Evidence for redundancy in cysteine biosynthesis in Rhizobium leguminosarum RL3841: analysis of a cysE gene encoding serine acetyltransferase

G. Parker1, D. Walshaw1, K. O’Rourke1, S. Broad1, A. Tingey1, P. S. Poole1 and R. L. Robson1

Microbiology Division, School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK1

Author for correspondence: R. L. Robson. Tel: +44 118 9316639. Fax: +44 118 9316562. e-mail: r.l.robson{at}reading.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified ~4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841. The gene exhibits >40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants. The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively. All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39. Insertion of Tn5-lacZ into cysE in the genome of R. leguminosarum (strain RU632) lowered SAT activity in crude extracts by >95%. However, RU632 was not a cysteine auxotroph, which suggests that R. leguminosarum possesses some redundancy in cysteine biosynthesis. Additional copies of cysE could not be detected in the genome when the R. leguminosarum cysE gene was used as a hybridization probe. Therefore it is possible that R. leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine. Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N2 fixation (measured by C2H2 reduction). Transcriptional activity of cysE was determined by measuring the ß-galactosidase arising from cysE::Tn5-lacZ fusions. Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate). However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures. Repression by ammonium was not seen in a strain defective for ntrC.

Keywords: sulphur metabolism, symbiotic nitrogen fixation, SAT, nodulation

Abbreviations: DTNB, 5,5'-dithobis-(2-nitrobenzoic acid); SAT, serine acetyltransferase

The GenBank accession number for the sequence reported in this paper is AJ271648.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Members of the soil-dwelling genus Rhizobium form specific N2-fixing symbioses with leguminous plants: the bacterium receives fixed carbon from the plant and supplies fixed nitrogen in return. This exchange of nutrients is highly co-ordinated and involves the induction of specific transport systems in the bacteroid. In the course of an investigation into the mechanisms by which fixed nitrogen compounds may be passed from Rhizobium leguminosarum to the plant, a general L-amino acid transporter was discovered which catalyses high rates of exchange between intracellular and extracellular amino acid pools (Walshaw & Poole, 1996 ). The genes encoding this transporter were originally identified on the basis that they increased [14C]glutamate uptake when supplied in additional copies in R. leguminosarum. One such clone which conferred this phenotype is pRU3024, a cosmid that contains a relatively large fragment (~30 kb) of the R. leguminosarum genome. Random Tn5 mutagenesis of pRU3024 led to the localization of an approximately 7 kb region that was responsible for the enhanced glutamate-uptake phenotype. Insertion mutations in this region abolish enhanced glutamate uptake. When this region was subcloned and sequenced it was found to contain a cluster of four genes (aapJ, aapQ, aapM and aapP) that encode polypeptides with a high degree of amino acid sequence identity to components of ATP-binding cassette (ABC) superfamily of transporters (Walshaw & Poole, 1996 ). Further analysis established that the aap genes in R. leguminosarum not only enhance the accumulation of a broad range of amino acids, including glutamate, but also affect the efflux of those amino acids directly or independently via a second efflux channel or transporter. The aapJQMP operon in R. leguminosarum was shown to be responsive to nitrogen regulation. In R. leguminosarum, nitrogen status is probably sensed by a mechanism similar to that known for Escherichia coli, in which a uridylyltransferase/uridylyl-removing enzyme senses the ratio of glutamine to {alpha}-ketoglutarate and changes the uridylylation state of the PII protein. This, in turn, alters the ability of NtrB to act either as a kinase or as a phosphatase for the phosphorylation or dephosphorylation of NtrC, which is a transcriptional activator of nitrogen-regulated operons (Patriarca et al., 1993 ). Mutants in ntrC caused derepression of the aap operon, which induced high rates of amino acid uptake even when the organisms were grown with ammonium as a nitrogen source. Therefore NtrC appears to negatively regulate the aap genes (Walshaw et al., 1997 ).

This work describes a second locus present on the cosmid pRU3024, in which Tn5 mutations lower the enhanced accumulation of glutamate but, unlike the mutations in the aap genes, do not abolish it completely. We show here that the mutations are located in a cysE gene encoding a serine acetyltransferase (SAT), an enzyme which catalyses the first unique step in cysteine biosynthesis.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Strains and media.
The strains and plasmids used in this work are listed in Table 1. E. coli strains were routinely grown at 37 °C on nutrient agar or broth. To test for cysteine auxotrophy, M9 minimal salts agar (Sambrook et al., 1989 ) was used and cysteine was added at 60 µg ml-1 for growth of cysteine auxotrophs. R. leguminosarum strains were grown at 28 °C on either tryptone yeast extract (TY) (Beringer, 1974 ) or acid minimal salts (AMS) medium (Poole et al., 1994a ). Antibiotics were added, where required, at the following concentrations (µg ml-1): for E. coli strains, ampicillin, 100; kanamycin, 20; and for R. leguminosarum strains, streptomycin, 500; kanamycin, 40, tetracycline, 2 in acid minimal salts medium and 5 in tryptone yeast extract medium.


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Table 1. Bacterial strains and plasmids used in this work

 
Molecular biology methods.
Methods used for preparation of genomic and plasmid/cosmid DNA, restriction analysis, agarose gel electrophoresis, cloning, production of digoxigenin-labelled probes, and automated DNA sequencing using a Pharmacia ALF Express and Cy5-labelled primers were as described by Walshaw & Poole (1996) or in Parker et al. (1999) . Hybridizations were carried out at various stringencies as stated in the text. The PCR reactions were routinely performed with the following cycle: a hot start at 94 °C for 2 min, then 30 cycles of 1 min at 94 °C for, 1 min at 60 °C and 1·5 min at 65 °C, after which samples were chilled at 4 °C. The cloning primers for the PCR were as follows: RLCEM1, 5'-CGAAAACATATGCTAAAAAACCATGGC-3'; RLCEM2, 5'-AACGAGGAGAGCGCATATGGTCGC-3'; RLCEM3, 5'-CCGCTGAAGCATATGGATCCC-3'; RLCEND1, 5'-GGCCCTCCCCGCTCGGATCCCGCCG-3'; RLCEND1B, 5'-GGCCCTCCCCGCTCAGATCTCGCCG’3' (restriction sites underlined). The DNA sequencing primers were as follows: RLCER, 5'-CAATGGGCGAGACGATGTGT-3'; RLCER2, 5'-TGGTGCCGACGCCGATCAGC-3'; RLCEF, 5'-CCAAGATCCTCGGCAATATC-3'; Tn5 primer, 5'-GGATCCATAATTTTTTCCTCC-3'.

Bacterial genetic methods.
The transformation of E. coli with plasmids, the conjugation in R. leguminosarum, the procedures for Tn5-lacZ mutagenesis, and the homogenotization of mutations into the genome of R. leguminosarum were as described by Walshaw & Poole (1996) .

Bioinformatics.
Protein-similarity searches were performed using BLAST or {psi}-BLAST (Altschul et al., 1997 ), using the server for The National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Multiple sequence alignments were carried out using MULTALIN (Corpet, 1988 ) at the Server of the Institut National de la Recherche Agronomique (http://www.jouy.inra.fr/) or CLUSTAL W (Thompson et al., 1994 ) at the server of the European Bioinformatics Institute (http://www2.ebi.ac.uk/clustalw/). Protein relationships were determined using the neighbour-joining method of Saitou & Nei (1987) , and the relational cluster diagram constructed from these analyses was drawn using TREEVIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Searches for protein targeting signals were conducted using SIGNALP (Nielsen et al., 1997 : http://www/cbs.dtu.dk/services/SignalP).

Measurement of enzyme activities and protein contents.
SAT activity was measured spectrophotometrically in a real-time assay at 30 °C by following the serine-dependent cleavage of acetyl-CoA in the presence of DTNB, essentially as described by Kredich & Tomkins (1966) . Units are defined as nmol (mg protein)-1 min-1. ß-Galactosidase activity was measured essentially as described by Miller (1972) but with the modifications (for R. leguminosarum) of Poole et al. (1994b ). Protein contents were measured using Folin–Ciocalteu reagent with BSA as the standard.

Determination of nodulation and nitrogen fixation.
Nodulation and N2 fixation in nodules was determined in triplicate as described by Trinick et al. (1976) . N2-fixation activity was expressed as µmol C2H2 reduced h-1 per plant.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Mutations in pRU3024 affecting enhancement of glutamate uptake in R. leguminosarum
Cosmid pRU3024 (Fig. 1) was described previously and contains the aap gene cluster within a segment of approx. 30 kb of the genome of R. leguminosarum strain 3841 cloned into pLAFR1. When introduced into R. leguminosarum, pRU3024 enhanced the accumulation of [14C]glutamate by more than sixfold in organisms grown in a nitrogen-sufficient medium. Only insertions of Tn5::lacZ into the aap cluster completely abolish the enhanced glutamate-uptake phenotype, but the insertions in plasmids pRU3031 and pRU3033 located 3·4 and 4 kb upstream of the aap gene cluster lower the enhancement of glutamate uptake by approximately 2·5-fold (Walshaw & Poole, 1996 ).



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Fig. 1. Location and cloning of the cysE gene from R. leguminosarum 3841. The figure shows the location and orientation of ORF1 and ORF2B (cysE) relative to the aapJQMP cluster previously described for cosmid pRU3024. The 3·4 kb EcoRI fragment of pRU3024 bearing these genes was cloned into pTZ18 to give plasmid pATRCE13 (shown in the lower part of the figure). The extent of the DNA sequence obtained for this fragment is shown by the continuous line. The sites of Tn5-lacZ mutations in cysE in the plasmids pRU3031 and pRU3033 are indicated. The mutation in pRU3031 was selected for most of the phenotypic analysis and for the construction of the chromosomal homegenote, since it is located closest to the N-terminal coding region of the gene.

 
Analysis of DNA sequences immediately flanking the Tn5-lacZ insertions in plasmids pRU3031 and pRU3033 indicated that both insertions disrupt a gene with high sequence identity to cysE genes from bacteria such as E. coli and Azotobacter sp. and from plants such as Arabidopsis. A 3·4 kb EcoRI fragment from pRU3024 covering the sites of insertions in pRU3031 and pRU3033 was subcloned into pTZ18 to give plasmid pATRLCE13. The DNA sequence for 2712 bp of this fragment was determined. Within this region, two ORFs were found which have codon usages consistent with other genes from R. leguminosarum. ORF1 is preceded by a good potential ribosome-binding site and encodes a polypeptide of 261 amino acid residues showing significant identity to a family of bifunctional enzymes with both esterase and halogenoperoxidase activities. A second ORF (ORF2A) of 936 bp was located 36 bp downstream of ORF1 (Fig. 2). ORF2A encodes a polypeptide of 311 amino acid residues with >40% identity to SATs from a wide variety of bacteria and plants (Fig. 3), the closest relatives being the CysE2 and CysE4 from Arabidopsis thaliana (Fig. 4). Alternative potential translation initiation sites are located 129 and 183 bp 3' to ORF1, giving ORF2B and ORF2C, which encode shorter polypeptides of 277 and 259 residues, respectively. Of the three potential translation initiation sites, only that of ORF2B (5'-AACGAGGAGAGCGGCGATG-3') is significantly purine rich and conforms well to the consensus sequence for potential ribosome- binding sites observed in other R. leguminosarum genes. Sequence analysis revealed a potential transcription terminator located 31 bp downstream of ORF2. No other significant matches at the DNA or protein levels were found in the sequences flanking ORFs 1 and 2. ORF1 and ORF2 are organized in such a way that they could be co-transcribed, but this remains to be established.



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Fig. 2. The nucleotide sequence for the region containing parts of ORF1 and ORF2. The sequence of a region extending from base 983 to base 1221 from within the 2712 bp of the sequenced portion of the insert in pATRLCE is shown. The deduced C-terminal amino acid sequence of ORF1 and potential alternative N-terminal sequences for ORF2 are shown using single-letter notation. The potential alternative translation starts for ORF2 are indicated by the arrows starting at positions 1041 (ORF2A), 1147 (ORF2B) and 1201 (ORF2C). Codon usage does not allow discrimination of the most likely translation product in vivo, but only ORF2B is preceded by a convincing potential ribosome-binding site starting at position 1135 (underlined).

 


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Fig. 3. Multiple alignments of amino acid sequences of SATs of bacterial and plant origin. The sequences are as follows: RleCysESS2, ORF2B from R. leguminosarum; HpyCysE, putative SAT from Helicobacter pylori (type 1 SAT); AchnifP, SAT from the N2-fixation gene cluster of Azotobacter chroococcum (type 3 SAT with C-terminal extension), EcoCysE, SAT from E. coli (type 2 SAT with N-terminal extension), AthSAT2, SAT 2 from A. thaliana (type 2 SAT with N-terminal extension including N-terminal targeting sequence). Amino acid residue identities are shown in bold.

 


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Fig. 4. Inter-relationships of serine acetyltransferases from different sources. The methods for multiple alignment and reconstruction of the unrooted phylogenetic tree are described in the text. The sources of the SAT amino acid sequences are as follows: Ace, Allium cepa (onion); Ach1, Azotobacter chroococcum nifP; Ath1, Ath2, Ath4 and AthSAT52, A. thaliana isoforms; Avi1 and Avi2, Azotobacter vinelandii; Bap, Buchnera aphidicola; Bsu, Bacillus subtilis; Cme, Cyanidioschyzon merolae (red alga, chloroplast isoform); Cla, Citrullus lanatus (water melon); Cje, Campylobacter jejuni; Cst, Clostridium sticklandii; Eco, Escherichia coli; Edi1 and Edi2, Entamoeba dispar isoforms; Ehi, Entamoeba histolytica; Hin, Haemophilus influenzae; Hpy, Helicobacter pylori; Mle, Mycobacterium leprae; Rle, Rhizobium leguminosarum ORF2B; S6803, Synechocystis sp. (strain 6803); S7942, Synechococcus sp. (strain 7942); S8801, Synechococcus sp. (strain 8801); Spo, Schizosaccharomyces pombe; Sty, Salmonella typhimurium; Sxy, Staphylococccus xylosus; Tma, Thermotoga maritima.

 
SAT catalyses the acetyl-CoA-dependent acetylation of serine, giving O-acetylserine, which serves as the acceptor for sulphide in the biosynthesis of cysteine catalysed by O-acetylserine-thiol lyase. SAT-encoding genes are found in eubacteria, microfungi, algae, higher plants and some protozoa but not in the Archaea or higher animals. In the enteric bacteria E. coli and Salmonella typhimurium, SAT is rate limiting for cysteine biosynthesis and is subject to feedback inhibition by cysteine. Also, O-acetylserine acts as a co-activator in the genetic control of the expression of proteins and enzymes involved in the assimilation of sulphur via the assimilatory sulphate-reduction pathway (Kredich, 1987 ). Irrespective of which of the three ORFs is correct, the SAT encoded by the R. leguminosarum gene clearly belongs to one of the three known SAT families. All members have a relatively high degree of identity extending over approximately 170 residues (which probably constitutes the catalytic core of the enzyme). Group 1 SATs contain little more than this conserved core, whereas group 2 SATs, including the example identified here, contain N-terminal extensions of between 40 and 200 amino acid residues. Group 3 SATs lack the long N-terminal extensions but have significant C-terminal extensions. The N-terminal domains of the group 2 SATs from plants are likely to be chloroplast target signals. This possibility was worth considering in the case of R. leguminosarum because of the plant–bacterium symbiosis, but when the three ORFs were subjected to analysis using the SIGNALP analytical tool, none appeared to encode N-terminal secretion signals.

Demonstration of SAT functionality
To test if pATRLCE13 contains a functional cysE gene, it was transformed into E. coli JM39, a cysE cysteine auxotroph. JM39(pATRLCE13) was able to grow on M9 minimal agar without cystine addition, whereas JM39 containing the parent vector, pTZ18, remained a cysteine auxotroph. To localize the cysE gene more precisely, the PCR was used to amplify the three alternative ORF2s (with pATRLCE13 as the template). Three different N-terminus-encoding primers were used (RLCEM1, RLCEM2 and RLCEM3) together with a common C-terminus-encoding primer (RLCEND1B) to amplify ORFs 2A, 2B and 2C, respectively. The amplified DNA segments were cloned between the NdeI and BamHI restriction sites in plasmid pT7-7 in order to exploit the strong translation initiation signals present in this expression vector for potential overexpression and further characterization of the R. leguminosarum SAT. The resultant plasmids, pSS1, pSS2 and pSS3, containing ORF2A, ORF2B and ORF2C, respectively, were transformed into E. coli JM39. All three plasmids complemented the mutation in JM39 after growth at 37 °C for 48 h on M9 minimal medium. Attempts to overexpress the three ORFs from pSS1, pSS2 and pSS3 in E. coli BL21(DE3) were unsuccessful. The SAT activities of crude extracts of the strains prepared after induction were not significantly above background in any case. This was surprising, since overexpression of CysE from E. coli and from Azotobacter sp. had been obtained in a similar vector/host system. We have observed previously that the minimum levels of SAT activity needed to complement the cysE mutation in JM39 are below the limits of detection of the biochemical assay in crude extracts. The native SAT in R. leguminosarum probably corresponds to the polypeptide of 277 amino acid residues encoded by ORF 2B, since it is preceded by the best potential ribosome-binding site. The question of the correct translation initiation start can only be resolved by purification and determination of the N-terminal sequence of SAT from R. leguminosarum.

Mutagenesis of cysE in R. leguminosarum
The Tn5-lacZ mutants in the cysE gene in cosmid pRU3024 were obtained by random mutagenesis. Plasmids pRU3031 and pRU3033 carry mutations in cysE (see Fig. 1). The mutation in pRU3031 was chosen for further work. Transfer of the insertion into the genome of RU3841 by homogenotization gave strain RU632. We were surprised to find that RU632 exhibited no requirement for cysteine or cystine for growth in the defined medium AMS. This suggests a potential redundancy in cysteine biosynthesis in R. leguminosarum RL3841. We measured enzyme activity in crude extracts prepared from the wild-type strain RU3841 and the mutant RU632. The specific activity for SAT observed in RU3841 was 1·38 nmol (mg protein)-1 min-1, which appears low, at only 17% of that observed in E. coli (Denk & Böck, 1987 ). However, such levels of activity are likely to be more than sufficient to satisfy cysteine needs because the R. leguminosarum cysE complemented the cysE mutation in E. coli JM39 even though the levels of SAT activity in extracts of the complemented strain were also below the detection limits of our assay. However, in the mutant, RU632, activity was not detectable above background. We estimated that the minimum detectable activity was not greater than 0·08 nmol (mg protein)-1 min-1. These data suggest that the cysE gene described here accounts for at least 95% of the SAT activity in R. leguminosarum.

Although we could detect no residual SAT activity in the mutant, a second cysE gene may nevertheless exist, especially since several organisms contain multiple copies of cysE genes, including A. thaliana, which has four cysE genes (Murillo et al., 1995 ; Howarth et al., 1997 ; Bogdanova & Hell, 1997 ), and Azotobacter species (which also fix N2) in which two cysE genes are known (Evans et al., 1991 ; Zheng et al., 1998 ). Therefore, DNA encoding the cysE gene cloned here was used to probe genomic DNA from R. leguminosarum under a variety of stringencies. In the EcoRI digest, a single 3·4 kb fragment was identified which probably corresponds to the EcoRI fragment cloned in pATRLCE13. No other hybridizing fragment was present in this digest. In the EcoRI/BamHI double digest, two hybridizing bands were seen: a more intense band at 0·8 kb, and a much less intense band at 1·2 kb. Since the cysE probe contains a BamHI site located 65 bp from the N-terminal coding region, it was anticipated that two fragments of different intensities would be detected. Also, the DNA sequence predicts that the probe should detect two EcoRI/BamHI fragments of 1205 and 804 bp, which agrees well with the observed results. Prolonged exposure of the blot and careful scrutiny provided no evidence for the existence of other hybridizing bands in R. leguminosarum.

Either a second copy of cysE exists in R leguminosarum RL3841 which is quite divergent from the gene cloned here and is expressed but unstable during extraction and/or assay, or cysteine can be made by a pathway avoiding O-acetylserine. One possibility is that cysteine can be synthesized from methionine; this appears to be the case in Pseudomonas aeruginosa (Foglino et al., 1995 ) and, more pertinently, Rhizobium etli (Taté et al., 1999 ). These organisms contain a methionine-biosynthesis pathway, first established for Saccharomyces cerevisiae, in which sulphide is incorporated not into O-acetylserine but into O-succinylhomoserine to form homocysteine, which is converted to methionine. Cysteine is formed from methionine via a trans-sulphurylation reaction involving cystathionine as an intermediate, obviating the need for O-acetylserine (Cherest & Surdin-Kerjan, 1992 ).

Evidence for regulation of the expression of CysE
The transcriptional activity of the R. leguminosarum cysE promoter was measured under a number of different growth regimes by following the ß-galactosidase activity arising from the Tn5-lacZ inserted into cysE. The experiments were performed with cosmid- or chromosomal-borne cysE::Tn5-lacZ fusions (Table 2). There was no significant difference between growth in sulphate-sufficient and sulphate-limited media. However, the nitrogen status influenced the expression of cysE, which was similar in degree in both chromosomal and cosmid-borne fusions. Ammonium, as opposed to glutamate, as a nitrogen source repressed transcription of cysE by ~50% in the chromosomal fusion strain RU632, and by ~35% in the cosmid-borne fusion strains RU502 and RU504. However, in an ntrC background, the repression by ammonium was not observed either in the chromosome (strain RU1027) or in the cosmid (strains RU981 and RU982). These results are similar to those obtained previously (Walshaw et al., 1997 ) for the aap operon, as illustrated here by new data for the aapJ::Tn5-lacZ fusion in the ntrC+ (strain RU543) and ntrC- (strain RU1017) backgrounds. Also, as reported earlier for the aap cluster, the repression of cysE appears, unusually, to require NtrC, since no repression by ammonium was observed in an ntrC- background. Since the cysE and aap genes are divergently transcribed, the repressive effects of NtrC appear to be mediated at two independent promoters.


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Table 2. Influence of nitrogen status and ntrC on transcription of the cysE and aapJ genes in R. leguminosarum RL3841, as measured in transcriptional Tn5::lacZ fusions

 
Influence of the cysE mutation on symbiotic nitrogen fixation
The cysE mutant, RU632, was indistinguishable from the parent strain in its ability to infect and nodulate peas. The mutation was stable in the host, since all 25 isolates from nodules infected with RU632 retained the kanamycin-resistance phenotype associated with the Tn5-lacZ insertion. Rates of C2H2 reduction for the parent and mutant strains were shown to be 0·45±0·02 and 0·83±0·01 µmol h-1 plant-1, respectively.

The finding of redundancy in cysteine biosynthesis and of a cysE gene in proximity to (and subject to) a similar pattern of regulation as the aap genes required for maximal symbiotic N2 fixation in R. leguminosarum RL3841 echoes the presence of cysE genes closely linked to nif genes in other organisms. Of the two cysE genes known for Azotobacter sp., one is intercalated into the major (nif) gene cluster (Evans et al., 1991 ; Zheng et al., 1998 ). A cysE gene is located adjacent to nifB in a large cluster of genes for N2 fixation in Synechococcus sp. strain RF-1 (Huang et al., 1999 ), but as yet there is no evidence for redundancy in cysteine biosynthesis in this cyanobacterium. It has been suggested that the link between cysteine metabolism and N2 fixation may be explained by a high requirement for inorganic sulphur obtained from cysteine for the synthesis of metal–sulphur clusters. Alternative or parallel pathways for cysteine biosynthesis may boost the provision of sulphur for the biosynthesis of nitrogenase often present in high levels (Evans et al., 1991 ). However, deletion of the nif-related cysE gene in Azotobacter has no marked effects on the development of nitrogenase activity. In R. leguminosarum RL3841, it is unclear how the bacteroid obtains sulphur for housekeeping purposes and for the synthesis of nitrogenase, but, like Azotobacter, RU632 (the strain defective in the cysE gene) was unaffected in terms of N2 fixation, though there may be conditions (e.g. nutrient stress) under which a phenotype is expressed.


   ACKNOWLEDGEMENTS
 
This work was supported, in part, by BBSRC grant CO4183 to R.L.R, and by The Nuffield Foundation (via a Vacation Studentship awarded to K.O.) We also like to thank Liz Pontin and Mike Taylor (both of AMSEQ) for the automated DNA sequencing.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 15 February 2001; revised 1 June 2001; accepted 12 June 2001.



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