SummaryBacteria 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.

Until now, peptidoglycan O-acetyl transferases (Oat) were only described for their peptidoglycan O-acetylating activity and for their implication in the control of peptidoglycan hydrolases. In this study, we show that a Lactobacillus plantarum mutant lacking OatA is unable to uncouple cell elongation and septation. Wild-type cells showed an elongation arrest during septation while oatA mutant cells continued to elongate at a constant rate without any observable pause during the cell division process. Remarkably, this defect does not result from a default in peptidoglycan O-acetylation, since it can be rescued by wild-type OatA as well as by a catalytic mutant or a truncated variant containing only the transmembrane domain of the protein. Consistent with a potential involvement in division, OatA preferentially localizes at mid-cell before membrane invagination and remains at this position until the end of septation. Overexpression of oatA or its inactive variants induces septation-specific aberrations, including asymmetrical and dual septum formation. Overproduction of the division inhibitors, MinC or MinD, leads to cell filamentation in the wild type while curved and branched cells are observed in the oatA mutant, suggesting that the Min system acts differently on the division process in the absence of OatA. Altogether, the results suggest that OatA plays a key role in the spatio-temporal control of septation, irrespective of its catalytic activity.

Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species.IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Lactococcus lactis is an ovoid bacterium that forms filaments during planktonic and biofilm lifestyles by uncoupling cell division from cell elongation. In this work, we investigate the role of the leading peptidoglycan synthase PBP2b that is dedicated to cell elongation in ovococci. We show that the localization of a fluorescent derivative of PBP2b remains associated to the septal region and superimposed with structural changes of FtsZ during both vegetative growth and filamentation indicating that PBP2b remains intimately associated to the division machinery during the whole cell cycle. In addition, we show that PBP2b-negative cells of L. lactis are not only defective in peripheral growth; they are also affected in septum positioning. This septation defect does not simply result from the absence of the protein in the cell growth machinery since it is also observed when PBP2b-deficient cells are complemented by a catalytically inactive variant of PBP2b. Finally, we show that round cells resulting from β-lactam treatment are not altered in septation, suggesting that shape elongation as such is not a major determinant for selection of the division site. Altogether, we propose that the specific PBP2b transpeptidase activity at the septum plays an important role for tagging future division sites during L. lactis cell cycle.

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal D-Ala-ended precursors were partially or totally replaced by D-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of D-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.Peptidoglycan (or murein) is a major stress-bearing component of the bacterial cell wall. This polymer is made of glycan chains interconnected by covalent cross-links between short peptides. It acts as an exoskeleton and plays a key role in determining and maintaining cell shape throughout the cell cycle.