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Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria
System. Appl. Microbiol. 26, 350–356 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria Martin Sievers1, Christina Uermösi1, Marc Fehlmann1, and Sibylle Krieger2 1 2
Hochschule Wädenswil, Molekularbiologie, Wädenswil, Switzerland Lallemand S.A., Blagnac, France Received: May 30, 3003
Summary The nucleotide sequences of the genes encoding the F1F0-ATPase β-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79–98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase β-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase β-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase β-subunit protein which is larger in size than the corresponding molecules from the investigated strains. Key words: F1F0-ATPase β-subunit – atpD – lactic acid bacteria – TA-cloning
Introduction The cytoplasmatic membrane-bound enzyme complex (F1F0) H+-ATPase catalyzes the formation of ATP driven by the proton gradient resulting from respiration in bacteria with a respiratory chain. The lactic acid bacteria lacking a respiratory chain have a proton-translocating F1F0-ATPase which does not generally synthesize ATP. The F1F0-ATPase regulates the cytoplasmic pH coupled with the extrusion of H+ via hydrolyses of ATP in anaerobic bacteria. The integral membrane proton channel F0 portion consists of three subunits: a (atpB), b (atpF), c (atpE). The F1 consists of five subunits: α (atpA), β (atpD), γ (atpG), δ (atpH), ε (atpC). The ORFs are organized in an operon arranged in the order atpBEFHAGDC in Lactobacillus acidophilus [9] and atpEBFHAGDC in Lactococcus lactis subsp. cremoris [8]. The operons contained no atpI gene in compare to the E. coli operon which consists of nine structural genes atpIBEFHAGDC [7, 22]. The βsubunit of the F1 portion of the ATPase is the catalytic site for ATP synthesis or hydrolysis. A structural model of the F1F0-ATPase and the ATP-binding domain of the F1β-subunit was described recently [1, 2, 5, 14].
In grape musts and wine, populations of lactic acid bacteria are able to grow at low pH values [10]. Increased activity and synthesis of F1F0-ATPase is a major strategy of bacteria to grow at acidic environments [6, 12, 20]. Recently, it was shown that stress compounds in wine have an inhibitory effect on the ATPase activity of O. oeni [3]. Wine lactic acid bacteria carrying out malolactic fermentation are able to synthesize ATP via the F1F0-ATPase from the uptake of malate, the decarboxylation of L-malate into L-lactate and CO2, and the excretion of L-lactate including a proton [13, 17, 21]. In this study, we focused on the DNA sequence of the beta-subunit (atpD) of the F1F0-ATPase from wine-relevant bacteria such as O. oeni, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii, and the non-acidophilic Leuconostoc mesenteroides. The coding sequences of the atpD genes were amplified by PCR and cloned in the vector pCRT7/CT-TOPO for expression in E. coli. We used the cloning procedure to determine the molecular weights of the recombinant ATPase β-subunit molecules by SDSPAGE and Western blot. Our cloning strategy will con0723-2020/03/26/03-350 $ 15.00/0
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria
firm the particular position of the atpD gene of O. oeni showing a molecular size which is larger than the corresponding ATPase β-subunit molecules of the other investigated strains of lactic acid bacteria.
Materials and Methods Bacterial strains Oenococcus oeni strain E656 was obtained from Lallemand, Blagnac, France. O. oeni was grown on MRS medium at pH 5.0 and Oenococcus oenos medium (MLO), Scharlau Chemie S.A., Barcelona, at 25 °C. Pediococcus damnosus DSM 20331, Pediococcus parvulus DSM 20332, Lactobacillus brevis DSM 2647, Lactobacillus hilgardii DSM 20051 and Leuconostoc mesenteroides subsp. mesenteroides DSM 20343 were cultured in appropriate media and at temperatures recommended in the DSMZ catalog. E. coli Top 10F’ cells and E. coli BL21(DE3)pLysS cells from Invitrogen were used as the hosts for the TA-cloning strategy. E. coli cells were grown at 37 °C in LB broth with shaking or on LB agar supplemented with ampicillin (60 µg/ml) and when necessary with chloramphenicol (34 µg/ml). PCR and DNA sequencing Isolation of chromosomal DNA was carried out by standard procedures [18]. Degenerate primers were designed on the basis of conserved motifs of atpG, atpD and atpC subunits of F1F0-ATPase. Primers were synthesized by Microsynth, Switzerland. The conserved sequences of forward and reverse primers, respectively, were: atpGf, 5′-ATTACIMMWSAAATTACIGAAAT-3′; atpDr, 5′TCTTCRTCRGAYAATTCATC-3′; atpDf, 5′-GTTGGTAARA CYGTTTTAATT-3′ and atpCr, 5′-TTDATYTCRATAATDC CACCRTT-3′. Isolated DNA of one strain as template (250 ng) was amplified in a 50 µl reaction volume containing 0.2 µM of the appropriate primer, 10 mM dNTP’s, one unit of HotStarTaq DNAPolymerase (Qiagen) and 5.0 µl of 10× PCR-buffer supplemented with MgCl2. PCR amplifications were performed in a Primus Multiblock TC-HTS thermocycler (MWG-Biotech). After initial denaturation for 15 min at 95 °C, a total of 40 cycles were run, each consisting of 1 min at 95 °C, 1 min at 40 °C and 5 min at 72 °C. The program was terminated with a 7-min incubation step at 72 °C prior to cooling at 8 °C. The amplification products were analysed by electrophoresis on a 1.0% agarose gel stained with ethidium bromide (1 µg/ml). Specific PCR-products were isolated from the agarose gel using the QIAquick gel extraction kit (Qiagen) procedure. Direct sequencing of the PCR-products was performed by the dideoxy-chain termination method using the Big-Dye Terminator chemistry version 1.1 (Applied Biosystems). Purification of the sequencing reaction was performed using the DyeEx spin kit (Qiagen) and analysed on an Applied Biosystems 310 DNA sequencer. Sequence comparison and phylogenetic analysis The deduced protein sequences of the atpD open reading frames were aligned by using the ClustalW computer program. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (http://www.megasoftware.net/). Recombinant DNA techniques Isolation of plasmid DNA and purification of plasmid DNA and PCR-products were carried out with the appropriate Qiagen (Hilden, Germany) kits.
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Amplified PCR products were purified, inserted into the pCRT7/CT-TOPO vector (Invitrogen) and transformed in E. coli Top 10F’ cells. The recombinant plasmid containing the insert was purified and digested with restriction enzyme (PvuI, HindIII, BamHI or DraI) to check the correct insertion orientation. Recombinant plasmids (pCRT7/CT(AtpD)) containing the ATPase β-subunit expression construct in the right orientation were used for further transformation steps in E. coli BL21(DE3)pLysS. Each expressed F1F0-ATPase β-subunit molecule is a fusion protein, which contains a C-terminal V5 epitope and the sequence coding for six histidine residues (6× His-tag). The V5 epitope was used for detection of the protein in western blot assay. Purification of the recombinant proteins E. coli BL21(DE3)pLysS transformed with pCRT7/CT(AtpD) was grown in 6 ml LB containing ampicillin and chloramphenicol to an OD600nm of 0.5. IPTG was then added to a final concentration of 1 mM and the cultures were grown for further 4 h at 37 °C. Cells were collected by centrifugation and resuspended in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0 for cell lysis. The recombinant hexahistidine-tagged ATPase β-subunit proteins were purified using the NI-NTA spin kit from Qiagen. SDS-PAGE and Western Blotting The cell extracts and the purified ATPase β-subunit proteins were examined by SDS-PAGE on 10% polyacrylamide gels. The gels were stained with Coomassie brilliant blue R-250, or electroblotted onto PVDF membrane for Western blotting. Immunodetection was performed with a monoclonal mouse anti-V5 antibody (Invitrogen). The WesternBreeze chromogenic detection kit-anti-mouse (Invitrogen) was used to develop the blot. The same antibody-conjugate reagent used to detect our recombinant proteins visualized also the proteins from the MagicMark standard (Invitrogen). Nucleotide sequence accession numbers The EMBL accession numbers of the F1F0-ATPase (β-subunit nucleotide sequences which we determined are AJ430065 (O. oeni E656), AJ508912 (L. mesenteroides subsp. mesenteroides DSM 20343), AJ508913 (L. brevis DSM 2647), AJ508914 (L. hilgardii DSM 20051) AJ515142 (P. damnosus DSM 20331), AJ515143 (P. parvulus DSM 20332), respectively.
Results and Discussion The atpD genes encoding the β-subunit of the F1F0ATPase from six wine lactic acid bacteria were amplified by PCR and sequenced using degenerate primers. Based on these sequences, specific primers for PCR amplification of the atpD open reading frames were designed (Table 1). The amplified fragments were cloned into the pCRT7/CT-TOPO vector (Invitrogen) for expression of the recombinant proteins in E. coli. The deduced amino acid sequences of the investigated atpD gene products were aligned to determine the homology of the F1F0ATPase β-subunit proteins among the different organisms. The distribution of highly and less conserved regions along the primary structure of the ATPase β-subunit molecules is visualized in the alignment given in Fig. 1. The amino acid sequence homologies of the ATPase βsubunit molecules from the investigated strains in this
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study are in the range of 79% to 98%. For example, the ATPase β-subunit protein of O. oeni is homologous to the corresponding proteins from L. mesenteroides subsp. mesenteroides (76% identity, 83% similarity,), L. brevis (72% identity, 81% similarity), L. hilgardii (72% identity, 79% similarity), P. damnosus (73% identity, 81% similarity) and P. parvulus (74% identity, 82% similarity). The ATPase β-subunit from L. brevis showed a similarity of 91% and an identity of 84% to the corresponding protein of L. hilgardii. The ATPase β-subunit proteins from P. damnosus and P. parvulus are 96% identical. A stretch of 38 residues 1 to 38 at the N-terminus of the F1F0-ATPase β-subunit protein from O. oeni is unique for this organism. A similarity search of the 38 additional amino acids against other sequences deposited in the EMBL and Genbank databases showed no similarity with any other sequence. The F1F0-ATPase β-subunit constitutes the catalytic sites of the enzyme and is characterized by mainly two nucleotide-binding sequence motifs. The nucleotide-binding sequence (motif A) for catalytic activity in nucleotide binding proteins is a glycine-containing loop with the motif GXXXXGKT (position 149–156 in E. coli atpD gene, methionine is not present in the mature protein [19]) [23]. Studies on ATPases with mutations in the βsubunit indicated that the conserved sequence GERXXE (β180–β185) is as well essential for catalytic activity. The residues of the catalytic site of the β-subunit around ATP are βLys-155, which binds to the β and γ phosphate moieties of ATP, βThr-156, βGlu-181, βArg-182 and βGlu185 [14, 16]. The two conserved motifs GGAGVGKT and GERTRE were found in all of the six ATPase β-subunit proteins of the investigated strains and are marked in the alignment given in Figure 1. The conserved sequence motif DDLTDP (β301–β306) in the F0F1-ATPase β-subunit seems to be a key region important for the stability of the assembly of the α3β3γ complex of the F1 sector [15].
The βLeu-303 of the described conserved sequence is replaced by βTyr-303 in the β-subunit sequences of our investigated lactic acid bacteria strains which have the sequence motif DDYTDP. A phylogenetic tree was reconstructed to determine the relationships of the homologous ATPase β-subunit molecules with known sequences available from the Genbank. The wine lactic acid bacteria investigated in this study formed one major cluster well separated in the ATPase tree shown in Figure 2. This cluster is composed of three groups. The first group comprises the sequences of L. brevis and L. hilgardii. The second group is composed of the closely related ATPase β-subunit molecules from the P. damnosus and P. parvulus. O. oeni formed together with L. mesenteroides subsp. mesenteroides a more distant group. O. oeni exhibits an acidophilic phenotype and is an example of a tachytelic (fast evolving) bacterium which forms a long 16S rRNA branch separating this species from Leuconostoc and Weissella [4, 24]. Phylogenetic trees based on ATPase β-subunit sequences generally support the topology of the 16S rRNA trees with the distinction that some branchings are better resolved using the rRNA data [11]. The atpD gene of O. oeni has an open reading frame of 1515 bp encoding 504 amino acid residues with a calculated molecular mass of 54.7 kDa. The calculated molecular masses of the ATPase β-subunit molecules of L. mesenteroides subsp. mesenteroides, P. damnosus, P. parvulus, L. brevis and L. hilgardii are 50.3 kDa, 50.8 kDA, 51.0 kDa, 51.1 kDa and 52,2 kDa, respectively. The expressed proteins obtained in this work are fusion proteins, which contain a C-terminal tag of six histidine residues and a V5 epitope, both used for purification and Western blot. Due to the unusual N-terminus of the ATPase β-subunit protein of O. oeni, a fusion at the Ctermini of the investigated proteins with the vector sequence was chosen.
Table 1. Specific PCR primers used for amplification of the atpD (F1F0-ATPase β-subunit) coding sequences of the wine lactic acid bacteria for cloning into pCRT7/CT-TOPO vector as fusion proteins. Species
strain
Accession No.
atpD cds, bp
calculated molecular mass, kDa
PCR primers (sequence 5′ to 3′) forward, f reverse, r
O. oeni
E656
AJ430065
1515
54.7
f: ATG GCA GAA AAG AAA ACA ACA r: GCT TTC CAG CTT TTT AGC C
L. mesenteroides DSM 20343 subsp. mesenteroides
AJ508912
1401
50.3
f: ATG AGT ACT GGA AAA GTC GT r: TTG GGC CAT TGT CTT GGC T
P. damnosus
DSM 20331
AJ515142
1413
50.8
f: ATG AGT ACT GGT AAA GTT GTT r: TTT ACT AGT GGC ACT AGT TAA
P. parvulus
DSM 20332
AJ515143
1413
51.0
f: ATG AGT ACT GGT AAA GTT GTC r: TTT ACT TGC AGC ACT AGT TAA
L. brevis
DSM 2647
AJ508913
1416
51.1
f: ATG AGT GCT GGT AAA ATT GTT r: GTT GTT GGC AAC GGC GTC
L. hilgardii
DSM 20051
AJ508914
1446
52.2
f: ATG AGT ACT GGT AAA GTT TTA C r: GTT TGC TGC ACT TTG CTT TT
Fig. 1. Alignment of the deduced amino acid sequences of the ATPase β-subunits from the wine-relevant lactic acid bacteria using the ClustalW program. Identical residues, well-conserved and less-conserved positions are indicated as * and :,., respectively. The three conserved motifs GGAGVGKT, GERTRE and DDYTDP are marked. Abbreviations: Oo, Oenococcus oeni; Lm, Leuconostoc mesenteroides subsp. mesenteroides; Pd, Pediococcus damnosus; Pp, Pediococcus parvulus; Lb, Lactobacillus brevis; Lh, Lactobacillus hilgardii
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Fig. 2. Phylogenetic tree showing relationships of ATPase β-subunit deduced protein sequences. The tree was reconstructed by the neighbor-joining method. Two major clusters compose this ATPase tree, one of ATPase β-subunit proteins from the wine-relevant lactic acid bacteria, the other of ATPase β-subunit proteins from Lactococcus lactis subsp. lactis, Streptococcus, Enterococcus faecalis and Lactobacillus species. The bar indicates 2% amino acid differences.
Fig. 3. SDS electrophoresis in 10% polyacrylamide gel of recombinant ATPase βsubunit proteins from wine-relevant lactic acid bacteria expressed as fusion proteins in E. coli BL21(DE3)pLysS harboring pCRT7/CT(AtpD). Gel was stained with Coomassie brilliant blue R-250. Lane 1, SDS-PAGE molecular weight standards, broad range (Bio-Rad); lane 2, total cellular fraction harboring recombinant ATPase β-subunit from O. oeni, no IPTG induction; lane 3, total cellular fraction harboring recombinant ATPase β-subunit from O. oeni, IPTG induction; lane 4 to lane 9, one-step purified ATPase β-subunit proteins using NI-NTA column and elution with imidazole from O. oeni (lane 4); L. mesenteroides subsp. mesenteroides (lane 5), P. damnosus (lane 6); P. parvulus (lane 7); L. brevis (lane 8) and L. hilgardii (lane 9); lane 10, SDS-PAGE molecular weight standards, broad range (Bio-Rad).
SDS-PAGE analysis of whole-cell lysates and of the purified recombinant proteins indicated that the ATPase βsubunit proteins were successfully expressed in E. coli. The expression of the recombinant proteins was optimal when induced for 4 h with 1 mM IPTG. A band corresponding to a 58 kDa protein was observed in SDS-PAGE after Coomassie staining of crude extracts of E. coli BL21(DE3)pLysS harboring pCRT7/CT(AtpD) containing the atpD insert from O. oeni after IPTG induction (Fig. 3, lane 3). Lanes 4 to 9 (Fig. 3) contain the one-step purified hexahistidine-tagged ATPase β-subunit proteins of the investigated strains migrating between 54 to 58 kDa due to their slightly different molecular weights. The thirty additional amino acid residues encoded by the used vector increased the molecular weight of the recombinant proteins by 3.3 kDa.
Expression of the recombinant fusion proteins was detected in Western blot analysis. The anti-V5 antibody (Invitrogen) used recognizes the V5 epitope sequence GKPIPNPLLGLDST. The antiserum raised against the V5-epitope reacted with the bands corresponding to the fused ATPase β-subunit proteins (Fig. 4). The purified recombinant ATPase β-subunit proteins appeared mainly as single bands of approximately 54 to 58 kDa which is in agreement with the results from the SDS-PAGE. The Western blot indicates a molecular weight for the recombinant protein from O. oeni of approximately 58 kDa (Fig. 4, lane 2) and confirmed that this organism has an ATPase β-subunit protein which is larger in size than the corresponding proteins from the investigated strains. Analysis of codon usage of the atpD sequence from O. oeni shows a preference of thymine in the third position.
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria
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the Swiss BioteCHnet and was supported by a grant from the Commission of Technology and Innovation of the Federal Office for Professional Education and Technology, Bern, Switzerland.
References
Fig. 4. Western blot for detection of the ATPase β-subunit proteins expressed as fusion proteins in E. coli BL21(DE3)pLysS. The recombinant proteins were purified using the Ni-NTA affinity purification strategy. Proteins were detected using a monoclonal mouse anti-V5 antibody and visualized using an alkaline phosphatase conjugated anti-mouse antibody. Lane 1, MagicMark Western Standard (Invitrogen); lane 2 to lane 7, purified recombinant ATPase β-subunit proteins from O. oeni (lane 2); L. mesenteroides subsp. mesenteroides (lane 3), P. damnosus (lane 4); P. parvulus (lane 5); L. brevis (lane 6) and L. hilgardii (lane 7); lane 8, MagicMark Western Standard (Invitrogen).
For example, codons CCT, CCG, CCA and CCC coding for proline were used with a frequency of 0.44, 0.36, 0.12 and 0.08, respectively and codons GTT, GTC, GTA and GTG coding for valine were used with a frequency of 0.82, 0.11, 0.07 and 0.00, respectively. CGT coding for arginine is used with a frequency of 0.667. AGA and AGG, which are rare codons in E. coli, are as well less frequently used codons in the wine lactic acid bacteria. TTA is the preferred codon used for leucine, ATT for isoleucine and CGT for arginine, respectively of the atpD gene from P. damnosus. The results of the present study indicate that the F1F0ATPase β-subunits from wine-relevant strains can be expressed as recombinant proteins in E. coli. The higher molecular weight band of the ATPase β-subunit of O. oeni confirms that this acidophilic bacterium has an ATPase βsubunit which is larger in size than the corresponding molecules of other lactic acid bacteria due to an additional stretch of amino acids at the N-terminus of the molecule. Further work will focus on the importance of the F1F0ATPase for the physiology of wine-relevant lactic acid bacteria and will explore the regulation of the atp operon in O. oeni exposed to certain environmental factors. The complete F1F0-ATPase sequence including the promotor region of O. oeni contribute to these issues. Acknowledgements The useful discussions and advices of Jürg Gafner, Sergio Schmid and Urs Wäspi during this study are very much appreciated. We would like to thank David Critchley for critical reading of the manuscript. This work is part of a network project from
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Corresponding author: Martin Sievers, Hochschule Wädenswil, Labor für Mikrobiologie und Molekularbiologie, Postfach 335, CH-8820 Wädenswil, Switzerland Tel.: ++41(0)17899716; Fax: ++41(0)17899950; e-mail: [email protected]
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria Martin Sievers1, Christina Uermösi1, Marc Fehlmann1, and Sibylle Krieger2 1 2
Hochschule Wädenswil, Molekularbiologie, Wädenswil, Switzerland Lallemand S.A., Blagnac, France Received: May 30, 3003
Summary The nucleotide sequences of the genes encoding the F1F0-ATPase β-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79–98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase β-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase β-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase β-subunit protein which is larger in size than the corresponding molecules from the investigated strains. Key words: F1F0-ATPase β-subunit – atpD – lactic acid bacteria – TA-cloning
Introduction The cytoplasmatic membrane-bound enzyme complex (F1F0) H+-ATPase catalyzes the formation of ATP driven by the proton gradient resulting from respiration in bacteria with a respiratory chain. The lactic acid bacteria lacking a respiratory chain have a proton-translocating F1F0-ATPase which does not generally synthesize ATP. The F1F0-ATPase regulates the cytoplasmic pH coupled with the extrusion of H+ via hydrolyses of ATP in anaerobic bacteria. The integral membrane proton channel F0 portion consists of three subunits: a (atpB), b (atpF), c (atpE). The F1 consists of five subunits: α (atpA), β (atpD), γ (atpG), δ (atpH), ε (atpC). The ORFs are organized in an operon arranged in the order atpBEFHAGDC in Lactobacillus acidophilus [9] and atpEBFHAGDC in Lactococcus lactis subsp. cremoris [8]. The operons contained no atpI gene in compare to the E. coli operon which consists of nine structural genes atpIBEFHAGDC [7, 22]. The βsubunit of the F1 portion of the ATPase is the catalytic site for ATP synthesis or hydrolysis. A structural model of the F1F0-ATPase and the ATP-binding domain of the F1β-subunit was described recently [1, 2, 5, 14].
In grape musts and wine, populations of lactic acid bacteria are able to grow at low pH values [10]. Increased activity and synthesis of F1F0-ATPase is a major strategy of bacteria to grow at acidic environments [6, 12, 20]. Recently, it was shown that stress compounds in wine have an inhibitory effect on the ATPase activity of O. oeni [3]. Wine lactic acid bacteria carrying out malolactic fermentation are able to synthesize ATP via the F1F0-ATPase from the uptake of malate, the decarboxylation of L-malate into L-lactate and CO2, and the excretion of L-lactate including a proton [13, 17, 21]. In this study, we focused on the DNA sequence of the beta-subunit (atpD) of the F1F0-ATPase from wine-relevant bacteria such as O. oeni, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii, and the non-acidophilic Leuconostoc mesenteroides. The coding sequences of the atpD genes were amplified by PCR and cloned in the vector pCRT7/CT-TOPO for expression in E. coli. We used the cloning procedure to determine the molecular weights of the recombinant ATPase β-subunit molecules by SDSPAGE and Western blot. Our cloning strategy will con0723-2020/03/26/03-350 $ 15.00/0
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria
firm the particular position of the atpD gene of O. oeni showing a molecular size which is larger than the corresponding ATPase β-subunit molecules of the other investigated strains of lactic acid bacteria.
Materials and Methods Bacterial strains Oenococcus oeni strain E656 was obtained from Lallemand, Blagnac, France. O. oeni was grown on MRS medium at pH 5.0 and Oenococcus oenos medium (MLO), Scharlau Chemie S.A., Barcelona, at 25 °C. Pediococcus damnosus DSM 20331, Pediococcus parvulus DSM 20332, Lactobacillus brevis DSM 2647, Lactobacillus hilgardii DSM 20051 and Leuconostoc mesenteroides subsp. mesenteroides DSM 20343 were cultured in appropriate media and at temperatures recommended in the DSMZ catalog. E. coli Top 10F’ cells and E. coli BL21(DE3)pLysS cells from Invitrogen were used as the hosts for the TA-cloning strategy. E. coli cells were grown at 37 °C in LB broth with shaking or on LB agar supplemented with ampicillin (60 µg/ml) and when necessary with chloramphenicol (34 µg/ml). PCR and DNA sequencing Isolation of chromosomal DNA was carried out by standard procedures [18]. Degenerate primers were designed on the basis of conserved motifs of atpG, atpD and atpC subunits of F1F0-ATPase. Primers were synthesized by Microsynth, Switzerland. The conserved sequences of forward and reverse primers, respectively, were: atpGf, 5′-ATTACIMMWSAAATTACIGAAAT-3′; atpDr, 5′TCTTCRTCRGAYAATTCATC-3′; atpDf, 5′-GTTGGTAARA CYGTTTTAATT-3′ and atpCr, 5′-TTDATYTCRATAATDC CACCRTT-3′. Isolated DNA of one strain as template (250 ng) was amplified in a 50 µl reaction volume containing 0.2 µM of the appropriate primer, 10 mM dNTP’s, one unit of HotStarTaq DNAPolymerase (Qiagen) and 5.0 µl of 10× PCR-buffer supplemented with MgCl2. PCR amplifications were performed in a Primus Multiblock TC-HTS thermocycler (MWG-Biotech). After initial denaturation for 15 min at 95 °C, a total of 40 cycles were run, each consisting of 1 min at 95 °C, 1 min at 40 °C and 5 min at 72 °C. The program was terminated with a 7-min incubation step at 72 °C prior to cooling at 8 °C. The amplification products were analysed by electrophoresis on a 1.0% agarose gel stained with ethidium bromide (1 µg/ml). Specific PCR-products were isolated from the agarose gel using the QIAquick gel extraction kit (Qiagen) procedure. Direct sequencing of the PCR-products was performed by the dideoxy-chain termination method using the Big-Dye Terminator chemistry version 1.1 (Applied Biosystems). Purification of the sequencing reaction was performed using the DyeEx spin kit (Qiagen) and analysed on an Applied Biosystems 310 DNA sequencer. Sequence comparison and phylogenetic analysis The deduced protein sequences of the atpD open reading frames were aligned by using the ClustalW computer program. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (http://www.megasoftware.net/). Recombinant DNA techniques Isolation of plasmid DNA and purification of plasmid DNA and PCR-products were carried out with the appropriate Qiagen (Hilden, Germany) kits.
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Amplified PCR products were purified, inserted into the pCRT7/CT-TOPO vector (Invitrogen) and transformed in E. coli Top 10F’ cells. The recombinant plasmid containing the insert was purified and digested with restriction enzyme (PvuI, HindIII, BamHI or DraI) to check the correct insertion orientation. Recombinant plasmids (pCRT7/CT(AtpD)) containing the ATPase β-subunit expression construct in the right orientation were used for further transformation steps in E. coli BL21(DE3)pLysS. Each expressed F1F0-ATPase β-subunit molecule is a fusion protein, which contains a C-terminal V5 epitope and the sequence coding for six histidine residues (6× His-tag). The V5 epitope was used for detection of the protein in western blot assay. Purification of the recombinant proteins E. coli BL21(DE3)pLysS transformed with pCRT7/CT(AtpD) was grown in 6 ml LB containing ampicillin and chloramphenicol to an OD600nm of 0.5. IPTG was then added to a final concentration of 1 mM and the cultures were grown for further 4 h at 37 °C. Cells were collected by centrifugation and resuspended in 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl, pH 8.0 for cell lysis. The recombinant hexahistidine-tagged ATPase β-subunit proteins were purified using the NI-NTA spin kit from Qiagen. SDS-PAGE and Western Blotting The cell extracts and the purified ATPase β-subunit proteins were examined by SDS-PAGE on 10% polyacrylamide gels. The gels were stained with Coomassie brilliant blue R-250, or electroblotted onto PVDF membrane for Western blotting. Immunodetection was performed with a monoclonal mouse anti-V5 antibody (Invitrogen). The WesternBreeze chromogenic detection kit-anti-mouse (Invitrogen) was used to develop the blot. The same antibody-conjugate reagent used to detect our recombinant proteins visualized also the proteins from the MagicMark standard (Invitrogen). Nucleotide sequence accession numbers The EMBL accession numbers of the F1F0-ATPase (β-subunit nucleotide sequences which we determined are AJ430065 (O. oeni E656), AJ508912 (L. mesenteroides subsp. mesenteroides DSM 20343), AJ508913 (L. brevis DSM 2647), AJ508914 (L. hilgardii DSM 20051) AJ515142 (P. damnosus DSM 20331), AJ515143 (P. parvulus DSM 20332), respectively.
Results and Discussion The atpD genes encoding the β-subunit of the F1F0ATPase from six wine lactic acid bacteria were amplified by PCR and sequenced using degenerate primers. Based on these sequences, specific primers for PCR amplification of the atpD open reading frames were designed (Table 1). The amplified fragments were cloned into the pCRT7/CT-TOPO vector (Invitrogen) for expression of the recombinant proteins in E. coli. The deduced amino acid sequences of the investigated atpD gene products were aligned to determine the homology of the F1F0ATPase β-subunit proteins among the different organisms. The distribution of highly and less conserved regions along the primary structure of the ATPase β-subunit molecules is visualized in the alignment given in Fig. 1. The amino acid sequence homologies of the ATPase βsubunit molecules from the investigated strains in this
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study are in the range of 79% to 98%. For example, the ATPase β-subunit protein of O. oeni is homologous to the corresponding proteins from L. mesenteroides subsp. mesenteroides (76% identity, 83% similarity,), L. brevis (72% identity, 81% similarity), L. hilgardii (72% identity, 79% similarity), P. damnosus (73% identity, 81% similarity) and P. parvulus (74% identity, 82% similarity). The ATPase β-subunit from L. brevis showed a similarity of 91% and an identity of 84% to the corresponding protein of L. hilgardii. The ATPase β-subunit proteins from P. damnosus and P. parvulus are 96% identical. A stretch of 38 residues 1 to 38 at the N-terminus of the F1F0-ATPase β-subunit protein from O. oeni is unique for this organism. A similarity search of the 38 additional amino acids against other sequences deposited in the EMBL and Genbank databases showed no similarity with any other sequence. The F1F0-ATPase β-subunit constitutes the catalytic sites of the enzyme and is characterized by mainly two nucleotide-binding sequence motifs. The nucleotide-binding sequence (motif A) for catalytic activity in nucleotide binding proteins is a glycine-containing loop with the motif GXXXXGKT (position 149–156 in E. coli atpD gene, methionine is not present in the mature protein [19]) [23]. Studies on ATPases with mutations in the βsubunit indicated that the conserved sequence GERXXE (β180–β185) is as well essential for catalytic activity. The residues of the catalytic site of the β-subunit around ATP are βLys-155, which binds to the β and γ phosphate moieties of ATP, βThr-156, βGlu-181, βArg-182 and βGlu185 [14, 16]. The two conserved motifs GGAGVGKT and GERTRE were found in all of the six ATPase β-subunit proteins of the investigated strains and are marked in the alignment given in Figure 1. The conserved sequence motif DDLTDP (β301–β306) in the F0F1-ATPase β-subunit seems to be a key region important for the stability of the assembly of the α3β3γ complex of the F1 sector [15].
The βLeu-303 of the described conserved sequence is replaced by βTyr-303 in the β-subunit sequences of our investigated lactic acid bacteria strains which have the sequence motif DDYTDP. A phylogenetic tree was reconstructed to determine the relationships of the homologous ATPase β-subunit molecules with known sequences available from the Genbank. The wine lactic acid bacteria investigated in this study formed one major cluster well separated in the ATPase tree shown in Figure 2. This cluster is composed of three groups. The first group comprises the sequences of L. brevis and L. hilgardii. The second group is composed of the closely related ATPase β-subunit molecules from the P. damnosus and P. parvulus. O. oeni formed together with L. mesenteroides subsp. mesenteroides a more distant group. O. oeni exhibits an acidophilic phenotype and is an example of a tachytelic (fast evolving) bacterium which forms a long 16S rRNA branch separating this species from Leuconostoc and Weissella [4, 24]. Phylogenetic trees based on ATPase β-subunit sequences generally support the topology of the 16S rRNA trees with the distinction that some branchings are better resolved using the rRNA data [11]. The atpD gene of O. oeni has an open reading frame of 1515 bp encoding 504 amino acid residues with a calculated molecular mass of 54.7 kDa. The calculated molecular masses of the ATPase β-subunit molecules of L. mesenteroides subsp. mesenteroides, P. damnosus, P. parvulus, L. brevis and L. hilgardii are 50.3 kDa, 50.8 kDA, 51.0 kDa, 51.1 kDa and 52,2 kDa, respectively. The expressed proteins obtained in this work are fusion proteins, which contain a C-terminal tag of six histidine residues and a V5 epitope, both used for purification and Western blot. Due to the unusual N-terminus of the ATPase β-subunit protein of O. oeni, a fusion at the Ctermini of the investigated proteins with the vector sequence was chosen.
Table 1. Specific PCR primers used for amplification of the atpD (F1F0-ATPase β-subunit) coding sequences of the wine lactic acid bacteria for cloning into pCRT7/CT-TOPO vector as fusion proteins. Species
strain
Accession No.
atpD cds, bp
calculated molecular mass, kDa
PCR primers (sequence 5′ to 3′) forward, f reverse, r
O. oeni
E656
AJ430065
1515
54.7
f: ATG GCA GAA AAG AAA ACA ACA r: GCT TTC CAG CTT TTT AGC C
L. mesenteroides DSM 20343 subsp. mesenteroides
AJ508912
1401
50.3
f: ATG AGT ACT GGA AAA GTC GT r: TTG GGC CAT TGT CTT GGC T
P. damnosus
DSM 20331
AJ515142
1413
50.8
f: ATG AGT ACT GGT AAA GTT GTT r: TTT ACT AGT GGC ACT AGT TAA
P. parvulus
DSM 20332
AJ515143
1413
51.0
f: ATG AGT ACT GGT AAA GTT GTC r: TTT ACT TGC AGC ACT AGT TAA
L. brevis
DSM 2647
AJ508913
1416
51.1
f: ATG AGT GCT GGT AAA ATT GTT r: GTT GTT GGC AAC GGC GTC
L. hilgardii
DSM 20051
AJ508914
1446
52.2
f: ATG AGT ACT GGT AAA GTT TTA C r: GTT TGC TGC ACT TTG CTT TT
Fig. 1. Alignment of the deduced amino acid sequences of the ATPase β-subunits from the wine-relevant lactic acid bacteria using the ClustalW program. Identical residues, well-conserved and less-conserved positions are indicated as * and :,., respectively. The three conserved motifs GGAGVGKT, GERTRE and DDYTDP are marked. Abbreviations: Oo, Oenococcus oeni; Lm, Leuconostoc mesenteroides subsp. mesenteroides; Pd, Pediococcus damnosus; Pp, Pediococcus parvulus; Lb, Lactobacillus brevis; Lh, Lactobacillus hilgardii
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Fig. 2. Phylogenetic tree showing relationships of ATPase β-subunit deduced protein sequences. The tree was reconstructed by the neighbor-joining method. Two major clusters compose this ATPase tree, one of ATPase β-subunit proteins from the wine-relevant lactic acid bacteria, the other of ATPase β-subunit proteins from Lactococcus lactis subsp. lactis, Streptococcus, Enterococcus faecalis and Lactobacillus species. The bar indicates 2% amino acid differences.
Fig. 3. SDS electrophoresis in 10% polyacrylamide gel of recombinant ATPase βsubunit proteins from wine-relevant lactic acid bacteria expressed as fusion proteins in E. coli BL21(DE3)pLysS harboring pCRT7/CT(AtpD). Gel was stained with Coomassie brilliant blue R-250. Lane 1, SDS-PAGE molecular weight standards, broad range (Bio-Rad); lane 2, total cellular fraction harboring recombinant ATPase β-subunit from O. oeni, no IPTG induction; lane 3, total cellular fraction harboring recombinant ATPase β-subunit from O. oeni, IPTG induction; lane 4 to lane 9, one-step purified ATPase β-subunit proteins using NI-NTA column and elution with imidazole from O. oeni (lane 4); L. mesenteroides subsp. mesenteroides (lane 5), P. damnosus (lane 6); P. parvulus (lane 7); L. brevis (lane 8) and L. hilgardii (lane 9); lane 10, SDS-PAGE molecular weight standards, broad range (Bio-Rad).
SDS-PAGE analysis of whole-cell lysates and of the purified recombinant proteins indicated that the ATPase βsubunit proteins were successfully expressed in E. coli. The expression of the recombinant proteins was optimal when induced for 4 h with 1 mM IPTG. A band corresponding to a 58 kDa protein was observed in SDS-PAGE after Coomassie staining of crude extracts of E. coli BL21(DE3)pLysS harboring pCRT7/CT(AtpD) containing the atpD insert from O. oeni after IPTG induction (Fig. 3, lane 3). Lanes 4 to 9 (Fig. 3) contain the one-step purified hexahistidine-tagged ATPase β-subunit proteins of the investigated strains migrating between 54 to 58 kDa due to their slightly different molecular weights. The thirty additional amino acid residues encoded by the used vector increased the molecular weight of the recombinant proteins by 3.3 kDa.
Expression of the recombinant fusion proteins was detected in Western blot analysis. The anti-V5 antibody (Invitrogen) used recognizes the V5 epitope sequence GKPIPNPLLGLDST. The antiserum raised against the V5-epitope reacted with the bands corresponding to the fused ATPase β-subunit proteins (Fig. 4). The purified recombinant ATPase β-subunit proteins appeared mainly as single bands of approximately 54 to 58 kDa which is in agreement with the results from the SDS-PAGE. The Western blot indicates a molecular weight for the recombinant protein from O. oeni of approximately 58 kDa (Fig. 4, lane 2) and confirmed that this organism has an ATPase β-subunit protein which is larger in size than the corresponding proteins from the investigated strains. Analysis of codon usage of the atpD sequence from O. oeni shows a preference of thymine in the third position.
Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria
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the Swiss BioteCHnet and was supported by a grant from the Commission of Technology and Innovation of the Federal Office for Professional Education and Technology, Bern, Switzerland.
References
Fig. 4. Western blot for detection of the ATPase β-subunit proteins expressed as fusion proteins in E. coli BL21(DE3)pLysS. The recombinant proteins were purified using the Ni-NTA affinity purification strategy. Proteins were detected using a monoclonal mouse anti-V5 antibody and visualized using an alkaline phosphatase conjugated anti-mouse antibody. Lane 1, MagicMark Western Standard (Invitrogen); lane 2 to lane 7, purified recombinant ATPase β-subunit proteins from O. oeni (lane 2); L. mesenteroides subsp. mesenteroides (lane 3), P. damnosus (lane 4); P. parvulus (lane 5); L. brevis (lane 6) and L. hilgardii (lane 7); lane 8, MagicMark Western Standard (Invitrogen).
For example, codons CCT, CCG, CCA and CCC coding for proline were used with a frequency of 0.44, 0.36, 0.12 and 0.08, respectively and codons GTT, GTC, GTA and GTG coding for valine were used with a frequency of 0.82, 0.11, 0.07 and 0.00, respectively. CGT coding for arginine is used with a frequency of 0.667. AGA and AGG, which are rare codons in E. coli, are as well less frequently used codons in the wine lactic acid bacteria. TTA is the preferred codon used for leucine, ATT for isoleucine and CGT for arginine, respectively of the atpD gene from P. damnosus. The results of the present study indicate that the F1F0ATPase β-subunits from wine-relevant strains can be expressed as recombinant proteins in E. coli. The higher molecular weight band of the ATPase β-subunit of O. oeni confirms that this acidophilic bacterium has an ATPase βsubunit which is larger in size than the corresponding molecules of other lactic acid bacteria due to an additional stretch of amino acids at the N-terminus of the molecule. Further work will focus on the importance of the F1F0ATPase for the physiology of wine-relevant lactic acid bacteria and will explore the regulation of the atp operon in O. oeni exposed to certain environmental factors. The complete F1F0-ATPase sequence including the promotor region of O. oeni contribute to these issues. Acknowledgements The useful discussions and advices of Jürg Gafner, Sergio Schmid and Urs Wäspi during this study are very much appreciated. We would like to thank David Critchley for critical reading of the manuscript. This work is part of a network project from
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Corresponding author: Martin Sievers, Hochschule Wädenswil, Labor für Mikrobiologie und Molekularbiologie, Postfach 335, CH-8820 Wädenswil, Switzerland Tel.: ++41(0)17899716; Fax: ++41(0)17899950; e-mail: [email protected]