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Lactobacillus casei can metabolize l-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of l-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on l-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for l-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on l-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of l-malic acid and repressed by glucose, whereas TC system expression was induced by l-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5′-TTATT(A/T)AA-3′] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.
The metabolism of l-malic acid by lactic acid bacteria (LAB) has brought about considerable interest because of its relevance in winemaking (24). The degradation of l-malate to l-lactate leads to a reduction of the acidity of wine, and it provides microbiological stability by preventing the secondary growth of LAB after bottling. Most LAB decarboxylate l-malate to l-lactate by an NAD+- and Mn2+-dependent malolactic enzyme (MLE) (Fig. (Fig.1);1); nevertheless, a few LAB species can also degrade l-malate to pyruvate by a malic enzyme (ME) (Fig. (Fig.1).1). This pathway was first detected in Enterococcus faecalis (20) and later in Lactobacillus casei (23, 33) and Streptococcus bovis (14). In contrast to the utilization of l-malate through MLE, the utilization of the ME pathway enables these organisms to grow with l-malate as a carbon source (22). However, whereas MLE has been the focus of an extensive research effort, the physiological role and the regulation of ME remain largely unknown.
L. casei is a facultative heterofermentative lactic acid bacterium frequently used as a cheese starter culture and which is also employed as a probiotic. Extensive research has been carried out on the study of sugar catabolism (28, 39-41); however, the knowledge of the utilization of organic acids has received less attention. As previously indicated, physiological and biochemical studies identified two l-malate dissimilation pathways in L. casei. Furthermore, these studies showed that ME expression was induced in the presence of l-malate and very low concentrations of glucose (4, 22). However, the regulatory system controlling ME expression was not identified. In other bacteria such as Bacillus subtilis (36) or Escherichia coli (11), utilization of some organic acids such as citrate, succinate, or malate is regulated by two-component (TC) systems. TC regulatory systems typically consist of a sensor kinase (histidine kinase [HK]) and a response regulator (RR) (34). Both proteins have a modular structure: HKs usually have two modules involved in the phosphorylation reaction, the kinase and H-box domains, and usually an N-terminal transmembrane sensory domain. On the other hand, the RR has a receptor domain of the phosphoryl group and a C-terminal effector domain. The domains involved in the phosphorylation reaction of both HKs and RRs are homologous in all TC systems (16), whereas the sensor and effector domains are specific to individual TC systems and determine their specificity. HKs monitor environmental signals and in response to a stimulus, autophosphorylates at a histidine residue (H-box). The high-energy phosphate group is subsequently transferred to an aspartyl residue on the RR receptor domain. Phosphorylation of the RR in turn modulates the activity of the RR effector domain. In many cases, the RR receptor domains are DNA-binding domains so that RR acts as transcriptional repressors or activators.
Our research group has initiated two studies focused on the physiological role of TC systems and the metabolism of organic acids in L. casei. In this work, we have identified in L. casei a gene cluster constituted by two putative operons. One of them encodes a TC system similar to other TC systems involved in the regulation of the utilization of organic acids in other bacteria. The other operon encodes a putative ME and an l-malate transporter. This work aimed to determine the possible regulatory role of the cognate TC system in the expression of ME. Results reported here show that this gene cluster accounts for l-malate utilization via the ME pathway and that their expression is under the control of the cognate TC system.
The strains and plasmids used in this study are listed in Table Table1.1. L. casei was routinely grown in MRS broth (Oxoid) at 37°C under static conditions. Escherichia coli DH5α strains were grown in LB medium at 37°C with aeration. The antibiotics used were 100 μg ml−1 ampicillin and 25 μg ml−1 kanamycin for E. coli and 5 μg ml−1 erythromycin for L. casei.
Growth assays, determination of l-malic acid degradation, and gene expression analyses were carried out at 30°C in malic enzyme induction (MEI) medium (33), modified as follows: tryptone replaced peptone, the medium was supplemented with cysteine at 0.5 g liter−1 and Tween 80 at 1 ml liter−1, and the pH was adjusted to 6.8. When required, filter-sterilized glucose or l-malate (adjusted to pH 6.8 with NaOH) was added at a final concentration of 5 g liter−1. Inoculation was performed with cells grown in MRS broth for 16 h and washed twice with 1 volume of sterile distilled water. Growth was monitored by measuring the optical density at 595 nm (OD595). At least three independent replicates of each growth curve were obtained. Results were expressed as averages ± standard deviations (SD).
Samples of cultures grown in MEI medium were taken at different times during growth. The samples were centrifuged, and the supernatant was filtered through 0.22-μm-pore-size Millex-GV syringe-driven filter units (Millipore) and stored at −80°C until use. Samples were analyzed using high-performance liquid chromatography (HPLC) equipment (Agilent series 1200) with an isocratic pump (Agilent G1310A) following the procedure described by Frayne (9) with minor modifications. The mobile phase consisted of a solution of 0.75 ml of 85% H3PO4 per liter of deionized water with a flow rate of 0.7 ml min−1. An Agilent G1322A degasser was employed. Samples (5 μl) were injected automatically (Agilent G1367B). The separation of the components was carried out using an Aminex HPX-87H precolumn (Bio-Rad) coupled with two ion exclusion columns of 300 mm by 7.8 mm Aminex HPX-87H (Bio-Rad) thermostatically controlled at 65°C (Agilent G1316A). Compounds were detected by a G1314B variable-wavelength detector (Agilent) set to 210 nm and a refractive index detector (Agilent G1362A) in series. External calibration was performed.
Bacterial genes encoding homologues of the products of maeK and maeR were retrieved from whole genomes by using BLASTP and TBLASTN (2, 3) and the genes carried by L. casei BL23 as query sequences. Representative species harboring maeK and maeR homologues of all taxonomic divisions were selected. Some sequences were modified as follows. A possible frameshift in the putative maeK homolog sequence of Carboxydothermus hydrogenoformans Z-2901 (GenBank accession no. NC_007503) was corrected by deleting one A at position 1227250. A possible frameshift in a Bacillus anthracis Ames ancestor maeR homolog (banth3; see Fig. S1 in the supplemental material) was corrected by inserting an A after position 568015 in the genomic sequence (accession no. NC_007530). Multiple alignments were obtained using ClustalW (37) and manually corrected where necessary. Positions of doubtful homology or that introduced phylogenetic noise were removed by using Gblocks software (6). The best-fit models of amino acid substitution were selected using the program ProtTest (1). The Akaike information criterion (AIC) was adopted to select the best model, which was “LG” (17), with a discrete gamma function with four categories plus invariant sites to account for substitution rate heterogeneity among sites for both protein sets. The selected model was implemented in PHYML 3.0 (12) to obtain maximum likelihood trees for the different alignments. Bootstrap support values were obtained from 500 pseudoreplicates.
Standard methods were used for cloning in E. coli (31). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. Taq DNA polymerase for PCR screening was from Biotools (B & M Labs, Madrid, Spain). Plasmids were isolated with the GFX Micro plasmid prep kit (GE Healthcare). DNA from L. casei was isolated with the UltraClean microbial DNA isolation kit (MoBio Laboratories, Solana Beach, CA). Southern hybridization analyses were carried out by transfer of DNA from agarose gels to Hybond-N membranes (GE Healthcare) (31). Probes were labeled with digoxigenin (DIG)-dUTP by using PCR DIG labeling mix (Roche) in standard PCRs and detected by using antidigoxigenin-AP and CDP-Star (Roche). Hybridization, washing, and detection were performed as instructed by the supplier. E. coli strains were transformed by electroporation with a Gene Pulser apparatus (Bio-Rad), as recommended by the manufacturer, and L. casei strains were transformed as described previously (30).
Total RNA was isolated from L. casei as described previously (42). Strains were grown in MEI medium (50 ml) supplemented with glucose and/or l-malate at 30°C. Unless otherwise indicated, samples were taken at mid-exponential phase, the beginning of the stationary phase, and at late stationary phase. Sample preparation, denaturing agarose gel electrophoresis, and RNA transfer were performed by standard methods (31). Internal fragments of the genes maeE and maeK were synthesized by PCR using primers MaeE1/MaeE2 and MaeKprob1/MaeKprob2 (Table (Table2)2) as probes and labeled with the PCR-digoxigenin labeling mix from Roche. Hybridization, washing, and detection with the CDP-Star (Roche) chemiluminescent reagent were performed as recommended by the supplier.
Transcription initiation sites were determined with a 5′→3′ rapid amplification of cDNA ends (RACE) kit (Roche), following the manufacturer's instructions. Reverse transcription (RT) reactions were performed with total RNA isolated from L. casei BL23 grown in MEI medium supplemented with 5 g/liter l-malic acid. To determine the transcriptional start site upstream from maeP, reverse transcription was performed with primer MalicR-1 (Table (Table2).2). The cDNA was dA tailed and then amplified by PCR using the primers oligo dT-anchor (supplied with the kit) and MalicR-2. The resulting PCR product was used in a second PCR with primers PCR-anchor (supplied with the kit) and MalicR-3. The amplified DNA fragment was purified and sequenced. For the determination of the transcriptional start site of maeK, the same strategy was followed by using the primers MaeA, MaeG, and RACEG2.
RNA samples purified as described above were treated with the Ambion Turbo DNA-free kit (Applied Biosystems) using the routine DNase I treatment outlined by the supplier in order to remove contaminating DNA. The quality and concentration of the RNA samples were subsequently evaluated by using the Experion automated electrophoresis system (Bio-Rad). Samples with 23S/16S ratios lower than 0.85 were discarded. First-strand cDNA was synthesized from 1 μg RNA using the SuperScript VILO cDNA synthesis kit (Invitrogen) as recommended by the manufacturer. Two retrotranscription reactions were performed for each RNA sample. Real-time PCR was performed using the Lightcycler 2.0 system (Roche) and LC Fast Start DNA Master SYBR green I (Roche). Primers were designed by using the Primer-BLAST service (http://www.ncbi.nlm.nih.gov/tools/primer-blast) in order to generate amplicons ranging from 100 to 150 bp in size (see Table S1 in the supplemental material). Real-time PCR was performed for each cDNA sample in triplicate in 10 μl of the reaction mixture containing 1 μl of 10× master mix, 1.2 μl of MgCl2 (25 mM), 0.5 μl of each primer (10 μM), and 1 μl of a 1/10-diluted sample from the cDNA synthesis reaction. Reaction mixtures without a template were run as controls. The cycling conditions were as follows: 95°C for 10 min, followed by 35 cycles of three steps consisting of denaturation at 95°C for 10 s, primer annealing at 55°C for 15 s, and primer extension at 72°C for 20 s. For each set of primers, the cycle threshold values (crossing point [CP]) were determined by the automated method implemented in the Lightcycler software 4.0 (Roche). In order to select appropriate reference genes, 10 housekeeping genes (fusA, ileS, lepA, leuS, mutL, pcrA, pyrG, recA, recG, and rpoB) were determined under all experimental conditions assayed and analyzed using the geNorm approach (38). As a result of the analysis, genes fusA, leuS, pyrG, and recG were selected as reference genes (see Fig. S2 in the supplemental material). The relative expression based on the expression ratio between the target genes and reference genes was calculated using the software tool REST (relative expression software tool) (29). Linearity and amplification efficiency were determined for each primer pair. Every real-time PCR was performed at least six times.
RNA and cDNA samples obtained above were used in PCR amplifications with oligonucleotides maeE1/maeE2 (Table (Table2)2) in 25-μl reaction mixtures containing 0.5 U of Paq5000 polymerase (Stratagene), 10 μM each oligonucleotide, 200 μM deoxynucleotide triphosphates (dNTPs), and 1 μl of a 1/10 dilution of each RNA or cDNA sample. The following PCR conditions were used: denaturation at 95°C for 5 min; 25 cycles of denaturation at 94°C for 15 s, annealing at 57°C for 30 s, and extension at 72°C for 60 s; and a final extension cycle at 72°C for 5 min. The amplification products were resolved by electrophoresis in 2% agarose gels.
To construct an maeE-defective mutant, an internal fragment of the maeE gene was amplified by PCR using the oligonucleotide pair maeE1/maeE2 (Table (Table2)2) (18). The PCR product was digested with HindIII/SacII and ligated to the integrative vector pRV300 (17), digested with the same enzymes, and transformed into E. coli DH5α. The resulting plasmid, pRVmaeE, was used to transform L. casei BL23, and single-crossover integrants were selected by resistance to erythromycin and confirmed by Southern analysis using the maeE internal fragment used for inactivation as a probe. One of these integrants was selected and named BL321 (maeE::pRV300).
In order to obtain BL23 derivative strains harboring complete deletions of either maeK or maeR, primer sets maeKup1/maeKup2 and maeKdown1/maeKdown2 and maeRup1/maeRup2 and maeRdown1/maeRdown2 (Table (Table2)2) were used to amplify the regions upstream and downstream of maeK and maeR (fragments maeKup, maeKdown, maeRup, and maeRdown, respectively) (Fig. (Fig.2A)2A) using Platinum Pfx DNA polymerase (Invitrogen). In order to generate single merged fragments (maeKdel and maeRdel), 100 ng of, on one hand, maeKup and maeKdown and on the other maeRup and maeRdown fragments were added to 50 μl of a PCR mixture, without primers, after which 20 cycles (94°C for 15 s, 60°C for 30 s, and 72°C for 150 s) were performed. Amplified fragments were separated in a 0.8% agarose gel, the bands corresponding to the expected sizes of the maeKdel and maeRdel fragments were excised from the gel, and the DNA was purified as indicated above. Using 50 ng of each purified fragment, the fragments were amplified using their corresponding primers maeKup1/maeKdown2 and maeRup1/maeRdown2. After digestion with SpeI and XhoI, the fragments were cloned in pRV300 digested with the same enzymes, resulting in plasmids pRVmaeK and pRVmaeR. L. casei was transformed with pRVmaeK or pRVmaeR, and for each plasmid, one erythromycin-resistant clone carrying the plasmid integrated by single crossover was grown in MRS broth without erythromycin for ~200 generations. Cells were plated on MRS and replica plated on MRS plus erythromycin. Antibiotic-sensitive clones were isolated and, among them, one was selected in which a second recombination event led to the deletion of either maeK (strain BL322) or maeR (strain BL315), as subsequently confirmed by sequencing of PCR-amplified fragments spanning the deleted regions. The resulting sequences of the derivative strains are shown in Fig. Fig.2D2D.
In order to complement the MaeK deficiency, a PCR fragment spanning the mae promoter region and maeK was amplified with Platinum Pfx DNA polymerase by using primers MaeS-1 and MaeS-2. The amplified fragment was digested with BglII and ligated to the expression vector pT1NX digested with BglII and EcoRI (made blunt with the Klenow fragment), resulting in plasmid pT1maeK. In this construct, maeK was expressed from its own promoter. The ligation mixture was used to transform Lactococcus lactis MG1363 by electroporation (13), and transformants were checked by restriction mapping and sequencing of the inserted fragment. Subsequently, plasmid pT1maeK was used to transform L. casei BL322.
The coding region of maeR was amplified by PCR using chromosomal DNA from L. casei BL23 as a template; primers MaeR-Nt and MaeR-Ct (Table (Table2),2), which added restriction sites to the 5′ and 3′ ends; and Platinum Pfx DNA polymerase. The PCR fragment was cleaved with BamHI/SacI and cloned into plasmid pQE80 (Qiagen) digested with the same enzymes. The resulting plasmid, pQEmaeR, was used to transform E. coli M15(pREP4), and the correct sequence of the inserts was confirmed by DNA sequencing. One clone was selected for expression of MaeR. Bacterial cells were grown in 0.5 liter of LB medium supplemented with ampicillin and kanamycin at 37°C with agitation. When the culture reached an OD550 of 0.5, 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added and incubation was continued for 1 h. Cells were harvested by centrifugation, washed with 10 ml of 100 mM Tris-HCl (pH 7.4), and resuspended in 5 ml of 100 mM Tris-HCl (pH 7.4), 1 mg ml−1 lysozyme, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol. The cell suspension was incubated for 30 min at 37°C and sonicated. The cell debris was removed by centrifugation at 12,000 × g for 20 min at 4°C. The cleared extract was directly loaded onto Ni-nitrilotriacetic acid agarose (1 ml) column (Qiagen) equilibrated with buffer A (50 mM Tris-HCl [pH 7.4], 50 mM Na2SO4, 15% glycerol). After the passage of the sample, the column was washed with 10 ml of buffer A and then 30 ml of buffer B (buffer A supplemented with 30 mM imidazole), and finally MaeR was eluted with buffer C (buffer A supplemented with 300 mM imidazole). Fractions were analyzed with SDS-PAGE gels, pooled, and dialyzed against 20 mM Bis-Tris-HCl (pH 6), 25 mM Na2SO4, and 3.75% glycerol. The dialyzed protein solution was applied to a RESOURCE S 1-ml column (GE Healthcare) equilibrated with 20 mM Bis-Tris HCl (pH 6) and 25 mM Na2SO4. The column was washed extensively with the same buffer, and proteins were eluted with a linear NaCl gradient of 0 to 500 mM in the same buffer (total volume, 60 ml). The protein eluted in one peak, and it was kept frozen at −80°C. Protein concentrations were determined with a Bio-Rad dye-binding assay.
Primers FP1 and FP2 (Table (Table2)2) were 5′ labeled with T4 polynucleotide kinase (USB) and [γ-32P]ATP and purified with MicroSpin G-25 columns (GE Healthcare). They were used in combination with the respective nonlabeled oligonucleotide to amplify a 219-bp DNA fragment spanning the intergenic region between maeP and maeK (fragment maepro3) (Fig. (Fig.2B).2B). In addition, labeled primer FP2 was used with primer FP4 and labeled primer FP1 with primer FP3 to generate fragments maepro2 and maepro1, containing two or one putative MaeR binding sites, respectively (Fig. (Fig.2B).2B). The amplified fragments were used in electrophoretic mobility shift assays with purified His-tagged MaeR. As negative controls, the coding region of maeR and internal fragments of maeK and maeE were used. The binding assay was carried out in 10 μl of binding buffer (2.5 mM Tris-HCl [pH 7.5], 100 mM NaCl, 25 mM MgCl2, 0.25 mM EDTA, 0.25 mM dithiothreitol, 1.5% glycerol) with 10 ng of target DNA (~10,000 cpm), 0.5 μg of salmon sperm DNA, and different amounts of His-tagged MaeR. The binding mixtures were incubated for 1 h at 37°C and separated on 6% nondenaturing polyacrylamide gels in 40 mM Tris-acetate (pH 8.2) and 1 mM EDTA buffer at 100 V for 1 h. The gel was dried and subjected to autoradiography.
For DNase I footprinting, binding reactions were performed as described for the gel retardation experiments in a total volume of 40 μl with 50,000 cpm of each radiolabeled fragment. Complexes were allowed to form for 1 h at 37°C, after which 10 μl of 10 mM MgCl2, 1 mM CaCl2, and 0.025 U DNase I were added. Digestion was allowed to proceed for 2.5 min, after which the reactions were terminated by adding 140 μl of stop solution (192 mM Na-acetate, 32 mM EDTA, 0.14% SDS, 64 μg ml−1 yeast tRNA). Samples were extracted twice with phenol-chloroform, and the DNase I digestion products were precipitated with ethanol. The precipitates were resuspended in 6 μl of loading buffer and loaded on a 6% polyacrylamide-urea gel. A+G Maxam and Gilbert reactions were run on the same gel to locate sequence positions and protected regions (31).
The inspection of the genomic sequences of L. casei ATCC 334 and L. casei BL23 led to the identification of two gene clusters possibly involved in l-malic acid metabolism. One cluster (see Fig. S3 in the supplemental material) is constituted by the genes Lsei_0739/LCABL_08050 (encoding a putative transcriptional regulator of the LysR family), Lsei_0740/LCABL_08060 (encoding a putative malolactic enzyme), and Lsei_0741/LCABL_08070 (encoding a putative malate permease). The second cluster is constituted by two diverging putative operons (Fig. (Fig.2).2). The first one consists of genes Lsei_2866/LCABL_30690 and Lsei_2867/LCABL30700, encoding a putative malic enzyme and a transport protein (Pfam 03390), respectively. The second operon consists of genes Lsei_2868/LCABL_30710 and Lsei_2869/LCABL_30720, encoding a putative two-component (TC) system closely related to TC systems involved in the regulation of the metabolism of dicarboxylic acids (8). On the basis of the similarities of the genes LCABL_30690 (77% identical, 86% conserved residues) and LCABL_30700 (77% identical, 88% conserved) to genes maeE and maeP, respectively, of Streptococcus bovis (14, 15), we propose to rename them maeE and maeP and to rename genes LCABL_30710 and LCABL_30720 maeK and maeR, respectively.
Putative rho-independent terminators could be identified downstream of genes LCABL_030690 (maeE) and LCABL_30720 (maeR), suggesting that both couples of diverging genes constitute two operons (Fig. 2A and C). Structurally identical gene clusters are present in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis (see Fig. S3 in the supplemental material). Lactobacillus brevis ATCC 367 also encodes a homologous TC system (Fig. S3); however, these genes constitute a cluster with genes encoding a putative membrane protein and a d-lactate dehydrogenase. In fact, L. brevis does not code for a malic enzyme (26).
In order to gain insight into the evolutionary relationships of these TC systems, a phylogenetic analysis of selected MaeK and MaeR homologs was carried out. The analysis showed a remarkable congruency with the gene content: both MaeK and MaeR homologs of L. casei, E. faecalis, and streptococci constitute strongly supported clusters, whereas L. brevis MaeK appears as distantly related and L. brevis MaeR is at a basal branch with low support (see Fig. S1 in the supplemental material). Furthermore, the phylogenetic reconstruction suggests a closer relationship to TC systems involved in the regulation of malate metabolism, such as Bacillus subtilis yufLM (36) and other TC systems of species belonging to the family Bacillaceae which form clusters with genes homologous to maeE and maeP (Fig. S1 and S3). In summary, the phylogenetic analyses indicate that the mae gene clusters present in LAB share an origin, although they cannot determine whether their distribution can be explained by horizontal gene transfer or lineage-specific gene losses and suggest a close relationship with mae clusters present in bacilli.
The growth behavior of L. casei BL23 in MEI medium supplemented with glucose and/or l-malate was investigated. As a control, L. casei BL23 was inoculated in MEI medium without glucose or l-malate. Under our experimental conditions, L. casei BL23 was able to grow in MEI medium without supplementation of glucose or l-malic acid to an OD of 0.28. When MEI medium was supplemented with l-malic acid, L. casei BL23 initially grew as in MEI medium, but after a lag phase, it resumed growth at a low rate (Fig. (Fig.3).3). In order to determine whether expression of gene maeE was responsible for the ability of L. casei BL23 to grow on l-malate, a mutant strain defective in maeE was constructed (BL321). The mutant strain grew in MEI medium supplemented with glucose or glucose and l-malic acid at growth rates similar to the wild-type strain, but it was not able to grow with l-malic acid alone (Fig. (Fig.3).3). Products of l-malic acid degradation were also analyzed. During the initial growth phase, the concentration of lactic acid exceeded that expected from l-malic acid consumption (Table (Table3),3), possibly due to fermentation of components of the MEI medium, as expected from the observed basal growth in MEI medium. Subsequently, the consumption of l-malic acid correlated with the accumulation of lactic acid. A small amount of acetic acid was only detected in samples corresponding to late stationary cells (data not shown), indicating the activation of pyruvate dissimilative pathways alternative to lactate dehydrogenase (Fig. (Fig.11).
The structure of the L. casei mae cluster suggested that MaeKR might be involved in the regulation of the expression of maePE. This hypothesis was in agreement with previous results obtained by other researchers. On one hand, a number of studies had shown that expression of ME in E. faecalis and L. casei was induced by l-malic acid when glucose was at a concentration lower than 0.2% (20, 22, 33). On the other hand, the expression in Bacillus subtilis of the malate transporter MaeN had been shown to be under the control of the YufLM TC system (36). YufLM shares significant similarity with MaeKR (35% identical and 56% conserved residues for MaeK and 39% identical and 57% conserved residues for MaeR), and the phylogenetic analyses suggested an evolutionary relationship between these two systems. In order to ascertain the possible role of the MaeKR TC system, two BL23 derivative strains with deletions of maeK (BL322) or maeR (BL315) were constructed as indicated in Materials and Methods, and their ability to grow with l-malic acid as a carbon source was assayed. Both derivative strains were unable to grow in MEI medium supplemented with 5 g/liter l-malic acid, whereas no significant differences were observed with the wild-type strain in MEI medium supplemented with glucose and l-malic acid (Fig. (Fig.3).3). In order to confirm that the loss of the ability to grow on l-malic acid was due to the inactivation of the MaeKR TC system, the mutant strain BL322 was transformed with plasmid pT1maeK. The expression of MaeK in the ΔmaeK strain restored the ability to grow with l-malic acid (Fig. (Fig.3).3). Therefore, the MaeKR TC system is essential for growth with l-malic acid.
In order to determine whether the MaeKR TC system is involved in the control of the expression of the maePE operon, Northern blot experiments were performed with RNA isolated from L. casei BL23 and the derivative strain BL315 (ΔmaeR) grown in MEI medium containing glucose, l-malic acid, or glucose plus l-malic acid. The results suggested that strain BL23 only expressed the maePE operon in the presence of l-malic acid and absence of glucose (Fig. (Fig.4A)4A) and only during active growth on l-malic acid since transcription was not detected in stationary phase. Transcription of maePE in the ΔmaeR strain was not observed under any condition. The hybridization signals were at the level of the ribosomal RNAs, likely due to degradation or comigration of the RNA. These results were confirmed by RT-PCR analysis of maeE, which showed that the maeE cDNA was preferentially amplified from strain BL23 actively growing in medium containing l-malic acid (Fig. (Fig.4B).4B). A faint signal was also observed in the cDNA sample obtained from cells of BL23 grown with glucose and l-malic acid (Fig. (Fig.4B),4B), possibly indicating a basal expression of maeE below the detection threshold of the Northern hybridization. Expression of the maeKR operon by using Northern blot analysis could only be detected after overexposure of the membrane (not shown), and changes in expression between different samples could not be reliably determined. Therefore, RT-quantitative PCR (qPCR) assays were carried out in order to determine the expression pattern of genes maeK and maeR. RNA samples from BL23 cultures grown for 24 h in MEI medium supplemented with glucose, glucose and l-malic acid, or l-malic acid were used. The results obtained are shown in Table Table4.4. Taking as a reference the transcript levels in MEI medium with glucose, a significant increase in expression of maeK and maeR was detected both in MEI medium with glucose and l-malic acid and in MEI medium with l-malic acid. Although both genes possibly are expressed as a single transcript, a difference in expression ratios was observed between maeK and maeR (Table (Table4).4). This was evidenced when the expression ratio in MEI medium with l-malic acid was determined using MEI medium with glucose and l-malic acid as the reference condition. Whereas no significant difference was observed for maeR, the expression of maeK was 1.8-fold lower in MEI medium with l-malic acid. Whether this result indicates a control system at the posttranscriptional level such as mRNA processing, it requires further confirmation. In any case, the results obtained indicate that expression of maeKR is induced in the presence of l-malic acid and, as a difference from the maePE operon, glucose does not repress expression of maeKR.
The transcriptional initiation site of the maePE operon was determined by 5′-RACE and shown to be the T or A located at position 25 or 26 upstream of the maeP translational start site (Fig. (Fig.4C).4C). The transcriptional initiation site could not be unequivocally identified due to an artifact of RACE when the start site is a T. On the other hand, the transcriptional initiation site of the maeKR operon could not be reliably identified by this technique, possibly because of the low levels of transcript present in the samples. The inspection of the sequence allowed identification of a putative −10 box (5′-TATGCT-3′) at −6 from the transcriptional initiation site of maePE (Fig. (Fig.4C),4C), although no −35 box could be identified. In concordance with the repressive effect of glucose on transcription, a sequence matching the consensus of the Gram-positive catabolite repression element (cre), the binding site of the CcpA regulator mediating carbon catabolite repression (27), was located upstream of the maeP −10 box. Finally, a second −10 box (5′-TTTACT-3′) was located 39 bases upstream of the putative translational start site of maeK (Fig. (Fig.4C4C).
In order to verify the role of MaeR in the regulation of the expression of the maePE operon, gel retardation experiments were performed using fragment maepro3, which spans the entire mae promoter region or nonspecific DNA fragments (MaeKup and MaeRdown) (Fig. (Fig.2A)2A) as controls of the specificity of the binding reaction. MaeR was able to bind to the maepro3 fragment (Fig. (Fig.5,5, left panel) in the presence of a large excess of unspecific DNA competitor but did not bind to unspecific fragments (not shown). Treatment of MaeR with up to 25 mM acetyl-phosphate, which is an in vitro phosphate donor able to phosphorylate different RRs (25), did not change its binding activity (data not shown).
DNase I footprinting on DNA fragment maepro3 was carried out to determine the sequences recognized by MaeR. Figure Figure66 shows that MaeR protects two regions within maepro3. These regions contain three AT-rich direct repeats (5′-TTATT(A/T)AA-3′) (Fig. (Fig.6),6), two of them separated by 1 base and the third one located 12 bases apart. Taking into account the contacts required by DNase I to cut the DNA at a certain position (35) and the presence of unprotected sites between the second and third repeats, it can be inferred that protection is centered in the repeats. A remarkable difference in protection could be appreciated between the forward strand (Fig. (Fig.6,6, right panel) and the reverse strand (left panel): whereas clear protected regions could be observed in the reverse strand, a change in the pattern of sites preferentially digested by DNase I was observed in the forward strand. This result suggests that MaeR binds to the reverse strand, leaving the forward strand relatively unprotected. Furthermore, the change in the DNase I digestion pattern indicates that MaeR binding results in an alteration of the structure of the forward strand.
In order to determine whether one or two MaeR binding sites would be enough for the formation of an MaeR:DNA stable complex, additional gel retardation experiments were performed using fragments maepro2 and maepro1. Under our experimental conditions, MaeR did not form stable complexes either with fragment maepro2 (Fig. (Fig.5,5, right panel) or maepro1 (not shown), thus indicating that the three direct repeats are required for stable MaeR DNA binding.
Whereas malolactic enzyme is widely distributed among LAB, ME is present in few species. The phylogenetic analysis reported here and the comparison of the respective mae gene clusters indicate that mae gene clusters present in LAB evolved from a common ancestor, although the data available do not allow determination of whether this cluster has been disseminated by horizontal gene transfer or the observed distribution is explained by multiple lineage specific gene losses. MLE decarboxylates l-malic acid to l-lactic acid which cannot be further utilized by most LAB. In contrast, biochemical characterization of L. casei ME showed that this enzyme yielded pyruvate and CO2 from malate, utilized preferentially NAD over NADP, and could also react with oxaloacetate (4). Pyruvate produced by l-malic acid can then be channeled to biosynthetic pathways via pyruvate phosphate dikinase or pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase (Fig. (Fig.1)1) or through the lactate dehydrogenase, pyruvate dehydrogenase, pyruvate oxidase, or pyruvate formate lyase pathways of dissimilation (Fig. (Fig.1).1). Therefore, L. casei possesses the required enzymatic complement to grow on l-malic acid as a carbon source. Our results confirm this hypothesis and show that the ME encoded by maeE is essential for growth with l-malic acid. The analysis of the final products of l-malic acid utilization showed that most l-malic acid was degraded to lactate so that only a minor part was used for growth which would agree with the slow growth rate and low maximal OD reached by the cultures. Furthermore, L. casei codes for a putative fumarase (EC 22.214.171.124; LCABL_25800) which might convert malate into fumarate; however, fumarate concentrations ranged between 0.8 and 4 μM and did not vary significantly during L. casei growth, indicating that fumarase activity did not have a significant role in l-malic acid metabolism under our experimental conditions.
Previous studies had shown that L. casei ME was inhibited by glycolytic intermediaries such as fructose-1,6-bisphosphate and 3-phosphoglycerate and that ME expression was induced in the presence of malic acid and a low concentration of glucose (22). Studies conducted with E. faecalis also showed that ME synthesis was induced by l-malic acid and inhibited by glucose (21). This study also showed that ME and the l-malic acid transport system were coordinately expressed. Our results, in agreement with the aforementioned studies, showed that expression of gene maeE was repressed in the presence of glucose, which is congruent with the presence of a cre sequence in its promoter sequence. Therefore, expression of maePE is possibly regulated by the global carbon catabolite repression transcriptional regulator CcpA (27). Furthermore, we have shown that the cognate TC system encoded by maeK and maeR plays a role in the induction mechanism of maePE in the presence of l-malic acid. The TC system MaeKR is then a functional homolog of the B. subtilis system YufLM, which was shown to be essential for growth on l-malic acid (36) and controls the expression of genes maeN and yflS encoding malate transporters (36) and ywkA (maeA) encoding an ME (7). Notwithstanding, there are some differences in the regulatory mechanisms operated by MaeKR and YufLM: deletion of the sensor-encoding gene yufL resulted in constitutive expression of the transporter encoded by maeN (36), whereas deletion of maeK or maeR resulted in inability to grow on l-malic acid or to express maeE and maeP in L. casei. There are also important differences in l-malic acid metabolism between B. subtilis and L. casei, since the latter lacks a functional tricarboxylic acid (TCA) cycle. L. casei possesses MLE and ME, whereas B. subtilis contains four putative ME-encoding genes, malS, mleA, ytsJ, and maeA (7). MaeE is essential for growth on l-malic acid; in contrast, absence of MaeA can be compensated by MalS or MleA in B. subtilis (7, 19). In fact, B. subtilis can grow on l-malic acid, albeit slowly, even in the absence of the four MEs since l-malic acid can be channeled into TCA via malate dehydrogenase (19).
The DNase I footprinting experiments showed that MaeR binds to a set of direct repeats in the mae promoter region, 5′-TTATT(A/T)AA-3′, two of them separated by 1 base and the third one 12 bases apart. In contrast, DNase I footprinting analyses of YufM binding sites identified two sets of direct repeats within the protected region of the maeA promoter, ATTAAAAAATTN11ATTAAAAAATT and TAAGTAN11TAAGTA (7), whereas no clear repeat sequences could be identified in the YufM-binding regions of the maeN and yflS promoter sequences (36). The inspection of the mae promoter regions in the homologous clusters of E. faecalis, Streptococcus agalactiae, S. pyogenes, and S. uberis showed the presence of highly conserved repeats (Fig. (Fig.7),7), although with significant differences. E. faecalis conserves a similar array of repeats (two repeats separated by 1 base and a third one 11 bases apart). In contrast, in Streptococcus the order of the repeats is inverted and the gap separating the repeats is larger (19 bp for S. agalactiae and 28 bp for S. pyogenes and S. uberis). Furthermore, within this intervening region several degenerated MaeR binding sites are present (Fig. (Fig.7).7). These differences, together with the different spacing between the MaeR binding sites and the putative −10 boxes suggest that regulation of the mae cluster by MaeR may differ in streptococci from the regulation in L. casei and, possibly, E. faecalis.
The distance (25 bp) (Fig. (Fig.7)7) between the repeats and the −10 boxes suggests that MaeR act as a class II activator (5) in streptococci and for the maePE promoter of E. faecalis and L. casei. From the DNase I footprinting data, it can be inferred that this is also the mechanism of activation of YufM (7). However, this seems not to be the case for the maeKR promoters of E. faecalis and L. casei. In these organisms, putative −10 boxes were identified but none of them is located at the expected distance from the most proximal maeR binding site (Fig. (Fig.7).7). Although we could not determine the transcriptional start site of the maeKR transcript, the transcriptional data indicate that expression of maeKR is induced by l-malic acid, thus strongly suggesting that MaeR also induces the expression of maeKR. If so, the mechanism of activation operated by MaeR must be different in the maePE promoter and the maeKR promoter, although further research will be required to ascertain this point.
In summary, our results have shown that expression of the maeP and maeE genes, required for growth on l-malic acid, is regulated by the cognate MaeKR TC system and repressed by glucose. We have shown that MaeR binds specifically to a set of three direct repeats in the mae promoter region which are conserved in promoter regions of homologous gene clusters in several bacteria. In vitro MaeR promoter binding activity did not require phosphorylation of the regulatory aspartate at the RR receptor domain. Further research is needed to determine how the environmental signal sensed by MaeK (most probably the presence of l-malic acid) is presumably transferred to MaeR.
This work was financed by AGL2007-60975 and Consolider Fun-C-Food CSD2007-00063 funds from the Spanish Ministry of Science and Innovation.
We thank Isidoro Olmeda and José Vicente Gil for technical assistance.
Published ahead of print on 6 November 2009.
†Supplemental material for this article may be found at http://aem.asm.org/.