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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 -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 FolinCiocalteu 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
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.
|
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 metalsulphur 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84, 188-198.[Medline]
Bogdanova, N. & Hell, R. (1997). Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana. Plant J 11, 251-262.[Medline]
Cherest, H. & Surdin-Kerjan, Y. (1992). Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130, 51-58.
Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16, 10881-10890.[Abstract]
Denk, D. & Böck, A. (1987). L-Cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyl transferase (cysE) gene from the wild-type and a cysteine excreting mutant. J Gen Microbiol 133, 515-525.[Medline]
Evans, D. J., Jones, R., Woodley, P. R., Wilborn, J. R. & Robson, R. L. (1991). Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM cluster, including a new gene (nifP) which encodes a serine acetyl transferase. J Bacteriol 173, 5457-5469.[Medline]
Foglino, M., Borne, F., Bally, M., Ball, G. & Patte, J. C. (1995). A direct thiolation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa. Microbiology 141, 431-439.[Abstract]
Glenn, A. R., Poole, P. S. & Hudman, J. F. (1980). Succinate uptake by free-living and bacteroid forms of Rhizobium leguminosarum. J Gen Microbiol 119, 267-271.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Howarth, J. R., Roberts, M. A. & Wray, J. L. (1997). Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph. Biochim Biophys Acta 1350, 123-127.[Medline]
Huang, T. C., Lin, R. F., Chu, M. K. & Chen, H. M. (1999). Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp. strain RF-1. Microbiology 145, 743-753.[Abstract]
Jones-Mortimer, M. C. (1968). Positive control of sulphate reduction in Escherichia coli. The nature of the pleiotropic cysteineless mutants of E. coli K12. Biochem J 110, 597-602.[Medline]
Kredich, N. M. (1987). Biosynthesis of cysteine. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 419428. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Kredich, N. M. & Tomkins, G. M. (1966). The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem 241, 4955-4965.
Mead, D. A., Long, S., Ruvkin, G., Brown, S. & Ausubel, F. (1982). Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti. J Bacteriol 149, 114-122.[Medline]
Mead, D. A., Szczesna-Skorupa, E. & Kemper, B. (1986). Single-stranded DNA blue promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng 1, 67-74.
Miller, J. H. (1972). Experiments in Molecular Genetics, pp. 352355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Murillo, M., Foglia, R., Diller, A., Lee, S. & Leustek, T. (1995). Serine acetyltransferase from Arabidopsis thaliana can functionally complement the cysteine requirement of a cysE mutant strain of Escherichia coli. Cell Mol Biol Res 41, 425-433.[Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of the cleavage sites. Protein Eng 10, 1-6.[Abstract]
Parker, G. F., Higgins, T. P., Hawkes, T. & Robson, R. L. (1999). Rhizobium (Sinorhizobium) meliloti phn genes: characterisation and identification of their protein products. J Bacteriol 181, 389-395.
Patriarca, E. J., Riccio, A., Taté, R., Colonna-Romano, S., Iaccarino, M. & Defez, R. (1993). The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation. Mol Microbiol 9, 569-577.[Medline]
Poole, P. S., Blyth, A., Reid, C. & Walters, K. (1994a). myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv. viciae. Microbiology 140, 2787-2795.
Poole, P. S., Schofield, N. A., Reid, C. J., Drew, E. M. & Walshaw, D. L. (1994b). Identification of chromosomal genes located downstream of dctD that affect the requirement for calcium and the lipopolysaccharide layer of Rhizobium leguminosarum. Microbiology 140, 2797-2809.[Abstract]
Saitou, N. & Nei, M. (1987). The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406-425.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Studier, F. W. & Moffatt, B. W. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[Medline]
Tabor, S. & Richardson, C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 82, 1074-1078.[Abstract]
Taté, R., Riccio, A., Caputo, E., Iaccarino, M. & Patriarca, J. (1999). The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris. Mol PlantMicrobe Interact 12, 24-34.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Trinick, M. J., Dilworth, M. J. & Grounds, M. (1976). Factors affecting the reduction of acetylene by root nodules of Lupinus species. New Phytol 77, 359-370.
Walshaw, D. L. & Poole, P. S. (1996). The general L-amino acid permease of Rhizobium leguminosarum is an ABC uptake system that influences efflux of solutes. Mol Microbiol 21, 1239-1252.[Medline]
Walshaw, D. L., Reid, C. J. & Poole, P. S. (1997). The general amino acid permease of Rhizobium leguminosarum is negatively regulated by the Ntr system. FEMS Microbiol Lett 152, 57-64.[Medline]
Zheng, L., Cash, V. L., Flint, D. H. & Dean, D. R. (1998). Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J Biol Chem 273, 13264-13272.
Received 15 February 2001;
revised 1 June 2001;
accepted 12 June 2001.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |