Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism.

AIMS: Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. METHODS AND RESULTS: A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis. CONCLUSIONS: The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.

An improved protocol for electroporation of Oenococcus oeni ATCC BAA-1163 using ethanol as immediate membrane fluidizing agent.

AIMS: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain. METHODS AND RESULTS: The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5.8 x 10(3) per microg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 125 kV cm(-1), under a resistance of 200 omega and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). CONCLUSIONS: An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA-1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a successful electroporation of O. oeni ATCC BAA-1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.

The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas.

AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated. METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents. Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV. The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7. CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc. mesenteroides physiology. The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc. mesenteroides in the fermentation process.

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase.

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

The Oenococcus oeni clpX homologue is a heat shock gene preferentially expressed in exponential growth phase.

Using degenerated primers from conserved regions of previously studied clpX gene products, we cloned the clpX gene of the malolactic bacterium Oenococcus oeni. The clpX gene was sequenced, and the deduced protein of 413 amino acids (predicted molecular mass of 45,650 Da) was highly similar to previously analyzed clpX gene products from other organisms. An open reading frame located upstream of the clpX gene was identified as the tig gene by similarity of its predicted product to other bacterial trigger factors. ClpX was purified by using a maltose binding protein fusion system and was shown to possess an ATPase activity. Northern analyses indicated the presence of two independent 1.6-kb monocistronic clpX and tig mRNAs and also showed an increase in clpX mRNA amount after a temperature shift from 30 to 42 degrees C. The clpX transcript is abundant in the early exponential growth phase and progressively declines to undetectable levels in the stationary phase. Thus, unlike hsp18, the gene encoding one of the major small heat shock proteins of Oenococcus oeni, clpX expression is related to the exponential growth phase and requires de novo protein synthesis. Primer extension analysis identified the 5' end of clpX mRNA which is located 408 nucleotides upstream of a putative AUA start codon. The putative transcription start site allowed identification of a predicted promoter sequence with a high similarity to the consensus sequence found in the housekeeping gene promoter of gram-positive bacteria as well as Escherichia coli.

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster.

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the alpha subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.

Molecular characterization of the gene encoding an 18-kilodalton small heat shock protein associated with the membrane of Leuconostoc oenos.

In Leuconostoc oenos, different stresses such as heat, ethanol, and acid shocks dramatically induce the expression of an 18-kDa small heat shock protein called Lo 18. The corresponding gene (hsp18) was cloned from a genomic library of L. oenos constructed in Escherichia coli. A 2.3-kb DNA fragment carrying the hsp18 gene was sequenced. The hsp18 gene encodes a polypeptide of 148 amino acids with a calculated molecular mass of 16,938 Da. The Lo18 protein has a significant identity with small heat shock proteins of the alpha-crystallin family. The transcriptional start site was determined by primer extension. This experiment allowed us to identify the promoter region exhibiting high similarity to consensus promoter sequences of gram-positive bacteria, as well as E. coli. Northern blot analysis showed that hsp18 consists of a unique transcription unit of 0.6 kb. Moreover, hsp18 expression seemed to be controlled at the transcriptional level. This small heat shock protein was found to be peripherally associated with the membrane of L. oenos.

D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus. II. Mutagenic analysis of catalytically important residues.

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos.

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum.

Most bacteria synthesize muramyl-pentapeptide peptidoglycan precursors ending with a D-alanyl residue (e.g., UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala). However, it was recently demonstrated that other types of precursors, notably D-lactate-ending molecules, could be synthesized by several lactic acid bacteria. This particular feature leads to vancomycin resistance. Vancomycin is a glycopeptide antibiotic that blocks cell wall synthesis by the formation of a complex with the extremity of peptidoglycan precursors. Substitution of the terminal D-alanine by D-lactate reduces the affinity of the antibiotic for its target. Lactobacillus plantarum is a lactic acid bacterium naturally resistant to vancomycin. It converts most of the glycolytic pyruvate to L- and D-lactate by using stereospecific enzymes designated L- and D-lactate dehydrogenases, respectively. In the present study, we show that L. plantarum actually synthesizes D-lactate-ending peptidoglycan precursors. We also report the construction of a strain which is deficient for both D- and L-lactate dehydrogenase activities and which produces only trace amounts of D- and L-lactate. As a consequence, the peptidoglycan synthesis pathway is drastically affected. The wild-type precursor is still present, but a new type of D-alanine-ending precursor is also synthesized in large quantities, which results in a highly enhanced sensitivity to vancomycin.

Pediococcus acidilactici ldhD gene: cloning, nucleotide sequence, and transcriptional analysis.

The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone.

Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene.

Recombinant plasmids containing the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(+)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAAT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici.

NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization.

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon.

Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression.

The genuine alpha-amylase gene from Bacillus licheniformis (amyL) is not expressed in Lactobacillus plantarum, but replacement of the amyL promoter by a strong L. plantarum promoter leads to efficient expression of the gene and secretion of more than 90% of the alpha-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (transcription and translation with or without secretion) were cloned by translation fusion of random DNA fragments to the silent amyL coding frame in the pGIP212 probe vector (P. Hols, A. Baulard, D. Garmyn, B. Delplace, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faecalis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated from L. plantarum led to a low level of amylase production in E. faecalis, suggesting that the L. plantarum signals are more species specific. Six transcriptional or translational fusions were constructed to express the Bacillus subtilis levanase gene (sacC) in L. plantarum. All of these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chromosomal integration procedure was used to achieve efficient stabilization of an amylase construction without any residual resistance marker or vector sequence.

Lactobacillus plantarum ldhL gene: overexpression and deletion.

Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.

Isolation and characterization of genetic expression and secretion signals from Enterococcus faecalis through the use of broad-host-range alpha-amylase probe vectors.

We have constructed two broad-host-range Gram+/Gram- probe vectors designed for the cloning of bacterial genetic expression and secretion signals. These vectors make use of a silent reporter gene encoding the mature alpha-amylase from Bacillus licheniformis whose reactivation can easily be monitored on iodine-stained starch plates. Shotgun cloning of Enterococcus faecalis DNA fragments allowed recovery of several cassettes directing transcription, translation of the reporter gene and secretion of alpha-amylase. Sequence analysis revealed, in each case, the presence of a putative promoter, ribosome-binding site and signal peptide similar to those described in other Gram+ bacteria.

Cloning of the D-lactate dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by complementation in Escherichia coli.

A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive cloned showed LDH activity and production of D(-)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).