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Environmental Microbiology (2013)
Inactivation of the SecA2 protein export pathway in
Listeria monocytogenes promotes cell aggregation,
impacts biofilm architecture and induces biofilm
formation 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*
1
INRA, UR454 Microbiologie, Saint-Genès-Champanelle
F-63122, France.
2
INRA, UMR1319 MicAliS, Massy F-91300, France.
3
Department of Food Science, University of
Copenhagen, Rolighedsvej 30, Frederiksberg C 1958,
Denmark.
4
Alimentary Pharmabiotic Centre & Microbiology
Department, University College Cork, Cork, Ireland.
5
Department of Applied Microbiology, Institute of
Microbiology, University of Warsaw, Warsaw, Poland.
Summary
Listeria monocytogenes has a dichotomous lifestyle,
existing as an ubiquitous saprophytic species and as
an opportunistic intracellular pathogen. Besides its
capacity to grow in a wide range of environmental and
stressful conditions, L. monocytogenes has the ability
to adhere to and colonize surfaces. Morphotype vari-
ation to elongated cells forming rough colonies has
been reported for different clinical and environmental
isolates, including biofilms. This cell differentiation is
mainly attributed to the reduced secretion of two
SecA2-dependent cell-wall hydrolases, CwhA and
MurA. SecA2 is a non-essential SecA paralogue
forming an alternative translocase with the primary
Sec 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 the
wild type strain, inactivation of the SecA2 pathway
Received 26 June, 2013; revised 12 August, 2013; accepted 16
August, 2013. *For correspondence. E-mail mickael.desvaux@
clermont.inra.fr; Tel. (+33) (0)4 73 62 47 23; Fax (+33) (0)4 73 62
45 81.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd
doi:10.1111/1462-2920.12257
promoted extensive cell aggregation and sedimenta-
tion. At ambient temperature, this effect was combined
with the abrogation of cell motility resulting in elon-
gated sedimented cells, which got knotted and entan-
gled together in the course of filamentous-biofilm
development. Such a cell differentiation provides a
decisive advantage for listerial surface colonization
under environmental condition. As further discussed,
this morphotypic conversion has strong implication
on listerial physiology and is also of potential signifi-
cance for asymptomatic human/animal carriage.
Introduction
Listeria monocytogenes is an opportunistic pathogen
Gram-positive bacterium associated with many foodborne
disease outbreaks. Infection mainly occurs after ingestion
of contaminated food (Gahan and Collins, 1991) and
affects predominantly pregnant women, neonates, the
elderly and immunocompromised patients (Farber and
Peterkin, 1991). In the food industry, L. monocytogenes
represents a major problem as a source of contamination
of raw and processed foods. Besides its ability to grow in a
wide range of conditions including pH (4.3 to 9.6), tempera-
ture (1 to 45°C), salt concentration (up to 10% NaCl) and
water activity (Aw down to 0.93), L. monocytogenes can
adhere to and colonizes abiotic surfaces, which contrib-
utes to its strong resistance to technological treatments
and environmental stresses (Carpentier and Cerf, 2011).
While different definitions can be found in the literature
(Dunne, 2002), a biofilm can be broadly defined as the
sessile development of microbial cells. The bacterial cells
adhering to each other and/or to a surface or interface are
generally surrounded by a matrix of extracellular polymers
(Kolter, 2005). In L. monocytogenes, the existence of such
an exopolysaccharide matrix has never been evidenced
(Nilsson et al., 2011; Renier et al., 2011). Extracellular
deoxyribonucleic acid (eDNA) could play a role in adhesion
and early stages of biofilm formation but only under certain
growth conditions (Harmsen et al., 2010). Instead, cell-
surface proteins are the major adhesion factors contribut-
ing to biofilm formation in L. monocytogenes (Smoot and
2 S. Renier et al.
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 proteins
need to be first translocated across the cytoplasmic mem-
brane (Desvaux et al., 2005a,b; 2006a; 2009). From
the most recent proteogenomic analyses and with 714
proteins exhibiting an N-terminal signal peptide (Renier
et al., 2012), the Sec (Secretion) pathway appears as the
main route for protein secretion in L. monocytogenes
(Desvaux and Hébraud, 2006; Desvaux, 2012). The Sec
translocase, composed of the transmembranar SecYEG
translocon and the peripheral SecA ATPase, is essential
for the passage of pre-proteins through the cytoplasmic
membrane (Du Plessis et al., 2011). As in several other
pathogenic Gram-positive bacteria, a paralogue of SecA,
named SecA2, has been identified in L. monocytogenes.
Upon deletion of the secA2 gene, the cell morphotype
changes from discrete cells forming smooth colonies in
L. monocytogenes wild type (wt) to long-chain cells
forming rough colonies and was associated to virulence
defect (Lenz and Portnoy, 2002; Lenz et al., 2003). Similar
morphotypes have been isolated from clinical patients,
food samples and environmental biofilms (Rowan et al.,
2000; Monk et al., 2004), and reversible conversion has
been observed upon acid, temperature and salt-induced
stresses (Brzin, 1975; Jørgensen et al., 1995; Bereksi
et al., 2002; Geng et al., 2003; Jydegaard-Axelsen et al.,
2005; Hazeleger et al., 2006; Giotis et al., 2007). While
secretion of several proteins is dependent on SecA2
(Lenz et al., 2003; Renier et al., 2013), this phenotypic
modification in L. monocytogenes ΔsecA2 is mainly attrib-
uted to reduced secretion of two extracellular cell-wall
hydrolases, 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-acetylmuramidase
A) (Lenz et al., 2003; Machata et al., 2005). Simultaneous
deletion of cwhA and murA is known to result in the rough
colony morphotype (Machata et al., 2005). A recent study
showed that strains lacking the divIVA gene also form
rough colonies (Halbedel et al., 2012). Actually, DivIVA is
involved in the recruitment of CwhA and MurA to the cell
poles prior to export in a SecA2-dependent manner.
While rough colony variants and/or related mutants
have been extensively studied with regards to their viru-
lence level, a few investigations examined their ability to
influence biofilm formation. Paradoxically, deletion of
divIVA leads to defective sessile development (Halbedel
et al., 2012) but rough colony variants would enhance
biofilm 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 the
rough colony morphotype, a contribution to biofilm forma-
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
tion has not been established. Here, we investigate the
role of the SecA2 protein export pathway at different
stages of biofilm formation and under different conditions
relevant to the physiology of L. monocytogenes, espe-
cially the growth temperature.
Results
Deletion of the SecA2 gene leads to the formation of
biofilm with a dramatically different architecture at 37°C
The involvement of the SecA2 pathway in L.
monocytogenes biofilm formation was first investigated at
temperatures relevant to its pathogenic lifestyles, i.e.
37°C, the temperature encountered in the course of a
human infection or animal carriage. As previously
observed (Lenz and Portnoy, 2002; Machata et al., 2005),
the isogenic L. monocytogenes secA2 mutant exhibited a
rough colony morphotype and elongated cells at 37°C
(Fig. 1); the discrete cells and smooth colony phenotypes
were restored upon complementation (Fig. S1). To inves-
tigate biofilm formation, all stages were considered, i.e.
initial adhesion, early (microcolony formation) and later
stages (mature sessile biomass) of biofilm development,
and evaluated by the BioFilm Ring Test (BRT) (BioFilm
Control, Saint-Beauzire, France) (Chavant et al., 2007)
and the crystal violet (CV) methods (Borucki et al., 2003).
Investigating bacterial cell aggregation at 37°C, it
clearly appeared that L. monocytogenes ΔsecA2 aggre-
gated and flocculated more rapidly than the wt strain
leading to sedimentation (Fig. 2A–C). Despite the fact that
initial bacterial adhesion was significantly reduced for the
secA2 mutant compared to the wt in both static and
dynamic conditions (Fig. 3A and Fig. S2), no difference in
the early stages of biofilm formation could be observed
since microcolonies from both the wt and secA2 mutant
strains blocked the microbeads [BioFilm Index (BFI) < 2]
from 6 h of incubation onwards (Fig. 3B). Investigating
later stages of biofilm formation, no significant difference
could be observed up to 48 h sessile growth (Fig. 3C).
However, the amount of sessile biomass for the secA2
mutant was significantly reduced from 54 h onwards but
was not associated with a lower maximum specific growth
rate (Fig. S3). These results were biased as it could be
visually observed that compared to the wt, significant
clumps of the sessile biomass from 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 the biofilm, the sessile
development L. monocytogenes wt and secA2 mutant
was investigated using confocal laser scanning micro-
scope (CLSM) under static conditions. After 24 h of
sessile growth at 37°C, the wt strain formed a biofilm that
covered the surface almost entirely and homogeneously
 SecA2 impacts biofilm formation in L. monocytogenes 3

Fig. 1. Microscopic analysis of colony and cell morphology of L. monocytogenes EGD-e wt and isogenic mutant strains grown in BHI at 37
and 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 colonies
remain 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 bacterial
cell forming chains. Listerial cells formed especially long filaments in L. monocytogenes ΔsecA2 and ΔmurAΔcwhA. Bars, 20 μm.

with an average thickness of 30 μm (Fig. 3D and E). In
contrast, L. monocytogenes ΔsecA2 formed a filamentous
biofilm with aerial structures (Figs 3D and S4) but the
surface coverage was significantly reduced and hetero-
geneous (Figs 3E and S4). While no significant difference
was evidenced with regards of the biofilm thickness, the
biofilm roughness for the secA2 mutant was significantly
higher than that of L. monocytogenes wt (Fig. 3G). Con-
sidering that the rough morphotype had previously been
reported and isolated from L. monocytogenes biofilms
(Monk et al., 2004), its contribution to sessile develop-
ment was addressed by co-culture experiments of
L. monocytogenes wt mixed with ΔsecA2 mutant strain
(Fig. S5). While the CV method was still quite inappropri-
ate to appreciate biofilm formation, CLSM observations
revealed that mixed biofilms displayed an aerial structure

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
4 S. Renier et al.

Fig. 2. Aggregation of L. monocytogenes EGD-e wt and isogenic mutant strains grown in BHI at 37°C and 20°C. The sedimentation assays
were performed at 37°C (A) and 20°C (D) with L. monocytogenes wt (□),ΔsecA2 (■), ΔmurA ( ), ΔcwhA ( ), and ΔmurAΔcwhA ( ). The
listerial cell aggregates were visualized by phase-contrast microscopy following sampling at 24 h incubation time from cultures at 37°C (B) and
20°C (E). Bars, 20 μm. Sedimentation was visualized after 24 h incubation from cultures at 37°C (C) and 20°C (F).

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
 SecA2 impacts biofilm formation in L. monocytogenes 5

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 Experimental
procedures.
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 (■).

formed by the secA2 mutant in whom the wt strain
was embedded (Fig. S5). Under dynamic conditions,
L. monocytogenes ΔsecA2 could also undergo sessile
development and similar results regarding the architec-
ture, surface coverage and roughness of the biofilm were
obtained 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, which
differ extensively in their architecture. Modification of
the bacterial morphology in L. monocytogenes ΔsecA2
promoted cell aggregates thereby leading to the formation
of a filamentous biofilm with aerial structures in both static
and dynamic conditions.
Simultaneous deletion of murA and cwhA genes
exacerbates the ΔsecA2 biofilm phenotype at 37°C
In order to investigate the contribution of the two major
proteins secreted by the SecA2 pathway and associated
with rough morphotype, adhesion and biofilm formation

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
6 S. Renier et al.
abilities of L. monocytogenes ΔmurA, ΔcwhA and
ΔmurAΔcwhA were compared to the wt. As previously
reported at 37°C (Machata et al., 2005), deletion of the
murA or cwhA gene also led to elongated cells but
to a much lesser extent than L. monocytogenes secA2
mutant (Fig. 1). Regarding colonies, those from L.
monocytogenes ΔcwhA retained a smooth morphotype,
whereas L. monocytogenes ΔmurA colonies were very
slightly rippled (Fig. 1); the wt phenotype for cell and colony
morphologies was restored upon complementation
(Fig. S1). Contrary to L. monocytogenes ΔmurA, which
behaved quite similarly to the wt strain, L. monocytogenes
ΔcwhA formed some cell aggregates and sedimented
slightly more rapidly (Fig. 2A–C). Regarding bacterial
adhesion, no difference with the wt strain was observed
for the murA mutant under static or dynamic conditions,
whereas it was significantly decreased upon deletion of
cwhA at 37°C (Figs 5A and S2). Nonetheless, the cwhA
mutant blocked the microbeads significantly earlier than
the wt strain (at 4 h against 6 h for the wt strain), whereas
microbead blockage by microcolonies occurred 2 h later
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Fig. 4. Biofilm architecture of
L. monocytogenes wt, ΔsecA2 and
ΔmurAΔcwhA under dynamic conditions at
37°C.
A. CLSM images of bacterial strains bearing
pNF8 after 24 h of sessile development in
flow cells as described in the Experimental
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.
Statistical significance of the results is
indicated by an asterisk (P < 0.05).
L. monocytogenes wt (□), ΔsecA2 (■) and
ΔmurAΔcwhA ( ).
with the murA mutant (Fig. 5B). These slight differences
were offset at later stages of biofilm formation since no
differences could be observed using the CV method when
comparing L. monocytogenes ΔcwhA or ΔmurA to the wt
(Fig. 5C). CLSM observations confirmed and indicated
L. monocytogenes ΔmurA and ΔcwhA formed homogene-
ous biofilm (Fig. 5D), in which surface coverage, thickness
and 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 the
formation of rough colonies with even more elongated
cells than the secA2 mutant (Fig. 1). L. monocytogenes
ΔmurAΔcwhA formed intricate and entangled cell aggre-
gates, which flocculated and sedimented more rapidly
than the secA2 mutant (Fig. 2A–C). In a manner similar to
the cwhA mutant, initial adhesion of L. monocytogenes
ΔmurAΔcwhA was significantly reduced and almost unde-
tectable under dynamic conditions (Figs 5A and S2).
Also similar to the cwhA mutant, L. monocytogenes
ΔmurAΔcwhA microcolonies blocked the BRT microbeads
 SecA2 impacts biofilm formation in L. monocytogenes 7

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 Experimental
procedures.
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 ( ) and
ΔmurAΔcwhA ( ).

earlier than the wt strains (Fig. 5B). However, the results
from the CV method were completely biased (Fig. 5C), in
that it showed a severely reduced sessile biomass for
L. monocytogenes ΔmurAΔcwhA compared to the wt.
Indeed, it was not just some significant clumps of the
sessile biomass that were removed at each washing
steps of the CV method as observed for the secA2 mutant
but nearly the entire L. monocytogenes ΔmurAΔcwhA
biofilm that detached at once. Clearly, the CV method was
not appropriate, and biofilm was further investigated by
CLSM.
As observed with L. monocytogenes ΔsecA2 under
static conditions, the simultaneous deletion of murA and
cwhA led to the formation of a filamentous biofilm with
aerial structures (Fig. 5D). Image analyses confirmed
that: (i) surface coverage was significantly reduced to less
than 20% of the wt strain (Fig. 5E); (ii) the average thick-
ness was higher than L. monocytogenes wt, reaching
up to 75 μm (Fig. 5F); and (iii) the biofilm roughness
was significantly increased (Fig. 5G). Interestingly and
despite its tendency to easily detach from the support,
L. monocytogenes ΔmurAΔcwhA could be maintained
under dynamic conditions (Fig. 4). This biofilm was thicker
and rougher than the wt but with a significantly reduced
surface coverage. In the end, the total absence of the
MurA and CwhA proteins in the double mutant resulted

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
8 S. Renier et al.
in an exaggerated effect over the L. monocytogenes
ΔsecA2 biofilm phenotype, where these proteins were just
secreted at lower level. Those especially elongated cells
promoted autoaggregation thereby leading to the forma-
tion of a filamentous biofilm, which was rougher than the
wt strain and even thicker than secA2 mutant in both static
and dynamic conditions.
Inactivation of the SecA2 pathway, or simultaneous
deletion of murA and cwhA, enhances the surface
colonization at 20°C
The involvement of the SecA2 pathway in
L. monocytogenes biofilm formation was further investi-
gated at a temperature relevant to its saprophytic
lifestyles in the environment. At a standard ambient tem-
perature of 20°C, all mutant strains presented similar cell
and colony phenotypes to those described at 37°C
(Fig. 1); L. monocytogenes ΔsecA2 and ΔmurAΔcwhA
exhibited elongated cells forming a rough colony
morphotype, whereas L. monocytogenes ΔmurA and
ΔcwhA formed very slightly rippled and smooth colonies
respectively.
L. monocytogenes ΔsecA2 and ΔmurAΔcwhA clearly
formed intricate and entangled cell aggregates, and
sedimentation was much more rapid than for L.
monocytogenes wt, ΔcwhA or ΔmurA (Fig. 2D–F). Initial
bacterial adhesion was significantly reduced for
L. monocytogenes ΔsecA2, ΔcwhA, ΔmurA and
ΔmurAΔcwhA in static conditions (Fig. 6A and B); this
contrasts with results at 37°C where only the murA mutant
was able to adhere similarly to the wt in both static and
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Fig. 6. Adhesion and biofilm formation of
L. monocytogenes EGD-e wt and the isogenic
mutants at 20°C.
A, B. Initial adhesion assay based on crystal
violet staining.
C, D. Biofilm formation at early stages of
sessile development assayed with the BRT.
Biased results from crystal violet method are
provided as supplementary material (Fig. S6).
Statistical significance of the results is
indicated by an asterisk (P < 0.05).
L. monocytogenes wt (□), ΔsecA2 (■), ΔmurA
( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ).
dynamic conditions. In dynamic conditions, the rate of
initial attachment (IAR) was significantly reduced for all
isogenic mutants, and adhered cells were almost unde-
tectable for L. monocytogenes ΔsecA2 and ΔmurAΔcwhA
(Fig. S2). Nonetheless, L. monocytogenes ΔsecA2 and
ΔmurAΔcwhA were completely blocked by the
microbeads within 24 h contrary to the wt strain or murA
and cwhA mutants (Fig. 6C and D). Results generated
from the CV method for L. monocytogenes ΔsecA2 and
ΔmurAΔcwhA were once again not representative since it
could be visually observed that the entire biofilms were
removed at once from the first washing steps (Fig. S6).
For L. monocytogenes wt, the sessile biomass formed
in 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 highly
motile and could not form a biofilm at 20°C (Fig. 7A, B and
Video S1). In agreement with aggregation and initial
adhesion results (Figs 2, 6, and S2), the very low but still
increasing sessile biomass measured over time by the CV
method (Fig. S6) was related to bacterial adhesion rather
than sessile growth. Similarly, the murA and cwhA
mutants were also motile at 20°C and did not form a
biofilm (Videos S2 and S3). L. monocytogenes ΔsecA2
and ΔmurAΔcwhA, however, were clearly non-motile at
20°C (Videos S4 and S5).
While no biofilm could be observed with
L. monocytogenes wt at 20°C, CLSM observations con-
firmed L. monocytogenes ΔsecA2 and ΔmurAΔcwhA
formed filamentous biofilms (Fig. 7A); compared to the
biofilms observed at 37°C in static conditions (Fig. 3 and
 SecA2 impacts biofilm formation in L. monocytogenes 9

Fig. 7. Biofilm architecture of L. monocytogenes wt and the isogenic mutants ΔsecA2 and ΔmurAΔcwhA strains under static and dynamic
conditions 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 the
Experimental 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 the
Experimental 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 ( ).

5), they were as rough (Fig. 7D) and even thicker in
average, i.e. between 60 μm and 77 μm (Fig. 7C) but with
a lower surface coverage not exceeding 10% (Fig. 7B).
When co-cultured with L. monocytogenes wt, biofilm at
20°C was exclusively formed by the secA2 mutant cells
(Fig. S5); in contrast to 37°C, the wt listerial cells were
thus not able to establish themselves within the biofilm.
Investigation of the biofilm formation under dynamic con-
ditions confirmed motile L. monocytogenes could not form
a biofilm contrary to L. monocytogenes ΔsecA2 and
ΔmurAΔcwhA, which could indeed be maintained within
flow cells (Fig. 7E). Again, L. monocytogenes ΔsecA2 (or
the simultaneous deletion of murA and cwhA) led to the
formation of filamentous biofilms with aerial structures.
Thus, inactivation of the SecA2 pathway allowed
L. monocytogenes to colonize a surface at 20°C.
Reduced secretion of its two major substrates MurA and
CwhA resulted in cell elongation, which abrogated motility
and propped up aggregation leading to cell sedimentation
then promoting sessile growth.
Discussion
The architecture of L. monocytogenes biofilms is
highly polymorphic ranging from monolayer, honeycomb,
mushroom-shaped and most recently knitted-chains
network (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 in
L. monocytogenes. Inactivation of SecA2 pathway in
L. monocytogenes systematically led to the formation of
filamentous biofilms with aerial and fluffy structures. They
are rougher and thicker than the wt biofilm and unevenly
covered the colonized surface. It clearly appeared the
phenotype of filamentous biofilm was associated with the
reduced secretion of the two major SecA2-dependent cell

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
10 S. Renier et al.
hydrolases, MurA and CwhA, which resulted in bacterial
cell elongation (Machata et al., 2005). Those cell-wall
hydrolases are important enzymes to maintain the cell
wall 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; Dumas
et al., 2009; Desvaux et al., 2010; Renier et al., 2013).
Actually, the total absence of the MurA and CwhA proteins
in L. monocytogenes ΔmurAΔcwhA resulted in an exag-
gerated effect as compared with inactivation of the SecA2
pathway. Bacterial cells were especially elongated,
autoaggregation was quicker and the biofilm was rougher
than the wt strain but also even thicker and more fragile
than the secA2 mutant. A similar reduction of adhered
biomass was also reported in a divIVA mutant, where
bacterial cells were elongated as a result of a down-
secretion of MurA and CwhA (Halbedel et al., 2012). As
for the virulence, it is most certainly an indirect and col-
lateral effect of the change in cell morphology (Desvaux
and Hébraud, 2006); the loss of bacterial septum forma-
tion is accompanied by mislocalization and misassembly
of some cell-surface proteins (Carroll et al., 2003; Pilgrim
et al., 2003), which can be of importance for bacterial
adhesion and biofilm formation as evidenced for the fla-
gella (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 biochemistry of the cell-wall hydrolases and
their respective implication in cell wall biogenesis in
L. monocytogenes (Popowska, 2004).
Nonetheless, L. monocytogenes could settle and colo-
nize a surface under both static and dynamic conditions
following SecA2 inactivation. The combinatory approach
here performed (i.e. cell aggregation, bacterial adhesion
in static and dynamic conditions, early and late stages of
sessile development using BRT and the crystal violet
method, CLSM in static and dynamic conditions) proved
a powerful approach to decipher biofilm formation in
L. monocytogenes. Also, the defect of biofilm formation
previously reported in divIVA mutant (Halbedel et al.,
2012) should be regarded with caution considering CV
method is not appropriate to observe filamentous biofilm.
Besides, temperature has a great influence on listerial
cell physiology in terms of genetic expression but it had
essentially been understood through the lens of patho-
genicity and virulence factors (Toledo-Arana et al., 2009;
de las Heras et al., 2011). Filamentous biofilms could
form under temperatures relevant to infection condition
(37°C) and environmental condition (20°C), albeit with a
major difference. It is well known that L. monocytogenes
motility is regulated by temperature, where flagella are
expressed at 20°C but not at 37°C (Renier et al., 2011).
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
At 37°C, L. monocytogenes wt could form a biofilm
and participate to filamentous-biofilm formation when
co-cultured with L. monocytogenes ΔsecA2. At 20°C, the
wt strain was motile and surface colonization arose
through adhesion rather than sessile growth; when
co-cultured with L. monocytogenes ΔsecA2, the wt strain
could not establish itself within the filamentous biofilm. In
environmental condition, SecA2 inactivation clearly gives
a competitive advantage for surface colonization over
L. 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 before
in the literature but reported only recently in L.
monocytogenes (Travier et al., 2013). Compared to the wt
strain, however, inactivation of the SecA2 pathway here
appeared to have a dramatic effect by promoting extensive
sedimentation following cell aggregation and flocculation,
which was unlike previously reported cell aggregation in
L. monocytogenes. Actually, down-secretion of MurA and
CwhA led to elongated cells, which further promoted
autoaggregation. At 20°C, this effect is combined with
the abrogation of cell motility resulting in elongated
sedimented cells, which got knotted and entangled
together 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 stressful
conditions (Jørgensen et al., 1995; Bereksi et al., 2002;
Hazeleger et al., 2006; Giotis et al., 2007), SecA2 inacti-
vation could contribute to asymptomatic animal/human
carriage at 37°C (Travier et al., 2013) and clearly partici-
pate in biofilm settlement in the environment at ambient
temperature. Here, a parallel can be drawn with the biofilm
network of knitted chains, where it was shown to be
dependent on RecA and YneA activated by the SOS
response, leading to the elongation of cells and allowing a
better stress resistance (van der Veen et al., 2010). There-
fore, it is tempting to hypothesize about a connection
between the SOS response in L. monocytogenes and the
SecA2 pathway, which could be implied in cell differentia-
tion in response to external conditions and thus biofilm
formation. The molecular mechanisms responsible for
SecA2 inactivation and the regulation of the reversible cell
differentiation/colony morphotype have yet to be clearly
established in L. monocytogenes (Lenz and Portnoy, 2002;
Rigel and Braunstein, 2008). In this context, phase-
variation is a promising research direction that would
deserve further in-depth investigations in Gram-positive
bacteria in general (Henderson et al., 1999; Lenz
and Portnoy, 2002). Differential regulation of biofilm forma-
tion in response to environmental conditions has been
previously described in different bacterial species
 SecA2 impacts biofilm formation in L. monocytogenes 11
Table 1. Strains and plasmids used in this study.

Name Relevant characteristics Source/Reference

Plasmids
pMAD 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 work
pMAD-ΔmurA AmpR, EmR, bgaB, ΔmurA construct This work
pIMK2-secA2 Site-specific listerial integrative vector, Phelp-secA2, KanR This work
pIMK2-murA Site-specific listerial integrative vector, Phelp-murA, KanR This work
pNF8 EmR, oriR pAMβ1, oriR pUC, Pdlt-gfpmut1 Fortinea and colleagues (2000)
L. monocytogenes strains
EGD-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 work
ΔcwhA 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 This work
cwhA genes
ΔsecA2::pIMK2-secA2 pIMK2secA2 integrated at tRNAArg with SecA2 expressed from This work
the Phelp promoter
ΔmurA::pIMK2-murA pIMK2murA integrated at tRNAArg with MurA expressed from the This work
Phelp promoter
ΔcwhA::pIMK2-cwhA pIMK2cwhA integrated at tRNAArg with CwhA expressed from the Monk and colleagues (2008)
Phelp promoter
EGD-e (pNF8) Green autofluorescent strain This work
ΔsecA2 (pNF8) Green autofluorescent strain This work
ΔmurA (pNF8) Green autofluorescent strain This work
ΔcwhA (pNF8) Green autofluorescent strain This work
ΔmurAΔcwhA (pNF8) Green autofluorescent strain This work

(Hall-Stoodley and Stoodley, 2002), namely in relation to
the expression of different exopolysaccharides (Franklin
et 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 eDNA
only expressed in some particular conditions and resulting
most certainly from cell lysis (Harmsen et al., 2010), a main
singularity of biofilm formation in L. monocytogenes is the
absence of a dense exopolymeric matrix as observed in
most 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 its
implication in colonization processes has been over-
looked (Lenz and Portnoy, 2002; Monk et al., 2004). In
this study, we demonstrated that the inactivation of the
SecA2 pathway provides a decisive advantage in listerial
surface colonization under environmental conditions. Cell
differentiation could be involved in the conversion from
rough morphotype in environmental conditions to virulent
smooth morphotype under infection conditions. The
morphotypic conversion in L. monocytogenes could be
further considered as a risk factor for contamination of
industrial production chain line and food products but also
of potential significance for asymptomatic human/animal
carriage. Understanding the regulation of the morphotypic
conversion would facilitate the eradication of listerial
biofilm in food plants and allow controlling listerial infec-
tion. Considering that biofilms are generally multispecies
rather than monospecies, this cell differentiation could
have consequences on L. monocytogenes implantation
and interaction with other microbial species in various
ecological 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 in
brain–heart infusion (BHI) broth or BHI agar plates at 20°C or
37°C. When necessary, X-Gal (100 μg ml−1) and/or antibiotics
were 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 all
cloning procedures (Sambrook and Russell, 2001). Growth
curves were obtained using a Bioscreen C (Labsystems).
Construction of in-frame ΔsecA2, ΔmurA and
ΔmurAΔcwhA L. monocytogenes mutants and gene
complementation
The genes encoding SecA2 (lmo0583) and MurA (lmo2691)
were deleted by allelic exchange using the pMAD vector
as previously described (Arnaud et al., 2004). From L.
monocytogenes EGD-e genomic DNA purified with Wizard
Genomic DNA Purification Kit (Promega), upstream and
downstream DNA fragments flanking the gene of interest

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
12 S. Renier et al.
were amplified by high-fidelity PCR using TaKaRa LA Taq
DNA 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 for
the SOE-PCR (splicing by overlapping extension PCR) using
Fw1/Rv4 primers (Desvaux et al., 2006c; 2007). Following
standard molecular cloning technique (Sambrook and
Russell, 2001), the resulting amplicon was cloned into pMAD
following 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 Plasmid
QuickPure (Macherey-Nagel), the resulting plasmid pMAD-
ΔsecA2 and pMAD-ΔmurA were electroporated into
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 et al., 2004), blue-white screening was applied to
select gene knockout events. The isogenic mutants were
identified by colony PCR with outFw/outRv primers using
GoTaq DNA polymerase (Promega) (Table S1) and were
further confirmed by DNA sequencing (GATC-Biotech) on
both strands using primers Fw1 and Rv4, respectively.
For gene complementation, the entire CDS (coding
sequence) was amplified from genomic DNA by PCR using
TaKaRa LA Taq DNA polymerase and the primers
SecA2BspHFw/SecA2PstRv and MurANcoFw/MurAPstRv
respectively. The amplicon was cloned into pIMK2 (Monk
et al., 2008) following DNA digest with NcoI/PstI restriction
enzymes, ligation, electroporation into E. coli TOP10 and
selection on LB agar with kanamycin (Rossiter et al., 2011).
After plasmid purification, the resulting pIMK2-secA2 and
pIMK2-murA were electroporated into L. monocytogenes
EGD-e ΔsecA2 and L. monocytogenes EGD-e ΔmurA
respectively. Site-specific integration of the plasmid was con-
firmed following plating on kanamycin BHI agar, and colony
PCR was performed using primers SecA2BspHFw/
SecA2PstRv and MurANcoFw/MurAPstRv respectively. For
the complemented L. monocytogenes strains, restoration of
the cell and colony morphotype was checked by microscopic
observations as detailed below (Fig. S1).
Microsocopic observation
Microscopic observations of bacterial colony and individual
listerial cells were performed with an inverted contrast phase
microscope (Olympus LH50A). For microscopic images of
bacterial colonies, the strains were grown on BHI agar plates
at 37°C and 20°C for 24 h, and resultant colonies were
observed at 150 × original magnification. For visualization of
bacterial cells, cultures grown in BHI broth, at 37 and 20°C,
were sampled during the exponential phase and fixed
onto glass slides for microscopic analysis at 600 × original
magnification.
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 in
BHI at 37°C or 20°C were adjusted to the same OD600 nm.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Briefly, chloramphenicol was added at a final concentration of
90 μg ml−1, and each suspension was placed vertically and
statistically in tubes at the relevant temperature up to 24 h. To
follow cell sedimentation, samples of 500 μl were taken from
the top of the tube at different time points to measure the
OD600 nm. To visualize cell aggregates, samples were taken at
the 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 medium
and loaded into the wells of a 96-well polystyrene microtiter
plate prior to static incubation at 20°C or 37°C. After 1 h, the
supernatant was removed from the wells, which were washed
with tryptone salt (TS) and directly stained with an aqueous
solution of crystal violet (0.1%). After washing, the bound dye
was solubilized in acetic acid (33%), then transferred to a
clean microtiter plate where the absorbance was finally
measured. At least five independent experiments with at least
two repeats each were performed for each strain.
Adhesion assay under liquid flow
Initial adhesion in dynamic conditions was assayed using a
recently described standard protocol (Szlavik et al., 2012).
Instead of glass, however, plastic cover slips (Agar Scientific,
dimension 22 mm × 22 mm) made of clear unbreakable
polystyrene were used. Briefly, cells were diluted in citric
acid-Na2HPO4 buffer (pH 6.6) to a final volume of 50 ml and
OD600 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 giving
a wall shear stress of 0.0505 pa. The chamber was mounted
on an inverted microscope (Zeiss, Axiovert 25) with an
attached camera (QImaging, MicroPublisher v3.3). After acti-
vation of the pumps, consecutive pictures were taken in three
separate vistas every 5 min for 30 min, and the adhered cells
were enumerated. The median was selected for each time
point, and the initial adhesion rate (IAR) was calculated using
linear regression from the medians. All adhesion tests were
performed at least in triplicates.
Biofilm formation assay at early stages of sessile
development
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 or
mutant strains adjusted at OD600nm = 0.01 (approximately
105 CFU ml−1) in sterile BHI medium. Briefly, a suspension of
paramagnetic microbeads (Ton5: 2.8 μm in diameter) was
added at 10 μl ml−1 final concentration, homogenized by
vortexing prior to 200 μl loading into 96-well BFC Polystyrene
Microtiter plates or 8-well BFC Polystyrene Strips (BioFilm
Control, Saint-Beauzire, France) and static incubation at
20°C or 37°C. Control wells were filled with sterile BHI and
Ton5. For reading at the different time points, wells of
microtiter plates were first covered with 100 μl of BFC

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 as
Biofilm Formation Index (BFI) (Chavant et al., 2007; Macé
et al., 2008). Basically, in the course of bacterial sessile
development, BFI decreases, and a BFI ≤ 2 indicates
a full immobilization of the paramagnetic microbeads by
L. 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 not
change anymore; still, the sessile biomass can continue to
grow, and later stages of biofilm formation were thus investi-
gated using the CV method described here below. At least
five independent experiments with at least two repeats each
were performed for each strain and incubation time.
Biofilm formation assay at late stages of
sessile development
The assay is based on the CV method (Djordjevic et al.,
2002; Borucki et al., 2003). Briefly, overnight cultures of
L. monocytogenes strains were adjusted at 0.01 (OD600nm) in
sterile BHI medium and 200 μl loaded into the wells of a
96-well polystyrene microtiter plate prior to static incubation
at 20°C or 37°C (de Luna et al., 2008). At different time
points, the supernatant was removed from the wells, which
were washed with TS. Absolute ethanol was then applied for
fixation (20 min). After emptying and air drying the wells,
200 μl of an aqueous solution of crystal violet (0.1%) was
added and left for 10 min. After washing with water, the bound
dye was solubilized with 200 μl of an aqueous solution of
acetic acid (33%). Contents of each well (150 μl) were trans-
ferred to a clean microtiter plate, and absorbance was finally
measured using a microtiter plate reader set to 595 nm. At
least five independent experiments with at least two repeats
each 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-well
polystyrene microtiter plate (Greiner Bio-One) which enables
high resolution fluorescence imaging. Then, the plates were
incubated at 20°C or 37°C. After 2 h, the medium was
removed, and 200 μl of fresh BHI was added. Biofilm devel-
opment was evaluated by microscopic observations after
24 h of incubation. At least three independent experiments
were 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. Flow
chambers were inoculated with overnight cultures of
L. monocytogenes EGD-e wt (pNF8), ΔsecA2 (pNF8) and
ΔmurAΔcwhA (pNF8) strains adjusted at 0.01 (OD600 nm) in
fresh BHI medium. After inoculation (2 ml), the medium flow
was stopped for 1 h to allow bacterial adhesion, and thereaf-
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
SecA2 impacts biofilm formation in L. monocytogenes 13
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 with
two replicates each were made.
Co-cultured assays. Static and flow cell biofilm experiments
were also performed in co-cultures. Overnight cultures of
L. monocytogenes EGD-e wt and ΔsecA2 (pNF8) or
L. monocytogenes EGD-e wt and ΔmurAΔcwhA (pNF8) were
mixed in equal quantity and then, adjusted at 0.01 (OD600 nm)
before being added in wells or inoculated in the flow cell
chamber. Then, sessile cells were stained with the red nucleic
acid stain SYTO 61 (0.01%) (Invitrogen) before microscopic
observation.
CLSM and image processing. Horizontal plane images of the
biofilms were acquired using a Leica SP2 AOBS CLSM
(Leica Microsystems) at the MIMA2 microscopy platform
(http://www6.jouy.inra.fr/mima2_eng). When necessary the
CLSM allowed simultaneous monitoring of GFP and SYTO
61 dyes. The excitation wavelength used for GFP was
488 nm, and emitted fluorescence was recorded within the
range of 500 nm to 550 nm. The red fluorescent nucleic acid
stain 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 immersion
objective (numerical aperture, 1.4).
3D projections were performed with IMARIS software
(Bitplane, Scientific Software, Zurich, Switzerland). The
biofilm structural parameters (thickness, roughness and sub-
stratum coverage) were evaluated using the PHLIP Matlab
program developed by J. Xavier (http://sourceforge.net/
projects/phlip/). For each experiment, at least three micro-
scopic fields were analyzed. Considering the heterogeneity of
the ΔsecA2 and ΔmurAΔcwhA, only images containing a
biofilm were considered for the analysis.
Statistical analysis
In order to test the significance of the differences observed in
each assay between the wt and the different mutants, a pair
Student’s t-test was performed. Differences were considered
significant from P < 0.05.
Acknowledgements
This work was supported in part by French National Institute
for Agronomical Research (INRA), the European Framework
Program 6 (FP6) with the ProSafeBeef (Advancing Beef
Safety and Quality through Research and Innovation)
research consortium (http://www.prosafebeef.eu), the
European Cooperation in Science and Technology (COST)
Action FA1202 BacFoodNet (a European network for mitigat-
ing bacterial colonization and persistence on foods and
food processing environments), the EGIDE Programme
Hubert Curien (PHC) France-Ireland ULYSSES 2010 from
the ‘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
14 S. Renier et al.
Denmark ‘Institut Français du Danemark’(IFD) (n°14/2012/
CSU.8.2.1). The authors are very grateful to Jens Bo
Andersen and Tine Rask Licht (Technical University of
Denmark, Soeborg) for kindly providing multiple fluorescence
labelling system in L. monocytogenes. The authors thank
Chantal Bizet (Institut Pasteur, Paris, France) for providing
pMAD under Material Transfer Agreement (MTA). The excel-
lent technical assistance of Marina Bjørklund (Copenhagen
University) was highly appreciated as well as Danièle
François and Amine Zorgani (INRA Clermont-Ferrand).
Caroline Chagnot is a PhD Research Fellow granted by
the ‘Région Auvergne – Fonds Européen de Développe-
ment Régional (FEDER)’. Dr Sandra Renier had a PhD
research fellowship granted by the French ‘Ministère de
l’Enseignement Supérieur et de la Recherche’.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Microscopic analysis of colony and cell morphology
of 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 left
side) 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 BHI
broth. Pictures of bacterial cells were taken in exponential
phase (phase-contrast microscopy, 600 × original magnifica-
tions) and show restoration of the discrete-cells phenotype.
Bars, 20 μm.
Fig. S2. Adhesion under dynamic conditions of
L. monocytogenes EGD-e wt and isogenic mutant strains at
37 and 20°C. Initial bacterial adhesion under liquid flow was
performed 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 colony
morphotype at both 37 and 20°C. From consecutive pictures
taken at regular time intervals, the adhered cells were enu-
merated and initial adhesion rate (IAR) was calculated using
linear regression from the median values.
Fig. S3. Growth curves of L. monocytogenes wt and
isogenic mutant strains performed in BHI at 37°C and 20°C.
Fig. S4. 3D reconstruction of L. monocytogenes ΔsecA2
biofilm at 37°C. Images were obtained by CLSM images after
24 h of sessile growth at 37°C in static conditions as
described in the Experimental Procedures.
Fig. S5. Biofilm architecture of mixed cultures of
L. monocytogenes EGD-e wt and ΔsecA2 mutant strains. (A)
CLSM images of mixed cultures of L. monocytogenes
wt/ΔsecA2 after 24 h growth at 37°C under static condition.
(B) CLSM images of mixed cultures of L. monocytogenes
wt/ΔsecA2 after 24 h growth at 20°C under static condition.
L. monocytogenes wt cells appear in red (stained with SYTO
61) and L. monocytogenes mutant cells strains appear in
green (bacterial strain bearing pNF8).
Fig. S6. Biofilm formation of L. monocytogenes EGD-e wt
and the isogenic mutants at late stages of sessile development
assayed with the crystal violet method at 20°C. (A) Results of
the CV method for L. monocytogenes wt (□) and ΔsecA2 (■).
(C) Results of the CV method for L. monocytogenes wt (□),
ΔmurA ( ), ΔcwhA ( ) and ΔmurAΔcwhA ( ). Most results
were not representative since it could be visually observed that
the entire biofilm was removed at once during washing steps of
the CV method for L. monocytogenes ΔsecA2, ΔmurA, and
ΔmurAΔcwhA.
Table S1. Oligonucleotides used in this study.
Video S1. Cell motility of L. monocytogenes EGD-e wt
recorded in contrast-phase microscopy after 24 h in static
biofilm culture at 20°C
 SecA2 impacts biofilm formation in L. monocytogenes 17
Video S2. Cell motility of L. monocytogenes ΔmurA Video S4. Cell motility of L. monocytogenes ΔsecA2
recorded in contrast-phase microscopy after 24 h in static recorded in contrast-phase microscopy after 24 h in static
biofilm culture at 20°C. biofilm culture at 20°C.
Video S3. Cell motility of L. monocytogenes ΔcwhA Video S5. Cell motility of L. monocytogenes ΔmurAΔcwhA
recorded in contrast-phase microscopy after 24 h in static recorded in contrast-phase microscopy after 24 h in static
biofilm culture at 20°C. biofilm culture at 20°C.

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