A new morphogenesis pathway in bacteria: unbalanced activity of cell wall synthesis machineries leads to coccus-to-rod transition and filamentation in ovococci


E-mail eric.guedon@jouy.inra.fr; Tel. (+33) 1 34 65 25 25; Fax (+33) 1 34 65 25 21.


Bacteria display a variety of shapes, which have biological relevance. In most eubacteria, cell shape is maintained by the tough peptidoglycan (PG) layer of the cell wall, the sacculus. The organization of PG synthesis machineries, orchestrated by different cytoskeletal elements, determines the specific shapes of sacculi. In rod-shaped bacteria, the actin-like (MreB) and the tubuline-like (FtsZ) cytoskeletons control synthesis of the sidewall (elongation) and the crosswall (septation) respectively. Much less is known concerning cell morphogenesis in cocci, which lack MreB proteins. While spherical cocci exclusively display septal growth, ovococci additionally display peripheral growth, which is responsible of the slight longitudinal expansion that generates their ovoid shape. Here, we report that the ovococcus Lactococcus lactis has the ability to become rod-shaped. L. lactis IL1403 wild-type cells form long aseptate filaments during both biofilm and planktonic growth in a synthetic medium. Nascent PG insertion and the division protein FtsK localize in multiple peripheral rings regularly spaced along the filaments. We show that filamentation results from septation inhibition, and that penicillin-binding proteins PBP2x and PBP2b play a direct role in this process. We propose a model for filament formation in L. lactis, and discuss the possible biological role of such morphological differentiation.


Life and persistence of microorganisms in their natural habitats require an adaptation of their cellular functions to the changing environment. One of the most visible adaptative strategies corresponds to changes in cell morphology, such as rod-to-coccus transition, filament formation, sporulation or extrusion of appendages (Young, 2006). A variety of factors can trigger these changes, which provide new features to bacterial cells (e.g. increased cell surface area: volume ratio, attachment or dispersal, and resistance to turgor pressure or predation). Environmentally induced pleomorphism has been well documented in different bacterial species (Young, 2006). Of particular interest are several pathogenic bacteria like uropathogenic Escherichia coli– UPEC, Mycobacterium or Salmonella ssp. that change their cell shape to escape the host immune system (Rosenberger and Finlay, 2002; Justice et al., 2004; 2008; Chauhan et al., 2006). Although the signalling pathways and molecular mechanisms underlying morphological changes are not well understood, they have been shown to be directly linked to shape determining elements. In most eubacteria, the highly cross-linked peptidoglycan (PG) meshwork of the cell wall (CW), namely the sacculus, and the bacterial cytoskeleton are the major determinants of cell shape (for recent reviews see Carballido-López and Formstone, 2007; den Blaauwen et al., 2008; Zapun et al., 2008a). The current model is that cytoskeletal elements orchestrate sacculi formation by controlling the activity and/or the localization of specific multi-enzyme complexes involved in CW biogenesis (Scheffers and Pinho, 2005). Thus, cell shape results from the co-ordinated action (in time and space) of the cytoskeleton and CW synthesis proteins such as penicillin-binding proteins (PBPs) – the enzymes that assemble the PG. Most studies of cell morphogenesis traditionally focused on the model rod-shaped bacteria E. coli and Bacillus subtilis (as examples of recent reviews see Osborn and Rothfield, 2007 and Carballido-López and Formstone, 2007), where two competing systems for CW synthesis coexist: one for crosswall formation (septation) and one for sidewall formation (elongation). Each one of these systems is associated to specific elements of the cytoskeleton and to specific PG biosynthetic machineries (den Blaauwen et al., 2008). The tubulin homologue FtsZ plays an essential role in septation. Assembly of FtsZ into a ring structure (the Z-ring) at the future division site (Bi and Lutkenhaus, 1991) is the earliest event in bacterial cell division, and subsequent recruitment of other division proteins to the Z-ring mediates septum formation (Errington et al., 2003; Aarsman et al., 2005; Harry et al., 2006; Adams and Errington, 2009). Depletion of FtsZ or of division-specific PBPs leads to the formation of long aseptated filamentous cells (Hirota et al., 1968; Wientjes and Nanninga, 1991; Daniel et al., 2000; Popham and Young, 2003), as sidewall synthesis continues while septal CW synthesis is inhibited. The actin homologue MreB is involved in elongation. MreB proteins are present in non-spherical bacteria, where they assemble into membrane-associated filamentous helical structures (Jones et al., 2001) that are believed to actively direct the synthesis and the maturation of the cylindrical sidewall (Jones et al., 2001; Carballido-Lopez and Errington, 2003; Daniel and Errington, 2003; Figge et al., 2004; Kruse et al., 2005; Carballido-López et al., 2006). Depletion of MreB or of elongation-specific PBPs leads to the loss of rod shape and rounding of cells (Ogura et al., 1989; Murray et al., 1997; Jones et al., 2001; Wei et al., 2003; Kruse et al., 2005).

Much less is known about the mechanisms of cell morphogenesis in cocci. Phylogenetic studies point to the hypothesis that modern cocci evolved from rods by loss-of-function of the machinery required for cylindrical elongation, and that this loss is evolutionarily irreversible (Siefert and Fox, 1998). As mreB-like genes are absent in the genomes of most coccoid bacteria (Jones et al., 2001), lack of an MreB cytoskeleton may be at the origin of their presumed inability to elongate their sidewalls. Two classes of cocci should be distinguished: spherical cocci with truly round shape and elongated ellipsoid organisms called ovococci (Zapun et al., 2008a). Spherical cocci, like staphylococci, only possess a CW synthesis system associated with septum formation and are unable to elongate under any condition (Lleo et al., 1990). Consistently, non-dividing cells of Staphylococcus aureus depleted of FtsZ increase their volume but remain spherical (Pinho and Errington, 2003). Ovococci, like streptococci, possess an additional division-specific mode of CW growth, namely peripheral growth (Tomasz, 2000), responsible of the slight longitudinal elongation of their sidewalls prior to division. Like in rod-shaped bacteria, each CW machinery is composed of specific PBPs. In Streptococcus pneumoniae, PBP2x and PBP2b have been shown to be directly involved in PG incorporation at the developing septum (septal growth) and into the sidewall (peripheral growth) respectively (Morlot et al., 2003; Zapun et al., 2008a). However, both septal and peripheral machineries are associated to FtsZ in ovococci (Zapun et al., 2008a).

In this paper, we show that the ovococcus Lactococcus lactis can naturally undergo coccus-to-rod transition and further filamentation during growth, both in planktonic conditions and in biofilms. This Gram-positive lactic acid bacterium is widely used for food fermentation and L. lactis resident biofilms on food surfaces could be used as ‘protective’ biofilms against food pathogens such as Listeria monocytogenese (Zhao et al., 2004; Habimana et al., 2009). L. lactis belongs to the Streptococcaceae family, whose members display a typical ovoid cell shape. Interestingly, unexplored cylindrical elongation had been reported earlier for other streptococcal species (Lorian and Atkinson, 1976; Tao et al., 1988; Lleo et al., 1990; Tao et al., 1993). Here, we analyse the mechanism underlying this intriguing, MreB-independent, sidewall elongation in L. lactis. We propose a model in which unbalanced activity of the peripheral and septal CW biosynthetic machineries upon arrest of cell division results in filament formation, and discuss the role of adaptative pleomorphism in ovococci.


Biofilm lifestyle generates morphological subpopulations of Lactococcus lactis

In natural, industrial and hospital settings, many bacterial species live in surface-associated microbial communities called biofilms (Costerton et al., 1999). In the course of a study aiming to characterize conditions promoting biofilm formation by wild-type L. lactis strain IL1403, two stratified morphologically distinct subpopulations were observed in biofilms grown in chemically defined medium (CDM) (Fig. 1A). The predominant subpopulation was located at the base of the biofilms and was composed of typical ovoid cells (Fig. 1C). The second subpopulation was restricted to the upper layers of the biofilms and contained mostly rod-shaped cells of various lengths (Fig. 1B). This shape alteration was striking because the ability of L. lactis to form long elongated cells was never reported before. Indeed, L. lactis displays an ellipsoid cell shape and forms diplococci or short chains during both biofilm and planktonic growth in all laboratory conditions reported so far. However, these observations suggested that L. lactis is a pleomorphic bacterium, and that stratified environmental niches present in biofilms can trigger morphological differentiation.

Figure 1.

Filamentation of L. lactis in biofilm and planktonic lifestyle.
A–C. Scanning electron micrographs of monospecific biofilms of L. lactis IL1403 formed after 16 h of growth at 30°C in a flow-cell system in CDM medium. Magnification of biofilm upper and lower regions containing elongated rods (B) and ovoid cells (C) respectively.
D and E. Brightfield images of representative fields of planktonic cells during growth in M17 medium (D) and CDM (E). OD600 was indicated for each time point. O.N., overnight growth. Scale bar, 2 µm.

Lactococcus lactis is a pleomorphic ovococcus

In order to determine whether filamentation of L. lactis cells was restricted to biofilm lifestyle, we examined the morphology of L. lactis IL1403 cells growing in planktonic cultures. Typical chains of ovococci were observed in M17 medium (Fig. 1D). The mean cell length slightly fluctuated between 1.25 ± 0.2 µm (n = 256) at early exponential phase (OD600 = 0.1) and 1.7 ± 0.3 µm (n = 261) at mid exponential phase (OD600 = 0.4) (Fig. S1A and B). However, cells growing in CDM transitorily adopted a filamentous morphology (Fig. 1E). During exponential growth, cells underwent coccus-to-rod transition and gradually elongated giving rise to very long filaments (up to 10-fold the mean length of cells at the same growth stage in M17 medium), which resolved in long chains of ovococcal cells at later growth points (Fig. 1E, Fig. S1A and B, Movies S1 and S2). At mid-exponential phase, 90% of the cells were longer than 2.5 µm and among these more than 50% were > 5 µm (Fig. S1A and B). The filamentous forms were viable (Fig. S1C) and membrane-staining indicated that they corresponded to individual long aseptated cells with a continuous chromosomal staining over the filament (Fig. S1D). To further demonstrate that these were true filaments, i.e. with a continuous cytoplasm, FLIP (fluorescence loss in photobleaching) experiments were performed. The FLIP technique consists in photobleaching a fluorophore in a small selected region of the cell by continuous application of a laser beam, which leads to complete extinction of the fluorescence in all regions of the same cellular compartment by exchange between the bleached and unbleached fluorophore populations. Figure 2A and Movie S3 show a typical FLIP experiment carried out in filamentous L. lactis cells constitutively expressing soluble GFP. After continuous application of the high-intensity laser beam to a small region of a filament (panel ii, arrow), the GFP fluorescence was extinguished throughout the filament (panel iii), indicating that the GFP molecules freely diffused inside. Similar experiments were performed in control ovococcal cells (Fig. 2B). When the laser beam was applied to a single cell in a chain (panel ii), the GFP fluorescence was lost in the bleached cell and remained constant in the neighbouring cells (panel iii), indicating that the fluorophore could not diffuse between adjacent cells. Taken together, these findings show that filaments, defined as long rod-shaped aseptate cells with continuous cytoplasm, are a transient morphological state in the cellular development of L. lactis. The term filament will be hereafter used to describe cells that are longer than 2.5 µm.

Figure 2.

L. lactis filaments are long rod-shaped aseptate cells.
A and B. FLIP experiment in filamentous (A) and coccoid (B) cells of strain JIM9173, which constitutively expresses the gfp, grown in CDM and M17 respectively. Panels represent cells before (i), during (ii) and after (iii) 315 s application of the high-intensity laser beam to a small circular region (arrows). Panel iv show the corresponding brightfield image.
C–E. Transmission electron micrographs of typical diplococci (C) and filamentous cells (D and E) of wild-type L. lactis IL1403, growing exponentially in M17 and CDM respectively. The arrows point to the invaginated regions (forming septa). Scale bar, 1 µm.

Filament formation results from transient arrest of cell division

To further investigate the filamentation process, planktonic cells were examined by transmission electron microscopy (TEM). Examples of CDM-induced filamentous cells are shown in Fig. 2D and Fig. S2A. The overall appearance of their CWs was indistinguishable from that of ovococcal cells grown in M17 (Fig. 2C). However, while dividing ovococcal L. lactis cells exhibited invaginations corresponding to nascent division septa exclusively at midcell (Fig. 2C, arrows), filaments presented several symmetric invaginations along their sidewalls, suggesting the presence of multiple nascent division sites (Fig. 2D and E, arrows and Fig. S2A). This indicated that filamentation may result from the transient arrest of cell division at an early stage of septum formation.

Cell wall synthesis occurs at multiple division-associated rings in filamentous cells

In order to elucidate the mechanism of sidewall elongation in the filamentous L. lactis cells, we used a fluorescent derivate of vancomycin (Van-FL) as a probe for nascent PG synthesis. This approach has been used to visualize the topology of insertion of new CW material in several Gram-positive bacteria, including rod-shaped bacteria (Daniel and Errington, 2003; Tiyanont et al., 2006), spherical cocci (Pinho and Errington, 2003) and ovococci (Daniel and Errington, 2003; Ng et al., 2004; Patel and Weaver, 2006). Cocci and ovococci synthesize CW material specifically from an equatorial, FtsZ-dependent, ring. Rod-shaped bacteria containing MreB homologues insert new PG into their lateral wall in a MreB-dependent helical manner during elongation, and at division sites (FtsZ-dependent) during septum formation. In rod-shaped bacteria lacking MreB homologues such as Corynebacterium glutamicum, only the septal machinery exists and cell elongation proceeds from the cell poles (ancient division sites) (Daniel and Errington, 2003).

Van-FL was used to stain exponentially growing cells of L. lactis. In typical ovococcal cells grown in M17 medium (Fig. 3A), the most prominent staining occurred in broad bands at midcell, corresponding to growing or newly formed division septa (arrowheads), as previously described for S. pneumoniae (Daniel and Errington, 2003). Staining was also observed at constriction sites between sister cells (arrow), which will form the new cell poles after division completion, but no staining was detected at the old cell poles. Strikingly, Van-FL staining of filamentous cells grown in CDM (Fig. 3B) revealed a regular pattern of parallel bands (corresponding to rings around the cylinder) perpendicular to the longitudinal axis of the cell. Like ovococcal cells, filamentous cells showed no polar staining. The number of rings increased with the length of the filaments; in the longest filaments (∼ 10 µm long), up to 10 Van-FL-stained rings were observed. This banded pattern was markedly differed from the typical helical pattern (Daniel and Errington, 2003; Tiyanont et al., 2006) obtained when FtsZ-depleted filamentous cells of B. subtilis were Van-FL-stained as control (Fig. S3). These findings indicated that PG synthesis takes place at different sites (rings) along the cylinder in L. lactis filaments.

Figure 3.

Van-FL staining of L. lactis cells and subcellular localization of FtsK–GFP.
A–C. Van-FL staining. Wild-type cells (strain IL1403) grown in M17 medium (A, i and ii), CDM medium (B, i and ii) and M17 upon incubation for 2 h with 1 µg ml−1 methicillin (C, i and ii). Arrowheads point to growing or newly formed division septa. The arrow points to a newly formed pole. Scale bar, 2 µm.
D and E. Subcellular localization of FtsK–GFP in L. lactis. Ovoid and filamentous cells of strain JIM9172 growing in M17 medium (D) and CDM (E) respectively. Cell membrane staining (i) and FtsK–GFP localization (ii). Scale bar, 2 µm.

To determine whether the Van-FL banded pattern reflected sites of division-specific CW growth in the filaments, we looked at the localization of a functional FtsK–GFP fusion (Le Bourgeois et al., 2007) in live L. lactis cells. FtsK is among the first divisome proteins that localize to the Z-ring (Wang and Lutkenhaus, 1998). As expected, FtsK–GFP localized in a single transverse band located at midcell in dividing ovococci (Fig. 3D, panel ii). In contrast, FtsK–GFP formed multiple bands distributed throughout filamentous cells (Fig. 3E, panel ii), in a pattern similar to that obtained by Van-FL staining (Fig. 3B, panels i and ii), indicating the presence of multiple division sites along the cylinder. Taken together, our findings suggest that sidewall elongation in L. lactis filaments results from simultaneous growth at multiple rings associated with temporally blocked division sites.

PBP2x and PBP2b play a key role in filament formation

PG is made up of long linear glycan chains interlinked by short peptide cross bridges, and PBPs catalyse the final stages of its synthesis: transglycosylation and transpeptidation of the PG precursors into the existing sacculus (Sauvage et al., 2008). L. lactis contains seven PBPs: three bifunctional class A PBPs with transpeptidase and transglycosylase activity (PBP1a, PBP1b, PBP2a), two class B PBPs with transpeptidase activity (PBP2b, PBP2x) and two carboxypeptidases (DacA, DacB) (Courtin et al., 2006; Zapun et al., 2008a). In S. pneumoniae, PBP2b and PBP2x are essential (Kell et al., 1993) and directly involved in peripheral and septal growth respectively (Morlot et al., 2003; Zapun et al., 2008a). Because CDM-induced filamentation of L. lactis results from a transient arrest of cell division, we analysed the role of PBP2x in this process. All our attempts to inactivate or deplete pbp2x were unsuccessful, suggesting that this gene is essential in L. lactis too. To bypass this, we analysed the effect of methicillin, a β-lactam antibiotic that inhibits septum formation in several ovococci (Williamson et al., 1980; Pucci et al., 1986; Lleo et al., 1990) presumably by targeting PBP2x (Laible and Hakenbeck, 1991; Jamin et al., 1993; Grebe and Hakenbeck, 1996; Dahesh et al., 2008; Zapun et al., 2008b). Upon addition of methicillin to wild-type L. lactis IL1403 growing exponentially in M17 medium, cultures rapidly stopped doubling (Fig. 4A) and cells filamented (Fig. 4B). Methicillin-induced filamentation was also observed for other L. lactis strains such as MG1363 (Fig. 5). Effect of methicillin on division inhibition was shown to be reversible (filaments to ovococcal cells conversion) after washing out methicillin in methicillin-treated cultures in M17 medium confirming that filaments corresponded to living cells (Movies S4–S6). Interestingly, the Van-FL staining pattern of L. lactis methicillin-induced filaments (Fig. 3C) was similar to that of CDM-induced filaments (Fig. 3B), suggesting a similar mechanism for cylindrical elongation.

Figure 4.

Methicillin-induced filamentation of L. lactis and inability of methicillin-resistant pbp2x mutants to filament.
A. Colony-forming units of L. lactis 1403 cultures growing exponentially in M17 in the absence (grey bars) and in the presence (black bars) of 1 µg ml−1 methicillin. Time points (h) upon addition of methicillin.
B. Membrane staining of wild-type L. lactis IL1403 cells exponentially growing in M17 medium 0, 30, 60, 120 and 180 minutes after addition of methicillin (1 µg ml−1). Scale bar, 1 µm.
C. Size-class distribution of wild-type IL1403 cells (WT) and cells of a representative methicillin-resistant pbp2x mutant (strain JIM9132) growing exponentially in CDM. Lengths of > 250 cells were measured for each strain and divided into three size classes: < 2.5 µm (white bars), 2.5–5 µm (black bars) and >5 µm (grey bars).
D. Brightfield images of representative fields of wild-type (WT) cells of L. lactis IL1403 and of methicillin-resistant mutants (JIM9132, JIM9133, JIM9134, JIM9162, JIM9163 and JIM9165) grown in M17 (i), in M17 supplemented with 1 µg ml−1 of methicillin (ii) and in CDM (iii). Scale bar, 2 µm.

Figure 5.

Role of PBP2b in cell morphology of L. lactis. Phase-contrast images of representative fields of cells of the wild-type strain MG1363 (WT) and the pbp1a-, pbp1b-, pbp2a-, pbp2b- and dacB- mutants grown in M17 (A) and in M17 supplemented of 1 µg ml−1 of methicillin (B). Scale bar, 2 µm.

To identify the genetic determinants of methicillin-induced filamentation, we next selected methicillin-resistant mutants of L. lactis IL1403 (see Experimental procedures section). Sequencing of the pbp2x and pbp2b genes from six independent mutants showed that all contained mutations in pbp2x and none in pbp2b. These mutations corresponded to single amino acid substitutions in the predicted transpeptidase domain of PBP2x (Zerfass et al., 2009) (Fig. S4). The strains bearing these mutations in the essential pbp2x gene grew and divided normally, indicating that their PBP2x variants were active. Furthermore, in addition to confer methicillin resistance, they were also found to inhibit both methicillin- and CDM-induced filamentation of strain IL1403 (Fig. 4C and D). To unambiguously demonstrate that the inhibition of filament formation in these spontaneous mutants was due to mutations in pbp2x and not to other uncharacterized mutations, two variants of pbp2x gene carrying the spontaneous F554V and A588D substitution were cloned on the high copy vector pJIM2278 and expressed in L. lactis IL1403. While the control IL1403 strain carrying the empty pJIM2278 plasmid forms filaments in CDM and in the presence of methicillin, the strains overproducing PBP2x F554V and A588D maintain their ovoid shape (Fig. S5). Taken together, these results show a direct functional link between PBP2x, resistance to methicillin and septum formation in L. lactis. Furthermore, they suggest that inhibition of PBP2x activity is at the origin of the division arrest that underlies filament formation in CDM.

We next searched at identifying the PBP(s) responsible of sidewall elongation in L. lactis filaments. To this end, we examined the cell morphology of different pbp mutants of L. lactis (Fig. 5). In M17 medium, only the pbp2b- mutant lost the typical ovoid shape to adopt a spherical morphology (panel A), indicating that PBP2b is involved in peripheral CW elongation in L. lactis, as previously described for S. pneumoniae (Morlot et al., 2003; Zapun et al., 2008a). Moreover, the pbp2b- mutant was the only PBP mutant that failed to elongate in the presence of methicillin (panel B), suggesting an essential role of PBP2b in sidewall elongation during filamentation.


Lactococcus lactis is a Gram-positive bacterium that belongs to the Streptococcaceae family, whose members display a typical ovoid cell shape. Here we show that wild-type L. lactis IL1403 naturally undergoes transient coccus-to-rod differentiation and further filamentation. When exponentially growing in CDM medium, planktonic cells gradually elongated to form long aseptate filaments (up to 10 times longer than ovoid cells) that later resolved into chains of ovococci (Fig. 1E, Movies S1 and S2). Filamentation was also triggered in the top layers of CDM-grown biofilms (Fig. 1A and B), and it was induced by the addition of methicillin (Figs 4B and 5B), a β-lactam antibiotic that specifically inhibits cell division in cocci (Pucci et al., 1986; Lleo et al., 1990). Methicillin-induced filamentation was not strain-specific, and it had been previously observed for other species such as S. agalactiae, S. bovis, S. sanguis and Enterococcus faecium (Lleo et al., 1990). Thus, cocus-to-rod transition after septum inhibition may be a general feature of Streptococci and other Gram-positive ovococci. CW elongation has been widely studied in rod-shaped bacteria where the MreB cytoskeleton is a pivotal actor (Carballido-López and Formstone, 2007; den Blaauwen et al., 2008; Zapun et al., 2008a). However, because of the differences in the geometry of the cells and in the composition of the CW synthetic machineries, significant differences in the mechanism(s) underlying sidewall elongation were expected in ovococci relative to rod-shaped bacteria. The aim of this study was to understand the molecular basis of this MreB-independent mechanism of elongation in L. lactis.

A functional FtsK–GFP fusion localized to multiple rings regularly distributed along the CDM-induced filamentous L. lactis cells (Fig. 3E, panel ii), indicating that the mechanisms of division-site selection and Z-ring formation are not disturbed. Aborted septa along the filaments sidewalls were observed by TEM (arrows in Fig. 2D and E and Fig. S2A), often associated with membrane, mesosome-like, complex invaginations (Fig. S2B) similar to those observed in B. subtilis cells when septum formation is inhibited (Daniel et al., 2000). Furthermore, the filaments later resolved into long chains of ovococcal cells of regular size (Fig. 1E, Movies S1 and S2). We concluded that these filaments contain multiple division sites where septation is temporarily arrested. To understand how these filamentous cells undergo cylindrical elongation, we used Van-FL to probe the regions of incorporation of new PG into the sacculus. In ovococcal L. lactis cells, Van-FL staining was associated to broad bands at midcell and to the newly formed poles, whereas old poles showed no staining (Fig. 3A). This staining was similar to that obtained for S. pneumoniae cells (Daniel and Errington, 2003) and indicates that PG synthesis occurs at division septa, as expected in ovococci. In both CDM- and methicillin-induced filamentous L. lactis cells, the Van-FL-pattern (Fig. 3B and C) was similar to that displayed by the FtsK–GFP fusion (Fig. 3E, panel ii), with multiple transversal bands distributed along the cylinder. Moreover, absence of polar staining indicated that filamentation is not achieved by polar growth, which is believed to be responsible for elongation in rod-shaped bacteria lacking MreB-like proteins (Daniel and Errington, 2003; Letek et al., 2008). In contrast, our results suggest a new mode of sidewall elongation in which PG insertion occurs simultaneously at multiple Z-ring-associated sites along the cylinder.

To identify actors involved in this new elongation mechanism, we examined the cell morphology of several PBP mutants. Only two of these, PBP2x and PBP2b, were found to be directly associated with filament formation. Cells of a pbp2b mutant were spherical, and unable to form filaments in the presence of methicillin (Fig. 5). These phenotypes are consistent with a direct role of PBP2b in peripheral growth and in filamentation. Inactivation of pbp2x could not be achieved, but use of methicillin allowed us to ascertain its function. Methicillin led to filamentation (see above) and spontaneous methicillin-resistant mutants were isolated, which all carried point mutations in the C-terminal transpeptidase domain of PBP2x (Fig. S4). These pbp2x mutants grew and divided normally, and were unable to form filaments in the presence of methicillin (Fig. 4D, Fig. S5). These findings indicated (i) that methicillin inhibits cell division by primarily targeting PBP2x activity in L. lactis; (ii) that PBP2x is involved in septal PG synthesis in L. lactis and (iii) that our methicillin-resistant PBP2x variants were still active. Furthermore, these pbp2x mutants failed to form filaments in CDM too (Fig. 4D, Fig. S5), suggesting that the transient inhibition of cell division that occurs in CDM may involve or result from a transient inhibition of PBP2x activity. Taken together, the findings reported here for L. lactis support the current model for CW synthesis in ovococci, which involves two distinct division-specific machineries associated with specific PBPs: one for septal growth (division, PBP2x-dependent) and one for peripheral growth (elongation, PBP2b-dependent). Furthermore, we show that their activity is independent to each other so that elongation can occur during division inhibition and vice versa.

On the basis of our findings, we propose a model in which cylindrical elongation in L. lactis results from the inhibition of septation (PBP2x activity) and the concomitant sustaining of peripheral growth (PBP2b activity) at division sites (Fig. 6). As the cell elongates (Fig. 6B, a–d), new Z-rings are assembled at future division sites and recruit components of the divisome (e.g. FtsK) and of the CW synthesis machineries (both septal and peripheral). The newly assembled septal machineries are also inhibited at an early stage of septum formation while the corresponding peripheral machineries remain active and synthesize new sidewall, thereby promoting cylinder elongation. At a later stage of growth, inhibition of PBP2x is released and septation resumes at the multiple immature septa present in the filament (Fig. 6B, e). Division terminates at these sites and filaments are resolved into chains of regular ovovocci (Fig. 6B, f). This new mechanism of elongation provides some important insights into the co-ordination of the competing activities of the peripheral and septal PG machineries in ovococci, and maybe in rods too. Indeed, recent evidence suggests that rods use both a FtsZ-independent (MreB-dependent) and a FtsZ-dependent mode of PG synthesis to elongate (Aaron et al., 2007; Varma et al., 2007; Varma and Young, 2009) and thus a new view of the cell cycle in rod-shape bacteria is emerging in which MreB-dependent dispersed elongation would be followed by a FtsZ-dependent pre-septal elongation (equivalent to peripheral growth in ovococci) prior FtsZ-dependent septum formation.

Figure 6.

Model for filament formation in L. lactis. Cell cycle of L. lactis determined by cell morphology during growth in M17 medium (A) and in CDM, with transient filamentation (B). Division rings are represented in green. Red circles represent the peripheral machinery (including PBP2b); blue circles represent the septal machinery (including PBP2x). Plain circles represent active machines and open circles represent inactive machineries. Red bands on the sidewall represent peripheral cell wall extension. Blue discs represent septum formation. See Discussion for a full description.

Switch from the ovococcal to a filamentous form appears to be part of a developmental process taking place under specific growing conditions in L. lactis. Filamentation of planktonic cells was medium- and growth phase-dependent, but not growth rate dependent (data not shown). In monospecific biofilms, filamentation was also medium-specific and filaments were present only in the upper layers of the biofilm, at the interface with the medium, where cells are presumably in a physiological state similar to exponentially growing planktonic cells (Lenz et al., 2008; Kim et al., 2009). The multiple inductive signals and cellular factors triggering filamentation in L. lactis are currently under study and will be reported elsewhere. Such morphological differentiation is expected to provide selective advantages under certain conditions. The presence of filaments in the upper layers of biofilms might confer ecological advantages to the microbial community such as resistance to predation (Pernthaler, 2005; Hilbi et al., 2007; Justice et al., 2008) or to the action of antimicrobials (Chen et al., 2005). As filamentation leads to an increase of the total bacterial cell surface area, it could also be a response to maximize nutritional uptake in the cells exposed to the flowing medium (Young, 2006).

Finally, several pathogenic rod-shaped bacteria and fungus (e.g. Salmonella typhimurium, Mycobacterium tuberculosis, UPEC and Candida albicans) undergo filament formation as a strategy for virulence and survival in stressful environments, including those of interaction with their hosts (Cutler, 1991; Lo et al., 1997; Saville et al., 2003; 2006; Justice et al., 2004; 2008). Interestingly, we found that wild-type cells of the opportunistic pathogen Streptococcus salivarius also form filaments during planktonic growth in CDM, similar to those formed by L. Lactis and which contained multiple aborted septa too (Fig. S2C). Thus, the filamentation mechanism reported here for L. lactis may be common to streptococci (see also above) and could be used by a number of clinically relevant pathogenic ovococci for survival and/or interactions. This study opens up the way for researches on adaptative pleomorphism in ovococci, which are major actors in the life of Human.

Experimental procedures

Bacterial plasmids, strains and growth conditions

Bacterial strains and plasmids used in this study are listed in Table 1. E. coli and B. subtilis strains were grown at 37°C in Luria–Bertani medium (Maniatis, 1982). Depletion of FtsZ in B. subtilis was performed as described previously (Carballido-Lopez and Errington, 2003). L. lactis was grown at 30°C in rich M17 medium (M17) or in Chemically Defined Medium (CDM) (Terzaghi and Sandine, 1975; Sissler et al., 1999). Methicillin (1 µg ml−1) was added to cultures of exponentially growing cells (OD600 = 0.1) in M17. When required, ampicillin (100 µg ml−1), chloramphenicol (5 µg ml−1) or erythromycin (5 µg ml−1 for L. lactis, 100 µg ml−1 for E. coli) was added to the medium.

Table 1.  Bacterial strains and plasmids.
Strain or plasmidRelevant genotype or descriptionSource
  • a. 

    INRA, UMR1319 Micalis, Jouy-en-Josas, France.

  • b. 

    and Kulakauskas S., INRA, UMR1319 Micalis, Jouy-en-Josas, France.

  • c. 

    and Maguin E., INRA, UMR1319 Micalis, Jouy-en-Josas, France.

  • d. 

    Laboratoire de Biotechnologie et Chimie Marines, EA 3884, Université de Bretagne Sud, BP92116, 56321 Lorient, France.

 E. coli  
  TG1E. coli supEΔthi(lac-proAB) hsdD5 (F′+traD36 proAB lacIqZΔM15)Gilson (1984)
 B. subtilis  
  168trpC2Kunst et al. (1997)
  1801trpC2 chr::pJSIZΔpble (Pspac-ftsZ ble)Marston et al. (1998)
 L. lactis  
  IL1403L. lactis ssp. lactis, His-, Iso-, Leu-, Val-Chopin et al. (1984)
  JIM4932IL1403 pJIM2278, EmrRenault et al. (1996)
  JIM7049IL1403 containing nisRK genes integrated at the his locusRenault P.a
  JIM9132Spontaneous IL1403 pbp2x methicillin-resistant mutant, A356V substitutionThis work
  JIM9133Spontaneous IL1403 pbp2x methicillin-resistant mutant, F554V substitutionThis work
  JIM9134Spontaneous IL1403 pbp2x methicillin-resistant mutant, Q569E substitutionThis work
  JIM9162Spontaneous IL1403 pbp2x methicillin-resistant mutant, A588T substitutionThis work
  JIM9163Spontaneous IL1403 pbp2x methicillin-resistant mutant, D419Y substitutionThis work
  JIM9165Spontaneous IL1403 pbp2x methicillin-resistant mutant, A588D substitutionThis work
  JIM9172IL1403 nisRK containing pKFLgfp, EmrThis work
  JIM9173IL1403 pES03, EmrThis work
  JIM9252IL1403 pPbp2xF554V, Emr (pbp2xF554V-overexpressing strain)This work
  JIM9253IL1403 pPbp2xA588D, Emr (pbp2xA588D-overexpressing strain)This work
  MG1363L. lactis ssp. cremoris, His-, Iso-, Leu-, Val-Gasson (1983)
  VES1842MG1363 pbp1A-Mercier et al. (2002)
  VES2054MG1363 pbp1B-Domakova E.b
  VI7146MG1363 pbp2A-Budin-Verneuil A.c
  VI7156MG1363 pbp2B-Budin-Verneuil A.c
  VES2065MG1363 dacB-Courtin et al. (2006)
 S. salivarius  
  JIM8777Oral cavity isolate, France.Delorme et al. (2007)
 pGEM-T easyApr, M13ori pBR322ori, linear T-overhangs vectorPromega
 pKFLgfpEmr, full-length lactococcal ftsK fused to gfpmut1and expressed from PnisALe Bourgeois et al. (2007)
 pES03Emr, gfp expressed from the constitutive Pldh on a lactococcal replicative vectorDufour A.d
 pJIM2278Emr, high-copy-number vectorRenault et al. (1996)
 pPBP2xF554VEmr, pJIM2278 derivative carrying pbp2x gene of L. lactis JIM9133 (pbp2xF554V)This work
 pPBP2xA588DEmr, pJIM2278 derivative carrying pbp2x gene of L. lactis JIM9165 (pbp2xA588D)This work

DNA manipulation procedures and isolation of methicillin-resistant variants of L. lactis

DNA manipulations, sequencing and transformation of E. coli and L. lactis were performed as described previously (Sperandio et al., 2005). Methicillin-resistant isogenic variants of L. lactis IL1403 were isolated upon liquid growth in M17 containing 2.5 µg ml−1 methicillin. After 3 days, about 10% of the cultures were still able to grow, and individual clones were isolated from plating.

Construction of pbp2xF554V and pbp2xA588D-overexpressing plasmids

DNA fragments containing the pbp2x gene of L. lactis IL1403 carrying F55AV and A588D substitution were PCR-amplified from JIM9133 and JIM9165 genomic DNA, respectively, using the high fidelity enzyme Pyrobest (Takara, Japan) and primers EG1136 (5′-GTGCTTGATACCGCGGATAAAGCTG-3′) and EG1126 (5′-AGCTCCACATAGTCTAGAGTCTGTGGA-3′, added restriction site XbaI underlined). The PCR fragments were cut by XbaI and inserted into the high copy vector pJIM2278, digested with EcoRV and PstI. The resulting plasmids were designated pPBP2xF554V and pPBP2xA588D and the IL1403 derivative strains carrying them (JIM9252 and JIM9253 respectively) overexpress the pbp2xF554V and pbp2xA588D genes, respectively, due to amplification on a multicopy plasmid.

Biofilm growth and ex situ SEM

Biofilms were grown in single-channel BST FC81 flow cells (Biosurface Technologies Corporation, Bozeman, MT, USA) as in (Habimana et al., 2009). For SEM, biofilms were cultivated, chemically fixated and dehydrated in the same flow cells (Habimana et al., 2009). The samples were air-dried and then coated with palladium for 210 s at 800 V and 10 mA. Ex situ high magnification imaging of biofilms was performed using a scanning electron microscope Hitachi S-4500.


Fluorescent staining of membranes was done using FM4-64 (Invitrogen) at 0.5 µg ml−1. For live-dead staining, 500 nM of Propidium Iodide (Invitrogen) was added to the culture and cells were incubated 5 min in the dark prior to observation. For Van-FL staining, vancomycin (SIGMA) was mixed 1:1 with fluorescein-labelled vancomycin (Van-FL, Invitrogen) at a final concentration of 0.5 mg ml−1, added to bacterial cultures at OD600 = 0.2 and incubated for 20 min at 30°C. Samples were either visualized directly or fixed for microscopic examination at a later time. For fixation, cells were mixed with 1.5% paraformaldehyde, incubated on ice for 1 h and washed three times in PBS. For FtsK–GFP localization, strain JIM9172 expressing the L. lactis full-length ftsK gene fused to gfpmut1 under control of the inducible PnisA promoter (Le Bourgeois et al., 2007) was grown to OD600 = 0.2. Nisin (20 ng ml−1) was added to the culture and cells were further incubated for 1 h prior to microscopical observation. Cells were visualized with a Leica DMRA2 microscope coupled to a Sony CoolSnap HQ camera (Roper Scientific). Images and videos were acquired and processed with METAMORPH V6.3r7 (Universal Imaging, PA, USA). Transmission electron microscopy was performed as described by Veiga et al. (2006) from samples of L. lactis cultures at OD600 = 0.2.

FLIP experiments

Lactococcus lactis JIM9173 strain carrying the gfp gene under control of the constitutive Pldh promoter was grown to an OD600 between 0.2 and 0.4. Cells were immobilized on agarose (0.05%)-coated slides and observed using a x63-oil objective (NA = 1.4) with a Leica SP2 AOBS confocal microscope. For GFP bleaching, an argon laser (488 nm) was used. Images were captured every 3 s over 315 s in total.


We thank Mariana Pinho, Dusko Ehrlich and Alexandra Gruss for critical reading of the manuscript. We thank Pascal Le Bourgeois and Alain Dufour for the gift of plasmids pKFLgfp and pES03 respectively. We also thank Emmamuelle Maguin and Saulius Kulakauskas for L. lactis pbp mutants, Thierry Meylheuc and Sophie Chat for SEM and TEM observations, respectively, at the MIMA2 microscopy platform (INRA) and Marie-Françoise Noirot-Gros for help with Pymol. This work was supported by the Commission of the European Communities Marie Curie Project LABHEALTH (MEST-CT-2004-514428), ANR (RCL, ANR-08-JCJC-0024–01) the National Foundation for Scientific Research (FNRS) and the Research Department of the Communauté Française de Belgique (Concerted Research Action). P.H. is Research Associate of the FNRS. D.P.N. was recipient of a Marie Curie fellowship for Early Stage Research Training (EST).