APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1997, p. 800–802 0099-2240/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 63, No. 2
Structural Gene (nirS) for the Cytochrome cd1 Nitrite Reductase of Alcaligenes eutrophus H16 ¨ STER,2 BEATE SCHNEIDER,2 EFFI REES,1 ROMAN A. SIDDIQUI,1 FRANK KO ¨ RBEL FRIEDRICH1* AND BA ¨r Pflanzenphysiologie und Institut fu ¨r Biologie der Humboldt-Universita ¨t zu Berlin,1 and Institut fu Mikrobiologie der Freien Universita ¨t Berlin,2 Berlin, Germany Received 10 June 1996/Accepted 27 November 1996
Denitrification by Alcaligenes eutrophus H16 is genetically linked to megaplasmid pHG1. Unexpectedly, the gene encoding the nitrite reductase (nirS) was identified on chromosomal DNA. The nirS product showed extensive homology with periplasmic nitrite reductases of the heme cd1-type. Disruption of nirS abolished nitrite-reducing ability, indicating that NirS is the enzyme essential for denitrification in A. eutrophus. The strictly respiratory proteobacterium Alcaligenes eutrophus H16 (ATCC 17699) is able to denitrify. During this process, nitrate and nitrite are converted to nitrous oxide or dinitrogen as the final products (12). We previously demonstrated that curing of the A. eutrophus megaplasmid correlates with loss of denitrifying ability (14). Subsequently, we identified the nitrous oxide reductase structural gene (nosZ) on megaplasmid DNA (25). Since the genes involved in denitrification are tightly clustered in the denitrifying bacteria studied (for a review, see reference 24), we expected to find additional denitrification genes linked to nosZ. The initial step of denitrification is catalyzed either by tetraheme cytochrome cd1 (EC 1.9.3.2) or by a copper-dependent nitrite reductase (EC 1.7.2.1). Based on immunological screening, hybridization studies, and H218O isotope exchange experiments, it was concluded that A. eutrophus contains a copperdependent nitrite reductase (2, 19, 22, 23). Nevertheless, we previously isolated a nitrite-inducible reductase (NirS) from denitrifying cells of A. eutrophus which showed features typical of those of a cytochrome cd1-type enzyme (17). The present study focused on two questions. (i) Is the structural gene for cytochrome cd1 (nirS) part of a megaplasmid-borne denitrification locus? (ii) In addition to cytochrome cd1, does A. eutrophus harbor a second denitrification-relevant nitrite reductase? Two experimentally determined internal peptides (peptide A [NQYNLDNLFSVTLRDAGEVALID] and peptide B [VNYSDLSNLKTTTIDSAKFL]) generated by cyanogen bromide cleavage of purified NirS from A. eutrophus provided the basis for the construction of a set of reverse-translated 59-32Plabeled oligonucleotides. These NirS-specific probes were used in hybridization experiments (15, 21) with isolated pHG1 DNA (11) and with total DNA (1, 10) from the wild type and from a megaplasmid-free derivative (Table 1). Surprisingly, positive signals were obtained with a 16-kb EcoRI fragment derived from the chromosomal DNA. No hybridization signal was detected with the megaplasmid. The 16-kb fragment was cloned from a genomic library of A. eutrophus, and nirS was identified within an internal 5-kb SmaI fragment subcloned into pBluescript KS1, yielding plasmid pCH489 (Table 1; Fig. 1). Both strands of a 2,205-bp segment were sequenced by primer walking by standard techniques (15, 16) to characterize
nirS (Fig. 1). The 1,662-bp nirS encodes a polypeptide of 554 amino acids (Mr, 60,757), which correlates with the size (60 kDa) determined for the purified NirS protein (17). The 18 amino acids determined by protein sequencing of the N terminus of the mature NirS (17) were identical with residues 26 through 43 of the deduced polypeptide (Fig. 2). Comparison of the amino terminus of the mature NirS with that of the predicted nirS product indicated that the 59 region of nirS codes for an export competent signal peptide (13) consisting of 25 amino acids (residues 1 through 25 [Fig. 2]). The sequences from the internal NirS peptides A and B perfectly matched those of residues 154 through 176 and residues 317 through 336, respectively (Fig. 2). Moreover, the calculated isoelectric point of 8.8 correlated well with the experimentally determined value of pI 8.6, which is unusually high in comparison with nitrite reductases from Pseudomonas strains (17). A sequence
TABLE 1. Bacterial strains and plasmids used Strain or plasmid
A. eutrophus H16 HF210 HF383 HF384 Plasmids pBluescript KS1 pVDZ92 pLO1 pLO2 pCH489 pCH491 pCH492 pGE345
* Corresponding author. Mailing address: Institut fu ¨r Biologie, Humboldt-Universita¨t zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany. Phone: 49-30-20938100. Fax: 49-30-20938102. E-mail:
[email protected].
Relevant characteristic(s)a
nirS, pHG1 pHG12, megaplasmid-free H16 nirSDR168 nirSDR783 Apr lacZ9 F1 ori; T7 promoter Tcr lacZ9 mob Kmr Tcr RP4 oriT ColE1 ori Kmr Tcr RP4 oriT ColE1 ori 5-kb SmaI chromosomal fragment of H16 harboring nirS in pBluescript KS1 0.7-kb SalI fragment of pCH489 carrying a 168-bp BsaAI deletion in pLO1 0.8-kb SacI-PstII fragment of pCH489 carrying a 783-bp XhoII deletion in pLO2 5-kb SmaI fragment of pCH489 in pVDZ92
Reference or source
DSM 428, ATCC 17699 7 This study This study Stratagene 4 8 8 This study This study This study This study
a Apr, ampicillin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance; mob, mobilizability; ori, origin of transfer.
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FIG. 1. Physical map of the nirS region of A. eutrophus H16. Black bar, the segment sequenced in the present study; open arrow, the gene encoding NirS; hatched bars, in-frame deletions within the structural gene. Plasmids used for construction of NirS mutants are listed at the left. Restriction sites: P, PvuI; S, SmaI; Sa, SacII; and Sl, SalI.
comparison of A. eutrophus NirS and five deduced NirS polypeptides showed extensive overall identity (specified in the legend to Fig. 2). It is remarkable that A. eutrophus NirS is missing a stretch of amino acids in the N-terminal domain, which is also the case for NirS of Pseudomonas stutzeri (5).
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Crystal structure analysis (5) of NirS from Thiosphaera pantotropha (identical with Paracoccus denitrificans [9]) revealed that this particular segment provides axial ligands for heme c (His-9) and d1 (Tyr-17), respectively, which are missing in the A. eutrophus NirS (Fig. 2). In this protein, a methionine residue (Met-99 or Met-116 [Fig. 2]) could replace His-9 in ligating heme c, as proposed for the Pseudomonas aeruginosa NirS protein (18, 20). To demonstrate that NirS is essential for denitrification in A. eutrophus, two nonpolar NirS mutants were constructed by allelic exchange (8). Starting with plasmid pCH489, nirS was disrupted by excision of a 168-bp BsaAI fragment and a 783-bp XhoII fragment (Fig. 1 and Table 1). The resulting mutants, HF383 and HF384, contained deletions of 56 (residues 206 through 261) and 261 (residues 53 through 313) amino acids, respectively (Fig. 2). Both mutants were impaired in anaerobic growth on fructose mineral medium containing 10 mM potassium nitrate (17). Like the wild type, the mutants converted nitrate to nitrite; however, nitrite accumulated in the medium without being further reduced to nitrogen oxides (Fig. 3). This deficiency was restored by introducing the nirS allele on plasmid pGE345, a derivative of the broad-host-range vector pVDZ92 (Table 1). This result clearly demonstrates that the chromosomally encoded cytochrome cd1 is the essential en-
FIG. 2. Alignment of heme cd1-dependent nitrite reductases. The protein sequences (identities with A. eutrophus NirS [scored as percentages], are given in parenthesis) are from A. eutrophus (Aeu), P. aeruginosa (Pae; 65% [18]), P. stutzeri ZoBell (PstZ; 57% [6]), P. stutzeri JM300 (PstJ; 53% [19]); P. denitrificans (Pde; 62% [3]), and T. pantotropha (Tpa; 62% [5]) (the Tpa sequence has been deduced from the crystal structure of the mature protein). Asterisks, identical residues; boxes, the heme c-binding site; arrows, ligands of functional importance (further discussed in the text).
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FIG. 3. Nitrite-reducing properties of the NirS mutant HF384 grown anaerobically on nitrate. ■, wild-type H16; Ç, NirS mutant HF384; å, HF384 (pGE345).
zyme for nitrite reduction during denitrification of A. eutrophus H16, and we conclude that it is very unlikely that a second copper-containing isoenzyme exists in this strain. The fact that NirS is chromosomally encoded leaves the following question open: why do megaplasmid-cured derivatives of A. eutrophus accumulate nitrite without further reducing this intermediate to gaseous nitrogen products? Thus, the megaplasmid must provide essential functions, e.g., regulatory components, factors involved in the biosynthesis of catalytically active nitrite reductase, and/or housekeeping gene products required for anaerobic metabolism. Nucleotide sequence accession number. The nirS nucleotide sequence has been deposited in the EMBL, GenBank, and DDBJ data banks under accession number X91394. ACKNOWLEDGMENTS We thank Rainer Cramm for valuable contributions to the manuscript and Botho Bowien and Oliver Lenz for providing the genomic cosmid library of A. eutrophus. The cyanogen bromide treatment and the amino acid sequencing by Susanne Kostka are gratefully acknowledged. We thank Werner Schro ¨der for oligonucleotide synthesis. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and from Fonds der Chemischen Industrie. REFERENCES 1. Chen, W.-P., and T.-T. Kuo. 1993. A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res. 21:2260. 2. Coyne, M. S., A. Arunakumari, B. A. Averill, and J. M. Tiedje. 1989. Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductase in denitrifying bacteria. Appl. Environ. Microbiol. 55:2924–2931. 3. de Boer, A. P., W. N. M. Reijnders, J. G. Kuenen, A. H. Stouthamer, and R. J. M. van Spanning. 1994. Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans. Antonie Leeuwenhoek 66:111–127.
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