[Show abstract][Hide abstract] ABSTRACT: Manganese serves an important function in Lactobacillus plantarum in protection against oxidative stress and this bacterium can accumulate Mn(2+) up to millimolar levels intracellularly. Although the physiological role of Mn(2+) and the uptake of this metal ion have been well documented, the only uptake system described so far for this bacterium is the Mn(2+)- and Cd(2+)-specific P-type ATPase (MntA). Recently, the genome of L. plantarum WCFS1 has been sequenced allowing in silico detection of genes potentially encoding Mn(2+) transport systems, using established microbial Mn(2+) transporters as the query sequence. This genome analysis revealed that L. plantarum WCFS1 encodes, besides the previously described mntA gene, an ABC transport system (mtsCBA) and three genes encoding Nramp transporters (mntH1, mntH2 and mntH3). The expression of three (mtsCBA, mntH1 and mntH2) of the five transport systems was specifically derepressed or induced upon Mn(2+) limitation, supporting their role in Mn(2+) homeostasis in L. plantarum. However, in contrast to previous reports, mntA expression remains below detection levels in both Northern and real-time RT-PCR analysis in both Mn(2+) excess and starvation conditions. Growth of WCFS1 derivatives mutated in mntA, mtsA or mntH2, or both mtsA and mntH2 appears unaffected under Mn(2+) excess or Mn(2+) limitation. Moreover, intracellular Mn(2+) concentrations remained unaltered in these mutants compared to the wild-type. This may suggest that this species is highly adaptive in response to inactivation of these genes or, alternatively, that other transporters that have not yet been identified as Mn(2+) transporters in bacteria are involved in Mn(2+) homeostasis in L. plantarum.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the genes encoding enzymes responsible for the incorporation of D-Ala into the cell wall of Lactococcus lactis affect autolysis. An L. lactis alanine racemase (alr) mutant is strictly dependent on an external supply of D-Ala to be able to synthesize peptidoglycan and to incorporate D-Ala in the lipoteichoic acids (LTA). The mutant lyses rapidly when D-Ala is removed at mid-exponential growth. AcmA, the major lactococcal autolysin, is partially involved in the increased lysis since an alr acmA double mutant still lyses, albeit to a lesser extent. To investigate the role of D-Ala on LTA in the increased cell lysis, a dltD mutant of L. lactis was investigated, since this mutant is only affected in the D-alanylation of LTA and not the synthesis of peptidoglycan. Mutation of dltD results in increased lysis, showing that D-alanylation of LTA also influences autolysis. Since a dltD acmA double mutant does not lyse, the lysis of the dltD mutant is totally AcmA dependent. Zymographic analysis shows that no degradation of AcmA takes place in the dltD mutant, whereas AcmA is degraded by the extracellular protease HtrA in the wild-type strain. In L. lactis, LTA has been shown to be involved in controlled (directed) binding of AcmA. LTA lacking D-Ala has been reported in other bacterial species to have an improved capacity for autolysin binding. Mutation of dltD in L. lactis, however, does not affect peptidoglycan binding of AcmA; neither the amount of AcmA binding to the cells nor the binding to specific loci is altered. In conclusion, D-Ala depletion of the cell wall causes lysis by two distinct mechanisms. First, it results in an altered peptidoglycan that is more susceptible to lysis by AcmA and also by other factors, e.g., one or more of the other (putative) cell wall hydrolases expressed by L. lactis. Second, reduced amounts of D-Ala on LTA result in decreased degradation of AcmA by HtrA, which results in increased lytic activity.
Journal of Bacteriology 02/2005; 187(1):114-24. DOI:10.1128/JB.187.1.114-124.2005 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.
[Show abstract][Hide abstract] ABSTRACT: The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen. Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract. Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers. Recombinant wild-type and mutant strains of L. plantarum NCIMB8826 and L. lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments. Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used. However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L. plantarum mutant strain. Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract. The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.
Infection and Immunity 06/2004; 72(5):2731-7. DOI:10.1128/IAI.72.5.2731-2737.2004 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have investigated the effect of overproducing each of the three cold shock proteins (CspL, CspP, and CspC) in the mesophilic lactic acid bacterium Lactobacillus plantarum NC8. CspL overproduction transiently alleviated the reduction in growth rate triggered by exposing exponentially growing cells to cold shock (8 degrees C), suggesting that CspL is involved in cold adaptation. The strain overproducing CspC resumed growth more rapidly when stationary-phase cultures were diluted into fresh medium, indicating a role in the adaptation and recovery of nutritionally deprived cells. Overproduction of CspP led to an enhanced capacity to survive freezing.
[Show abstract][Hide abstract] ABSTRACT: Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 184.108.40.206), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.
[Show abstract][Hide abstract] ABSTRACT: Fragments of the cspL promoter region were fused to the gusA reporter and reintroduced into Lactobacillus plantarum cells, either on multicopy plasmids or through single-copy chromosomal integration. beta-Glucuronidase activity and primer extension data demonstrate that the cspL promoter is induced in response to cold shock and that multicopy constructs quench the induction of the resident cspL gene.
Journal of Bacteriology 11/2002; 184(19):5518-23. DOI:10.1128/JB.184.19.5518-5523.2002 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus helveticus is used as starter in Swiss cheese manufacture. To characterize the technological behavior of lysogenic L. helveticus, 21 strains were screened for prophage inducibility using mitomycin C. Eight out of the 11 inducible strains, i.e. CNRZ 32, CNRZ 243, CNRZ 303, IL1, T649, T650, T651, T653, were assayed in experimental Swiss cheeses, using the same Streptococcus thermophilus and Propionibacterium freudenreichii industrial starters. Six strains, CNRZ 32, CNRZ 243, CNRZ 303, T649, T650, and T651 grew well during pressing, exhausting galactose as expected. They lysed then extensively and early during the ripening as shown by the detection of d-Lactate dehydrogenase in the cheese aqueous extracts. Phages were detected at day 1 in four cases out of six, indicating a spontaneous release in situ. Except CNRZ 32, renaturing SDS-PAGE of the cheese extracts revealed the presence of a lytic activity (30kDa). For strains IL1 and T653, growth was not sufficient during pressing leading to a higher pH (5.4 versus 5.2) and to residual galactose (about 600mg/100g) that had a strong impact on the subsequent growth of propionibacteria and non-starter lactic acid bacteria. No free d-Lactate dehydrogenase, no phages and no lytic activity were detected at day 1. Both strains lysed later between days 20 and 36 concomitantly with galactose exhaustion.Interestingly, decrease of viability and lysis of S. thermophilus during the ripening were observed and were correlated to those of the L. helveticus strain, except for CNRZ 32. Thus, lysis of L. helveticus can seemingly induce S. thermophilus death and lysis during ripening. Such an in situ interaction, whatever the mechanism involved, was further supported by scanning electron microscopy showing large mixed colonies of L. helveticus and S. thermophilus.
International Dairy Journal 01/2002; 12(7):591-600. DOI:10.1016/S0958-6946(02)00051-1 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.
[Show abstract][Hide abstract] ABSTRACT: Molecular genetics of thermophilic lactic acid bacteria has advanced in several directions: exploitation of the milk proteins and sugars; primary and secondary metabolism; stress response; and molecular ecology of bacteria and their phages. These have singularly contributed to open new avenues of scientific interest in the field: comparative phage genomics; horizontal gene transfer events in bacterial or phage populations; and genetics of external polysaccharide production.
Current Opinion in Biotechnology 11/2000; 11(5):497-504. DOI:10.1016/S0958-1669(00)00134-8 · 8.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and alpha-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via alpha-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.
[Show abstract][Hide abstract] ABSTRACT: An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.
Journal of Bacteriology 10/2000; 182(18):5105-13. DOI:10.1128/JB.182.18.5105-5113.2000 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cell wall of lactic acid bacteria has the typical gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attributes of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.
Antonie van Leeuwenhoek 10/1999; 76(1-4):159-84. DOI:10.1023/A:1002089722581 · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-(13)C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase.
Journal of Bacteriology 10/1999; 181(17):5521-6. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.
[Show abstract][Hide abstract] ABSTRACT: The intracellular expression of the B subunit of cholera toxin (CTB) was first achieved in Lactobacillus paracasei LbTGS1.4 with an expression cassette including the P25 promoter of Streptococcus thermophilus combined with the translation initiation region from the strongly expressed L. pentosus D-lactate dehydrogenase gene (ldhD). Secretion of CTB was next attempted in L. paracasei LbTGS1.4 and L. plantarum NCIMB8826 with four different signal sequences from exported proteins of lactic acid bacteria (Lactococcus lactis Usp45 and PrtP, Enterococcus faecalis unknown protein and S. pyogenes M6 protein). Host-dependent secretion of CTB was clearly observed: whereas none of the secretion cassettes led to detectable CTB in the extracellular fraction of L. paracasei LbTGS1.4, secretion of CTB molecules was clearly achieved with three of the selected signal sequences in L. plantarum NCIMB8826.
[Show abstract][Hide abstract] ABSTRACT: Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an alpha-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg l-1.
[Show abstract][Hide abstract] ABSTRACT: The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.
Journal of Bacteriology 07/1997; 179(11):3804-7. · 2.69 Impact Factor