School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK1
Author for correspondence: Stephen Spiro. Tel: +44 1603 593222. Fax: +44 1603 592250. e-mail: s.spiro{at}uea.ac.uk
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ABSTRACT |
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Keywords: Paracoccus denitrificans, nitric oxide, transcription, denitrification
Abbreviations: FNR, fumarate and nitrate reductase regulator; NNR, nitrite and nitric oxide reductase regulator; NO, nitric oxide
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INTRODUCTION |
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In P. denitrificans, NO reductase is encoded in the six-gene norCBQDEF operon, which has a predicted NNR (nitrite and nitric oxide reductase regulator)-binding site upstream of norC (de Boer et al., 1996 ; Fig. 1
). The norC and norB genes encode the enzymically active NorCB complex that can be purified from P. denitrificans (de Boer et al., 1996
; Hendriks et al., 1998
). The remaining four genes have unknown functions, but are required for the synthesis of an active NO reductase in vivo (de Boer et al., 1996
). Transcription of the nor genes is activated by NNR under anaerobic growth conditions in response to NO or a related species (van Spanning et al., 1995
, 1999
). NNR also activates the transcription of the divergent nirI and nirS genes that are required for nitrite reductase activity (Saunders et al., 1999
). Another FNR family member, FnrP, activates expression of nitrate reductase in P. denitrificans, probably in response to anoxia and/or a redox signal (van Spanning et al., 1997
). NNR and FnrP appear to bind to identical or very similar DNA sequences, yet activate their target genes specifically with little or no cross-talk (van Spanning et al., 1997
). FnrP appears to be a true orthologue of FNR, in that it contains the cysteine residues that are conserved in FNR-like proteins and are believed to provide ligands to an oxygen/redox-sensitive ironsulphur cluster. NNR, on the other hand, does not have these residues and so is thought to function by a different mechanism (Baker et al., 1998
).
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METHODS |
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Primer extensions.
Total RNA was isolated from P. denitrificans using RNeasy spin columns (Qiagen) according to the manufacturers instructions. RNA integrity was checked by agarose gel electrophoresis and concentrations were determined by measuring the absorbance at 260 nm. RNA (10 or 20 µg) was added to a 1·5 ml microfuge tube containing 10 µl 10 mM DTT, 0·1 M KCl, 4 mM Tris/HCl (pH 7·9), 40 units RNasin (Promega) and 0·2 pmol 32P-end-labelled primer RNA1 (5'-CGTAGATATGGCTGTGCACGGTCAATGCGC-3'). The tube was heated to 80 °C for 5 min, incubated at 30 °C for 3 h and then cooled on ice. Then 4 µl 5x MMLV reverse transcriptase buffer, 2 µl dNTP mix (50 mM), 2 µl DTT (0·1 M), 0·5 µl actinomycin D and 200 units MMLV reverse transcriptase were added and the volume made up to 20 µl with water. The reaction was incubated at 37 °C for 1 h and stopped by addition of 4 µl Sequenase stop solution (Amersham-Pharmacia). Products (5 µl) were separated on an 8% sequencing gel alongside a sequencing ladder generated using primer RNA1.
PCR methods.
The nor promoter fragments (norP175, norP133, norP98 and norP57) were generated using the reverse primer norP1B (5'-CGGGCGTAGATATGGCTGTGCACG-3') and either P175 (5'-TTTGCCTGATGCCGCCCAAGGCCGC-3'), P133 (5'-GTCTGCCGGGCTGCCGTATCGAGCT-3'), P98 (5'-CGCAGGACGCGGAATTTCCGGACA-3') or P57 (5'-CCTTCACTTGACTTTCATCAATGAG-3'). Each reaction contained 25 ng template DNA (pEG8HI; van Spanning et al., 1997 ), 2 µl each primer (25 µM), 5 µl Pwo buffer (MgCl2 included), 1·5 µl dNTP mix (50 mM), 0·5 µl Pwo (5 units µl-1) in a total volume of 50 µl. Reaction conditions were: 94 °C for 5 min, then 25 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 30 s, followed by 72 °C for 5 min. Products were separated on a 1·5% agarose gel, purified using a Qiaex Gel extraction kit (Qiagen) and ligated into SmaI-cut, phosphatase-treated pUC18 (Pharmacia). Ligation reactions were then used to transform competent JM83 and plated on L agar + X-Gal and ampicillin. The orientations of the inserts were determined by colony PCR and then by sequencing of both strands. Each fragment was excised with KpnI and XbaI and ligated into pMP220 cut with the same enzymes, to generate plasmids designated pnorP57, pnorP98, pnorP133, pnorP175 and pnorP133NN (see Fig. 1b
). For colony PCR, white colonies were picked and transferred to a 0·2 ml PCR tube (the toothpick was then used to inoculate 5 ml L broth + ampicillin). The tubes were microwaved at full power (1000 W), with the lids open, for 1 min. PCR mix contained (for eight reactions) 20 µl Taq buffer, 12 µl dNTP mix (50 mM), 16 µl each primer (5 µM), 6 µl MgCl2 (50 mM), 1 µl Taq and 129 µl H2O; 25 µl was added to each tube. The cycling conditions were as described above and products were separated on a 1·5% agarose gel. The primers used were the pUC18 Universal primer and the appropriate promoter-specific forward primer (P57, P98, P123 or P175).
PCR mutagenesis.
To incorporate single or double point mutations into the nor promoter, appropriate complementary primers were used in an amplification reaction with plasmid DNA as the template. The template DNA was removed by treatment with DpnI (which digests only methylated DNA) and the remaining DNA was used to transform JM83. Each reaction mix contained: 25 ng template DNA (the P133 promoter cloned in pUC18), 5 µl Pwo buffer (MgCl2 included), 1·5 µl dNTP mix (50 µM), 4 µM each primer (NN1, 5'-TTGCCTTCACCTGACTTTCATCAGTGAGCGACTC-3' and NN2, 5'-GAGTCGCTCACTGCTGAAAGTCAGGTGAAGGCAA-3', or TT1, 5'-CGACTCACGCGCGCCGGACAGT-3' and TT2, 5'-ACTGTCCGGCGCGCGTGAGTCG-3'), 0·5 µl Pwo (5 units µl-1), in a total volume of 50 µl. Reaction conditions were: 94 °C for 5 min, then 25 cycles of 94 °C for 30 s, 67 °C for 30 s, 72 °C for 10 min, followed by 72 °C for 15 min. The reaction mixtures were transferred to 1·5 ml tubes and treated with 10 units DpnI for 30 min at 37 °C, then 72 °C for 30 min. After cooling on ice, each reaction was treated with 2 units T4 DNA ligase for 1 h and then used to transform competent JM83. Control reactions contained no primers. Mutant DNAs were sequenced using an ABI Prism automated sequencer. Other general recombinant DNA techniques were as described by Sambrook et al. (1989) .
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RESULTS AND DISCUSSION |
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Promoter alignments
All four promoters appear to be class II, with the factor-binding site centred 41·543·5 bp upstream of the transcription-start site, which is the preferred spacing for FNR-dependent promoters (Wing et al., 1995 ). Otherwise, the alignment of the nirI, nirS and norC promoters reveals very little similarity (Fig. 2
). Only one sequence appears to be conserved, a TTGC motif positioned 1420 bp upstream of each transcript start site, as recently noted by Saunders et al. (2000)
for the nirS promoters of both P. pantotrophus and P. denitrificans. This sequence, and its position, are reminiscent of the conserved -12 recognition sequence for the alternative sigma factor,
N (Wang et al., 1999
). However, the NNR-dependent promoters have only weak matches to the -24 element consensus, the second
N recognition element (Barrios et al., 1999
), and so are unlikely to be dependent on RNA polymerase containing
N. Neither is there any obvious similarity to the -10 and -35 recognition elements for the major (
70 type) sigma factor found in many promoters from other bacteria, although it has been proposed that these motifs are likely to be conserved in the
-Proteobacteria (Gruber & Bryant, 1997
; Baker et al., 1998
). Hence, the role of the TTGC motif, if any, is uncertain and was tested by mutagenesis (see below).
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The transcript start site of the nor operon of Pseudomonas stutzeri has also been determined and is positioned 40·5 bp downstream of a binding site for a regulatory protein of the FNR family (Zumft et al., 1994 ). Transcription initiation from this promoter is independent of
N (Härtig & Zumft, 1998
). At least in these respects, the nor promoters of Pse. stutzeri and Par. denitrificans appear to be similar.
In vivo analysis of the nor promoter
To investigate the norC promoter further, a series of 5' deletions was constructed by PCR. Deletions were designed with the aim of identifying any regions of DNA that may be required for promoter activity and regulation. Each promoter fragment was cloned into the low-copy-number promoter-probe vector pMP220 and the resulting constructs were conjugated into P. denitrificans Pd1222. A Pd1222 derivative containing a single-copy chromosomal norPlacZ fusion (van Spanning et al., 1997 ) was used to check that there were no major copy-number effects associated with the use of pMP220. The largest promoter construct (in pnorP175) contains both the P1 and P2 start sites, with 54 bp of DNA upstream of P2 (Fig. 1b
). The second deletion (in pnorP133) contains P1 and is truncated immediately upstream of the P2 start site, having only 12 bp of DNA upstream of P2. This deletion was designed to remove the RNA polymerase-binding site presumably associated with P2. Subsequent truncations removed the palindrome (pnorP98) and the AT-rich sequence (pnorP57) in turn (Fig. 1b
). The full-length plasmid-borne construct (pnorP175) exhibited only approximately twofold higher activity than the chromosomal-borne, single-copy fusion and was regulated in a similar fashion (Fig. 3
). In both cases there is a substantial increase in promoter activity in cultures grown under anaerobic, denitrifying conditions. Removal of the sequences immediately upstream of P2 (pnorP133) had a relatively small effect, but caused a greater decrease in nor promoter activity under anaerobic conditions than under aerobic conditions (Fig. 3
). This suggests that the P2 transcript makes a small contribution to the total nor promoter activity, which is more significant under anaerobic conditions; this conclusion is consistent with the results of the mRNA analysis. Removal of the 10 bp palindrome (in pnorP98) had no significant effect on nor promoter activity. Removal of the AT-rich sequence (in pnorP57) caused a further small decrease in the aerobic activity of the promoter, and a small increase in anaerobic activity, compared to the previous two deletions (Fig. 3
). This suggests that the AT-rich sequence may have a negative effect on transcription from norP1. The pnorP57 construct contains a promoter deleted to within 7 bp of the putative NNR-binding site, yet retains the anaerobic inducibility of the full-length nor promoter. Thus, the P1 promoter is the most important contributor to the activation of the nor promoter, which presumably occurs through NNR binding to the putative binding site retained in pnorP57.
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The high anaerobic activity of the shortest promoter construct (pnorP57) is not consistent with norP1 being dependent on N, since in all known cases
N has an absolute requirement for an additional transcription factor (upstream activator) bound to a remote site 100 bp or more upstream of the transcript start (Shingler, 1996
). This conclusion was further supported by the finding that alteration of the TTGC motif to TTTT had no significant effect on nor promoter activity in the full-length construct (results not shown). Thus, norP1 cannot be
N dependent, since at
N-dependent promoters the GC nucleotides of the -12 element are essential for promoter activity (Barrios et al., 1999
). Mutation of the TTGC motif to GCGC resulted in a 20% reduction in promoter activity anaerobically, demonstrating that this motif, though conserved at a similar position at all four NNR-dependent promoters, is not essential for promoter activation (results not shown).
To show unequivocally that the suggested NNR-binding site does indeed play this role, the sequence (TTGACtttcATCAA) was altered to the neutral NN sequence (CTGACtttcATCAG), which is recognized by neither FNR nor CRP of E. coli (Spiro et al., 1990 ), and which it was assumed would not be recognized by NNR. Mutagenesis was undertaken on the promoter fragment containing only 12 bp of DNA upstream of P2 (Fig. 1a
) since in the fusion vector containing this fragment (pnorP133) only P1 is active anaerobically (Fig. 3
). Introduction of the NN sequence into the putative NNR-binding site (in pnorP133NN) eliminated promoter activity under anaerobic conditions and abolished the residual aerobic activity seen in pnorP57 (Fig. 3
). Hence, norP1 activity is completely dependent on the putative NNR-binding site and it is very likely that NNR recognizes the same sequence, or a very similar sequence, as does E. coli FNR. These results also suggest that at least some of the nor promoter activity in aerobic cultures depends on NNR, indicating that there may be some active NNR present in aerobic cells. Attempts to confirm the NNRDNA interaction biochemically using purified protein in gel-retardation assays (in the presence and absence of NO generators) have so far proved inconclusive.
Concluding remarks
The physiological role of the minor NNR-independent promoter of the nor operon is unclear. Transcription from this promoter was detected both aerobically and anaerobically, in vivo and in vitro, and is consistent with previous reports of a low level of activity of the nor promoter in aerobic cultures (van Spanning et al., 1999 ). Furthermore, there is substantial residual NOR activity in an nnr mutant, and the NorC polypeptide is detectable in aerobically grown cells (van Spanning et al., 1997
). All of these observations point towards there being a significant NNR-independent activity of the nor promoter under both aerobic and anaerobic conditions. Aerobic expression of NO reductase may safeguard cells against sudden exposure to NO, and may have a role in the reported ability of P. denitrificans to denitrify in the presence of oxygen (Davies et al., 1989
). However, the low level of aerobic activity observed with the chromosomal fusion strain and with the plasmid-borne fusions was reduced but not completely abolished by removal of norP2 and must therefore be partly due to norP1. Mutation of the NNR-binding site completely abolished both anaerobic and aerobic activity of norP1, which suggests that at least some of the activity detected aerobically is NNR dependent. The key to understanding NO signalling and NNR-dependent regulation in P. denitrificans lies in identifying the sigma factor(s) which recognize NNR-dependent promoters and on elucidating the exact mechanism of NNR activation by NO.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 April 2000;
revised 6 July 2000;
accepted 12 July 2000.