Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK1
Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QT, UK2
Author for correspondence: Stuart J. Ferguson (Dept of Biochemistry). Tel: +44 1865 275240. Fax: +44 1865 275259. e-mail: ferguson{at}bioch.ox.ac.uk
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ABSTRACT |
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Keywords: Paracoccus pantotrophus, nitrite reductase, transcription, denitrification, promoter
Abbreviations: Fnr, fumarate nitrate reduction transcriptional activator; Nnr, nitrate nitrite reduction transcriptional activator; 5'-RACE, rapid amplification of cDNA 5' ends; TSS, transcript start site
The GenBank accession number for the sequence in this paper is U75413.
a Present address: Department of Microbial Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, the Netherlands.
b Present address: Oxford Centre for Environmental Biotechnology, NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, and Department of Engineering Science, Parks Road, Oxford OX1 3PJ, UK.
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INTRODUCTION |
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The transcription of nirS has been shown to be activated by Fnr-like proteins. These are Nnr in the case of Pa. denitrificans (van Spanning et al., 1997 ), Anr in Ps. aeruginosa (Arai et al., 1997
; Hasegawa et al., 1998
) and the set of regulatory proteins named DnrE, DnrS and DnrD in Ps. stutzeri (Vollack et al., 1999
). All these proteins bear a high similarity to the transcriptional activator Fnr of Escherichia coli, but lack the N-terminal cysteines which allow the formation of an oxygen-labile ironsulphur cluster in Fnr (Khoroshilova et al., 1995
). The functions associated with oxygen sensing Fnr in E. coli are performed by another protein in Pa. denitrificans, FnrP, which is distinct from Nnr, and likewise Ps. stutzeri possesses FnrA. It is tempting to assume that the regulation of denitrification in Ps. aeruginosa and Ps. stutzeri is very similar to that in Pa. denitrificans and Pa. pantotrophus. However, not only is the gene organization within the nir loci different (Zumft, 1997
; Baker et al., 1998
), but the environmental signals which trigger denitrification are different as well. It would appear that in Ps. stutzeri nitrate and the absence of oxygen are sensed, directly or indirectly, by DnrE, DnrS and DnrD (Vollack et al., 1999
) and these proteins then activate the nir and nor operons. In Pa. denitrificans and Ps. aeruginosa, the transcription of the Nnr or Dnr proteins is dependent on nitric oxide (van Spanning et al., 1999
; Arai et al., 1999
), and nirS in these organisms is thus subject to product regulation rather than the more normal substrate regulation.
In Pa. denitrificans, nitric oxide and Nnr also serve to activate promoters upstream of the nor (nitric oxide reductase) operon and nirI (van Spanning et al., 1999 ), a gene encoding a putative DNA-binding protein of unknown function. The site at which Nnr binds is presumably the inverted repeat, reminiscent of an Fnr box, found upstream of nirS, nirI and norC. These regulatory sequences differ by only a few base pairs from the consensus E. coli motif for Fnr binding, and are also very similar to the anaeroboxes found upstream of Pa. denitrificans FnrP regulated genes. Selection between FnrP and Nnr may not solely be on the basis of the sequence of the DNA-binding site and secondary factors are presumably involved. It has yet to be shown whether Pa. pantotrophus has multiple Nnr-like proteins (as in Ps. stutzeri), but knockouts of Nnr suggest it is the major contributor to the activation of nirS transcription in Pa. denitrificans (van Spanning et al., 1997
).
The transcription of nirS in Pa. pantotrophus [note that this organism was formerly designated Thiosphaera pantotropha (Rainey et al., 1999 )] requires study for two important reasons. First, Pa. pantotrophus has been described as an aerobic denitrifier (Arts et al., 1995
; Robertson et al., 1995
) yet Moir et al. (1995)
failed to detect cytochrome cd1 in aerobically grown cells. Second, this organism has served as the source of protein for the first crystal structure of cd1 nitrite reductase (Fülöp et al., 1995
; Baker et al., 1997
). An understanding of how the structural gene is transcribed with the genes responsible for the manufacture of the d1 haem prosthetic group is essential if gene knockout strains are to be constructed, in which mutant nirS genes can be efficiently manufactured by the cell in a holo form. Results obtained with Pa. pantotrophus can be extrapolated to Pa. denitrificans as the two organisms are closely related to one another (Rainey et al., 1999
) although are distinct species. To begin to understand the activation of transcription of nirS we have sequenced part of the nir locus from Pa. pantotrophus LMD 92.63, determined the transcription start site of the gene and estimated the length of transcripts from the nirS promoter.
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METHODS |
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Bacterial strains, media and vectors.
RNA and DNA were isolated from Paracoccus pantotrophus LMD 92.63. Escherichia coli JM83 or DH5 was used for routine cloning. Pa. pantotrophus was grown on either the minimal medium described by Robertson & Kuenen (1983)
or LuriaBertani (LB) medium. LB was also used for the growth of E. coli. Plasmid pTNIR3 was kindly supplied by T. de Boer, BioCentrum Amsterdam, Vrije Universiteit, the Netherlands (T. de Boer, R. J. M. van Spanning & A. H. Stouthamer, unpublished results).
Extraction of total RNA from Pa. pantotrophus.
Total RNA was isolated from cells of Pa. pantotrophus LMD 92.63 using a method based on that of Cuypers & Zumft (1993) . Cultures (50 ml) were grown to mid-exponential phase, harvested by centrifugation and lysed in 4 ml lysis buffer (1% SDS, 1 mM EDTA). The lysate was extracted with 4 ml phenol/chloroform, saturated with 2 M sodium acetate (pH 4·0) and centrifuged at 10000 g for 10 min at 4 °C. The aqueous phase was then re-extracted with 4 ml chloroform and recovered by centrifugation. Propan-2-ol (0·8 vol.) was added and the mixture was incubated on ice for 30 min, then centrifuged at 15000 g for 15 min at 4 °C. The precipitated nucleic acids were redissolved in 200 µl 1xconcentration DNase buffer (40 mM Tris/HCl, pH 8·0; 10 mM NaCl; 6 mM CaCl2) containing 100 units RNase-free DNase and incubated at 37 °C for 30 min. After DNase treatment, the mixture was extracted once more with an equal volume of phenol/chloroform and the RNA was ethanol-precipitated by incubating at -70 °C, followed by centrifugation at 15000 g for 15 min, 4 °C. The pellet was washed with 100 µl ice-cold 70% (v/v) ethanol, air-dried and redissolved in 100 µl RNase-free water. RNA quality was analysed by the absorbance ratio A260:A280 and by formaldehyde-agarose gel electrophoresis.
Northern blotting.
Northern blotting was used to detect specific mRNA transcripts from denitrification genes in Pa. pantotrophus. The method used closely followed that of Engler-Blum et al. (1993) . The membrane was pre-hybridized for 1 h at 68 °C in a high SDS phosphate buffer (250 mM sodium phosphate, pH 7·2; 1 mM EDTA; 20%, w/v, SDS). A digoxigenin-labelled dsDNA probe was hybridized to the RNA in 10 ml of the same buffer at 68 °C overnight. The membrane was washed three times for 20 min each at 65 °C in 50 ml H-wash buffer (20 mM sodium phosphate, pH 7·2; 1%, w/v, SDS; 1 mM EDTA), then once for 5 min in W-wash buffer (100 mM maleic acid, pH 8·0; 3 M NaCl; 0·3%, v/v, Tween 20) and blocked for 1 h in blocking buffer (W-wash buffer plus 0·5%, w/v, blocking reagent; Roche). Digoxigenin antibody conjugate was diluted 1:10000 in blocking buffer and the membrane was incubated with 20 ml of this buffer for 30 min. The membrane was then washed four times for 10 min each with W-wash buffer and equilibrated for 5 min in substrate buffer (100 mM Tris/HCl, pH 9·5; 100 mM NaCl; 50 mM MgCl2). Hybridized probe was detected by chemiluminescence, using the alkaline phosphatase substrate CSPD (Roche) diluted 1:100 in 1 ml substrate buffer and pipetted onto the membrane. Following a 5 min incubation in the dark, excess solution was removed and the membrane was incubated in a sealed hybridization bag at 37 °C for 15 min. The membrane was then exposed to X-ray film (Kodak), initially for 1 h, or until exposed bands appeared on the film.
DNA probes used in this study were made from internal PCR products of nirS or nirE. The 141 bp nirS probe was amplified from genomic Pa. pantotrophus DNA using primers NIRSF (CTGGCCCTTGTCCTTGGGC) and 228R (TACGTCCTGCTGTGCAAGGT), which were subsequently labelled with dUTP-digoxigenin-alkaline phosphate conjugate (Roche). A 419 bp probe for nirE was made in the same manner using the primers 2431F (AGACGGTGACGAACGGGG) and 2849R (GGGCACATAGTCGCAGGG).
Primer extension analysis.
Primer extension was used to determine the 5' end of mRNA transcripts. The experiment is divided into three stages: oligonucleotide labelling, primer-RNA annealing and cDNA extension. Ten picomoles of oligonucleotide was end-labelled in a 20 µl reaction exactly as described in Sambrook et al. (1989) . The labelled oligonucleotide was redissolved in 100 µl TE buffer and could be stored at -20 °C for up to 1 week. One microlitre (approx. 105 c.p.m.) of labelled primer was annealed to 40 µg heat-denatured RNA in 20 µl annealing buffer (2 mM Tris/HCl, pH 7·9; 250 mM KCl; 0·2 mM EDTA) at 50 °C for 90 min. The annealed primer RNA was then ethanol-precipitated, redissolved in 20 µl reverse transcription mix (50 mM Tris/HCl, pH 8·3; 75 mM KCl; 3 mM MgCl2; 10 mM DTT; 1·3 mM each dNTP; 20 units RNasin inhibitor; 50 µg actinomycin D ml-1; 33 units AMV reverse transcriptase) and incubated at 37 °C for 2 h. cDNA synthesis was stopped with 25 mM EDTA. The mixture was treated with RNase at 37 °C for 20 min, extracted with phenol/chloroform, ethanol-precipitated and redissolved in 4 µl TE buffer. Six microlitres of sequencing stop solution was added and the labelled cDNA was run on a 6% acrylamide sequencing gel alongside a reference sequencing ladder generated using the same primer.
5'-RACE (Rapid amplification of cDNA 5' ends).
5'-RACE was used as an alternative method to determine the 5' end of mRNA transcripts. The procedure used closely followed that of Frohman (1993) . Ten micrograms of total RNA was reverse-transcribed using the nirS sequence specific primer 284R (5'-GACATCCTGCTGCGCAAGGT-3'). The mixture was then diluted with TE buffer, excess primer and nucleotides were removed by centrifugation through a Centricon-100 filter and the cDNA was recovered on a Millipore spin filter in 10 µl TE buffer. Addition of the poly-dATP tail and the two subsequent nested PCR amplifications were performed exactly as described by Frohman (1993)
, using the forward primers QTF, Q0F and Q1F (detailed in Frohman, 1993
) and the sequence-specific reverse primers 242R (5'-ATAGTCGGTGCGCGTCTT-3') and 228R (5'-TCTTGTGGTCCTCCAGCG-3'). PCR products were analysed by electrophoresis on 1·2% agarose gels, cloned into the pGEM-T vector and sequenced using M13 universal primers.
Sequencing.
Sequencing was performed manually. The standard Sequenase protocol (Amersham International) was modified by the addition of DMSO to the reaction to aid the resolution of stops caused by the high G+C mol% content of the DNA.
Alignment of sequences and DNA analysis.
Sequence data were analysed using GCG9 (Genetics Computer Group, USA) and the GenBank and EMBL databases held at Oxford University Molecular Biology Data Centre, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK).
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RESULTS AND DISCUSSION |
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The nirS stop codon is not immediately followed by the initiation codon of the adjacent gene nirE. Instead, there is a 73 bp intergenic region, which contains an inverted repeat which would form a strong stemloop, followed by a region rich in adenosine and thymine (Fig. 2). These features indicate a rho-independent terminator, but in other denitrifiers nirS is co-transcribed with the genes required for d1 biosynthesis (Härtig & Zumft, 1999
; Kawasaki et al., 1997
). To determine if nirS of Pa. pantotrophus is transcribed separately or as an operon including at least nirE, Northern blots were used to determine the size of transcript(s) arising from the nirS promoter.
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The monocistronic nature of the nirS transcript agreed with the presence of a putative downstream transcript termination structure (Fig. 2), and no larger band could be seen even if the Northern blot was overexposed. The length of the nirS transcript suggests that transcription terminates around 55 bp upstream of the nirE translation initiation codon, within 10 bp of the stemloop structure found in the nirSnirE intergenic region. This supports the proposal that this structure does indeed function as a rho-independent terminator. When the same RNA preparation was blotted to a nirE probe (which hybridized successfully to a plasmid-borne nirE gene) no hybridization was seen to any RNA molecule, confirming that the previously observed transcript did not contain nirE mRNA. This observation suggests that the nirE transcript was in an abundance too low to be visualized by the methods used.
The presence of Pa. pantotrophus nirS as a monocistronic mRNA is in contrast to the nirSMCFDLGHJEN nitrite reductase operon of Ps. aeruginosa, in which a single transcript provides both the structural nitrite reductase gene and the genes for d1 haem biosynthesis (Kawasaki et al., 1997 ). Northern blot analysis of the nirSTM operon of Ps. stutzeri (ZoBell) showed that two transcripts originated from the nirS promoter, one containing nirS alone, the other nirS, T and M (Härtig & Zumft, 1999
). In neither pseudomonad is the nirS gene followed by a terminator such as that identified in Pa. pantotrophus (Fig. 2
).
Initiation of transcription of nirS
The transcription start site (TSS) of nirS was initially determined by primer extension analysis (Fig. 4), and this suggested a guanidine 29 bp upstream of the translation initiation codon. Since high GC content mRNA (Pa. pantotrophus has a genome of approximately 66 mol% G+C; Rainey et al., 1999
) has proved to be susceptible to artefactual termination of the primer extension reaction (S. C. Baker & S. J. Ferguson, unpublished results), the TSS was further analysed using 5'-RACE. This again showed that, under denitrifying conditions, the nirS transcript commenced from a guanidine 29 bp upstream of the translation initiation codon (Fig. 5
).
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An Fnr-like box, equivalent to that seen in Pa. denitrificans (Fig. 2), has previously been suggested to be the binding site of Nnr (van Spanning et al., 1997
). Nnr is in the Fnr/Crp family of proteins and is induced in Pa. denitrificans during denitrification. Insertion of a kanamycin cassette into the gene encoding Nnr leads to a nitrite reductase minus phenotype, characterized by the absence of a NirS polypeptide, but conversely an Fnr mutant is unaffected in nitrite reduction (de Boer et al., 1996
). The combination of these results and the presence of an Fnr-like motif in an appropriate position upstream of the TSS of nirS strongly suggests that TTAACaatgGTCAA is the binding site of Nnr.
The position of a single Fnr-like box between the nirS transcription start site (this work) and that of nirI (Saunders et al., 1999 ) suggests that, unusually, a single motif controls the transcription of nirS and nirI in a bi-directional manner. The nirI promoter has previously been shown to be Nnr-dependent (van Spanning et al., 1999
). This may have implications in the co-regulation of these two genes, as sterically the binding of a holo RNA polymerase to enable transcription of nirS would prevent transcription towards nirI.
Pa. pantotrophus promoter regions controlled by Nnr (the regions upstream of nirS and nirI) were aligned with respect to their TSSs to identify common motifs between them. It is striking that the sequence T-T-G/C-C-G/C-G/C occurs in the region -14 to -19 with respect to the transcription start site in both the nirS and nirI promoter regions. This motif differs considerably from the 70-like -10 motif (TATAAT in E. coli; Hawley & McClure, 1983
) or any other known RNA polymerase motifs and cannot be found in the same position upstream of any other transcript start sites of the alpha Proteobacteria (as judged by a GCG FINDPATTERNS examination of prokaryotic sequence databases using T-T-G/C-C-G/C-G/C-N20100-ATG as a search term and subsequently examining sequence annotations for defined transcript start sites within the search results). The one promoter recovered in this search was that of Pa. denitrificans norC, again positively regulated by Nnr. This suggests that there may be other DNA-binding protein(s) involved in the Nnr-dependent transcription of nirS and nirI, but the motif at -16 cannot be found in the promoter regions of nirK [encoding an Nnr-controlled copper-containing nitrite reductase (Bartnikas et al., 1997
; Tosques et al., 1997
)] of the closely related bacterium Rhodobacter sphaeroides. Such motifs are also absent from Ps. stutzeri nirS and dnrS (the latter gene is the functional equivalent of Pa. denitrificans nnr) promoters, which conform more closely to the
70-mediated E. coli Fnr consensus promoter sequence with a clear -10 hexamer (Vollack et al., 1999
).
Although Pa. pantotrophus and Pa. denitrificans are very closely related, albeit separate, species (Rainey et al., 1999 ), the nirS promoter regions of the two organisms do differ from one another (Fig. 2
), and a notable feature is that one motif has retained its structure while altering its sequence. In Pa. pantotrophus, an imperfect inverted repeat (TTGCGCGCAA) appears between the Nnr box and the T-T-G/C-C-G/C-G/C motif discussed above. This inverted repeat also appears in the Pa. denitrificans nirS promoter region, but the flanking TT/AA nucleotides are replaced with G and C so that the sequence appears as CCGCGCGCGG. The position of this sequence may indicate the binding site of another protein, but the purpose of retaining the integrity of the repeat between species remains obscure. Such inverted repeats cannot be found in the upstream region of nirI.
The monocistronic nature of transcription from the promoter upstream of Pa. pantotrophus nirS, and the presence of a rho-independent terminator in the nirSnirE intergenic region, suggest that genes downstream of nirS are transcribed from a separate promoter. A gap of approximately 70 bp between the termination codon of nirS and the initiation codon of nirE is a plausible location for this promoter. A sequence much like that seen in the nirS and nirI promoters (TTCGCG) can be seen in a plausible position relative to the nirE translation start codon (40 bp upstream), but no binding site for Nnr can be distinguished. Extensive attempts to determine the nirE TSS experimentally by both techniques used for nirS in this paper were unsuccessful, perhaps because of the low abundance of nirE-hybridizing mRNA noted during Northern blotting. Since nirE and the genes downstream encode proteins involved in the production of the specialized d1 haem, fewer transcripts would be required relative to the structural gene for nitrite reductase (nirS). Thus the presence of two promoters to control the structural gene and accessory proteins may provide a means of co-regulating the abundance of nitrite reductase polypeptide with its specialized d1 haem.
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ACKNOWLEDGEMENTS |
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Received 23 July 1999;
revised 8 October 1999;
accepted 8 November 1999.