Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J11
Author for correspondence: L. C. Vining. Tel: +1 902 494 2040. Fax: +1 902 494 3736. e-mail: Leo.Vining{at}Dal.Ca
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ABSTRACT |
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Keywords: cysteine auxotroph, sulfur metabolism, gene disruption, streptomycetes
Abbreviations: CBL, cystathionine ß-lyase; CBS, cystathionine ß-synthase; CGL, cystathionine -lyase; CGS, cystathionine
-synthase; CS, cysteine synthase; OAH, O-acetylhomoserine; OSH, O-succinylhomoserine
b The GenBank accession number for the sequence reported in this paper is AF319543.
a Present address: Department of Microbiology, University of Minnesota, 1030 Mayo Building, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
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INTRODUCTION |
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Studies of the biosynthesis and metabolism of sulfur-containing amino acids in Escherichia coli and numerous other micro-organisms have established that bacteria form cysteine from serine by a two-step process initiated by serine acetyltransferase (Fig. 1). The O-acetylserine intermediate reacts with H2S to give cysteine by the action of O-acetylserine thiolase. Although both enzyme activities should properly be included in the term cysteine synthase (CS), this designation will be used below, as elsewhere (Kredich, 1997
), to refer only to the second reaction, releasing cysteine. In enterobacteria, isozymic forms (A and B) of the thiolase are encoded by cysK and cysM respectively (Hulanicka et al., 1986
; Sirko et al., 1987
). Isozyme B accepts thiosulfate as an alternative substrate to sulfide, forming S-sulfocysteine in the initial condensation, but this eventually yields cysteine (Nakamura et al., 1984
). Although cysK has been cloned from a wide variety of bacteria in addition to enterics: e.g., Rhodobacter sphaeroides 2.4.1 (OGara et al., 1997
), Flavobacterium K3-15 (Muller et al., 1996
), Synechococcus sp. (Nicholson et al., 1995
) and Streptococcus suis (Osaki et al., 2000
), cysM has a more limited distribution (Hensel & Trüper, 1976
). Also, some bacteria (e.g., Pseudomonas aeruginosa) possess alternative reactions, commonly called reverse transsulfuration, that generate cystathionine and then cleave it with cystathionine
-lyase (CGL) to form cysteine (see Fig. 1
; Gunther et al., 1979
). In Saccharomyces cerevisiae cystathionine is available from the cystathionine ß-synthase (CBS)-catalysed condensation of homocysteine and serine (Cherest & Surdin-Kerjin, 1992
), whereas in bacteria cystathionine is usually formed, along with the homocysteine required for methionine biosynthesis, in a cystathionine
-synthase (CGS)-catalysed transsulfuration reaction between cysteine and O-acylhomoserine (reviewed by Greene, 1997
).
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METHODS |
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Preparation of cell extracts.
The mycelium from a 50 ml Str. venezuelae culture was collected by centrifugation (10000 g at 4 °C for 10 min) and washed with TEPD buffer (100 mM Tris/HCl, 1 mM EDTA, 0·1 mM pyridoxal phosphate, 0·1 mM DTT, pH 8·0). For homocysteine synthase assays, PEPD buffer (TEPD with Tris replaced by 100 mM phosphate, pH 7·6) was used. The washed mycelium was disrupted with a Branson Sonifier (model 210) for 6x15 s at 4 °C. The sonicate was centrifuged (12000 g for 20 min at 4 oC and the supernatant solution was used as the cell extract. Protein was measured with the bicinchoninic acid reagent (Pierce Chemical). Cell extracts of strains compared for enzyme activity were adjusted with TEPD or PEPD buffer to the same protein concentration.
Assay of CS activity.
The reaction mixture (500 µl) contained cell extract (250 µl), 30 mM O-acetylserine and 20 mM Na2S, all prepared in TEPD buffer. After incubation at 30 °C for 30 min, activity was stopped by adding 50 µl 50% (w/v) trichloroacetic acid. The mixture was clarified by centrifugation and its cysteine content was determined by the specific colorimetric method described below.
Assay of CBS and CGS.
Cystathionine can be formed by CBS-catalysed condensation of homocysteine with serine, or by CGS-catalysed condensation of O-acyl (succinyl- or acetyl-) homoserine with cysteine. To measure CBS activity, the reaction mixture (500 µl) contained 250 µl cell extract, 20 mM homocysteine and 20 mM serine, all in PEPD buffer. CGS activity in mycelial extracts of Str. venezuelae was measured in a reaction mixture containing 20 mM O-succinyl-DL-homoserine, synthesized as described by Nagai & Flavin (1967) , and 20 mM cysteine as substrates, again all in PEPD buffer. Both assay mixtures were incubated for 30 min at 30 °C and terminated by freezing (-20 °C); the cystathionine formed was measured by HPLC using the procedure described below for amino acid analysis.
Assay of CBL and CGL.
Cystathionine is cleaved to form pyruvic acid, homocysteine and ammonia by cystathione ß-lyase (CBL), or -ketobutyric acid, cysteine and ammonia by CGL. The reaction mixture (500 µl) for either assay consisted of 250 µl cell extract, 200 µl cystathionine (10 mM in 10 mM HCl) and 50 µl TEPD buffer. After incubation at 30 °C for 30 min, reactions were terminated by acidification with 500 µl 2 M HCl and clarified by centrifugation for analysis. Pyruvic and
-ketobutyric acids in enzyme incubation mixtures were derivatized by reaction with 1,2-diamino-4,5-dimethoxybenzene, synthesized as described by Ohmori et al. (1992)
. The 3-methyl-2-hydroxy-6,7-dimethoxyquinoxaline formed from pyruvic acid, and 3-ethyl-2-hydroxy-6,7-dimethoxyquinoxaline from
-ketobutyric acid were measured by HPLC with a detector monitoring absorbance at 360 nm (Ohmori et al., 1992
).
Specific measurement of cysteine.
The colorimetric method of Gaitonde (1967) was modified as follows: the cysteine-containing solution (0·5 ml) was mixed in a test tube with 0·5 ml glacial acetic acid and 0·5 ml ninhydrin reagent. The solution was heated in boiling water for exactly 90 s, cooled on ice for 2 min and diluted with 2 ml 95% ethanol before the absorbance was measured at 560 nm.
Measurement of amino acids by HPLC.
Each sample (40 µl) was neutralized and mixed with 40 µl o-phthaldialdehyde reagent (Pierce Chemical). After 1 min, 120 µl 0·1 M sodium acetate buffer (pH 6·2) was added and a portion (20 µl) of the mixture was injected on to a 4·6x45 mm Beckman Ultrasphere ODS column. Elution of fluorescent amino acid derivatives by a programmed methanol/solvent A gradient was monitored with a Beckman fluorescence detector (excitation 305395 nm, emission 420650 nm). Solvent A contained 100 ml of a 19:1 mixture of methanol and tetrahydrofuran, brought to 1000 ml with 0·1 M sodium acetate, pH 6·2. The percentage of solvent A in the gradient was decreased from 100% to 25% in 6·74 min, then to 0% in 0·26 min, and kept at 0% for an additional 0·5 min. To reequilibrate the column for another injection, solvent A was increased to 100% in 0·5 min and kept at 100% for another 2 min.
Chloramphenicol assays.
Production of chloramphenicol was bioassayed as described by Doull et al. (1985) . For specific and more precise measurement of chloramphenicol produced in liquid cultures, the HPLC procedure of Brown et al. (1996)
was used.
DNA manipulation.
The procedures of Sambrook et al. (1989) were used to isolate or modify DNA, and to prepare and transform competent E. coli cells. For rapid screening of E. coli plasmids, the Sekar (1987)
procedure was adopted. DNA fragments were recovered from agarose gels with the QIAEX II Gel Extraction Kit (Qiagen) using the suppliers protocol. Routine plasmid isolation from streptomycetes was based on the alkaline lysis method for isolating E. coli plasmids, but Solution I was replaced with P-buffer (Hopwood et al., 1985
) containing 2 mg lysozyme ml-1 (Solution I*), and the mycelium was incubated for 30 min at 37 °C instead of on ice. Isolation of streptomycete genomic DNA and protoplast procedures for transforming streptomycetes followed the protocols of Hopwood et al. (1985)
.
Gene disruption.
To inactivate ORF1, a disruption plasmid was prepared by inserting into pJV208 at its SalI site near the middle of ORF1, a fragment (both orientations) conferring apramycin resistance (AmR) that had been excised with SalI from pJV225. From the resultant plasmid (pJV213) a PstI fragment conferring AmR was recloned in pHJL400 to give the disruption plasmid pJV218. This was passaged through E. coli ET12567 to avoid restriction barriers (MacNeil et al., 1992 ) during its subsequent use in transforming protoplasts of Str. venezuelae ISP5230 and VS263. From these transformants strains resistant to both thiostrepton and apramycin (AmR TsR), and therefore presumed to contain pJV218 either as the free plasmid or integrated into chromosomal DNA by a single crossover, were isolated (VS1025 derived from the wild-type, and VS1030 derived from the cys-28 mutant). To promote allele replacement in single-crossover strains by a second crossover between the integrated disruption plasmid and homologous chromosomal DNA, strains VS1025 and VS1030 were grown through two rounds of sporulation on MYM agar without antibiotic selection. Spores from single colonies were screened to detect strains resistant to apramycin and sensitive to thiostrepton (VS1028 arising from VS1025, and VS1032 from VS1030). To disrupt ORF2 the apramycin resistance gene from pUC120A was inserted into an NcoI site near the centre of the ORF in pJV220, giving pJV222. The plasmid was used to transform ISP5230 and AmR colonies were selected.
PCR amplification of Str. venezuelae genomic DNA.
Primers ch3 (5'-GAGACCATCGGCAACACCCC-3') and ch4 (5'-GTGATCGTGCCGCCGGTGCC-3') were designed from a CLUSTAL W alignment of the cysK sequences in a variety of Gram positive and Gram negative bacteria. Consensus sequences in two conserved regions 500 bp apart (Fig. 3) were modified for streptomycete codon usage (Wright & Bibb, 1992
) and used to amplify a DNA fragment from the Str. venezuelae ISP5230 genome by PCR. The procedure in the first 6 cycles involved denaturing at 96 °C for 1 min, annealing at 67 °C for 1 min and extension at 72 °C for 1 min. Denaturing and annealing were each reduced to 45 s for the next 30 cycles. Fragments of DNA amplified in the reaction were cloned in the SmaI site of pUC18 with the SureClone kit (Pharmacia Biotech) and sequenced.
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Sequencing and sequence analysis.
Nested deletions (Sambrook et al., 1989 ) were introduced into DNA fragments cloned in pBluescript II SK+, and both strands of the DNA were sequenced. Restriction sites were located with Gene Runner (Hastings Software). Potential ORFs were detected with CODONPREFERENCE (GCG), and the internet-based FramePlot 2.3 (Ishikawa & Hotta, 1999
). CLUSTAL W (Thompson et al., 1994
) was used for multiple sequence alignments, and BLAST searches (Altschul et al., 1997
) were used to compare cloned sequences with DNA and protein sequences in GenBank.
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RESULTS |
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In BLAST P searches of GenBank the deduced amino acid sequence of the 48·82 kDa (463 aa) product of ORF1 resembled CBS and CS sequences in both prokaryotes and eukaryotes. It most closely matched (82% identity) the putative CBS (CAB89449) encoded by the Str. coelicolor A3(2) gene cloned in cosmid E25; next in similarity was the cysM2 product (CAA17193) of Mycobacterium tuberculosis (62% identity). Mammalian CBSs were much less similar (3742% identity), but in general CBS sequences ranked higher than CS sequences. The closest CS match (42% identity) was with the cysK product (GenBank T43792) of Clostridium perfringens. Although CBS genes are more common in eukaryotes than prokaryotes, similarities between the sequence of the ORF1 product and sequences in the genomes of Str. coelicolor A3(2) (http://www.sanger.ac.uk/Projects/S_coelicolor/), M. tuberculosis (Cole et al., 1998 ), Mycobacterium leprae (Cole et al., 2000
) and P. aeruginosa (Stover et al., 2000
) indicate that a CBS gene in Str. venezuelae would not be unique. However, none of the prokaryote CBSs postulated from genome sequencing has been functionally characterized.
A multiple sequence alignment of CBSs and CSs from various organisms (Fig. 3) indicated that CBSs are about 150 aa longer than CSs, with the extra length in the C-terminal region. The sequence of the ORF1 product (CBS-Str. venezuelae) matched CBS sequences in length (
450 aa) and overall alignment, whereas only the N-terminal region aligned with CS sequences. All of the sequences contained a conserved pyridoxal phosphate-binding motif. The highly conserved lysine 42 in both the SVKCRI motif of the E. coli cysK product (Rege et al., 1996
) and the SIKDRI motif of the Flavobacterium sp. cysK (Muller et al., 1996
) corresponded to lysine 44 in the SVKDRI motif of the Str. venezuelae ORF1 product. The results implied that the ORF1 product, like other transsulfuration enzymes, is pyridoxal phosphate-dependent, and supported assignment of the GTG at nt 4460 as the start codon for ORF1. On the evidence from sequence analysis, ORF1 was predicted to be a CBS rather than a CS gene, but confirmation was sought from complementation, gene disruption and enzyme analysis.
Test for complementation of the cys-28 mutation with ORF1
The cysteine auxotroph (VS263) obtained by mutagenesis with ethyl methanesulfonate of a cml- strain of Str. venezuelae ISP5230 (Vats et al., 1987 ) was examined for growth requirements imposed by the unidentified cys-28 mutation. When colonies on MM agar were supplemented with substances potentially involved in cysteine biosynthesis, positive responses were obtained with cysteine, methionine, homocysteine and cystathionine (Table 2
). Thiosulfate restored weak growth, but sulfite, sulfide, serine, O-acetylserine, homoserine, O-acetylhomoserine (OAH) and O-succinylhomoserine (OSH) had no effect. These results implied that the step in cysteine biosynthesis converting O-acetylserine to cysteine, catalysed by CS (also called O-acetylserine thiolase) was blocked in VS263, and pointed to a mutation in either a gene encoding CS or a regulatory gene as the cause of the cys-28 phenotype. To determine whether ORF1 could complement the mutation, the 4·0 kb PstI insert from pJV208 was subcloned in the shuttle vector pHJL400 to give pJV215 and pJV216 (alternative orientations). Transformation of VS263 with pJV215, in which ORF1 had the same orientation as the vector promoter, and screening for growth on MM agar did not detect prototrophs, indicating that either the cys-28 mutation was not in the CS gene, or that the DNA cloned in ORF1 lacked a functional CS gene.
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Enzyme assays
The activities of five enzymes that participate in the biosynthesis and interconversion of sulfur-containing amino acids were assayed in mycelium extracts of Str. venezuelae. CS activity was determined in Str. venezuelae strains ISP5230, VS1028, VS263 and VS1032. Cultures were grown in the nutrient-poor MMY medium supplemented, for those strains carrying the cys-28 mutation (VS263 and VS1032), with methionine and cystathionine to allow growth comparable to that in ISP5230 and VS1028 cultures. Assays directly measuring the amount of cysteine synthesized showed similar CS activity in ISP5230 and VS1028 (Fig. 5), indicating that disruption of cbs had no effect, and thereby confirming that ORF1 does not encode CS. The assays also showed that CS activities in VS263 and VS1032 were 10-fold lower than in ISP5230 and VS1028, respectively. That the lower activities were not due to cystathionine or methionine in the supplement used to improve growth in the nutrient-poor MMY medium was shown by the similar, low CS activities [0·07, 0·04 and 0·03 mM cysteine (mg protein)-1] in VS263 mycelium grown in MMY medium alone or with cystathionine or methionine supplements, respectively. The high activities in MYM medium [1·21·3 mM cysteine (mg protein)-1; see Fig. 5
] were observed in both of the cys-28 mutant strains (VS263 and VS1032), and were well above activities in the wild-type strains (ISP5230 and VS1028), which were not influenced by the culture medium. Low CS activity in the nutrient-poor MMY medium was consistent with the cysteine requirement of cys-28 mutant strains tested for auxotrophy on MM agar. Since enzyme assays with extracts from mycelium grown in MYM medium indicated that the structural CS gene in the cys-28 mutant was intact, the results suggested that auxotrophy was caused by mutation of a regulatory gene controlling CS expression. Systematically varying the composition of MYM medium showed that yeast extract and malt extract in combination strongly stimulated CS activity in mycelium extracts, but neither substance alone was effective. The presence of glucose markedly lowered activity.
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DISCUSSION |
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Cysteine biosynthesis in streptomycetes
The presence of CS activity in a streptomycete has been demonstrated here for the first time by a direct enzyme assay. The cysteine auxotrophy of the Str. venezuelae mutant VS263 was correlated with low intracellular CS activity, and the resulting impaired output of cysteine from the thiolation pathway. Evidence that CS activity in the mutant depended markedly on growth conditions, and was high in the nutrient-rich MYM medium, indicated that the gene encoding CS in Str. venezuelae VS263 was intact, and suggested that the cys-28 mutation unmasks a repressor preventing gene expression. The possibility that a gene regulating cysteine biosynthesis might be involved in the cys-28 mutation is also suggested by restoration of slow growth with thiosulfate. Regulation of CS by thiosulfate varies from the strong repression exerted in Rhodopseudomonas palustris to the derepression of enzyme synthesis found in Rhodopseudomonas sulfidophila (Hensel & Trüper, 1976 ). Thus thiosulfate might have a role as an effector in the cysteine biosynthesis pathway of Str. venezuelae. However, an alternative explanation for the ability of thiosulfate to support slow growth of Str. venezuelae VS263 is the potential existence of the S-sulfocysteine pathway for biosynthesis of cysteine in this streptomycete. Actinomycetes vary in the contributions of the direct thiolation and S-sulfocysteine pathways to cysteine biosynthesis: whereas in Streptomyces griseus the S-sulfocysteine pathway is the major contributor (Kitano et al., 1985
), in Str. venezuelae direct thiolation appears to be dominant, and both pathways are present in Sacp. erythraea. This versatility in cysteine biosynthesis has been associated (Donadio et al., 1990
) with differences in the distribution of the CSB isozyme (cysM product) in actinomycete species. Like the cysM product in enterobacteria, CSB exhibits dual functions supporting both the direct thiolation and S-sulfocysteine pathways. The exclusive presence of cysM and absence of cysK in Str. venezuelae may account for the amplification of a cbs fragment by PCR with primers ch3 and ch4, which contained consensus sequences of both cysteine synthase isozyme A and B genes. Cloning and sequencing 100 fragments amplified from Str. venezuelae ISP5230 DNA with these primers did not yield a single product that matched the sequence of cysK (data not shown).
CBS and other transsulfuration enzymes in Str. venezuelae
Since disrupting cbs in wild-type Str. venezuelae did not prevent growth on minimal medium, the gene appeared at first to have no role in primary metabolism. However, a function was revealed by insertional inactivation of cbs in the cys-28 mutant, which in minimal medium cannot synthesize cysteine by thiolation of O-acetylserine. Disrupting cbs created cysteine auxotrophy by interrupting the alternative supply of this amino acid via reverse-transsulfuration. This route to cysteine relies on the availability of cystathionine from CBS-catalysed condensation of homocysteine and serine, and is lost when cbs is inactivated. Besides confirming the function of the gene, the disruption provided evidence unobtainable from enzyme assays for reverse transsulfuration in a streptomycete. Although assay values for CBS activity in mycelial extracts were below the limits of detection by the procedure used, the activity presumably meets the needs of the organism in vivo. Overall, the results indicated that, although cysteine is mainly synthesized de novo in Str. venezuelae by thiolation of O-acetylserine, interconversion of pre-existing sulfur-containing amino acids has a supplementary role that can become important under some circumstances.
Methionine biosynthesis
In the transsulfuration reaction between cysteine and O-acylhomoserine for the biosynthesis of methionine, the acyl group and the fate of the acylated product can vary. In Gram-negative bacteria, with some exceptions, OSH is the substrate, whereas in Gram-positive bacteria such as bacilli, corynebacteria and brevibacteria, the substrate is OAH. The evidence that OSH but not OAH is the substrate for CGS in Str. venezuelae is consistent with previous observations that streptomycetes differ from other Gram-positive bacteria, and resemble Gram-negative bacteria, in using OSH for this reaction (Kanzaki et al., 1986 ). Some micro-organisms convert O-acylhomoserine directly to homocysteine in an alternative route to methionine. Genes for the thiolase (homocysteine synthase) catalysing this reaction have been cloned and sequenced from P. aeruginosa, where this is the main route (Foglino et al., 1995
), and the gene products have been identified from genome sequencing of several bacterial genomes, including that of the actinomycete M. tuberculosis (GenBank CAA17112). The presence of CGL in Str. venezuelae was predictable in view of the report (Nagasawa et al., 1984
) that this enzyme is widely distributed in streptomycetes. Since CBL activity was also detected in Str. venezuelae, a dual-function cystathionine ß,
-lyase of the type discovered in Lactococcus lactis (Dobric et al., 2000
) is not excluded. This enzyme catalyses
,ß and
,
-elimination reactions equally well, and could have been responsible for the similar levels of the two lyases detected in Str. venezuelae. However, information currently available does not distinguish between the activities of a dual function enzyme or two individual enzymes. The similar amounts of CBL and CGL in mycelial extracts suggest that conversion of cystathionine to cysteine must be tightly regulated. The high level of CGS activity is consistent with this reaction supplying the relatively large amount of cystathionine needed for conversion to methionine. Since the formation of cysteine from cystathionine is not a principal supply route in Str. venezuelae, most of the cystathionine would be expected to provide homocysteine by the action of CBL, and then be S-methylated to methionine.
Relationships among streptomycete genes for reactions involving sulfur amino acids
Although cysK is widely distributed in bacteria, it has not been identified in the completely sequenced genome of Str. coelicolor A3(2). Its apparent absence, and our failure to obtain a cysK fragment by PCR amplification from the Str. venezuelae ISP5230 genome, suggest that this gene is not universally present in streptomycetes. The importance of transsulfuration reactions supporting cysteine and methionine biosynthesis in these organisms is implied by the putative assignment of six genes in the Str. coelicolor A3(2) genome to such functions (Redenbach et al., 1996 ). They include the cysM cloned in cosmid E19A, the CBS gene in cosmid E25, the CGS gene in cosmid K13, the CGL gene in cosmid Q11, and probably also the putative lyase gene in cosmid G20A responsible for the CBL activity that forms homocysteine from the cystathionine generated by CGS. BLAST searches of GenBank showed close similarity between protein sequences deduced from these genes and their counterparts in other organisms. A BLASTX search of the Str. coelicolor A3(2) genome with the cloned Str. venezuelae cbs showed a striking 82% similarity in deduced amino acid sequences to a gene in the Str. coelicolor cosmid E25, indicating that cbs is well conserved in streptomycetes. Examination of sequence relationships suggests early divergence of CBS and CS groups. The CBSs from Str. venezuelae and Str. coelicolor A3(2) cluster with the cysM2 products of M. tuberculosis and M. leprae to form a prokaryotic subgroup that has diverged from the subgroup of eukaryotic CBSs. Probably because eukaryotic CBSs have received more attention than those from prokaryotes, the Str. venezuelae CBS gene is the first in the latter group to have been cloned and characterized.
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Received 19 September 2001;
revised 15 February 2002;
accepted 26 February 2002.
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