Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK1
Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, UK2
Department of Biochemistry, University of Oxford, Oxford, UK3
Author for correspondence: David J. Richardson. Tel: +44 1603 593250. Fax: +44 1603 592250. e-mail: d.richardson{at}uea.ac.uk
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ABSTRACT |
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Keywords: Periplasmic nitrate reductase, oxygen regulation, transcription, repression, Paracoccus
Abbreviations: MV+, reduced methyl viologen; NAP, periplasmic nitrate reductase; NAR, membrane-bound nitrate reductase
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INTRODUCTION |
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NAP is present in a wide range of , ß,
, and
Proteobacteria (Berks et al., 1995
). The role of NAP, and consequently the pattern of nap gene expression, varies amongst the systems that have thus far been investigated. In Haemophilus influenzae, NAP is the only nitrate reductase present and the role for NAP presumably is to support anaerobic growth in the presence of nitrate. NAP is also the sole nitrate reductase in the denitrifying bacterium Pseudomonas sp. strain G179. Mutation in the NAP region rendered the cells incapable of growth under anaerobic conditions with nitrate as the electron acceptor, showing that the primary role of NAP in this bacterium is in respiratory nitrate reduction (Bedzyk et al., 1999
). Mutation of NAP in Rhodobacter sphaeroides f. sp. denitrificans also clearly demonstrated that it is the only nitrate reductase involved in anaerobic denitrification (Liu et al., 1999
).
In Escherichia coli, the situation is more complex since the organism possesses two isozymes of the NAR in addition to NAP. Transcription of the nap genes is induced by anaerobiosis in the presence of nitrate or nitrite (Darwin & Stewart, 1995 ). Expression of the nap operon is under the positive control of the transcription factors FNR and NarP; however, it is antagonized by NarL, which binds to the same site as NarP. Recent work has suggested that the NAP system has a high affinity for nitrate in the intact cell, and that the nap genes are transcribed only at low nitrate concentrations (Potter et al., 1999
; Wang et al., 1999
). This has suggested a role for NAP in anaerobic respiration in nitrate-limited environments.
The denitrifying bacterium Ralstonia eutropha also possesses membrane-bound, periplasmic and assimilatory nitrate reductases and, as in P. pantotrophus, synthesis of NAP is confined to aerobic growth conditions (Siddiqui et al., 1993 ). It is probable that the fundamental principles of NAP regulation will be similar in bacteria that synthesize the enzyme during aerobic growth and an understanding of the molecular basis of this regulation will complement physiological studies on the function of the enzyme. As a first step towards this, the organization and transcriptional regulation of the nap operon in P. pantotrophus has been investigated. Our findings reveal that expression of the nap operon is subject to induction aerobically, but only when the organism is growing on highly reduced carbon substrates, such as butyrate. Remarkably, operon expression is also negatively regulated in response to anaerobiosis. This unusual control of gene expression is intriguing as it may represent regulation exerted at the level of the oxidation state of the carbon source that only occurs in the presence of dioxygen.
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METHODS |
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Recombinant DNA techniques.
Standard methods of recombinant DNA technology were used (Sambrook et al., 1989 ).
Primer extension analysis.
Total RNA was isolated from aerobic and anaerobic cultures grown to mid-exponential phase using the Qiagen RNeasy kits. Primer extension analysis of the specific transcripts generated from promoters P1 and P2 was performed essentially as described by Sawers & Böck (1989 ) using 15 µg total RNA and 0·1 pmol specific 32P-labelled oligonucleotide NapEpx (5'-CGCTTGCGGTGCTTGGGAC-3'), which annealed within the napE gene (Berks et al., 1995
). Sequencing reactions were performed according to the procedure of Sanger et al. (1977
) using the same labelled primer and plasmid pHJS110.
Construction of HJS101 (napE::Kmr).
The first step in the construction of a P. pantotrophus napE mutant strain involved PCR to generate fragments of napE with suitable restriction sites to facilitate cloning of a kanamycin cassette. The primer pairs hsp86 (5'-TCGTTCCCGGGCCTGTCCACC-3')/hsp87 (5'-TCGCGGATCCGATCATTTCTGG-3') and hsp88 (5'-CGGCGGATCCGGCTTCCTGGT-3')/hsp89 (5'-GGCGCCATGCATCGTCTTGGACA-3') were used to amplify 476 bp and 701 bp fragments, respectively, from cos154. The 476 bp fragment was restricted with SmaI/BamHI and ligated into SmaI/BamHI-restricted pUC18 to generate pHJS100. The 710 bp fragment was restricted with BamHI/SphI and ligated into BamHI/SphI-restricted pUC18 to generate pHJS101. The SphIBamHI insert from pHJS101 was cloned into pHJS100 to generate pHJS102, which has a 111 bp in-frame deletion in napE. The deletion is marked by a BamHI site into which the kanamycin-resistance cassette from pUC4K was cloned to generate pHJS103. The complete insert was then cloned into pGRPD1 as a SphIEcoRI fragment to construct pHJS104. This plasmid was mobilized into P. pantotrophus in a triparental mating using pRK2013 as a helper plasmid (Figurski & Helinski, 1979 ). Kanamycin-resistant exconjugants were isolated and the presence of the kanamycin cassette in napE was verified by Southern analysis of genomic DNA. The resulting strain was called HJS101.
Construction of HJS105 (nap).
To construct a strain deleted for the nap operon, plasmid derivatives carrying appropriate DNA fragments from the operon first had to be constructed. Plasmid pHJS200 was constructed by cloning a 1·25 kb FspIXbaI fragment from pSPHI into PmlI/XbaI-restricted pSPH22, generating a deletion of the nap operon and flanking DNA from 306 bp upstream of the napE start codon to 217 bp downstream of the napC stop codon. The pHJS200 insert was excised with SmaI/SphI and ligated into pRVS1 to generate pHJS201. This plasmid was mobilized into P. pantotrophus HJS101 in a triparental mating, and kanamycin- and spectinomycin-resistant exconjugants were selected. One of these colonies was resuspended in 1 ml LB and serial dilutions were plated onto LB. Resultant colonies were resuspended in LB and serially diluted onto LB plates containing X-Gal at 80 µg ml-1. The white colonies of recombinant cells were plated in duplicate in the absence and presence of kanamycin. Mutant strains, in which the insertionally inactivated gene had been replaced by the one with a deletion showed a kanamycin-and spectinomycin-sensitive phenotype. The deletion of napEDABC was verified by Southern analysis and the strain was called HJS105.
Construction of pHJS304.
The primer pairs Hind826N (5'-GGCGCTTTAAAGCTTCTAGGCCCG-3')/NapEpx were used in PCR to amplify a 263 bp fragment from the napE promoter. The fragment was cloned into pCR-Script to generate pHJS300. Plasmid pHJS302 was made by three-way ligation. The first fragment was the insert of plasmid pHJS300 excised with HindIII/ClaI, the second fragment was the 4·2 kb ClaIXhoI fragment from pHJS4 and the third fragment was derived from pBluescript II SK(+) by digestion with HindIII/XhoI. The pHJS302 insert was excised with SacI and ligated into SacI-restricted pJB3Km1 to generate pHJS304. In pHJS304 the insert is oriented in the opposite direction to the lacZ promoter such that expression of the napEDABC operon is controlled by its own promoter, together with 178 bp of nap regulatory DNA. Plasmid pHJS304 was introduced into P. pantotrophus strain HJS105 by triparental mating. Exconjugants were selected on kanamycin plates.
Construction of pHJS308.
The aim of this construction was to make a derivative of pHJS304, but which included the point mutation in the nap regulatory region. To do this plasmid oligonucleotides Hind826N and NapEpx were used to amplify a 263 bp DNA fragment from the promoter of the nap operon in mutant M6, which was cloned into pCR-Script to generate pHJS306. The HindIIIClaI insert from pHJS306 was then cloned in a three-way ligation with the 4·2 kb ClaIXhoI from pHJS4 and HindIII/XhoI-digested pBluescript II SK(+) to generate pHJS307. Finally, the insert from pHJS307 was isolated as a SacI fragment, which was cloned into SacI-digested pJB3Km1 to generate pHJS308. pHJS308 was then used to study nap operon expression in P. pantotrophus.
Construction of BCB12 (narI::).
Strain BCB12 was constructed by inserting the omega element (Prentki & Kritsch, 1984 ) into the BamHI site of the narI gene. Transfer of the mutation to the P. pantotrophus chromosome was performed using the same methods as described for M6
(Sears et al., 1997
).
Sequence determination of P. pantotrophus M6 promoter.
Primers hsp86/hsp87 were used to amplify a 476 bp DNA fragment from P. pantotrophus M6 genomic DNA. The fragment was restricted with SmaI/BamHI and ligated into SmaI/BamHI-restricted pUC18 to generate pHJS100M6. The sequence of the pHJS100M6 insert was determined using Universal and Reverse primers, as described below.
Enzyme assays.
MV+ was used to measure nitrate reductase enzyme activity. MV+-dependent nitrate reductase assays and chlorate reductase assays were performed exactly as described by Bell et al. (1990 ).
Automated DNA sequencing.
Automated dye terminator sequencing was carried out using ABI Big Dye terminators and the reaction products were analysed on Applied Biosystems 377 automated sequencers. All sequence alignments, manipulations and analyses were performed using programs in the Wisconsin GCG software package version 10.0 for UNIX.
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RESULTS |
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Previous studies had shown that NAP enzyme activity was highest in cells grown aerobically with reduced carbon sources such as butyrate and was significantly lower in cells grown with more oxidized carbon sources such as malate or succinate (Richardson & Ferguson, 1992 ). The results of the primer extension experiment described above suggest that the use of the P1 and P2 initiation sites is determined by the oxidation state of the carbon source. To test this hypothesis, total RNA was isolated from wild-type aerobic cells grown with malate, succinate, acetate or butyrate as the carbon source (Fig. 2b
). The results indicate that the P1 site is indeed preferentially used when cells are grown on the oxidized carbon sources malate and succinate, whilst the more reduced substrate acetate results in P1 and P2 being used more or less equally. Cells grown on the most reduced substrate (butyrate) use P2 almost exclusively.
CA transversion in the nap regulatory region causes anaerobic derepression of nap operon transcription
Under anaerobic growth conditions in wild-type cells, NAP enzyme activity is present at low levels (Table 2). Strains of P. pantotrophus lacking the membrane-associated NAR enzyme are unable to grow anaerobically with nitrate (B. Berks & D. Richardson, unpublished results), which indicates that under these conditions NAP activity is too low to support growth. In a previous study we isolated a P. pantotrophus narH::Tn5 insertion mutant, termed M6, that is deficient in NAR, yet after selection had regained the ability to grow anaerobically with nitrate (Bell et al., 1993
). Thus, this mutant appeared to have become derepressed for the NAP enzyme, since nitrate reduction was supported under anaerobic conditions in the absence of a functional NAR. A more detailed analysis of NAP enzyme activity present in the periplasmic fraction of M6 cells grown aerobically or anaerobically with butyrate or succinate as carbon source is presented in Table 2
. After aerobic growth in the presence of succinate, NAP activity was increased approximately 7·5-fold relative to the wild-type. Aerobic growth with butyrate as the carbon source resulted in a more than 50-fold increase in NAP enzyme activity in the wild-type compared with the activity observed after growth with succinate. This result is in agreement with previous findings (Richardson & Ferguson, 1992
). Interestingly, NAP activity levels were similar in the wild-type and the mutant after aerobic growth in the presence of butyrate, which indicates that these values probably represent the maximum attainable NAP activity under these particular conditions. Anaerobic growth of the M6 mutant with either succinate or butyrate as the carbon source revealed a 17- to 20-fold increase in NAP activity relative to the values observed with the wild-type (Table 2
).
Unlike the membrane-associated NAR enzyme, NAP does not accept chlorate as a substrate (Craske & Ferguson, 1986 ). In wild-type cells grown anaerobically, a substantial chlorate reductase activity was detectable (Table 2
), suggesting that the nitrate reductase observed may have been due in large part to NAR and not NAP. Significantly however, no chlorate reductase activity in the periplasmic fractions from anaerobically grown M6 cells was detected (Table 2
), indicating that the nitrate reductase activity determined was exclusively due to NAP.
To investigate whether the substantial increase in NAP activity observed in the M6 mutant was a result of altered transcriptional regulation of the nap operon, mRNA prepared from M6 grown under aerobic and anaerobic conditions was analysed by primer extension (Fig. 2a). The results clearly demonstrate that nap operon transcription had become derepressed under all four growth conditions tested. The P1 and P2 transcripts were both present at roughly equal levels.
To ensure that the increase in nap expression was independent of the secondary nar mutation in M6, we constructed a different nar insertion mutant BCB12 (narI::), which lacked the ability to grow anaerobically with either succinate and nitrate or butyrate and nitrate. This nar mutant also exhibited MV+-dependent NAP specific activities for aerobic succinate- and butyrate-grown cells, which were in a similar range to those observed for the wild-type. Furthermore, primer extension analysis of nap mRNA from this mutant showed a pattern of transcription indistinguishable from the wild-type (data not shown). Taken together, these data suggest that the M6 mutant carries a secondary mutation that lies outside the nar locus and which resulted in derepression of nap transcription.
All of the nap operon structural genes from the M6 mutant were sequenced and no mutation was found. However, sequence analysis of the 268 bp of 5' regulatory DNA immediately upstream of the napE translation initiation codon revealed a single CA substitution (see Fig. 1b
). The mutation lies 16 bases upstream from the napE translation start codon and 13 bp downstream from the P2 transcription start site. Amplification and sequencing of this DNA fragment was performed a number of times to exclude the possibility of the substitution having arisen through a PCR error.
To ensure that the CA transversion was solely responsible for the derepression of nap operon transcription, two broad-host-range plasmids in which the complete napEDABC operon including 217 bp of 5' regulatory region were constructed (see Table 1
and Methods). Plasmid pHJS304 included the wild-type operon regulatory region, while pHJS308 included the C
A substitution. These plasmids were introduced into the nap deletion mutant HJS105 (Table 1
). Direct analysis of P1 and P2 transcription by primer extension demonstrated that, as anticipated, the C
A transversion resulted in derepression of nap operon expression (Fig. 3
), yielding a pattern similar to that seen with M6.
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DISCUSSION |
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The transcriptional control of the P. pantotrophus nap operon features apparent dual regulation that combines control exerted by the nature of the carbon source and the requirement for aerobiosis. It is clear from the strong deregulation of nap transcription in the P. pantotrophus M6 mutant that at least part of this regulation involves a repression mechanism. The CA point mutation in the nap operon regulatory region in M6 lies in a near perfect inverted heptamer repeat, TGaGACA-N3-TGTCgCA, which overlaps the lower of the two transcription start sites (Fig. 1b
). This could be the recognition sequence for a repressor protein. The fact that transcription is also derepressed anaerobically suggests that this may be the binding site for an oxygen-dependent repressor. The obvious candidate for this would be an FNR-like protein; however, we were unable to identify a DNA sequence similar to the recognition sequence (TTGAT-N4-ATCAA) for FNR, which is common for FNR-like proteins from a number of micro-organisms (Spiro, 1994
; Sawers, 1999
). Another possible candidate for an anaerobic repressor would be ArcA. Although ArcA has yet to be identified in a micro-organism outside the enterobacteria, the regulation exhibited at the P. pantotrophus nap promoters is reminiscent of ArcAs mode of action at a number of E. coli promoters (Sawers, 1999
).
It was notable that the nature of the carbon source had no apparent effect on transcription in the M6 mutant, whereas in the wild-type promoter it had a marked effect, with transcription increasing in the presence of butyrate. This may indicate either that the oxygen-dependent regulation overrides the carbon-source control or that both oxygen and carbon control may be mediated by the same repressor protein. The former is perhaps more likely, since it is difficult to reconcile how a single repressor protein could mediate both signals to which the nap operon promoters respond.
Transcription of the napEDABC operon initiates at two distinct sites, which are separated by 7 bp (Fig. 1). There are no obvious -10 and -35 promoter sequences showing similarity to those recognized by E. coli RNA polymerase; however, it should also be noted that RNA polymerase recognition sequences have yet to be defined in P. pantotrophus or its close relative Paracoccus denitrificans. The proximity of the start sites, coupled with the fact that they are used differentially depending on the carbon source, suggests that both are primary transcription start sites. Formally, this promoter arrangement is similar to that of the two promoters of the E. coli gal operon, which are separated by 5 bp (Choy & Adhya, 1996
). Expression of the gal promoters is controlled both positively and negatively by the GalR repressor. Depending on repressor concentration, GalR can repress one promoter and activate the other or repress transcription from both. Whether a similar control mechanism operates at the nap promoter must await further analysis.
In wild-type cells we determined that transcription from the P2 promoter was enhanced approximately fivefold during growth with butyrate as a substrate compared to the level of expression when the organism was grown with succinate. Clearly, however, this does not account for the greater than 50-fold increase in enzyme activity observed in the mutant under these conditions. Moreover, despite the high levels of transcript in the M6 mutant grown anaerobically with succinate and nitrate or with butyrate and nitrate, NAP enzyme activity did not attain the level observed in the M6 mutant grown aerobically with butyrate. This indicates that one or more of the processes of translation, cofactor insertion, or export limit NAP biosynthesis under these conditions, but not when the cells are grown aerobically with butyrate. This suggests that there is a further level of regulation controlling NAP enzyme synthesis, which is distinct from the transcriptional control.
During anaerobic growth of wild-type P. pantotrophus, NAP enzyme activity was reduced to the level observed when the cells were grown aerobically with oxidized carbon sources. This is a reflection of the fact that both promoters are essentially inactive during anaerobic growth. This is a logical bioenergetic rationale for the bacterium, as electron transport to nitrate via the FNR-dependent NAR is more highly coupled than that via NAP and so it represents a more efficient means of energy conservation under anaerobic conditions. In contrast, during aerobic growth, because butyrate is more reduced than the average cell biomass of P. pantotrophus, the presence of NAP allows excess reductant to be readily dissipated during oxidative metabolism.
The molecular nature of the putative transcriptional repressor involved in nap regulation remains to be determined, but the work presented in this paper has provided the first insight into what is a new regulatory mechanism in bacteria that express nap aerobically and repress it anaerobically. Significantly, this regulatory mechanism must be distinct from that controlling nap expression in E. coli, and reflects the different physiological roles for NAP in these different bacterial species (Potter et al., 1999 ). Given that a number of other proteins appear to become up-regulated during aerobic growth on butyrate (H. J. Sears & D. J. Richardson, unpublished observations) it is likely that this control system will have additional targets to the nap gene cluster. We now have the genetic and biochemical tools available to permit elucidation of this novel regulatory mechanism.
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ACKNOWLEDGEMENTS |
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Received 6 April 2000;
revised 24 July 2000;
accepted 1 August 2000.