Applied and Environmental Microbiology, April 2003, p . 1973-1979, Vol . 69, No . 4

Absence of Malolactic Activity Is a Characteristic of H⁺-ATPase-Deficient Mutants of the Lactic Acid Bacterium Oenococcus oeni

Delphine Galland, Raphaëlle Tourdot-Maréchal,^* Maud Abraham, Ky Son Chu, and Jean Guzzo

Laboratoire de Microbiologie, UMR INRA 1232, Équipe PG2MA, ENSBANA, Université de Bourgogne, 21000 Dijon, France

Received 19 December 2002/ Accepted 8 January 2003

In order to withstand the low external pH values (pH_ex) of environments, bacteria have developed different strategies . When the pH_ex decreases, maintenance of a neutral cytoplasmic pH is essential for survival of the fermentative bacterium Enterococcus faecalis (23) . Many acid-tolerant fermentative bacteria use another strategy: the internal pH (pH_in) decreases as the pH_ex decreases in order to maintain a constant transmembrane pH gradient rather than a constant pH_in (24) .

Whatever the strategy, the most important mechanism by which fermentative bacteria can regulate pH_in is the proton-translocating ATPase . For Enterococcus hirae, this enzyme has been shown previously to be similar to the F₁-F₀ ATPase of aerobic bacteria, which synthesizes ATP coupled with the proton motive force (16, 23) . However, the unique function of the H⁺-ATPase of enterococci is regulation of the cytoplasmic pH (9) . This enzyme is also involved in regulation of pH_in in other lactic acid bacteria (18) . An acid-sensitive variant of Lactobacillus helveticus was isolated as an H⁺-ATPase-deficient strain (27) . More recently, Kullen and Klaenhammer (12) characterized the pH-inducible F₁F₀-ATPase operon of Lactobacillus acidophilus . An increase in atp mRNA was induced by low pH and was correlated with an increase in the activity of the H⁺-ATPase in membrane extracts . Genes encoding F₁F₀-ATPase in Lactococcus lactis have also been cloned and sequenced (11) . Results obtained with a mutant strain in which expression of H⁺-ATPase on the chromosome is under control of the nisA promoter clearly demonstrated a requirement for nisin for growth of the mutant . The H⁺-ATPase was therefore essential for growth of this bacterium . Moreover, the acid sensitivity of a mutant of L . lactis with reduced membrane-bound H⁺-ATPase activity confirmed the major role of this enzyme in regulation of the cytoplasmic pH (1, 28) .

Other mechanisms involving decarboxylase systems are thought to participate in pH_in regulation . Decarboxylation of amino acids may protect bacterial cells against intracellular and extracellular acidification, particularly when the main carbon and energy sources have been consumed (4, 25) . Decarboxylation of carboxylic acids also leads to internal consumption of protons . This is the case for Oenococcus oeni, the lactic acid bacterium responsible for malolactic fermentation in wine . Studies of the energetics of L-malic acid metabolism (5, 19, 21) have demonstrated that malolactic fermentation is an energy-producing pathway based on the electrogenic uptake of L-malate, intracellular decarboxylation of this compound by the malolactic enzyme (MLE), and efflux of the decarboxylation product L-lactic acid . Not only does this metabolic pathway result in the generation of a proton motive force sufficient to drive ATP synthesis via the membrane-bound F₁F₀-ATPase, but the proton consumption during the decarboxylation of L-malate also participates in the regulation of pH_in (22) . We previously cloned and characterized the mleA and mleP genes encoding the MLE and the malate permease, respectively, in O . oeni (13) . These two genes are organized in an mle locus . Upstream of the mleA gene, an open reading frame likely to encode a LysR-type regulatory protein was found (14) . The role of this regulatory protein in malolactic gene expression in O . oeni has not been determined yet . On the other hand, it has been shown that activation of the malolactic system in L . lactis is mediated by mleR (20) . The protein encoded by mleR is homologous to a class of positive regulatory proteins belonging to the LysR family of proteins .

In our laboratory, we isolated spontaneous ATPase-deficient mutants of O . oeni (26) . The acid sensitivity of these mutants suggested a role for H⁺-ATPase in the acid tolerance of O . oeni, like that demonstrated for other lactic acid bacteria . However, all of the mutants isolated lacked malolactic activity, suggesting that there is linkage between the ATPase and malolactic activities in O . oeni . In order to investigate more precisely the nature of the relationship between these two activities, we analyzed the absence of malolactic activity at the molecular level in ATPase-deficient mutant strains of O . oeni . Moreover, the lack of information about the influence of a deficiency in H⁺-ATPase activity on L-malic acid metabolism in other malolactic bacteria led us to isolate ATPase-deficient mutant strains of L . lactis and Leuconostoc mesenteroides by using neomycin resistance as a positive marker . The metabolism of L-malic acid in these mutant strains under optimal or acidic growth conditions was also investigated .

Isolation of neomycin-resistant mutants of L . lactis and L . mesenteroides.
Spontaneous neomycin-resistant mutants of L . lactis and L . mesenteroides were isolated by the method of Yokota et al . (28), with some modifications . Bacteria were grown in 40 ml of MRS medium until the exponential phase was reached (optical density at 600 nm [OD₆₀₀], 0.3) . Cells were harvested by centrifugation, concentrated in 2 ml of fresh medium, and spread onto MRS medium plates containing 300 µg of neomycin sulfate ml^-1 . The plates were then incubated at 30°C for 48 h . Neomycin-resistant colonies were selected and inoculated onto plates containing 5 ml of MRS medium supplemented with 300 µg of neomycin sulfate ml^-1 . A first screening, based on the bacterial growth characteristics, was conducted with MRS medium at pH 6.5 . The specific growth rates were calculated during the exponential phase (i.e., between 10 and 15 h for L . lactis strains and between 5 and 12 h for L . mesenteroides strains) . Biomass concentrations were calculated for an initial OD₆₀₀ of 0.06 after 27 and 38 h of growth for L . lactis and L . mesenteroides, respectively . Cell biomass was deduced based on a preliminary calibration, as follows: 1 OD₆₀₀ unit = 0.4 mg (dry weight) . Specific L-malate consumption was calculated by determining the amount of L-malate metabolized, expressed as millimoles per milligram of biomass at the end of culture .

Preparation of membranes and ATPase activity assay.
Membranes were prepared and ATPase activity assays were performed as described by Tourdot-Maréchal et al . (26) . The methods were adapted to L . lactis and L . mesenteroides as follows: the buffer used to break the cells was Tris-HCl buffer (50 mM Tris, 10 mM MgSO₄; pH 7.0); and ATPase activities were measured in Tris-HCl buffer (pH 7.0) with 6.25 mM ATP . ATPase activity was expressed as micromoles of P_i produced per minute per milligram of protein .

Western immunoblot analysis.
Protein extraction was performed as described by Labarre et al . (15) . Proteins were transferred from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to nitrocellulose membranes (PROTEAN; Schleicher and Schuell) by using a trans-blot semidry transfer cell (Bio-Rad, Richmond, Calif.), and immunoblot analysis was performed with a specific rabbit polyclonal antiserum directed against the 60-kDa MLE protein from O . oeni (15) .

RNA isolation.
O . oeni cells cultured at pH 5.3 were harvested by centrifugation for10 min at 5,000 x g and resuspended in 1 ml of Tri reagent (Sigma) . Cells were mechanically broken with 200 mg of glass beads (diameter, 70 to 110 µm) by using six 40-s periods of homogenization with 30-s intervals between the periods with a FastPrep cell disintegrator (FP120; Instrument Savant; Bio 101) . Samples were then treated as recommended by the manufacturer .

RT-PCR.
RNA samples were treated with RNase-free DNase (1 U µl^-1) as recommended by the manufacturer (Life Technologies, Gibco BRL) . Reverse transcription (RT) was done in a 25-µl mixture containing 4 µl of 5x reverse transcriptase buffer, 2 µl of 0.1 M dithiothreitol, and 4 µl of a preparation containing each deoxynucleoside triphosphate at a concentration of 2.5 mM . The reaction mixture was incubated at 42°C for 2 min . Then 200 U of Escherichia coli SuperScript II Rnase H^- reverse transcriptase (Life Technologies, Gibco BRL) was added . Incubation was continued for 50 min at 42°C and then for 15 min at 70°C for enzyme denaturation . Amplification was performed by using a 50-µl (final volume) reaction mixture containing 50 ng of cDNA, 30 pmol of each primer, each deoxynucleoside triphosphate at a concentration of 2 µM, and 2.5 U of Taq DNA polymerase . Specific cDNA was amplified for 40 cycles consisting of denaturation for 45 s at 92°C, annealing for 45 s at 55°C, and elongation for 1 min at 72°C . The PCR products were analyzed on a 1.6% agarose gel . The primers used for this experiment were 23- and 24-mer oligonucleotides for the mleA gene fragment (mleA probes 60440 [5'-ACC AAA ATG GTC GGG TGG ACA GC-3'] and 43406 [5'-GGA AGA TTT TGG CCG TTC GAA TGC-3']) and 18- and 22-mer oligonucleotides for the mleR-like gene fragment (mleR4 [5'-CGA TAA AAC CAA TCC CGG-3'] and Reg1 [5'-GGT TTG GAA ACA ATT GAA ATC G-3']) . The gene encoding the thioredoxin gene trxA, used as an internal control, was amplified by using 20- and 21-mer oligonucleotides (Trx1 [5'-TTG CCG AAT TTA ACC CTC GA-3'] and Trx5 [5'-AGG AGG AAT TAT ATG GCA AT-3']) .

Other procedures.
Protein concentration was determined by the Bradford method (3) by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, Calif.) with bovine serum albumin as the standard . L-Malic acid concentrations were determined enzymatically by using Boehringer Mannheim kits .

 
Transcriptional analysis of malolactic gene expression.
In order to detect mleA mRNA, RT-PCR experiments were performed . The RT-PCR experiments were performed with total RNA from exponentially growing cells of strains IOB84.13 and 9.01.07 . Probes for mleA were used to amplify a 417-bp DNA fragment of the MLE gene (Fig . 2) . The thioredoxin gene, trxA, was used as a PCR positive control . This gene was detected at a significant level and was estimated to be at roughly the same level during the exponential phase (10) . A DNA strand of the expected length (417 bp) was amplified by PCR from the reverse transcriptase product only for the parental strain (Fig . 2, lane 1) . No amplification was observed for mutant strain 9.01.07 (Fig . 2, lane 3) . The positive results observed for amplification of a 170-bp DNA fragment of the trxA gene (control) from the parental strain and mutant 9.01.07 validated our experiments . A lack of amplification with the mleA probes was also observed with strain 6.27.06 (data not shown) . These data suggest that either there was no transcription or the mleA messenger was more unstable in the two mutants .

 
An RT-PCR analysis was conducted in order to detect mleR mRNA in the parental strain and the H⁺-ATPase-deficient mutants . The RT-PCR experiment was performed with total RNA from exponentially growing cells . Primers mleR4 and Reg1 were used to amplify the 262-bp DNA fragment of the mleR-like gene . A DNA strand of the expected length was amplified only from the parental strain (Fig . 2, lane 2) . No amplification was obtained for mutant strain 9.01.07 (Fig . 2, lane 4) . The same result was obtained with mutant strain 6.27.06 (data not shown) .

According to Labarre et al . (14), the mleR-like gene of O . oeni is transcribed divergently from the mle operon (Fig . 3), and the promoter regions of the mleR gene and the mle operon certainly overlap . To further investigate the lack of putative mleR gene expression in H⁺-ATPase-deficient mutants, the mleR-like gene and the hypothetical region of overlapping promoters were sequenced . No change in the nucleotide sequence of the promoter region and the mleR-like gene was observed . Moreover, the complete mle operon was sequenced for both mutant strains . No mutation was observed (data not shown) . These data led us to conclude that the malolactic deficiency phenotype is not due to a mutation in the malolactic locus in H⁺-ATPase-deficient mutants .

 
Isolation of H⁺-ATPase-deficient mutants of L . lactis and L . mesenteroides.
The effect of a reduction in the H⁺-ATPase activity on L-malate metabolism in other lactic acid bacteria was also investigated . We used L . lactis and L . mesenteroides for this analysis . In L . lactis, the mleS gene encoding the MLE was cloned and sequenced (2, 7), and activation of the malolactic system was shown to be mediated by mleR (20) . Moreover, mutants of L . lactis with reduced membrane-bound ATPase activity were previously obtained by selection for neomycin resistance (1) . In contrast, little information is available about genes implicated in malolactic activity in L . mesenteroides . However, this bacterium is phylogenetically closely related to O . oeni (8), and it is one of the heterofermentative cocci of wines (17) . To isolate spontaneous neomycin-resistant mutants of L . lactis and L . mesenteroides, two sets of cultures containing 2 x 10¹⁰ CFU for each strain were treated with a lethal concentration of neomycin sulfate (300 µg ml^-1) . A total of 1,682 spontaneous neomycin-resistant mutants were obtained from L . lactis with a frequency of 1.9 x 10^-7 . A total of 345 mutants were obtained from L . mesenteroides with a frequency of 4.5 x 10^-6 . Fifty L . lactis colonies and 30 L . mesenteroides colonies were randomly picked and first analyzed to determine their growth profiles under optimal growth conditions . We selected neomycin-resistant mutants of L . lactis and L . mesenteroides whose growth profiles were most affected at pH 6.5 . Mutant 5.16 of L . lactis and mutant 5.15 of L . mesenteroides had the lowest final biomass (35 and 44% of the wild-type final biomasses, respectively) (Table 1) . The membrane-bound ATPase activities of the mutants were also determined . As shown in Table 1, the membrane-bound ATPase activities of mutant strain 5.16 (L . lactis) and mutant strain 5.15 (L . mesenteroides) were reduced to 54 and 70%, respectively, of the activities of the parental strains .

 
Growth of ATPase-deficient mutants of L . lactis and L . mesenteroides under acidic conditions.
The parental strains, the L . lactis mutant, and the L . mesenteroides mutant were cultured in MRS medium with an initial pH of 5.0 for L . lactis and an initial pH of 4.5 for L . mesenteroides . Under these conditions, the specific growth rates of the L . lactis and L . mesenteroides parental strains were 0.16 and 0.12 h^-1, respectively, compared with specific growth rates of 0.46 and 0.45 h^-1, respectively, at pH 6.5 (Fig . 4) . The 5.16 mutant strain of L . lactis did not grow at pH 5.0 . Mutant strain 5.15 of L . mesenteroides had an 85% lower specific growth rate than the parental strain when the organisms were cultured at pH 4.5 . A significant decrease (43%) in the final biomass obtained after 38 h of growth was also observed (Fig . 4B) .

 
L-Malate metabolism in H⁺-ATPase-deficient mutants of L . lactis and L . mesenteroides.
The residual malate was assayed enzymatically after growth on MRS medium under optimal growth conditions or under acidic conditions (Table 2) . At pH 6.5, the ATPase-deficient mutants of L . lactis and L . mesenteroides and the wild-type strains used approximately 90% of the 37 mM L-malic acid initially present in MRS medium . Moreover, the H⁺-ATPase-deficient mutants exhibited greater consumption of L-malate (expressed as millimoles of L-malate consumed per milligram of final biomass) than the parental strains exhibited . The L . lactis and L . mesenteroides mutants consumed three and two times more L-malate than the wild-type strains consumed, respectively .

 
Similar measurements were obtained after growth under acidic conditions (Table 2) . At pH 5.0, the wild-type strain of L . lactis metabolized 90% of the L-malate, whereas for the ATPase-deficient mutant, in spite of an absence of significant growth, 50% of the L-malate was still present in MRS medium after 27 h of culture . At pH 4.5, the wild-type strain of L . mesenteroides metabolized 90% of the L-malate, compared with 70% for the ATPase-deficient mutant, after 38 h of growth . Compared with the results obtained at pH 6.5, the consumption of L-malate increased four- and fivefold for the L . lactis and L . mesenteroides wild-type strains, respectively . The same results were obtained with the ATPase-deficient mutant strains . Moreover, for the mutant strains of L . lactis and L . mesenteroides, consumption of L-malate increased 3.5- and 1.5-fold, respectively, compared with consumption by the wild-type strains when the organisms were cultured under acidic conditions .

These results show that the ATPase-deficient mutants of L . lactis and L . mesenteroides metabolized L-malic acid, in contrast to the ATPase-deficient mutant of O . oeni . Moreover, the consumption of L-malate was greater than that calculated for wild-type strains . In the same way, under acidic growth conditions the L-malate consumption by the parental strains and also by the ATPase-deficient mutants clearly increased .

It has been proven that in L . lactis induction of the genes necessary to perform malolactic fermentation occurs only in bacteria with a functional copy of mleR . This gene encodes a LysR-type regulatory protein that acts as a positive regulator of the expression of the mleA gene (20) . The mleR gene of L . lactis is not clustered with the mleA gene . In contrast, in O . oeni, an mleR-like gene was previously found upstream of the mle operon . This gene is transcribed divergently from the operon (14) . The translation initiation sites of mleR and mleA are very close to each other, and the promoter region of the mleR gene and the mle operon certainly overlap (14) . The role of mleR in malolactic gene expression in O . oeni remains unknown . Results obtained in this study show that the mleR-like transcript was not detected by RT-PCR in H⁺-ATPase-deficient mutants . Moreover, sequencing data proved that there is no mutation in the mleR-like gene and promoter region or in the mle operon in the H⁺-ATPase-deficient mutants . Taken together, these results are in accordance with positive regulation of mle operon expression by the mleR-like gene in O . oeni, like the situation in L . lactis . Further genetics experiments are needed to confirm the role of the mleR gene in mle operon regulation . Disruption of the mleR gene in O . oeni would be of great interest and would certainly allow us to understand regulation of malolactic gene expression . Unfortunately, DNA transfer techniques are only at the experimental stage at this time for this type of genetic approach with O . oeni . Mainly for this reason we chose to investigate L-malate metabolism in H⁺-ATPase-deficient mutants in another lactic acid bacterium, L . lactis . The MLE of this bacterium and the MLE of O . oeni are encoded by similar genes . Spontaneous neomycin-resistant mutants of L . lactis with reduced membrane-bound ATPase activity were obtained previously (1, 28) . We likewise chose L . mesenteroides . This bacterium, in contrast to L . lactis, is one of the heterofermentative cocci of wines (17), and it is phylogenetically closely related to O . oeni .

First, investigations were carried out to select neomycin-resistant mutants of L . lactis and L . mesenteroides on the basis of their growth profiles under optimal conditions . Selected mutants were found to be acid sensitive and had ATPase activities that were significantly reduced . Similar results were obtained previously with spontaneous neomycin-resistant mutants of O . oeni that were acid sensitive and exhibited twofold-reduced H⁺-ATPase activity (26) . The acid sensitivity of the ATPase-deficient mutants of L . lactis and L . mesenteroides confirms the suggestion that in lactic acid bacteria, the major role of the H⁺-ATPase is maintenance of pH_in (1, 27) .

Previous data indicated that there is a relationship between a reduction in ATPase activity and a lack of malolactic activity in O . oeni (26) . This study showed that there were unexpected increases in L-malate consumption in H⁺-ATPase-deficient mutants of L . lactis and L . mesenteroides under both optimal and acidic growth conditions compared to the metabolism of the parental strains . In contrast to ATPase-deficient mutants of O . oeni, activation of L-malate metabolism for pH_in homeostasis could be a compensatory consequence of the H⁺-ATPase deficiency in these mutants .

In conclusion, the relationship between the H⁺-ATPase deficiency and the lack of expression of the malolactic system seems to be a characteristic of mutants of O . oeni . It has been shown previously that expression of the MLE in O . oeni is not effectively modified by the presence of L-malate in the medium (14) . In contrast, the malolactic activity in L . lactis, like that in L . mesenteroides, is inducible by L-malate (15) . It appears that in O . oeni regulation of the expression of the malolactic operon may involve another regulatory factor that is presumably linked to the metabolic energy . Determination of the amount of ATP in relation to the level of transcription of malolactic genes should certainly help us elucidate the link between the ATPase and malolactic activities in O . oeni .

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