Sylviane Furlan

Factors Affecting Exocellular Polysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus Grown in a Chemically Defined Medium

ABSTRACT We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains ofLactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp.bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricusCNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricusstrains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.

Cell Surface of Lactococcus lactis Is Covered by a Protective Polysaccharide Pellicle

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

SpxB regulates O-acetylation-dependent resistance of Lactococcus lactis peptidoglycan to hydrolysis

Endogenous peptidoglycan (PG)-hydrolyzing enzymes, the autolysins, are needed to relax the rigid PG sacculus to allow bacterial cell growth and separation. PGs of pathogens and commensal bacteria may also be degraded by hydrolases of animal origin (lysozymes), which act as antimicrobials. The genetic mechanisms regulating PG resistance to hydrolytic degradation were dissected in the Gram-positive bacterium Lactococcus lactis. We found that the ability of L. lactis to counteract PG hydrolysis depends on the degree of acetylation. Overexpression of PG O-acetylase (encoded by oatA) led to bacterial growth arrest, indicating the potential lethality of oatA and a need for its tight regulation. A novel regulatory factor, SpxB (previously denoted as YneH), exerted a positive effect on oatA expression. Our results indicate that SpxB binding to RNA polymerase constitutes a previously missing link in the multistep response to cell envelope stress, provoked by PG hydrolysis with lysozyme. We suggest that the two-component system CesSR responds to this stress by inducing SpxB, thus favoring its interactions with RNA polymerase. Induction of PG O-acetylation by this cascade renders it resistant to hydrolysis.

Comparison of the thickening properties of four Lactobacillus delbrueckii subsp. bulgaricus strains and physicochemical characterization of their exopolysaccharides.

It is now well established that physicochemical properties of exopolysaccharides (EPS) can vary between strains of a given species and according to growth conditions. The EPS production of four strains of Lactobacillus bulgaricus was monitored during growth in milk and in a chemically defined media. All strains, including the non-ropy one, produced EPS. The monosaccharide composition, molar mass (M(w)), and intrinsic viscosity of these EPS were determined and compared. Further characterization using high-performance size-exclusion chromatography revealed the presence of two fractions in all EPS: one fraction exhibited a high M(w) and a high intrinsic viscosity while the other had a low M(w) and a low intrinsic viscosity. Strikingly, the EPS synthesized by the non-ropy strain was mainly composed of the low-M(w) fraction while for the ropy strains, the fraction of high M(w) varied between 43 and 90%. According to our results, we propose that the ratio between the high-M(w) and low-M(w) fractions is critical for the texturing properties of L. bulgaricus EPS.

Identification of an essential gene responsible for d‐Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge

Bacteria such as Lactococcus lactis have d‐aspartate (d‐Asp) or its amidated derivative d‐asparagine (d‐Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing d‐amino acids in their PG crossbridge, but absent from those that instead insert l‐amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the PG crossbridge. Our results show that (i) yxbA encodes d‐Asp ligase responsible for incorporation of d‐Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of d‐Asp may be complemented by l‐Ser‐l‐Ala or l‐Ala‐l‐Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side‐chain residues, and (iv) lactococcal strains having l‐amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG side‐chain composition.

Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.

Nutritional value of the non-protein N that accumulates during growth of proteinase-positive strains of Lactococcus lactis in milk for dairy lactococcal and leuconostoc isolates

To estimate the suitability of cultured milk for the subsequent growth of dairy lactic acid bacteria in cheese manufacturing, control milk was first cultured until the end of the exponential phase with one of four proteolytic (Prt + ) strains of Lactococcus lactis differing in their proteolytic enzymes, and pasteurized after readjusting the pH to its initial value. Nineteen non-proteolytic (Prt − ) strains of Lc. lactis and nine Leuconostoc mesenteroides strains of dairy origin were then grown in the precultured milk until the stationary phase and their growth was compared with that in control milk. Despite the accumulation of non-protein N (NPN) during preculture, the growth of most Prt − strains of Lc. lactis in precultured milk was either reduced or unchanged whereas that of Ln. mesenteroides strains was unchanged or slightly stimulated. This reduction in growth was reversed by adding an NPN source to precultured milk, indicating that it was due to the exhaustion of assimilable NPN in precultured milk. Thus, preculturing milk with Prt + strains of Lc. lactis could not be recommended for promoting the subsequent growth of starter cultures in cheese manufacturing.

Regulation of cell wall plasticity by nucleotide metabolism in Lactococcus lactis

To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of l-aspartate (l-Asp) to N-carbamoyl-l-aspartate by PyrB may reduce the amount of l-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of l-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that l-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.