INTRODUCTION

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Nitrate can be used as an alternative to oxygen in respiration by many bacteria and by the mitochondria of certain fungi (1, 17). In most cases, nitrate reduction is the first step in a pathway catalyzed by a series of membrane-bound reductases whose final products are dinitrogen and ammonia (1, 17).

We have recently described the presence of a nitrate reductase operon in the extreme thermophile Thermus thermophilus HB8, a bacterium formerly considered a strict aerobe (11). Interestingly, the absence of a nitrite reductase in this bacterium results in the long-term accumulation of nitrite in the medium of anaerobically grown cultures. Accordingly, a very active nitrite export system should function in this bacterium to escape from its high toxicity.

Genes encoding polytopic membrane proteins belonging to the major facilitator superfamily of transporters (9) have been found close to the nar gene cluster for all such genes so far sequenced (1, 17). The role of such proteins is still controversial as nitrate/nitrite or H⁺/nitrite antiporters and nitrate/H⁺ symporters (17). There is experimental evidence that the NarK protein, encoded upstream of the narGHJI operon of Escherichia coli, is essentially implicated in nitrite export by using the electrochemical gradient as the energy source (an H⁺/nitrite antiporter) (14). On the other hand, and despite their similarity to the E. coli NarK, the NarT and NasA proteins from Staphylococcus carnosus and Bacillus subtilis, respectively, have been proposed to function as nitrate/H⁺ symporters (5, 10). Thus, the role of such membrane transporters in different bacteria is still unclear due to the intrinsic difficulties in measuring the transport of these anions and also because of the ability of these bacteria to overcome mutations in the corresponding genes through secondary transporters (17). Moreover, the ability of most denitrifiers to use alternative anaerobic pathways for growth increases the difficulties in analyzing the effects of mutations in the corresponding nitrate/nitrite transporters.

Since T. thermophilus HB8 does not have any alternative way for anaerobic growth than nitrate respiration, and keeping in mind that this bacterium lacks a nitrite reductase, a requirement for nitrite extrusion proteins seems crucial for its viability. Thus, any mutation in the putative nitrite transporters should have a strong phenotypic consequence in this bacterium. Here we describe two NarK homologues (named NarK1 and NarK2) encoded by genes downstream of the narGHJI operon of T. thermophilus HB8, one of which (narK2) overlaps the replicative origin of the nar-carrying conjugative plasmid (13). We demonstrate that mutations in each of the coding genes (narK1 and narK2) have minor effects on the ability to grow anaerobically, whereas double narK1 narK2 mutants are unable to grow under these conditions. This, and the analysis of nitrite excretion, led us to conclude that both proteins are implicated in nitrate import and nitrite export during anaerobic growth.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. T. thermophilus HB8 (ATCC 27634) was obtained from the American Type Culture Collection (Rockville, Md.). Its nitrate reductase (NR) deletion derivative (narGH::kat) was described previously (12). E. coli strains TG1 [supE (nsdM mcrB) 5(r_Km_K mcrB) thi (lac-proAB) F' (traD36 proAB⁺ laqI^qZM15)] and DH5F' [F' supE44 (lacZYA-argF)U169 80lacZM15 hsdR17 recA1 endA1 gyrA96 thi-1 relA1] (Bethesda Research Laboratories, Gaithersburg, Md.) were used as hosts for plasmid construction. Plasmids pNIT5 and pNIT9 (12) were pUC119 derivatives containing the narK1 and narK2 genes, respectively. Plasmid pKT1 was the source of the kat gene, encoding thermostable resistance to kanamycin (7).

Mutant isolation. Insertional mutagenesis was done through electroporation of T. thermophilus HB8 (3) with HindIII-linearized forms of defined plasmids, followed by selection on kanamycin-containing plates.

Genetic analysis. General methods were used for DNA manipulation (15). Southern blots of total DNA from putative mutants digested with the appropriate enzyme(s) were hybridized with fluorescein-labeled oligonucleotides O-70 (5'CGGAGAGGAAGATGCCG3'), which hybridized to the sequence of narK2, and Okat-2 (5'GAAACTTCTGGAATCGC3'), directed against the 3' region of the kat gene, and revealed with the ECL kit (Amersham Ibérica SA). DNA was sequenced by automatic methods (Applied Biosystems) with synthetic primers (Isogen Bioscience, Maarssen, The Netherlands). Partial sequences were assembled with the software of the University of Wisconsin Genetics Computer Group (4). DNA amplification from colonies of the putative mutants was developed in an MJ Research minicycler (MJ Research Inc., Watertown, Mass.). Oligonucleotides O-28 (5'CACCCTCATGTTCGCCG3'), O-54 (5'CCACCCTCCTCCTTCTC3'), O-65 (5'CGGGCCGATGAACTTGG3') and O-70 (see above) were used for the amplification of specific fragments. Their approximate hybridization sites are labeled in Fig. 2A.

NR activity and nitrite analysis. For induction of the NR, cells of T. thermophilus HB8 were grown at 70°C in a shaker bath to an OD₅₅₀ of 0.5. After addition of potassium nitrate (40 mM), the cultures were incubated at 70°C without stirring for an additional 2 h. Cells were then recovered by centrifugation, washed twice with phosphate buffer (50 mM, pH 7) by centrifugation (10,000 × g, 5 min, room temperature), and disrupted by sonication at a cell density of ~24 OD₅₅₀ units/ml. Intracellular concentrations of nitrite were measured in the above cell extracts by assuming a cell volume and a ratio of OD₅₅₀ to cell mass similar to those of E. coli (10¹² ml/cell and 10⁹ cells per OD₅₅₀ unit per ml). The NR activity was measured as described before (12) after 5 min of incubation at 80°C with methyl viologen as the electron donor (16) and nitrate as the electron acceptor. The nitrite excreted was measured in cell-free samples of the growth medium.

	RESULTS

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Sequence of the region downstream of the narGHJI operon. Analysis of the sequence downstream of the narGHJI operon from T. thermophilus HB8 (accession number AJ237974) revealed the presence of two open reading frames encoding 435 and 443 amino acids, both of them preceded by putative Shine-Dalgarno sequences. Due to their similarities to the sequences for nitrite transporters found close to nar operons from other bacteria (see below), the corresponding encoding genes were named narK1 and narK2, respectively.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Average-distance tree for NarK proteins. The sequences of the putative nitrate and nitrite transporters were aligned through the CLUSTAL program, and the average distance was represented through the JALview editor. In addition to the NarK1 and NarK2 proteins from T. thermophilus HB8 (Tth), the proteins used in the alignment included NarK (P10903) and NarU (P37958) from E. coli (Eco), NarK1 and NarK2 from P. aeruginosa (accession no. Y15252) (Pae), NarT from S. carnosus (accession no. U40014) (Sca), and NarK (P46907) and NasA (P42432) from B. subtilis (Bsu).

Insertional inactivation of narK1 and narK2. The analysis described above suggested a role for NarK1 and NarK2 in nitrate transport and nitrite extrusion, respectively, suggesting that both proteins were required for nitrate respiration. To analyze this hypothesis, we isolated individual narK1::kat and narK2::kat mutants as well as a double narK1K2::kat mutant by using the plasmids shown in Fig. 2A (see Materials and Methods for construction details). After transformation and selection for kanamycin resistance, the presence of the expected mutations was confirmed by DNA amplification with primer pairs that hybridized at the approximate positions shown in Fig. 2A. As shown in Fig. 2B, the use of primers O-28 and O-65 allowed the amplification of a 0.4-kbp fragment in the wild-type strain (lane 1), whereas in the narK1::kat mutant, this fragment was replaced by a 1.3-kbp fragment as a result of insertion of the kat gene (lane 2). In the narK2::kat mutant, the use of primers O-54 and O-70 resulted in amplification of the expected 1.29-kbp fragment (Fig. 2B, lane 4) instead of the 0.69-kbp DNA obtained from the wild type (lane 3). Finally, the 2.2-kbp fragment amplified from the wild type with primers O-28 and O-70 (Fig. 2B, lane 5) was replaced by a 1-kbp fragment in the double mutant as a consequence of replacement with the kat cassette of most of the sequence from both genes (lane 6). These results were further confirmed by Southern blot analysis (not shown).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Isolation and analysis of narK::kat mutants. (A) Restriction maps of the plasmids used to get single and double narK::kat mutants. Enzymes: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; Sp, SphI. (B) PCR analysis of narK::kat mutants. PCR amplification products from the DNA of single narK1 (lane 2) and narK2 (lane 4) and double narK1 narK2 (lane 6) mutants were compared with those obtained from the DNA of the parental strain (lanes 1, 3, and 5). Primers used for amplification: lanes 1 and 2, O-28 and O-65; lanes 3 and 4, O-54 and O-70; lanes 5 and 6, O-28 and O-70. Arrows indicate the product expected from the corresponding amplifications. The approximate positions at which these primers hybridize are shown in panel A.

Phenotypic analysis of narK::kat mutants. As could be expected, the growth of single and double mutants under aerobic conditions was indistinguishable from that of the wild-type strain (not shown), indicating that neither of these genes is required for the aerobic metabolism of the bacterium.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Anaerobic growth and nitrite export of narK::kat mutants. (A) Anaerobic growth. Growth curves under anaerobic conditions were performed as described in Materials and Methods. Tubes were taken at the indicated times to measure the OD550. (B) External concentration of nitrite at different nitrate concentrations. Samples from induced cultures of single and double narK mutants were incubated in preheated culture medium containing increasing concentrations of nitrate. After 10 min, the nitrite produced was measured and plotted against the external concentration of nitrate (see the text for details). Symbols: , wild type; , narK1::kat; , narK2::kat; , narK1K2::kat; , narGH::kat.

	DISCUSSION

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The narK1 and narK2 genes described in this study are predicted to encode proteins belonging to the major facilitator superfamily of transporters. Proteins from this family have a broad substrate spectrum that includes H⁺, sugars, and antibiotics, and its members share 12 membrane-spanning -helix and diverse sequence motifs (9). This fact, and the location of the genes downstream of the narGHJI cluster, suggested that NarK1 and NarK2 were involved in the transport of nitrate and/or nitrite. The average-distance tree shown in Fig. 1, in which each protein clusters in a different group, suggested a role in nitrate transport for NarK1 and a more specific role in nitrite extrusion for NarK2.

The results shown in Fig. 3A demonstrate that a double mutation in narK1 and narK2 abolishes the ability of T. thermophilus HB8 to grow anaerobically. Thus, either the substrate (nitrate) or the product (nitrite), or both, has no alternative way to cross the membrane than by using the NarK1 or NarK2 protein. On the other hand, as the presence of either of these proteins suppresses this defect in anaerobic growth, it follows that they share at least one of these functions. Whether this function is nitrate transport or nitrite excretion cannot be deduced from this experiment. However, a detailed analysis of our present results led us to support a nitrate/nitrite antiporter capability for both proteins.

The arguments in favor of this interpretation are various. First, if both proteins shared only the ability to extrude nitrite, the presence of an alternative way to bring nitrate into the cell should result in a dramatic intracellular accumulation of nitrite because of the presence of a normal level of NR. Most probably, such levels of intracellular nitrite would be lethal to the cell. In fact, in the double mutant, intracellular nitrite remains at concentrations similar to or even lower than that of the wild type (0.5 to 1 mM) in all the experiments in which it was checked. Thus, we concluded that there is not an alternative way to transport nitrate into T. thermophilus HB8 than the NarK1/NarK2 proteins. In consequence, both proteins should have the ability to act as nitrate transporters into the cell.

On the other hand, the inability of the double mutant to excrete a detectable amount of nitrite at the highest nitrate concentration used (40 mM) in the experiment shown in Fig. 3B could be expected from the absence of nitrate transporters. In addition, the plateau of nitrite secretion reached by each single mutant could reflect the existence of a limiting step only in nitrate transport. Alternatively, it could be related to a limitation in the excretion of nitrite that could be expected from the absence of nitrite transporters outside the nar cluster. In this sense, the fact that the nar cluster of T. thermophilus HB8 is located within a self-mobilizable element, along with the small size of its chromosome, strongly argues against the existence of nitrite extrusion transporters outside this genetic element. In fact, no homologous genes could be detected either with a labeled probe of narK1 and narK2 in Southern blot analysis or by comparison with the unfinished genome sequence of T. thermophilus HB27, a closely related aerobic strain to which the nar cluster can be transferred and expressed. Thus, although the inability of the double mutant to excrete a detectable amount of nitrite (Fig. 3B) does not exclude it, the putative existence of an alternative nitrite extruder other than the NarK1/NarK2 proteins seems unlikely.

As noted above, the T. thermophilus HB8 chromosome is quite small (1.8 Mbp) (2), and consequently, genetic redundancy is rare (e.g., there are only two copies of DNA encoding rRNA). Thus, a good but yet-unknown reason should justify the presence of two genes encoding functionally redundant proteins in the nar cluster for anaerobic respiration of T. thermophilus. Despite the above discussion, the low similarity between the two proteins suggests different roles for each protein in the natural environment in which these microorganisms live. For instance, the extrusion of nitrite and the transport of nitrate would require the proton motive force at low nitrate concentrations. In this scenario, one of the proteins could have the ability to function as an H⁺/nitrate symporter and the other as an H⁺/nitrite antiporter, and only at higher concentrations of nitrate could they function as nitrate/nitrite antiporters. Alternatively, the presence of two enzymes with redundant enzymatic activities could be related to putative differential expression between them. In this sense, the distance between the genes (20 bp) and the existence of a T-rich sequence overlapping the C-encoding region of narK1 could be related to differential expression of each protein.

Interestingly, the two narK genes identified upstream of the nitrate respiration cluster of P. aeruginosa encode proteins with high similarity to those described here from T. thermophilus (Fig. 1). Moreover, this similarity is conserved at the DNA sequence level, not only between the narK genes but also along many stretches of the whole nar cluster. Keeping in mind the plasmidic nature of the genetic element that encodes anaerobic respiration in T. thermophilus HB8 and the ubiquity, respiratory character, and similarity in codon usage of Thermus spp. and Pseudomonas spp., it is tempting to speculate about a common origin for both groups of genes. Meanwhile, future work with P. aeruginosa would confirm the functional relationship between the tandemly organized narK genes of these bacteria.