms ishighly polymorphic ranging from monolayer, honeycomb,mushroom-shaped and most recently knitted-chainsnetwork (Chae and Schraft, 2000; Chavant et al., 2002;Borucki et al., 2003; Rieu et al., 2008). The present inves-tigation reveals a new kind of biofilm structural design inL. monocytogenes. Inactivation of SecA2 pathway inL. monocytogenes systematically led to the formation offilamentous biofilms with aerial and fluffy structures. Theyare rougher and thicker than the wt biofilm and unevenlycovered the colonized surface. It clearly appeared thephenotype of filamentous biofilm was associated with thereduced secretion of the two major SecA2-dependent cell
Fig. 7. Biofilm architecture of L. monocytogenes wt and the isogenic mutants ΔsecA2 and ΔmurAΔcwhA strains under static and dynamicconditions at 20°C.On the left side: A. CLSM images of bacterial strains bearing pNF8 after 24 h of sessile development in static conditions as described in theExperimental procedures.B. Surface coverage calculated from analyses of CLSM images.C. Thickness calculated from analyses of CLSM images.D. Roughness calculated from analyses of CLSM images.On the right side: E. CLSM images of bacterial strains bearing pNF8 after 24 h of sessile development in flow cells as described in theExperimental procedures.F. Surface coverage calculated from analyses of CLSM images.G. Thickness calculated from analyses of CLSM images.H. Roughness calculated from analyses of CLSM images.Statistical significance of the results is indicated by an asterisk (P < 0.05). L. monocytogenes wt (□), ΔsecA2 (■) and ΔmurAΔcwhA ( ).
hydrolases, MurA and CwhA, which resulted in bacterialcell elongation (Machata et al., 2005). Those cell-wallhydrolases are important enzymes to maintain the cellwall integrity and murein sacculus turnover (Popowska,2004).
Contrary to MurA, the secretion of CwhA is signifi-cantly reduced but not completely abolished in L.monocytogenes ΔsecA2 (Lenz and Portnoy, 2002; Dumaset al., 2009; Desvaux et al., 2010; Renier et al., 2013).Actually, the total absence of the MurA and CwhA proteinsin L. monocytogenes ΔmurAΔcwhA resulted in an exag-gerated effect as compared with inactivation of the SecA2pathway. Bacterial cells were especially elongated,autoaggregation was quicker and the biofilm was rougherthan the wt strain but also even thicker and more fragilethan the secA2 mutant. A similar reduction of adheredbiomass was also reported in a divIVA mutant, wherebacterial cells were elongated as a result of a down-secretion of MurA and CwhA (Halbedel et al., 2012). Asfor the virulence, it is most certainly an indirect and col-lateral effect of the change in cell morphology (Desvauxand Hébraud, 2006); the loss of bacterial septum forma-tion is accompanied by mislocalization and misassemblyof some cell-surface proteins (Carroll et al., 2003; Pilgrimet al., 2003), which can be of importance for bacterialadhesion and biofilm formation as evidenced for the fla-gella (Desvaux et al., 2006b; Lemon et al., 2010; Renieret al., 2011) or more recently for ActA (Travier et al.,2013). This also stressed that much remains to be learnedabout the biochemistry of the cell-wall hydrolases andtheir respective implication in cell wall biogenesis inL. monocytogenes (Popowska, 2004).
Nonetheless, L. monocytogenes could settle and colo-nize a surface under both static and dynamic conditionsfollowing SecA2 inactivation. The combinatory approachhere performed (i.e. cell aggregation, bacterial adhesionin static and dynamic conditions, early and late stages ofsessile development using BRT and the crystal violetmethod, CLSM in static and dynamic conditions) proveda powerful approach to decipher biofilm formation inL. monocytogenes. Also, the defect of biofilm formationpreviously reported in divIVA mutant (Halbedel et al.,2012) should be regarded with caution considering CVmethod is not appropriate to observe filamentous biofilm.Besides, temperature has a great influence on listerialcell physiology in terms of genetic expression but it hadessentially been understood through the lens of patho-genicity and virulence factors (Toledo-Arana et al., 2009;de las Heras et al., 2011). Filamentous biofilms couldform under temperatures relevant to infection condition(37°C) and environmental condition (20°C), albeit with amajor difference. It is well known that L. monocytogenesmotility is regulated by temperature, where flagella areexpressed at 20°C but not at 37°C (Renier et al., 2011).
At 37°C, L. monocytogenes wt could form a biofilmand participate to filamentous-biofilm formation whenco-cultured with L. monocytogenes ΔsecA2. At 20°C, thewt strain was motile and surface colonization arosethrough adhesion rather than sessile growth; whenco-cultured with L. monocytogenes ΔsecA2, the wt straincould not establish itself within the filamentous biofilm. Inenvironmental condition, SecA2 inactivation clearly givesa competitive advantage for surface colonization overL. monocytogenes wt.
All-in-all, this investigation provided further signifi-cance of morphotypic conversion to rough colony in L.monocytogenes biofilm formation (Monk et al., 2004).Listerial cell aggregation was rarely addressed beforein the literature but reported only recently in L.monocytogenes (Travier et al., 2013). Compared to the wtstrain, however, inactivation of the SecA2 pathway hereappeared to have a dramatic effect by promoting extensivesedimentation following cell aggregation and flocculation,which was unlike previously reported cell aggregation inL. monocytogenes. Actually, down-secretion of MurA andCwhA led to elongated cells, which further promotedautoaggregation. At 20°C, this effect is combined withthe abrogation of cell motility resulting in elongatedsedimented cells, which got knotted and entangledtogether in the course of filamentous-biofilm development.Considering that the rough colony morphotype was iso-lated from clinical to environmental samples (Rowan et al.,2000; Monk et al., 2004) and further occur under stressfulconditions (Jørgensen et al., 1995; Bereksi et al., 2002;Hazeleger et al., 2006; Giotis et al., 2007), SecA2 inacti-vation could contribute to asymptomatic animal/humancarriage at 37°C (Travier et al., 2013) and clearly partici-pate in biofilm settlement in the environment at ambienttemperature. Here, a parallel can be drawn with the biofilmnetwork of knitted chains, where it was shown to bedependent on RecA and YneA activated by the SOSresponse, leading to the elongation of cells and allowing abetter stress resistance (van der Veen et al., 2010). There-fore, it is tempting to hypothesize about a connectionbetween the SOS response in L. monocytogenes and theSecA2 pathway, which could be implied in cell differentia-tion in response to external conditions and thus biofilmformation. The molecular mechanisms responsible forSecA2 inactivation and the regulation of the reversible celldifferentiation/colony morphotype have yet to be clearlyestablished in L. monocytogenes (Lenz and Portnoy, 2002;Rigel and Braunstein, 2008). In this context, phase-variation is a promising research direction that woulddeserve further in-depth investigations in Gram-positivebacteria in general (Henderson et al., 1999; Lenzand Portnoy, 2002). Differential regulation of biofilm forma-tion in response to environmental conditions has beenpreviously described in different bacterial species
SecA2 impacts biofilm formation in L. monocytogenes 1185
(Hall-Stoodley and Stoodley, 2002), namely in relation tothe expression of different exopolysaccharides (Franklinet al., 2011; Colvin et al., 2012; Young et al., 2012), cell-surface proteins and/or pili (Korea et al., 2010; Heilmann,2011; Giraud and de Bentzmann, 2012). Except for eDNAonly expressed in some particular conditions and resultingmost certainly from cell lysis (Harmsen et al., 2010), a mainsingularity of biofilm formation in L. monocytogenes is theabsence of a dense exopolymeric matrix as observed inmost other microbial biofilms (Renier et al., 2011). Instead,extracytoplasmic proteins are the major determinants con-tributing to biofilm formation in L. monocytogenes.
SecA2 has been previously shown and essentially con-sidered as critical for listerial virulence (Lenz et al., 2003;Rigel and Braunstein, 2008; Halbedel et al., 2012) but itsimplication in colonization processes has been over-looked (Lenz and Portnoy, 2002; Monk et al., 2004). Inthis study, we demonstrated that the inactivation of theSecA2 pathway provides a decisive advantage in listerialsurface colonization under environmental conditions. Celldifferentiation could be involved in the conversion fromrough morphotype in environmental conditions to virulentsmooth morphotype under infection conditions. Themorphotypic conversion in L. monocytogenes could befurther considered as a risk factor for contamination ofindustrial production chain line and food products but alsoof potential significance for asymptomatic human/animalcarriage. Understanding the regulation of the morphotypicconversion would facilitate the eradication of listerial
biofilm in food plants and allow controlling listerial infec-tion. Considering that biofilms are generally multispeciesrather than monospecies, this cell differentiation couldhave consequences on L. monocytogenes implantationand interaction with other microbial species in variousecological niches (Sasahara and Zottola, 1993).
Experimental procedures
Bacterial strains and culture conditions
The bacterial strains used in this study are listed in Table 1.Routinely, cells of L. monocytogenes were cultivated inbrain–heart infusion (BHI) broth or BHI agar plates at 20°C or37°C. When necessary, X-Gal (100 μg ml−1) and/or antibioticswere added at the following concentrations: erythromycin(5 μg ml−1), kanamycin (50 μg ml−1). Escherichia coli TOP10(Invitrogen) was used as the standard plasmid host for allcloning procedures (Sambrook and Russell, 2001). Growthcurves were obtained using a Bioscreen C (Labsystems).
Construction of in-frame ΔsecA2, ΔmurA andΔmurAΔcwhA L. monocytogenes mutants and genecomplementation
The genes encoding SecA2 (lmo0583) and MurA (lmo2691)were deleted by allelic exchange using the pMAD vectoras previously described (Arnaud et al., 2004). From L.monocytogenes EGD-e genomic DNA purified with WizardGenomic DNA Purification Kit (Promega), upstream anddownstream DNA fragments flanking the gene of interest
Table 1. Strains and plasmids used in this study.
Name Relevant characteristics Source/Reference
PlasmidspMAD AmpR, EmR, bgaB Arnaud and colleagues (2004)pIMK2 Site-specific listerial integrative vector, phelp, KanR Monk and colleagues (2008)pMAD-ΔsecA2 AmpR, EmR, bgaB, ΔsecA2 construct This workpMAD-ΔmurA AmpR, EmR, bgaB, ΔmurA construct This workpIMK2-secA2 Site-specific listerial integrative vector, Phelp-secA2, KanR This workpIMK2-murA Site-specific listerial integrative vector, Phelp-murA, KanR This workpNF8 EmR, oriR pAMβ1, oriR pUC, Pdlt-gfpmut1 Fortinea and colleagues (2000)
L. monocytogenes strainsEGD-e L. monocytogenes wt (wild type) Mackaness (1964)ΔsecA2 Isogenic mutant of L. monocytogenes EGD-e deleted of secA2 (lmo0583) Renier and colleagues (2013)ΔmurA Isogenic mutant of L. monocytogenes EGD-e deleted of murA (lmo2691) This workΔcwhA Isogenic mutant of L. monocytogenes EGD-e deleted of ΔcwhA (lmo0582) Monk and colleagues (2008)ΔmurAΔcwhA Isogenic mutant of L. monocytogenes EGD-e deleted of both murA and
cwhA genesThis work
ΔsecA2::pIMK2-secA2 pIMK2secA2 integrated at tRNAArg with SecA2 expressed fromthe Phelp promoter
This work
ΔmurA::pIMK2-murA pIMK2murA integrated at tRNAArg with MurA expressed from thePhelp promoter
This work
ΔcwhA::pIMK2-cwhA pIMK2cwhA integrated at tRNAArg with CwhA expressed from thePhelp promoter
Monk and colleagues (2008)
EGD-e (pNF8) Green autofluorescent strain This workΔsecA2 (pNF8) Green autofluorescent strain This workΔmurA (pNF8) Green autofluorescent strain This workΔcwhA (pNF8) Green autofluorescent strain This workΔmurAΔcwhA (pNF8) Green autofluorescent strain This work
were amplified by high-fidelity PCR using TaKaRa LA TaqDNA polymerase with two pairs of primers (Guedon et al.,1999; 2000), i.e. Fw1/Rv2 and Fw3/Rv4 respectively(Table S1). The two PCR products then served as a matrix forthe SOE-PCR (splicing by overlapping extension PCR) usingFw1/Rv4 primers (Desvaux et al., 2006c; 2007). Followingstandard molecular cloning technique (Sambrook andRussell, 2001), the resulting amplicon was cloned into pMADfollowing DNA restriction digestion with NcoI and MluI, liga-tion, transformation into E. coli TOP10 (Invitrogen) and selec-tion on lysogeny broth (LB) agar with ampicillin (100 μg ml−1).After purification from E. coli using Nucleospin PlasmidQuickPure (Macherey-Nagel), the resulting plasmid pMAD-ΔsecA2 and pMAD-ΔmurA were electroporated intoL. monocytogenes EGD-e (Monk et al., 2008) and also in thein-frame ΔcwhA mutant with the selection performed on BHIagar-containing erythromycin. As previously described(Arnaud et al., 2004), blue-white screening was applied toselect gene knockout events. The isogenic mutants wereidentified by colony PCR with outFw/outRv primers usingGoTaq DNA polymerase (Promega) (Table S1) and werefurther confirmed by DNA sequencing (GATC-Biotech) onboth strands using primers Fw1 and Rv4, respectively.
For gene complementation, the entire CDS (codingsequence) was amplified from genomic DNA by PCR usingTaKaRa LA Taq DNA polymerase and the primersSecA2BspHFw/SecA2PstRv and MurANcoFw/MurAPstRvrespectively. The amplicon was cloned into pIMK2 (Monket al., 2008) following DNA digest with NcoI/PstI restrictionenzymes, ligation, electroporation into E. coli TOP10 andselection on LB agar with kanamycin (Rossiter et al., 2011).After plasmid purification, the resulting pIMK2-secA2 andpIMK2-murA were electroporated into L. monocytogenesEGD-e ΔsecA2 and L. monocytogenes EGD-e ΔmurArespectively. Site-specific integration of the plasmid was con-firmed following plating on kanamycin BHI agar, and colonyPCR was performed using primers SecA2BspHFw/SecA2PstRv and MurANcoFw/MurAPstRv respectively. Forthe complemented L. monocytogenes strains, restoration ofthe cell and colony morphotype was checked by microscopicobservations as detailed below (Fig. S1).
Microsocopic observation
Microscopic observations of bacterial colony and individuallisterial cells were performed with an inverted contrast phasemicroscope (Olympus LH50A). For microscopic images ofbacterial colonies, the strains were grown on BHI agar platesat 37°C and 20°C for 24 h, and resultant colonies wereobserved at 150 × original magnification. For visualization ofbacterial cells, cultures grown in BHI broth, at 37 and 20°C,were sampled during the exponential phase and fixedonto glass slides for microscopic analysis at 600 × originalmagnification.
Bacterial cell aggregation assay
Based on a previously described assay (Chagnot et al., 2013),listerial cell suspensions from overnight cultures (early station-ary phase) of L. monocytogenes strains previously grown inBHI at 37°C or 20°C were adjusted to the same OD600 nm.
Briefly, chloramphenicol was added at a final concentration of90 μg ml−1, and each suspension was placed vertically andstatistically in tubes at the relevant temperature up to 24 h. Tofollow cell sedimentation, samples of 500 μl were taken fromthe top of the tube at different time points to measure theOD600 nm. To visualize cell aggregates, samples were taken atthe bottom of the tubes after 24 h of incubation for observa-tions in phase-contrast microscopy as described above.
Initial adhesion
This assay is based on the CV method as described below.Briefly, were adjusted at 1.5 (OD600 nm) in sterile BHI mediumand loaded into the wells of a 96-well polystyrene microtiterplate prior to static incubation at 20°C or 37°C. After 1 h, thesupernatant was removed from the wells, which were washedwith tryptone salt (TS) and directly stained with an aqueoussolution of crystal violet (0.1%). After washing, the bound dyewas solubilized in acetic acid (33%), then transferred to aclean microtiter plate where the absorbance was finallymeasured. At least five independent experiments with at leasttwo repeats each were performed for each strain.
Adhesion assay under liquid flow
Initial adhesion in dynamic conditions was assayed using arecently described standard protocol (Szlavik et al., 2012).Instead of glass, however, plastic cover slips (Agar Scientific,dimension 22 mm × 22 mm) made of clear unbreakablepolystyrene were used. Briefly, cells were diluted in citricacid-Na2HPO4 buffer (pH 6.6) to a final volume of 50 ml andOD600 nm = 0.1 (cell density of 108 CFU ml−1). The tested bac-terial solution was connected to a peristaltic pump (Spectec,Perimax 16/1) with a pumping velocity of 0.76 ml min−1 givinga wall shear stress of 0.0505 pa. The chamber was mountedon an inverted microscope (Zeiss, Axiovert 25) with anattached camera (QImaging, MicroPublisher v3.3). After acti-vation of the pumps, consecutive pictures were taken in threeseparate vistas every 5 min for 30 min, and the adhered cellswere enumerated. The median was selected for each timepoint, and the initial adhesion rate (IAR) was calculated usinglinear regression from the medians. All adhesion tests wereperformed at least in triplicates.
Biofilm formation assay at early stages of sessiledevelopment
The assay was conducted using the BRT (Chavant et al.,2007) following BioFilm Control (BFC) supplier recommenda-tions from overnight cultures L. monocytogenes EGD-e wt ormutant strains adjusted at OD600nm = 0.01 (approximately105 CFU ml−1) in sterile BHI medium. Briefly, a suspension ofparamagnetic microbeads (Ton5: 2.8 μm in diameter) wasadded at 10 μl ml−1 final concentration, homogenized byvortexing prior to 200 μl loading into 96-well BFC PolystyreneMicrotiter plates or 8-well BFC Polystyrene Strips (BioFilmControl, Saint-Beauzire, France) and static incubation at20°C or 37°C. Control wells were filled with sterile BHI andTon5. For reading at the different time points, wells ofmicrotiter plates were first covered with 100 μl of BFC
SecA2 impacts biofilm formation in L. monocytogenes 1187
Contrast Liquid prior to scanning before and after 1 min mag-netization using a BFC Magnetic Rack (BioFilm Control,Saint-Beauzire, France). Results were expressed asBiofilm Formation Index (BFI) (Chavant et al., 2007; Macéet al., 2008). Basically, in the course of bacterial sessiledevelopment, BFI decreases, and a BFI ≤ 2 indicatesa full immobilization of the paramagnetic microbeads byL. monocytogenes EGD-e microcolonies (Chavant et al.,2007). The BRT is restricted to early stages of biofilm forma-tion since once the microbeads are blocked, the BFI does notchange anymore; still, the sessile biomass can continue togrow, and later stages of biofilm formation were thus investi-gated using the CV method described here below. At leastfive independent experiments with at least two repeats eachwere performed for each strain and incubation time.
Biofilm formation assay at late stages ofsessile development
The assay is based on the CV method (Djordjevic et al.,2002; Borucki et al., 2003). Briefly, overnight cultures ofL. monocytogenes strains were adjusted at 0.01 (OD600nm) insterile BHI medium and 200 μl loaded into the wells of a96-well polystyrene microtiter plate prior to static incubationat 20°C or 37°C (de Luna et al., 2008). At different timepoints, the supernatant was removed from the wells, whichwere washed with TS. Absolute ethanol was then applied forfixation (20 min). After emptying and air drying the wells,200 μl of an aqueous solution of crystal violet (0.1%) wasadded and left for 10 min. After washing with water, the bounddye was solubilized with 200 μl of an aqueous solution ofacetic acid (33%). Contents of each well (150 μl) were trans-ferred to a clean microtiter plate, and absorbance was finallymeasured using a microtiter plate reader set to 595 nm. Atleast five independent experiments with at least two repeatseach were performed for each strain and incubation time.
Biofilm growth conditions for CLSM
Static biofilm experiments. Overnight cultures of L.monocytogenes strains carrying the pNF8 plasmid express-ing the green fluorescent protein GFPmut1 (Fortinea et al.,2000), were adjusted at 0.01 (OD600 nm) in sterile BHI medium.200 μl of these cultures were pipetted into the wells of 96-wellpolystyrene microtiter plate (Greiner Bio-One) which enableshigh resolution fluorescence imaging. Then, the plates wereincubated at 20°C or 37°C. After 2 h, the medium wasremoved, and 200 μl of fresh BHI was added. Biofilm devel-opment was evaluated by microscopic observations after24 h of incubation. At least three independent experimentswere performed for each strain.
Flow cell biofilm experiments. Biofilms under dynamic con-ditions were performed in flow cells (DTU Systems Biology)with individual channel dimensions of 1 x 4 x 40 mm. Flowchambers were inoculated with overnight cultures ofL. monocytogenes EGD-e wt (pNF8), ΔsecA2 (pNF8) andΔmurAΔcwhA (pNF8) strains adjusted at 0.01 (OD600 nm) infresh BHI medium. After inoculation (2 ml), the medium flowwas stopped for 1 h to allow bacterial adhesion, and thereaf-
ter the medium was pumped through the flow cells at 4 ml h−1
by using a peristaltic pump (Watson-Marlow, Model 205S,Wilmington, MA, USA). Two independent experiments withtwo replicates each were made.
Co-cultured assays. Static and flow cell biofilm experimentswere also performed in co-cultures. Overnight cultures ofL. monocytogenes EGD-e wt and ΔsecA2 (pNF8) orL. monocytogenes EGD-e wt and ΔmurAΔcwhA (pNF8) weremixed in equal quantity and then, adjusted at 0.01 (OD600 nm)before being added in wells or inoculated in the flow cellchamber. Then, sessile cells were stained with the red nucleicacid stain SYTO 61 (0.01%) (Invitrogen) before microscopicobservation.
CLSM and image processing. Horizontal plane images of thebiofilms were acquired using a Leica SP2 AOBS CLSM(Leica Microsystems) at the MIMA2 microscopy platform(http://www6.jouy.inra.fr/mima2_eng). When necessary theCLSM allowed simultaneous monitoring of GFP and SYTO61 dyes. The excitation wavelength used for GFP was488 nm, and emitted fluorescence was recorded within therange of 500 nm to 550 nm. The red fluorescent nucleic acidstain SYTO 61 was excited at 633 nm, and the emitted fluo-rescence was collected in the range of 650 nm to 700 nm.Images were collected through a 63x Leica oil immersionobjective (numerical aperture, 1.4).
3D projections were performed with IMARIS software(Bitplane, Scientific Software, Zurich, Switzerland). Thebiofilm structural parameters (thickness, roughness and sub-stratum coverage) were evaluated using the PHLIP Matlabprogram developed by J. Xavier (http://sourceforge.net/projects/phlip/). For each experiment, at least three micro-scopic fields were analyzed. Considering the heterogeneity ofthe ΔsecA2 and ΔmurAΔcwhA, only images containing abiofilm were considered for the analysis.
Statistical analysis
In order to test the significance of the differences observed ineach assay between the wt and the different mutants, a pairStudent’s t-test was performed. Differences were consideredsignificant from P < 0.05.
Acknowledgements
This work was supported in part by French National Institutefor Agronomical Research (INRA), the European FrameworkProgram 6 (FP6) with the ProSafeBeef (Advancing BeefSafety and Quality through Research and Innovation)research consortium (http://www.prosafebeef.eu), theEuropean Cooperation in Science and Technology (COST)Action FA1202 BacFoodNet (a European network for mitigat-ing bacterial colonization and persistence on foods andfood processing environments), the EGIDE ProgrammeHubert Curien (PHC) France-Ireland ULYSSES 2010 fromthe ‘Ministère des Affaires Etrangères et Européennes’(n°23755ZD), EGIDE PHC France-Poland POLONIUM 2013(n°28298ZE) and ‘Coopération Scientifique Universitaire’(CSU) France-Denmark 2012 from the Embassy of France in
Denmark ‘Institut Français du Danemark’(IFD) (n°14/2012/CSU.8.2.1). The authors are very grateful to Jens BoAndersen and Tine Rask Licht (Technical University ofDenmark, Soeborg) for kindly providing multiple fluorescencelabelling system in L. monocytogenes. The authors thankChantal Bizet (Institut Pasteur, Paris, France) for providingpMAD under Material Transfer Agreement (MTA). The excel-lent technical assistance of Marina Bjørklund (CopenhagenUniversity) was highly appreciated as well as DanièleFrançois and Amine Zorgani (INRA Clermont-Ferrand).Caroline Chagnot is a PhD Research Fellow granted bythe ‘Région Auvergne – Fonds Européen de Développe-ment Régional (FEDER)’. Dr Sandra Renier had a PhDresearch fellowship granted by the French ‘Ministère del’Enseignement Supérieur et de la Recherche’.
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Supporting information
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Fig. S1. Microscopic analysis of colony and cell morphologyof complemented L. monocytogenes mutant strains, i.e.L. monocytogenes ΔsecA2::pIMK2-secA2, ΔmurA::pIMK2-murA and ΔcwhA::pIMK2-cwhA in BHI at 37°C. (On the leftside) Morphology of colonies grown in BHI agar plates(phase-contrast microscopy, 150 × original magnification)showing the smooth morphotype was restored. Bars,100 μm. (On the right side) Bacterial cells were grown in BHIbroth. Pictures of bacterial cells were taken in exponentialphase (phase-contrast microscopy, 600 × original magnifica-tions) and show restoration of the discrete-cells phenotype.Bars, 20 μm.Fig. S2. Adhesion under dynamic conditions ofL. monocytogenes EGD-e wt and isogenic mutant strains at37 and 20°C. Initial bacterial adhesion under liquid flow wasperformed against polystyrene coverslips at a low flow rate(0.76 ml min-1) for L. monocytogenes wt, ΔsecA2, ΔmurA,ΔcwhA and ΔmurAΔcwhA exhibited a rough colonymorphotype at both 37 and 20°C. From consecutive picturestaken at regular time intervals, the adhered cells were enu-merated and initial adhesion rate (IAR) was calculated usinglinear regression from the median values.Fig. S3. Growth curves of L. monocytogenes wt andisogenic mutant strains performed in BHI at 37°C and 20°C.Fig. S4. 3D reconstruction of L. monocytogenes ΔsecA2biofilm at 37°C. Images were obtained by CLSM images after24 h of sessile growth at 37°C in static conditions asdescribed in the Experimental Procedures.Fig. S5. Biofilm architecture of mixed cultures ofL. monocytogenes EGD-e wt and ΔsecA2 mutant strains. (A)CLSM images of mixed cultures of L. monocytogeneswt/ΔsecA2 after 24 h growth at 37°C under static condition.(B) CLSM images of mixed cultures of L. monocytogeneswt/ΔsecA2 after 24 h growth at 20°C under static condition.L. monocytogenes wt cells appear in red (stained with SYTO61) and L. monocytogenes mutant cells strains appear ingreen (bacterial strain bearing pNF8).Fig. S6. Biofilm formation of L. monocytogenes EGD-e wtand the isogenic mutants at late stages of sessile developmentassayed with the crystal violet method at 20°C. (A) Results ofthe CV method for L. monocytogenes wt (□) and ΔsecA2 (■).(C) Results of the CV method for L. monocytogenes wt (□),ΔmurA ( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ). Most resultswere not representative since it could be visually observed thatthe entire biofilm was removed at once during washing steps ofthe CV method for L. monocytogenes ΔsecA2, ΔmurA, andΔmurAΔcwhA.Table S1. Oligonucleotides used in this study.Video S1. Cell motility of L. monocytogenes EGD-e wtrecorded in contrast-phase microscopy after 24 h in staticbiofilm culture at 20°C
SecA2 impacts biofilm formation in L. monocytogenes 1191
Video S2. Cell motility of L. monocytogenes ΔmurArecorded in contrast-phase microscopy after 24 h in staticbiofilm culture at 20°C.Video S3. Cell motility of L. monocytogenes ΔcwhArecorded in contrast-phase microscopy after 24 h in staticbiofilm culture at 20°C.
Video S4. Cell motility of L. monocytogenes ΔsecA2recorded in contrast-phase microscopy after 24 h in staticbiofilm culture at 20°C.Video S5. Cell motility of L. monocytogenes ΔmurAΔcwhArecorded in contrast-phase microscopy after 24 h in staticbiofilm culture at 20°C.
