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210 Biochemical Society Transactions (2005) Volume 33, part 1 Nitrate respiration in the actinomycete Streptomyces coelicolor G. van Keulen, J. Alderson, J. White and R.G. Sawers1 Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, U.K. Abstract Streptomyces coelicolor is an obligate aerobic, filamentous soil-dwelling bacterium. Remarkably, the genome of S. coelicolor has three copies of the narGHJI operon that encodes respiratory nitrate reductase. This review summarizes our current views on the requirements for multiple nitrate reductases in S. coelicolor. Introduction Streptomycetes are ubiquitous actinomycetes that have had a major impact on the biotechnological and pharmaceutical industries. They undergo a complex developmental life-cycle and exhibit a remarkable metabolic diversity. As well as having a wealth of genes encoding products involved in secondary metabolic pathways, analysis of the 8.6 Mb genome of Streptomyces coelicolor has revealed the presence of some rather unexpected operons, in particular three operons encoding respiratory NARs (respiratory nitrate reductases) [1]. Despite being classified as an obligate aerobe, the presence of an enzyme in anaerobic respiration suggests that S. coelicolor has at least the capacity to grow, or generate energy, using nitrate as an alternative electron acceptor and under oxygenlimiting conditions in standing liquid culture [2]. Indeed, nitrate reduction has been demonstrated for a number of other Streptomyces spp. ([3], and references therein). Members of the NAR class of nitrate reductase are predicted to form a membrane-associated enzyme complex, which has the site of nitrate reduction on the cytoplasmic face of the membrane [4]. The energy-conserving enzyme complex comprises three different protein subunits and the structure of the enzyme from Escherichia coli has been recently determined [5,6]. The NarG subunit harbours the bis-MGD (bis-molybdenum guanine dinucleotide) containing catalytic site, while the NarH subunit is an electron transfer component which binds 4 iron–sulphur clusters. The whole complex is anchored to the membrane via the di-bhaem integral membrane NarI quinol dehydrogenase subunit. The fourth protein encoded in the operon, NarJ, is proposed to have a chaperone function, necessary for maturation of the enzyme. Nitrate respiratory capacity in S. coelicolor Although originally characterized extensively in Gram negative bacteria, the characteristic narGHJI operon structure Key words: anaerobic respiration, energy conservation, nitrate reductase, phylogeny, soil bacteria, Streptomycetes. Abbreviations used: bis-MGD, bis-molybdenum guanine dinucleotide; NAR, respiratory nitrate reductase. 1 To whom correspondence should be addressed (email [email protected]). C 2005 Biochemical Society is also highly conserved in the genomes of a number of Gram positive bacteria (Figure 1). Perhaps unsurprisingly, the degree of amino acid sequence conservation between the three NarG polypeptides of S. coelicolor and that of the actinomycete Mycobacterium tuberculosis and that of Bacillus subtilis is, respectively, 65 and 50%. However, the S. coelicolor NarG proteins also share 45–48% amino acid similarity with the E. coli NarG polypeptide. Remarkably, when compared against each other, the NarG polypeptides of S. coelicolor only share approx. 65% amino acid identity. As well as having three narGHJI operons, S. coelicolor also has two NarK orthologues. NarK belongs to the major facilitator superfamily of transmembrane transporters. Phylogenetic analyses suggest that there are two classes of NarK: the type I class is proposed to catalyse nitrate/proton symport and nitrate/nitrite antiport, while the type II class probably functions in nitrite efflux [7]. The narK gene is often, but not always, found upstream of the narGHJI operon. The narK2 gene in S. coelicolor is found near the nar2 operon (Figure 1) and NarK2 falls within the type I subgroup along with NarK2 from M. tuberculosis and NarK from B. subtilis [7]. The narK1 gene product from S. coelicolor falls within the type II class along with NarK1, NarK3 and NarU from M. tuberculosis and NarK and NarU from E. coli. Molybdenum cofactor biosynthesis A major requirement for the synthesis of catalytically active nitrate reductase is the machinery for the biosynthesis of the bis-MGD cofactor. The S. coelicolor genome encodes all of the proteins required for the uptake of molybdenum and the biosynthesis of bis-MGD. Differential expression of the nar operons We have determined through transcript analysis that all three nar operons in S. coelicolor are expressed. Although we have not identified conditions in the laboratory that allow S. coelicolor to grow anaerobically, our transcript analysis suggests that at least one of the operons (nar2) is regulated in response to fluctuations in oxygen levels. Surprisingly, however, we have not yet identified conditions under which expression of any of the nar operons responds to nitrate. 10th Nitrogen Cycle Meeting 2004 Figure 1 Schematic representation of the nar gene operons in S. coelicolor Shown is the organization of the nar operons in S. coelicolor, M. tuberculosis and B. subtilis. Genes whose products exhibit a high degree of amino acid identity (>60%) within and between species share the same colour. Genes shown in white or light blue and without associated names are not related to the nar genes. The transcriptional orientation of the genes in the respective chromosomes is depicted by the orientation of the arrows. The location of the S. coelicolor nar operons on the 8.6 Mb genome is shown in brackets. The narK orthologues (narK1 in S. coelicolor, and all four in M. tuberculosis) shown in isolation are located in distinct and unrelated regions of the respective genomes. Phylogenetic relationships An alignment of full-length NarG sequences obtained from the sequenced and annotated genomes in the databases was used to construct a phylogenetic tree using distance matrix methods (Figure 2). A number of major clades were supported by the analysis and reflect reasonably well the findings reported recently for partial NarG sequences [8,9]. The three NarG sequences from Streptomyces coelicolor cluster together and form a clade with other Actinobacteria, in particular NarG from the Mycobacteria. Genome sequence projects for other actinomycetes, for example S. scabies and Nocardia farcinica, indicate the presence of at least partial narG genes, suggesting that nitrate reduction is widespread in this group of bacteria. Interestingly, NarG sequences from Rhodococcus sp. RHA1 do not cluster with those from other Actinomycetes, but form a deeply branched cluster, which indicates that the NARs from these bacteria diverged from a common ancestor in the distant evolutionary past. Perspectives Future challenges will be to determine the roles of the respective nitrate reductases in S. coelicolor physiology and to identify the source of the reducing power that drives nitrate reduction. Preliminary data indicate that the three narGHJI operons are differentially regulated. A detailed analysis of expression levels throughout the growth cycle may Figure 2 Phylogenetic relationship of translated, full-length narG gene products The phylogenetic tree was constructed using the Treecon v 1.3b program [10] inferred from alignments created by the ClustalX program [11]. Phylogenetic distances were determined by neighbour-joining analysis; numbers on the branching points are bootstrap values with 100 replicates. C 2005 Biochemical Society 211 212 Biochemical Society Transactions (2005) Volume 33, part 1 reveal the conditions under which each NAR is required. Clearly, the construction and analysis of specific narGHJI operon knock-out mutants will aid functional assignment of the NAR enzyme complexes. It remains to be established whether there is functional significance in the fact that the NarG polypeptides in S. coelicolor share only 65% amino acid sequence similarity. Indeed, the NarH and NarI polypeptides share even less similarity. The complex nature of the soil implies that oxygen supply is often limited for the growth of ‘aerobes’. Consequently, Streptomyces spp. will have had to evolve a broad range of metabolic pathways that deliver sufficient energy to maintain the membrane potential, or to sustain growth, when oxygen gets depleted [2]. The capacity to respire with nitrate may give Streptomycetes a selective advantage in this complex environment. The work in our laboratory is supported by the BBSRC (EGH16080) and through a Marie Curie Postdoctoral Fellowship to G.v.K. (MEIFCT-2004-506056). C 2005 Biochemical Society References 1 Bentley, S.D., Chater, K.F., Cerdeño-Tárraga, A.-M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D. et al. (2002) Nature (London) 417, 141–147 2 van Keulen, G., Jonkers, H.M., Claessen, D., Dijkhuizen, L. and Wösten, H.A.B. (2003) J. Bacteriol. 185, 1455–1458 3 Kumon, Y., Sasaki, Y., Kato, I., Takaya, N., Shoun, H. and Beppu, T. 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