Characterization of the homthrCthrB cluster in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331

Mónica Fernández1, Yolanda Cuadrado1, Eliseo Recio1, Jesús F. Aparicio1,2 and Juan F. Martín1,2

Institute of Biotechnology INBIOTEC, Parque Científico de León, Avda. del Real, no 1, 24006 León, Spain1
Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain2

Author for correspondence: Juan F. Martín. Tel: +34 987 210308. Fax: +34 987 210388. e-mail: degjmm{at}unileon.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Three genes from the aminoethoxyvinylglycine (AVG)-producing Streptomyces sp. NRRL 5331 involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes appear to be organized in a bicistronic operon as deduced by disruption experiments. The thrB gene, however, is transcribed as a monocistronic transcript. The encoded proteins are quite similar to the HDH, HK and TS proteins from other bacterial species. The overall organization of these three genes, in the order homthrCthrB, differs from that in other bacteria and is similar to that reported in the Streptomyces coelicolor genome sequence. This is the first time in which the gene cluster for the three last steps of threonine biosynthesis has been characterized from a streptomycete. Disruption of thrC indicated that threonine is not a direct precursor for AVG biosynthesis in Streptomyces sp. NRRL 5331 and suggested that the branching point of the aspartic acid-derived biosynthetic route of this metabolite should lie earlier on the threonine biosynthetic route.

Keywords: threonine biosynthesis, homoserine dehydrogenase, homoserine kinase, vinylglycine, actinomycetes

Abbreviations: AVG, aminoethoxyvinylglycine; HDH, homoserine dehydrogenase; HK, homoserine kinase; PLP, pyridoxal phosphate; TS, threonine synthase

The GenBank accession number for the sequence reported in this paper is AJ312095.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Vinylglycines have been traditionally studied as phytotoxins responsible for the foliar chlorosis resulting from plant infection by certain micro-organisms. Examples include rhizobitoxine [L-2-amino-4-(2-amino-3-hydroxypropoxy)-trans-but-3-enoic acid] produced by the root-nodulating bacterium Bradyrhizobium japonicum (La Favre & Eaglesham, 1986 ) and the broad host range plant pathogen Burkholderia andropogonis (Mitchell & Frey, 1988 ), methoxyvinylglycine (L-2-amino-4-methoxy-trans-but-3-enoic acid) produced by the opportunistic human pathogen Pseudomonas aeruginosa (Goodwin & Mercer, 1983 ), and aminoethoxyvinylglycine [AVG; L-2-amino-4-(2-amino-ethoxy)-trans-but-3-enoic acid] produced by the soil dweller Streptomyces sp. NRRL 5331 (Fig. 1). All of them are enol ether amino acids derived from aspartic acid (Mitchell & Coddington, 1991 ). Vinylglycines are analogues of cystathionine and constitute potent inhibitors of ß-cystathionase (Giovanelli et al., 1971 ; Owens et al., 1971 ) in the methionine biosynthesis pathway. Additionally, their characteristic vinyl group makes these metabolites irreversible inhibitors of other pyridoxal phosphate-dependent enzymes (Rando, 1974 ; Gehring et al., 1977 ; Soper et al., 1977 ). Vinylglycines thus interfere, at very low concentrations, with the biosynthesis of the senescence hormone ethylene in plants through mechanism-based inhibition of two key enzymes on ethylene production, namely, the 1-aminocyclopropane-1-carboxylate synthase (Satoh & Yang, 1989 ; Feng & Kirsch, 2000 ), and the 1-aminocyclopropane oxidase (Barry et al., 1996 ; Have & Woltering, 1997 ). Therefore, the normal function of the plant, and in particular ethylene biosynthesis, should be drastically affected in the presence of vinylglycines.



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Fig. 1. (a) Biosynthesis pathway of threonine showing the putative branch leading to AVG. The threonine synthase gene (encoded by thrC) that has been disrupted is underlined. (b) Chemical structures of natural vinylglycines. Note that the vinyl portion of the molecule is shared in the different vinylglycines.

 
The biosynthetic pathway of vinylglycines has been only studied in the case of rhizobitoxine. In Bur. andropogonis it has been established that aspartate is the first precursor of the pathway, homoserine being an essential intermediate (Mitchell & Coddington, 1991 ). This result suggests that the vinylglycine pathway should branch somewhere from the threonine biosynthetic pathway. Additionally, hydroxythreonine has been proposed as a likely biosynthetic intermediate on the pathway from homoserine to rhizobitoxine (Mitchell & Frey, 1988 ). On the other hand, studies with Bra. japonicum have led to the cloning of two genes whose products are putatively involved in the other branch of the route, namely RtxA, a dehydroxyacetone phosphate aminotransferase involved in serinol production, and RtxB, the dihydrorhizobitoxine synthase responsible for the condensation of serinol and homoserine (Ruan et al., 1993 ). The biosyntheses of AVG and methoxyvinylglycine, however, remain totally obscure although they should have some steps in common since the main part of the molecule is shared.

To gain knowledge about AVG biosynthesis in Streptomyces sp. NRRL 5331 we decided to study the threonine biosynthetic genes of this bacterium. Threonine biosynthesis from aspartate has been well characterized in Escherichia coli, Bacillus, corynebacteria and other bacteria (for a review see Malumbres & Martín, 1996 ), but not from Streptomyces. Here we report the cloning and characterization of the genes encoding the enzymes involved in the conversion of aspartate semialdehyde to threonine in the AVG-producing Streptomyces strain NRRL 5331. Promoter analysis and transcript disruption techniques were used to reveal their transcriptional organization.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, cloning vectors and cultivation.
Streptomyces sp. NRRL 5331 was used as the source of DNA in the construction of the genomic library. Escherichia coli strain XL-1 Blue MR was used for obtaining SuperCos 1 cosmid (Stratagene) recombinant derivatives, and also served as a host for subcloning in plasmids pBluescript (Stratagene), pUC18 and pUC19. Streptomyces sp. NRRL 5331 was routinely grown in TSB, YEME medium without sucrose (Kieser et al., 2000 ) or YEPEG medium [glucose, 10 g l-1; Bacto peptone, 5 g l-1; yeast extract, 3 g l-1; Fe(NH4)2(SO4)2.6H2O, 0·03 g l-1]. Sporulation was achieved in TBO medium (Aparicio et al., 2000 ) at 30 °C. Auxotrophs were tested on Streptomyces minimal medium (Hopwood, 1967 ). L-threonine or L-homoserine (0·4 mM) were added, when required, to the minimal medium.

Genetic procedures.
Standard genetic techniques with E. coli and in vitro DNA manipulations were as described by Sambrook & Russell (2001) . Recombinant DNA techniques in Streptomyces species and isolation of Streptomyces total and plasmid DNA were performed as previously described (Kieser et al., 2000 ). For construction of the genomic library, Streptomyces sp. NRRL 5331 genomic DNA was partially digested with Sau3AI and fragments in the 35–40 kb size range were cloned into SuperCos 1 digested with BamHI and XbaI. The ligation mixture was packaged with Gigapack III XL (Stratagene) and used to transfect E. coli XL-1 Blue. Southern hybridization was carried out with probes labelled with digoxigenin by using the DIG DNA labelling kit (Roche Biochemicals).

Generation of DNA probes.
DNA probes for screening of the library of size-fractionated genomic DNA were obtained by PCR amplification of Streptomyces sp. NRRL 5331 chromosomal DNA by using oligonucleotides derived from conserved stretches of several microbial homoserine dehydrogenases (HDHs), homoserine kinases (HKs) and threonine synthases (TSs). The oligonucleotide pairs used were the following: HDH1 (5'-GTSGTSACCGCSAACAAGGC-3') (where S is G or C) and HDH2 (5'-GATGAAGTTGGTGGTGCCGTT-3') for the hom gene (encoding HDH); HK1 (5'-AACCTSGGCCCSGGCTTCGAC-3') and HK2 (5'-GCGTTGTCSGGGTGGCCCTC-3') for the thrB gene (encoding HK); TS1 (5'-CCSACCGGCTCSTTCAAGGAC-3') and TS2 (5'-TCGTCGTCGAAGTTGCCGTC-3') for the thrC gene (encoding TS). DNA fragments of the expected size (198 nt for hom; 300 nt for thrB; 210 nt for thrC) were obtained.

DNA sequencing and analysis.
Sequencing templates were obtained by random subcloning of fragments generated by controlled partial HaeIII digestions. DNA sequencing was accomplished by the dideoxynucleotide chain-termination method (Sanger et al., 1977 ) using the Perkin Elmer AmpliTaq Dye-terminator sequencing system on double-stranded DNA templates with an Applied Biosystems model 310 sequencer. Each nucleotide was sequenced a minimum of three times on both strands. Alignment of sequence contigs was performed using the DNAStar Seqman program. DNA and protein sequences were analysed with the University of Wisconsin Genetics Computer Group software programs (Devereux et al., 1984 ) and the NCBI Worldwide Web BLAST server (www.ncbi.nlm.nih.gov/blast).

AVG determination.
AVG production was determined by a bioassay using Bacillus subtilis as the test organism. Solutions of pure AVG (Sigma) were used as reference values for halo formation.

AVG assay in cell-free extracts.
Intracellular levels of AVG during growth was determined by analysing supernatants prepared by high-speed centrifugation of sonicated mycelial pellets. Sonication was carried out at 14 µm frequency for 30 s periods, until complete rupture, in the extraction buffer (50 mM Tris/HCl pH 8·0, 100 mM NaCl, 1 mM EDTA). The ratio between the weight of the mycelium sample and the volume of the extraction buffer was kept constant throughout the experiment. The disrupted mycelium was centrifuged (40000 g for 1 h at 4 °C) and the supernatant used for analysis.

Transcript disruption.
The transcript responsible for hom and thrC expression was disrupted by pHZ1351Km plasmid-mediated single-crossover integration as follows. A 1·3 kb BamHI fragment encompassing the neomycin phosphotransferase gene from transposon Tn5 (Beck et al., 1982 ) was cloned into a BamHI-digested pHZ1351 vector to yield pHZ1351Km. This plasmid retained the broad host-range and high copy number properties of the parental vector isolated from Streptomyces sp. FR-005 (Bao et al., 1997 ), and is prone to chromosomal integration mediated by homologous recombination. Although pHZ1351 was originally described as useful for gene replacement, and its replicon was never found to be integrated into the Streptomyces hygroscopicus KMP3 chromosome (Bao et al., 1997 ), we have observed that the replicon of pHZ1351Km is stable upon integration into Streptomyces sp. NRRL 5331 chromosome (not shown). pHZ1351Km was therefore used as a vector for DNA delivery into Streptomyces sp. NRRL 5331 by transformation.

Subcloning in promoter-probe vectors.
Promoter activity of selected DNA fragments was assessed by cloning them upstream of the promoterless kanamycin resistance gene present in the promoter-probe vector pIJ486 (Kieser et al., 2000 ). In particular, for the putative thrB promoter two DNA fragments were used, a 708 bp SmaI fragment including the 3' end of thrC and the 5' end of thrB genes, and a 275 bp NruI–SmaI fragment extending 205 bp upstream from the thrB start codon. Both DNA fragments were subcloned in the Ecl136II site of pIJ486. Following transformation of Streptomyces lividans 66 (Kieser et al., 2000 ) with the resulting plasmids, promoter strength was assessed with increasing concentrations of kanamycin.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and identification of hom, thrB and thrC genes forming a single gene cluster
Despite the high degree of conservation among microbial HDHs, HKs and TSs, we were unable to identify their corresponding genes in Streptomyces sp. NRRL 5331 by hybridization using heterologous probes internal to the homologous genes from Nocardia lactamdurans and Corynebacterium lactofermentum. Therefore, the three biosynthetic genes had to be identified by using homologous DNA probes PCR-derived from Streptomyces sp. NRRL 5331 chromosomal DNA (see Methods). A cosmid library was constructed in the SuperCos 1 vector and several positively hybridizing cosmids were selected independently with the three probes. Further characterization of all of them by cross-hybridization suggested that the three genes were linked.

Two of the cosmids (Cos 27 and Cos 13) were further selected and mapped with restriction enzymes NotI, SacI, BamHI and EcoRI. The two cosmids covered a contiguous 60 kb region of the Streptomyces sp. NRRL 5331 chromosome. Interestingly, the three genes were linked in a 3·7 kb region, most of it covered by a BamHI fragment (Fig. 2). Internal BamHI and SacI fragments of the cosmids were the same size as their homologous fragments of Streptomyces sp. NRRL 5331 total DNA, suggesting that the cloned DNA was not rearranged.



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Fig. 2. Organization of Streptomyces sp. NRRL 5331 threonine gene cluster. The transcriptional direction and the relative sizes of the predicted ORFs are indicated by pointed boxes. Only BamHI (B), HpaI (H), SmaI (S) and NruI (N) restriction sites are indicated. The cosmid clones studied are shown.

 
The complete sequence of the 3·7 kb region, which encompasses the three genes, was determined. Computer-assisted analysis of the sequenced region revealed three potential complete ORFs (Fig. 2), corresponding to the hom, thrB and thrC genes sought. The G+C content of the nucleotide sequence is 72·2 mol%, well within the range of the reference values for Streptomyces DNA (Wright & Bibb, 1992 ).

Seventy-nine base pairs from the 5' end of the BamHI fragment (Fig. 2) lies the ATG start codon of the hom gene (1290 bp), whose product showed a very high end-to-end sequence identity with bacterial HDHs, enzymes that catalyse the NADP-dependent reduction of aspartate ß-semialdehyde into homoserine. These values ranged from 39·6% for the HDH of P. aeruginosa (SWALL accession no. P29365) to 89% for the S. coelicolor HDH (SWALL accession no. Q9ADB4) (Table 1). An alignment of the regions of bacterial HDH proteins that show the greatest degree of sequence conservation is shown in Fig. 3(a). These include the N-terminal end fingerprint region GXGXXG common to the vast majority of NAD(P)H binding sites (Wierenga et al., 1986 ) and the central consensus pattern for these enzymes (Thomas et al., 1993 ).


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Table 1. Percentage identity between the HDHs, TSs and HKs of Streptomyces sp. NRRL 3551 and other micro-organisms

 


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Fig. 3. Comparison of conserved motifs in HDHs, TSs and TKs. Numbers indicate amino acid residues from the N terminus of the protein. Amino acids matching the consensus are shown in bold. The consensus sequences for both regions are shown below. (a) NADP-binding sites and consensus patterns from Streptomyces sp. NRRL 5331 and other bacterial HDHs. (b) Pyridoxal attachment sequences of nine TS enzymes from Streptomyces sp. NRRL 5331 and other bacteria and archaea. The asterisk shows the pyridoxal-binding Lys residue. The consensus sequence shown below corresponds to the Serine/threonine dehydratases pyridoxal-phosphate attachment fingerprint sequence [DESH]-X(4/5)-[STVG]-X-[AS]-[FYI]-K*-[DLIFSA]-[RVMF]-[GA]-[LIVMGA]. (c) ATP-binding regions of Streptomyces sp. NRRL 5331 and other bacterial HKs. Amino acids matching the fingerprint sequence [LIVM]-[PK]-X-[GSTA]-X(0/1)-G-L-[GS]-S-S-[GSA]-[GSTAC] characteristic of galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase enzymes are shown in bold.

 
Located 8 bp downstream from the TAA stop codon of hom lies the ATG start codon of thrC, an ORF encoding a protein of 356 aa. This gene product was easily assigned as the enzyme that catalyses the transformation of homoserine phosphate into threonine because of its significant sequence similarity over its entire length (up to 93% identity) to other TS enzymes from both bacteria and archaea, including S. coelicolor (SWALL accession no. Q9ADB3), Mycobacterium leprae (SWALL accession no. P45837), Aquifex aeolicus (SWALL accession no. O66740) and Archaeoglobus fulgidus (SWALL accession no. O28953) (Table 1). The enzyme shows in its N-terminal end the characteristic lysine residue that constitutes the pyridoxal phosphate attachment site (Fig. 3b), a feature also shared by other pyridoxal phosphate-dependent enzymes like serine and threonine dehydratases (Parsot, 1986 ).

Downstream (347 bp) from the TGA stop codon of thrC is the ATG start codon of the ORF (thrB) needed to phosphorylate homoserine, which in turn will serve as substrate for TS. thrB encodes a protein of 305 aa, with a molecular mass estimated to be 31352 Da as a monomer. HK showed extensive homology to other bacterial HKs. Thus HK showed 86% sequence identity along its full length with the HK from S. coelicolor (accession no. Q9ADB2) and over 40% sequence identity with Mycobacterium tuberculosis (accession no. Q10603), and Brevibacterium lactofermentum (accession no. P07128), among others. Fig. 3(c) shows an alignment of the serine/glycine-rich region of the N-terminal section of HK proteins probably involved in the binding of ATP (Tsay & Robinson, 1991 ).

Chromosome walking from both ends of the 3·7 kb DNA region allowed the identification of the genes present upstream and downstream from the threonine cluster. Upstream (95 bp) from the ATG start codon of hom lies the stop codon of an incomplete ORF whose deduced product showed extensive homology to bacterial diaminopimelate decarboxylases (LysA). Similarly, 385 bp from the TAA stop codon of thrB is the GTG start codon of an ORF whose partial product showed significant sequence similarity to Rho transcriptional termination factors (not shown).

A strong promoter controls the expression of thrB
Computer-assisted analysis of the 347 bp intergenic region between thrC and thrB revealed a putative transcription terminator loop downstream from thrC and also a sequence that matched exactly the -10 and -35 regions of the amy-p2 promoter sequence of the {alpha}-amylase gene from Str. hygroscopicus (Hoshiko et al., 1987 ) followed by a conserved ribosome-binding site sequence (AAGGAG; Fig. 4), thus suggesting that the thrC and thrB genes belong to different operons. This finding was corroborated when a 708 bp DNA fragment including the whole intergenic region, and a shorter version of it (275 bp) including just the putative promoter region (Fig. 4) were subcloned in the promoter-probe vector pIJ486 (see Methods) and the resulting plasmids were transformed in Str. lividans. All the recombinants obtained were resistant to 250 µg kanamycin ml-1 whereas control transformants with pIJ486 were sensitive to 30 µg kanamycin ml-1, indicating that a strong promoter activity was associated with both fragments.



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Fig. 4. Nucleotide sequence of the intergenic region between thrC and thrB in Streptomyces sp. NRRL 5331. The translation initiation codon (Met) and the putative -10 and -35 regions for the potential promoter of thrB are shown in black bold type and underlined (see text). The potential RBS is boxed. The possible terminator (inverted repeat) is indicated by convergent arrows. The SmaI and NruI sites used for promoter activity assessment are also indicated.

 
This result indicates that the expression of thrB in Streptomyces takes place from a different transcriptional unit to that of thrC, a situation already observed in Pseudomonas (Clepet et al., 1992 ) and Methylobacillus (Marchenko et al., 1999 ).

hom and thrC are organized as a single transcriptional unit
The closeness of hom and thrC in the Streptomyces sp. NRRL 5331 chromosome, only 8 bp apart, suggested that the two genes were transcriptionally linked. Since the mRNA isolated from the parental strain turned out to be highly unstable for primer extension or S1 mapping studies, the study of that putative transcriptional linkage was addressed by chromosomal gene disruption of the possible bi-cistronic transcript controlling the expression of both genes. A 870 bp SmaI DNA fragment, encompassing the 3' end of hom and the 5' end of thrC genes, was cloned into the Ecl136II site of pHZ1351Km (a vector with a highly unstable ori, see Methods), and the resulting plasmid used to transform strain NRRL 5331.

Several transformants were obtained by selection for thiostrepton and kanamycin resistance and tested for their inability to grow on minimal medium. One of these disrupted mutants was randomly selected and named HDH-TS. The identity of the mutant was confirmed by Southern hybridization (Fig. 5). Chromosomal DNAs isolated from Streptomyces sp. NRRL 5331 and mutant HDH-TS were digested with BamHI and probed with the 870 bp SmaI fragment used to construct the pHZ1351Km derivative utilized for gene disruption. A hybridizing band of 3·5 kb was found for the wild-type as expected (Fig. 5). However, in the disrupted mutant, two new bands of 11 kb and 1·6 kb were detected, indicating that a single crossover event had occurred. The observed hybridizing bands corresponded exactly to those bands expected according to the integration shown in Fig. 5.



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Fig. 5. Disruption of the transcript that controls the expression of thrC. (a) Predicted restriction enzyme polymorphism caused by gene disruption. The BamHI restriction pattern before and after disruption is shown. The probe is indicated by thick dashed lines. B, BamHI; S, SmaI. Note that only the SmaI sites relevant for obtaining the fragment used in the gene disruption are shown. (b) Southern hybridization of the BamHI-digested chromosomal DNA of the wild-type (lane 1) and the mutant (lane 3). Digoxigenin labelled lambda DNA digested with HindIII is shown in lane 2.

 
As a result of plasmid integration into the chromosome, the disrupted mutant retains intact copies of hom and thrC genes, although a putative bi-cistronic transcript governing the expression of both genes would be interrupted. Therefore, in such a case, the expression of thrC would be blocked and the mutant would display a threonine auxotrophy phenotype. To test this possibility the mutant strain was grown in minimal medium with and without added threonine, showing an absolute requirement for added threonine to sustain growth. Furthermore, the disrupted mutant could not be complemented with homoserine. These results indicate that plasmid integration affected the transcription of thrC and suggest that both genes form part of the same operon.

Production of AVG in the thrC (TS) mutant and the wild-type
To determine the growth phase where we could detect a maximum of AVG production, Streptomyces sp. NRRL 5331 cells were grown for 72 h in TSB medium and used as inoculum (1 ml in 50 ml) of cultures in YEPEG medium. Cultures were then incubated for different periods of time and the AVG content of the broth was measured (see Methods). A clear peak of maximum yield [80 µg ml-1; 1·25 µg AVG (mg dry weight)-1] was observed at 48 h of incubation, during the second half of the exponential phase, decreasing rapidly afterwards. Such conditions were then used to test AVG levels in the mutant strain. No significant differences were found from the production of the wild-type strain (not shown).

To determine if AVG was retained in the cell, AVG levels were also measured in cell-free extracts of the wild-type and the thrC mutant grown in the same media. No traces of AVG were found in the cytoplasm or the cell-wall fractions, even at a threefold concentration relative to the broth. Therefore, AVG does not seem to be retained inside the cells either in the wild-type or in the thrC mutant.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This paper constitutes the first characterization of the threonine biosynthetic gene cluster for a Streptomyces species. The first conclusion from sequence analysis of the Streptomyces sp. NRRL 5331 cluster is that the overall organization of the genes, in the order homthrCthrB, is identical to the one displayed by M. tuberculosis (Cole et al., 1998 ) and to that found recently after completion of the S. coelicolor genome. Furthermore, the genes upstream and downstream from this cluster, lysA and rho, respectively, also match the genes found in both bacteria. Such an organization would imply a different strategy for the regulation of gene expression in these two genera of actinomycetes as compared with enterobacteria such as E. coli (Théze & Saint-Girons, 1974 ) or Serratia marcescens (Komatsubara et al., 1979 ), where the thrA gene, which encodes a polypeptide with aspartate kinase and homoserine dehydrogenase activities, is organized in an operon along with the thrB and thrC genes. The organization in Streptomyces sp. NRRL 5331 is also different from what has been observed in lower actinomycetes such as Brevibacterium lactofermentum (Malumbres et al., 1994 ) [renamed as Corynebacterium lactofermentum (Amador et al., 1999 )] or Corynebacterium glutamicum (Peoples et al., 1988 ), or in other Gram-positive bacteria such as Lactococcus lactis (Madsen et al., 1996 ), where hom and thrB are linked and thrC is located elsewhere in the chromosome. However, a similar gene arrangement has been found in other, more distantly related, micro-organisms such as Bacillus (Parsot & Cohen, 1988 ; Malumbres et al., 1995 ), where the three genes are linked together in the same operon, or Pseudomonas (Clepet et al., 1992 ) and Methylobacillus (Motoyama et al., 1994 ; Marchenko et al., 1999 ), where the hom and thrC genes seem to be co-transcribed while the thrB gene is not linked to the other two (see Malumbres & Martín, 1996 for a review).

Functional promoter analysis and chromosomal disruption have been used in this work to prove that Streptomyces sp. genes are organized in two operons, a situation resembling that observed in P. aeruginosa (Clepet et al., 1992 ) or Methylobacillus flagellatus (Marchenko et al., 1999 ), but in these Gram-negative bacteria the thrB gene is located far apart from the other two. Although a similar overall arrangement of the threonine genes has been found in Myc. tuberculosis (Cole et al., 1998 ) and S. coelicolor genomes, no studies have been undertaken to establish whether the three genes are transcriptionally linked or not.

The other two genes needed to complete the threonine biosynthetic route from aspartate, namely ask and asd, encoding aspartate kinase and aspartate semialdehyde dehydrogenase, respectively, are not within the 60 kb DNA stretch covered by cosmids 27 and 13 (unpublished results). This separate arrangement of the genes responsible for threonine biosynthesis from aspartate in two different clusters has been also observed in Myc. tuberculosis (Cole et al., 1998 ). A cluster containing the askasd genes unlinked to the threonine genes has been found recently in Amycolatopsis lactamdurans (Hernando-Rico et al., 2001 ). In the S. coelicolor genome ask and asd are also organized in an operon separated from homthrCthrB (Redenbach et al., 1996 ); it is therefore tempting to speculate that the same situation will be found in other Streptomyces species. However a discrete asd gene has been reported in Streptomyces akiyoshiensis (Le et al., 1996 ).

If AVG biosynthesis in Streptomyces follows a route equivalent to that of rhizobitoxine formation in Bur. andropogonis, homoserine would constitute an essential intermediate (Mitchell & Coddington, 1991 ), thus suggesting that the route should branch somewhere from the threonine biosynthetic pathway (Fig. 1). Furthermore, vinylglycine has been described as substrate for TS in the formation of threonine (Laber et al., 1994 ).

The levels of AVG displayed by the thrC mutant suggest that threonine is not a direct precursor for AVG in Streptomyces sp. 5331 since disruption of the thrC gene should have resulted in the absence of AVG formation as compared to the wild-type. The branching should, therefore, lie earlier in the threonine route.

The availability of the threonine genes should prove useful in the future in elucidating the AVG biosynthetic route in Streptomyces sp. NRRL 5331, and therefore establish the basis for a rational manipulation of AVG production.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the European Union (FAIR-CT97-3140) and a generic project from the Agencia de Desarrollo Económico of the Junta de Castilla y León to INBIOTEC (08-2/99/LE/0001). M.F., Y.C. and E.R. received fellowships from the University of León, Junta de Castilla y León and Diputación de León, respectively. We thank M. Corrales for the preparation of the manuscript, and the excellent technical assistance of J. Merino and B. Martín.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Amador, E., Castro, J. M., Correia, A. & Martín, J. F. (1999). Structure and organization of the rrnD operon of ‘Brevibacterium lactofermentum’: analysis of the 16S rRNA gene. Microbiology 145, 915-924.[Abstract]

Aparicio, J. F., Fouces, R., Mendes, M. V., Olivera, N. & Martín, J. F. (2000). A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol 7, 895-905.[Medline]

Bao, K., Zhou, X., Kieser, T. & Deng, Z. (1997). pHZ1351, a broad host-range plasmid vector useful for gene cloning and for gene replacement in Streptomyces hygroscopicus KMP3. In Abstracts of the 11th International Symposium on Biology of Actinomycetes, Beijing. Beijing: Chinese Society for Microbiology.

Barry, C. S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, A. J. & Grierson, D. (1996). Differential expression of the 1-aminocyclopropane-1-carboxylate oxidase gene family of tomato. Plant J 9, 525-535.[Medline]

Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. & Schaller, H. (1982). Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19, 327-336.[Medline]

Clepet, C., Borne, F., Krishnapillai, V., Baird, C., Patte, J. C. & Cami, B. (1992). Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes. Mol Microbiol 6, 3109-3119.[Medline]

Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[Medline]

Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]

Feng, L. & Kirsch, J. F. (2000). L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase. Biochemistry 39, 2436-2444.[Medline]

Gehring, H., Rando, R. R. & Christen, P. (1977). Active-site labeling of aspartate aminotransferases by the beta, gamma-unsaturated amino acid vinylglycine. Biochemistry 16, 4832-4836.[Medline]

Giovanelli, J., Owens, L. D. & Mudd, S. H. (1971). Mechanism of inhibition of spinach ß-cystathionase by rhizobitoxine. Biochim Biophys Acta 227, 671-684.[Medline]

Goodwin, T. W. & Mercer, E. I. (1983). Introduction to Plant Biochemistry. New York: Pergamon.

Have, A. T. & Woltering, E. J. (1997). Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence. Plant Mol Biol 34, 89-97.[Medline]

Hernando-Rico, V., Martín, J. F., Santamarta, I. & Liras, P. (2001). Structure of the ask–asd operon and formation of aspartokinase subunits in the cephamycin producer ‘Amycolatopsis lactamdurans’. Microbiology 147, 1547-1555.[Abstract/Free Full Text]

Hopwood, D. A. (1967). Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriol Rev 31, 373-403.[Medline]

Hoshiko, S., Makabe, O., Nojiri, C., Katsumata, K., Satoh, E. & Nagaoka, K. (1987). Molecular cloning and characterisation of the Streptomyces hygroscopicus alpha-amylase gene. J Bacteriol 169, 1029-1036.[Medline]

Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. (2000). Practical Streptomyces Genetics. Norwich: John Innes Foundation.

Komatsubara, S., Kisumi, M. & Chibata, I. (1979). Transductional construction of a threonine production strain of Serratia marcescens. Appl Environ Microbiol 38, 1045-1051.[Medline]

Laber, B., Gerbling, K-P., Harde, C., Neff, K.-H., Nordhoff, E. & Pohlenz, H-D. (1994). Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors. Biochemistry 33, 3413-3423.[Medline]

La Favre, J. S. & Eaglesham, A. R. J. (1986). Rhizobitoxine: a phytotoxin of unknown function which is commonly produced by bradyrhizobia. Plant Soil 92, 443-452.

Le, Y., He, J. & Vining, L. C. (1996). Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids. Microbiology 142, 791-798.[Abstract]

Madsen, S. M., Albrechtsen, B., Hansen, E. B. & Israelsen, H. (1996). Cloning and transcriptional analysis of two threonine biosynthesis genes from Lactococcus lactis MB1614. J Bacteriol 178, 3689-3694.[Abstract]

Malumbres, M. & Martín, J. F. (1996). Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: redirecting carbon flow. FEMS Microbiol Lett 143, 103-114.[Medline]

Malumbres, M., Mateos, L. M., Lumbreras, M. A., Guerrero, C. & Martín, J. F. (1994). Analysis and expression of the thrC gene of Brevibacterium lactofermentum and characterization of the encoded threonine synthase. Appl Environ Microbiol 60, 2209-2219.[Abstract]

Malumbres, M., Mateos, L. M., Guerrero, C. & Martín, J. F. (1995). Molecular cloning of the hom-thrC-thrB cluster from Bacillus sp. ULM1: expression of the thrC gene in Escherichia coli and corynebacteria, and evolutionary relationships of the threonine genes. Folia Microbiol 40, 595-606.

Marchenko, G. N., Marchenko, N. D., Tsygankov, Y. D. & Chistoserdov, A. Y. (1999). Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus. Microbiology 145, 3273-3282.[Abstract/Free Full Text]

Mitchell, R. E. & Coddington, J. M. (1991). Biosynthetic pathway to rhizobitoxine in Pseudomonas andropogonis. Phytochemistry 30, 1809-1814.

Mitchell, R. E. & Frey, E. J. (1988). Rhizobitoxine and hydroxythreonine production by Pseudomonas andropogonis strains, and the implications to plant disease. Physiol Mol Plant Pathol 32, 335-341.

Motoyama, H., Maki, K., Anazawa, H., Ishino, S. & Teshiba, S. (1994). Cloning and nucleotide sequence of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the Gram-negative obligate methylotroph Methylobacillus glycogenes. Appl Environ Microbiol 60, 111-119.[Abstract]

Owens, L. D., Lieberman, M. & Kunishi, A. (1971). Inhibition of ethylene production by rhizobitoxine. Plant Physiol 48, 1-4.

Parsot, C. (1986). Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase. EMBO J 5, 3013-3019.[Abstract]

Parsot, C. & Cohen, G. N. (1988). Cloning and nucleotide sequence of the Bacillus subtilis hom gene encoding homoserine dehydrogenase: structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II. J Biol Chem 263, 14654-14660.[Abstract/Free Full Text]

Peoples, O. P., Liebl, W., Bodis, M., Maeng, P. J., Folletie, J. T., Archer, J. A. & Sinskey, A. J. (1988). Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon. Mol Microbiol 2, 63-72.[Medline]

Rando, R. R. (1974). Irreversible inhibition of aspartate aminotransferase by 2-amino-3-butenoic acid. Biochemistry 13, 3859-3863.[Medline]

Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. A. (1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, 77-96.[Medline]

Ruan, X., Zhang, C. & Peters, K. (1993). Bradyrhizobium japonicum rhizobitoxine genes and putative enzyme functions: expression requires a translational frameshift. Proc Natl Acad Sci USA 90, 2641-2645.[Abstract]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]

Satoh, S. & Yang, S. F. (1989). Inactivation of 1-aminocyclopropane-1-carboxylate synthase by L-vinylglycine as related to the mechanism-based inactivation of the enzyme by S-adenosyl-L-methionine. Plant Physiol 91, 1036-1039.

Soper, T. S., Manning, J. M., Marcotte, P. A. & Walsh, C. T. (1977). Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine. J Biol Chem 252, 1571-1575.[Abstract]

Théze, J. & Saint-Girons, I. (1974). Threonine locus of Escherichia coli K12: genetic structure and evidence for an operon. J Bacteriol 118, 990-998.[Medline]

Thomas, D., Barbey, R. & Surdin-Kerjan, Y. (1993). Evolutionary relationships between yeast and bacterial homoserine dehydrogenases. FEBS Lett 323, 289-293.[Medline]

Tsay, Y. H. & Robinson, G. W. (1991). Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase. Mol Cell Biol 11, 620-631.[Medline]

Wierenga, R. K., Terpstra, P. & Hol, W. G. J. (1986). Prediction of the occurrence of the ADP-binding ß{alpha}ß-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187, 101-107.[Medline]

Wright, F. & Bibb, M. J. (1992). Codon usage in the G+C-rich Streptomyces genome. Gene 113, 55-65.[Medline]

Received 21 May 2001; revised 13 January 2002; accepted 29 January 2002.