INTRODUCTION

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The (F₁F_o) H⁺-ATPase complex plays an important role in the free energy metabolism of virtually all living cells. The structures of F₁F_o-ATPase complexes from different sources are very similar and consist of two parts: a membrane integral part, F_o, which forms a proton channel, and a soluble part, F₁, which contains the catalytic site for ATP hydrolysis. In bacteria, the enzyme is located in the cytoplasmic membrane, where it catalyzes the interconversion of ATP and the transmembrane proton gradient. Depending on the particular organism and on the conditions for growth, the enzymes function in the direction of either ATP synthesis or ATP hydrolysis (14). In organisms which contain a respiratory chain, such as Escherichia coli and Bacillus subtilis, the primary role of the enzyme is to synthesize ATP driven by the proton gradient that results from respiration, when these organisms are supplied with an electron acceptor. In organisms that lack a respiratory chain, or in the absence of electron acceptors, the enzyme generates a transmembrane proton gradient, and this process is then driven by ATP hydrolysis. The anaerobic bacterium Lactococcus lactis also possesses an F₁F_o-ATPase complex. This bacterium lacks the respiratory chain, and the enzyme here is involved in the extrusion of protons driven by ATP hydrolysis to generate the necessary driving force for solute transport and to maintain an acceptable intracellular pH value (21, 38). The latter function is supported by the fact that the activity of the F₁F_o-ATPase in these anaerobic bacteria is enhanced at low external pH (2, 23).

The anaerobic bacteria have an alternative route to generate a proton gradient across the cytoplasmic membrane, namely, through end product excretion. In the so-called energy recycling model, which was first demonstrated by Michels et al. (27), it was suggested that carrier-mediated excretion of end products can occur in symport with protons, and this contributes to the generation of the transmembrane proton gradient. This mechanism has been thoroughly investigated in Lactococcus lactis by Otto et al. (30), and ten Brink et al. (40), who demonstrated that the energy recycling by lactate efflux makes a significant contribution to the generation of the proton gradient in this organism, particularly at high external pH and low external lactate concentrations. An interesting question is then whether this contribution would be sufficient to allow growth of L. lactis in the absence of the H⁺-ATPase.

In this paper we report the cloning, sequencing, and characterization of the genes that encode the H⁺-ATPase in L. lactis subsp. cremoris MG1363. A mutant strain was constructed in which the expression of H⁺-ATPase on the chromosome is under control of the nisA promoter. The strain was completely dependent on nisin for growth on GM17 plates, which demonstrates that the H⁺-ATPase is an essential enzyme for growth of L. lactis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains. The plasmid-free L. lactis subsp. cremoris strain MG1363 (16) was used to study the atp operon in L. lactis. E. coli K-12 strain BOE270 is highly competent with respect to transformation and was derived from strain MT102, which is an hsdR derivative of strain MC1000 [araD139 (ara-leu)7679 galU galK (lac)174 rpsL thi-1] (7). BOE270 was used as a host for plasmids in the cloning procedures and for propagation of plasmid DNA in E. coli.

Oligonucleotides and enzymes. Oligonucleotides were obtained from Hobolth DNA Synthesis (Hillerød, Denmark). Restriction enzymes (Gibco BRL, Pharmacia), Taq and Pfu DNA polymerases (Pharmacia and AH Diagnostics, respectively), calf intestine alkaline phosphatase (Pharmacia), and T4 DNA ligase (Gibco BRL) were used as recommended by the manufacturers.

Sequencing and sequence analysis of the H⁺-ATPase operon. The DNA sequencing was carried out either by the dideoxy nucleotide chain termination method (33) with [-³³P]ddNTP (500 Ci/mmol) (Pharmacia) or by autosequencing by capillary electrophoresis with the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer).

Transformation. Cells of E. coli were made competent by the Ca²⁺ method (32). Plasmid DNA was used to transform the cells by a standard transformation procedure (28), and the transformation mixtures were plated at 30°C on Luria-Bertani agar plates supplemented with either ampicillin (100 µg/ml) or erythromycin (200 µg/ml). Cells of L. lactis (16) were made competent by growth in GM17 medium containing 1% glycine and resuspended in 10% glycerol and 0.5 M sucrose as described by Holo and Nes (18). Plasmid DNA was used to transform the cells by electroporation (18), and the cells were allowed to regenerate in SGM17 medium for 2 h and then plated onto Schmidt-Ruppin plates containing the appropriate selective antibiotic.

Cloning of the atp operon from L. lactis subsp. cremoris MG1363. Fragments of the atp operon were cloned as PCR products or by the plasmid rescue technique (see below). Chromosomal DNA from L. lactis MG1363 was used as a template for amplification of DNA. Several primer sets were used to amplify different regions of the atp operon (Fig. 1). Here we took advantage of the fact that in an unrelated project, the first part of the atp operon from the closely related bacterium L. lactis subsp. lactis B1014 was accidentally discovered in a clone from a gene library, which allowed us to design primers for the amplification of the genes that encode the F_o part of the enzyme complex. PCR amplification was carried out in a total volume of 100 µl and in the presence of 0.4 mM concentrations of each deoxynucleoside triphosphate (Boehringer), 3 to 5 µM concentrations of each primer DNA, 0.1 µg of chromosomal DNA, 2.5 U of Taq polymerase, and the buffer recommended by the manufacturer (Pharmacia). The reactions were carried out for 25 cycles (1 min at 94°C for denaturing, 1 min at 55°C for annealing, and 2 min at 72°C extension step) by use of a DNA thermal cycler. The resulting PCR products were cloned in pMOSBlue (Amersham) and sequenced. To confirm the correctness of the cloned product, the sequence was also determined directly on the PCR products.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Genetic organization of the L. lactis atp operon and DNA fragments used in this work. The open reading frames are shown as boxes, and the designations of the atp genes are shown below the boxes in italic letters. The designations of the H+-ATPase subunits are shown above the boxes. The arrow indicates the direction of transcription of the atp operon, and the stem loop indicates the putative terminator. The cloned fragments, which are referred to in the text, are indicated in the boxes below the scale.

Cloning of atpC by plasmid rescue. Plasmid pQ1, which harbors the DNA sequence from position 4177 to position 6394 (the C-terminal part atpG of the product and the N-terminal part atpD of the product) and was obtained by cloning a PCR fragment obtained with primers 3987 (5'-TTGGTGGTGGATCAATGACGGC) and 3991 (5'-TTNCCNTCACGAGTACGNTCNCC), was inserted into pMOSBlue. This plasmid was used to construct a plasmid for cloning the remaining part of the atp operon by the plasmid rescue technique as follows. A 3.2-kb EcoRI fragment from pCP12 carrying the erm gene and the strong artificial constitutive promoter CP12 (19) was cloned into pQ1 digested with EcoRI, resulting in pBK105, in which the atpGD' genes (sequence from position 5268 to 6394) had been placed under control of the CP12 promoter. The plasmid pBK105 was then used to transform L. lactis MG1363 to erythromycin resistance (2 µg/ml). This plasmid is unable to replicate in L. lactis, and only cells with the plasmid integrated into the chromosome will become resistant to erythromycin. If the plasmid integrates into the atp operon by a Campbell-type event, the genes atpDC will come under control of the CP12 promoter. Chromosomal DNA of some transformants was prepared, and the appropriate integration of pBK105 was verified by PCR techniques. The chromosomal DNA was digested with SalI, ligated at a low DNA concentration, and transformed in E. coli, which resulted in pRESDC, in which approximately 4.5 kb downstream of the atp operon was cloned. Plasmid pRESDC was more extensively characterized and sequenced.

Primer extension. Total RNA was extracted from exponentially growing L. lactis (30°C, optical density at 600 nm [OD₆₀₀] = 0.5) in GM17 (1% glucose) by the FastRNA kit, BLUE (Bio 101), as recommended by the manufacturer.

Northern blotting. A single-stranded RNA probe labeled with [-³²P]CTP was derived from a PCR product (primer 5883, 5'-CAACGTGTCCTTCAACGC, and primer T7atpC, 5'-TAATACGACTCACTATAGATAAACCACACCAGCAGGGG), which contains atp'DC' (position 6918 to 7462) and the T7 promoter, by in vitro transcription using T7 RNA polymerase (Promega). A total RNA preparation (12 µg) was dried in a vacuum drier and resuspended in 4.5 µl of H₂O, 2 µl of 5× formaldehyde gel running (FGR) buffer (0.1 M MOPS [morpholinepropanesulfonic acid] [pH 7], 40 mM sodium acetate, 5 mM EDTA), 3.5 µl of formaldehyde (final concentration, 7% [vol/vol]), and 10 µl of formamide (final concentration, 50% [vol/vol]). The RNA molecules were denatured by incubation for 15 min at 60°C and separated by electrophoresis in a 1.2% (wt/vol) agarose gel containing 2.2% formaldehyde, which was run at 5 V/cm with FGR buffer as the electrophoresis buffer. The gel was then washed in H₂O for 20 min at room temperature. The RNA was transferred to a Zeta-Probe GT membrane (Bio-Rad) by overnight capillary blotting with 50 mM NaOH as the transfer buffer. The membrane was air dried and prehybridized for 2 h at 42°C in hybridization buffer (1 mM NaCl, 4 mM Na₄P₂O₇, 5× Denhardt's solution, 1% sodium dodecyl sulfate [SDS], 10% [wt/vol] polyethylene glycol 6000, 50 mM Tris-HCl [pH 7.5], 50% [vol/vol] formamide) before the -³²P-labeled riboprobe was added. After overnight hybridization at 42°C, the membrane was washed twice for 5 min at room temperature in 2× SSC, twice at 30 min at 65°C in 0.2× SSC-1% SDS, and twice for 30 min at 65°C in 0.1× SSC before being used for autoradiography (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The 0.24- to 9.5-kb RNA ladder from Gibco BRL was used as a molecular size standard.

Replacement of the chromosomal atp promoter in L. lactis by the nisin-inducible nisA promoter. A PCR fragment that harbors the DNA sequence from position +998 to position 1850 (the atpEB' genes) was amplified using Taq polymerase. After polishing the DNA ends with Pfu polymerase, the fragment was cloned into the SfrII site on the vector pCR-Script Amp SK(+) (Stratagene) (Fig. 2). A plasmid was isolated in which the fragment was inserted in the orientation opposite to that of lacZ (pRI13). A 1.5-kb SalI-PstI fragment from pNZ8010 (12) that carries the cat-194 gene and the nisA promoter was then cloned into pRI13 digested with SalI-PstI, which yielded the plasmid pATP1, in which the atpEB' genes had been placed under the control of the (nisin-inducible) nisA promoter. A 2.4-kb ApaI-NotI fragment from pATP1, which contains the cat-194 gene, the nisA promoter, and the atpEB' genes, was cloned into pRC1 digested with ApaI-NotI, which gave rise to plasmid pNIS-ATP2. pRC1 is a 3.5-kb derivative of pBluescript II KS in which the bla gene has been replaced by the ermAM genes to allow for selection of erythromycin resistance in L. lactis (25). The strain NZ9000 (12) is a derivative of strain MG1363 (16) in which the nisR and nisK genes (required for induction of the nisA promoter) are integrated into the pepN locus on the chromosome. Plasmid pNIS-ATP2 was introduced into strain NZ9000 with selection for erythromycin resistance (2 µg/ml) on plates that contained nisin (5 ng/ml). Since this plasmid is unable to replicate in L. lactis, only cells in which the plasmid has integrated into the chromosome should become resistant to erythromycin. If the plasmid integrates into the atpEB locus, the transcription of the entire atp operon will be placed under the control of the nisA promoter. The clones were verified by PCR with primers positioned upstream of the nisA promoter and immediately downstream of position 1850. 

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Cloning strategy used in the replacement of the native atp promoter with the nisin-inducible nisA promoter. (a) A 1.5-kb SalI-PstI fragment from pNZ8010 (12) carrying the cat-194 gene and the nisA promoter was cloned into pRI13 digested with SalI-PstI (pATP1). (b) A 2.4-kb ApaI-NotI fragment from pATP1, containing the cat-194 gene, the nisA promoter, and the atpEB' genes, was then cloned into pRC1 digested with ApaI-NotI (pNIS-ATP2). (c) Plasmid pNIS-ATP2 was integrated into the atp operon in L. lactis strain NZ9000 with selection for erythromycin resistance (2 µg/ml) on plates containing nisin (5 ng/ml), resulting in replacement of the native atp promoter with the inducible nisA promoter. The designation of the genes is shown above the boxes in italic letters. See Materials and Methods for further details. S, SalI; P, PstI; A, ApaI; N, NotI.

Nucleotide sequence accession number. The sequence of the lipA gene, the sequence of the atp operon of L. lactis subsp. cremoris strain MG1363, and the sequence downstream of the atp operon (8,912 bp) have been deposited in the NCBI data bank with the accession no. AF059739, and the numbers used in the present paper refer to the numbering used in this sequence.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

The genes encoding the H⁺-ATPase in L. lactis. The genes encoding the subunits of the H⁺-ATPase were cloned on a series of overlapping fragments, and the complete sequence of the atp operon was determined and analyzed for the presence of open reading frames (Fig. 1). Within a 7-kb region we identified eight open reading frames with putative ribosome binding sites. The deduced amino acid sequences of the eight gene products of the L. lactis atp operon were aligned with the corresponding amino acid sequences from other organisms, and the sequences of the L. lactis ATPase subunits showed good homology with those of other bacteria (Table 1). The homologies were particularly high between L. lactis, Streptococcus mutans, and Streptococcus bovis, which confirms the close evolutionary relationships of these bacteria. Among the ATPase subunits, the , , and  subunits from the cytoplasmic domain, F₁, were especially highly conserved. The consensus nucleotide-binding domains, Walker motifs A (GXXXXGKT) and B (L-hydrophobic-hydrophobic-hydrophobic-D) (1, 42), were also conserved in the deduced sequences of the  and  subunits. Significantly lower homologies were seen for the subunits of the membrane-bound domain, F_o. The  subunit, a part of the F₁ domain, exhibited the lowest subunit homology in the comparison.

Transcription of the atp genes. A standard promoter with 35 (TTGACA) and 10 (TAGAAT) consensus boxes separated by 17 nucleotides was identified in the region upstream of atpE (Fig. 3). The presence of the 35 and 10 consensus sequences suggests that the promoter is recognized by the L. lactis ³⁹ transcription factor (3), and primer extension analysis confirmed the existence of a transcript that corresponds to this promoter (Fig. 3). In comparison with other lactococcal promoters, the similarities were particularly high between the atp promoter and the rrnA promoter (rRNA operon) (8). The region upstream of the 35 region of the atp promoter sequence (position 853 to 963) has a higher A+T content (75%) than the average value reported for L. lactis DNA (62.8%), which may contribute to the activity of the atp promoter, due to curvature of the A+T-rich sequences (5, 15).

View larger version (26K): [in this window] [in a new window]   FIG. 3.   The atp promoter and the transcriptional initiation site for the atp operon. (a) Determination of the transcription initiation site of the atp operon. Primer extension analysis was carried out using primer 5264 labeled by 33P as described in Materials and Methods. A sequence ladder was made by sequencing with primer 5264 on a PCR product as described in Materials and Methods. (b) Comparison of the promoter region of related organisms. Letters in bold indicate conserved bases. The putative 35 and 10 consensus boxes of the atp promoter for L. lactis upstream of atpE are underlined. The transcription initiation site (TS) at +1 bp is indicated with an asterisk. Note the extensive homology, particularly around position +1.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Northern blot analysis. Total RNA was extracted from L. lactis, and Northern blot analysis was performed as described in Material and Methods. A ribonucleotide probe labeled with [-32P]CTP containing the C-terminus-encoding part of atpD and the N-terminus-encoding part of atpD (position 7915 to 8459) was used as a probe. The 0.24- to 9.5-kb RNA ladder from Gibco BRL was used as a molecular size standard.

The F₁F_o-ATPase is essential for growth of L. lactis. In the anaerobic bacterium L. lactis, the role of the H⁺-ATPase is to maintain the electrochemical proton gradient across the cytoplasmic membrane, and it has been proposed that the H⁺-ATPase functions to regulate the internal pH (4, 11, 21, 24). Is the H⁺-ATPase then essential for growth? In principle, the anaerobic bacteria have the option to generate a proton gradient through carrier-mediated excretion of end products in symport with protons (30).

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Colonies of strain L. lactis PJ4700, in which the native atp promoter had been replaced by a nisA promoter. The strain was streaked on GM17 plus 2 µg of erythromycin/ml at various nisin concentrations (0, 0.25, 0.5, 1, 2, 4, 8, and 16 ng of nisin/ml), and the graph illustrates the average diameter of colonies obtained with the different nisin concentrations.