INTRODUCTION

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The biosynthetic pathways of sulfur amino acids are well documented. Two alternative methionine biosynthetic pathways exist in microorganisms (Fig. 1). One, called the transsulfuration pathway, involves cystathionine formation, and the other bypasses cystathionine via direct sulfhydrylation of O-acylhomoserine to homocysteine (29).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Biosynthetic pathways of sulfur amino acids in E. coli (A) and S. cerevisiae (B). Enzyme steps: 1, O-succinylhomoserine transferase (metA); 1', O-acetylhomoserine transferase; 2, cystathionine -synthase (metB); 3, cystathionine -lyase (metC); 4, O-acetylhomoserine sulfhydrylase (Met17 [or Met25]); 5, cystathionine -synthase; 6, cystathionine -lyase. Genes shown in parentheses in this legend are the corresponding E. coli genes; for step 4 the S. cerevisiae gene is indicated.

In enteric bacteria, the sulfur atom is incorporated first into a serine ester (O-acetylserine) to yield cysteine (16). Sulfur is then transferred from cysteine to homocysteine via transsulfuration. In Escherichia coli, it requires the sequential action of cystathionine -synthase (EC 4.2.99.9), the product of the metB gene (7), and cystathionine -lyase (EC 4.4.1.8), the metC gene product (1), with the intermediary formation of cystathionine (Fig. 1A, steps 2 and 3).

The direct sulfhydrylation pathway has been reported to be the main pathway for homocysteine biosynthesis in Saccharomyces cerevisiae (5) and bacteria such as Brevibacterium flavum and Pseudomonas aeruginosa (10, 23). In S. cerevisiae, which is the best-studied example, the direct synthesis of homocysteine is catalyzed by an O-acetylhomoserine sulfhydrylase, the Met25 (or Met17) product (Fig. 1B, step 4) (5, 34). The resulting homocysteine is used as a direct precursor for methionine and is converted to cysteine via the reverse transsulfuration pathway (Fig. 1B, steps 5 and 6).

In addition, it should be kept in mind that the ester of homoserine used for homocysteine biosynthesis differs depending on the organisms: enteric bacteria use O-succinylhomoserine, while fungi and most gram-positive bacteria use O-acetylhomoserine (Fig. 1, steps 1 and 1') (for a review, see reference 33).

Little is presently known about the regulation of the metabolite flux of the methionine pathway. However, it has been reported that the control at the enzymatic level in bacteria and S. cerevisiae occurred at an early step of the methionine biosynthetic pathway. The first enzyme of the methionine biosynthetic pathway in E. coli, O-succinylhomoserine transferase, is feedback inhibited by methionine and S-adenosylmethionine (20), while the activity of O-acetylhomoserine transferase from S. cerevisiae is inhibited only by S-adenosylmethionine (6). In previous work, we demonstrated that O-acetylhomoserine transferase activity in Leptospira meyeri is not regulated by methionine and/or S-adenosylmethionine (2).

Our goal was to investigate the evolution of sulfur metabolism in L. meyeri. We report here the construction of a representative cosmid L. meyeri DNA library and the cloning of a biosynthetic gene, metY, which complements E. coli metB mutants. Analysis of the inferred L. meyeri MetY amino acid sequence, growth impairment of E. coli mutants carrying metY, and results of enzymatic assays allow us to propose a direct sulfhydrylation pathway catalyzed by an O-acetylhomoserine sulfhydrylase.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and media. L. meyeri serovar semaranga strain Veldrat S.173 (National Reference Center, Paris, France), isolated from a rat, was grown in EMJH medium at 30°C (8, 14).

Construction of an L. meyeri genomic DNA library in a cosmid. Total DNA from L. meyeri was prepared as described previously (11) from 500 ml of culture at 5 × 10⁸ bacteria/ml. Genomic DNA partially cleaved with restriction endonuclease Sau3A and sized on a 10 to 40% sucrose density gradient was ligated into the BamHI-digested and alkaline-phosphatase-treated cosmid vector pill₂₀₀ (1 µg). The ligated mix was packaged into phage lambda particles as described by the supplier (Gigapack III Gold 11 kit; Stratagene) and used to infect E. coli HB101 harboring helper plasmid pRK212.1 (9).

Complementation of E. coli methionine auxotrophs. Two different strategies were used for complementation of E. coli methionine auxotrophs: (i) direct infection of E. coli methionine auxotrophs with lambda phage particles from the cosmidic library at 30°C and (ii) mobilization of the recombinant cosmids as follows. Individual clones of the L. meyeri gene library (HB101 harboring the IncP helper plasmid plus the hybrid plasmid to be mobilized) were grown in LB broth at 30°C with kanamycin. Aliquots (2 µl) of each clone were transferred to L agar plates spotted with the recipient cells (2 µl of E. coli spontaneous Rif^r AB1932 and WA802 methionine auxotrophs).

DNA analysis, DNA sequencing, and computer analysis. Restriction endonucleases purchased from Boehringer Mannheim were used as described in the manufacturer's instructions. Calf intestinal alkaline phosphatase, T4 DNA ligase (high concentration), and the Wizard DNA cleanup system were purchased from Promega, Madison, Wis. TaqI DNA polymerase was from Cetus. Ligations, agarose gel electrophoresis, and electroporation were performed by standard procedures (27).

Overexpression of metY. A plasmid allowing expression of the metY structural gene under the control of the T7 promoter was constructed by oligonucleotide mutagenesis. The final construct (pETmetY), verified by nucleotide sequencing, contained the whole metY gene (starting at the ATG and continuing to the BamHI site located 44 bp beyond the metY stop codon) inserted into pET20b⁺ (Novagen, Madison, Wis.). The pETmetY plasmid was transformed into E. coli BL21 (DE3) (Novagen), which carries the T7 RNA polymerase gene on DE3 integrated on the chromosome of BL21. The conditions of overexpression of metY under the control of the T7 promoter were as described previously for overexpression of L. meyeri metX (2).

Enzymatic assays. O-Acetylhomoserine sulfhydrylase and O-acetylserine sulfhydrylase activities were assayed as described by Ravanel et al. (25). The amount of homocysteine or cysteine formed in a 0.1-ml reaction mixture was determined by the nitroprusside reaction (32) or by the procedure described by Kredich and Becker (17). Since MetY does not contain cysteine, dithiothreitol was omitted from the reaction mixture. The reaction was started by addition of 2 mM sodium sulfide (Na₂S). The reaction mixtures overlaid with 50 µl of paraffin oil were incubated at 30°C for 10 and 30 min. When aliphatic thiols were determined with the nitroprusside test, the incubation was stopped by 3 min of heating at 100°C instead of acid precipitation (which can cause formation of thiolactone from homocysteine at a lower pH). The assays were found to be reproducible.

	RESULTS

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Cloning of an L. meyeri DNA fragment able to complement E. coli metB and metA mutants. A cosmid library with 25- to 40-kbp inserts of L. meyeri DNA was obtained. Km^r clones were screened for complementation of the metB1 E. coli mutant (WA802) at 30°C. Seven recombinant cosmids were found in two separate experiments using either mobilization (1,152 clones) or direct infection of WA802 with transducing phage particles (5,760 clones). Several restriction fragments were common to the seven cosmids. Cosmid pb10 containing a 25-kbp insert was kept for further study and was shown to also complement E. coli EC972 carrying the metB185 allele. No major rearrangement had occurred during the cloning experiment since BamHI restriction fragments from cosmid pb10 were the same size as fragments of the genomic DNA of L. meyeri as determined by Southern blotting (data not shown).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   (A) Subcloning of L. meyeri DNA able to complement an E. coli metB mutant (WA802) and/or an E. coli metA mutant (AB1932). Large arrows indicate the orientations of the metX and metY genes. Names of the recombinant plasmids (or cosmid for pb10) are indicated on the left. Restriction site abbreviations: Ac, AccI; Ba, BamHI; Bg, BglII; Cfr, Cfr101; E, EcoRI; H, HindIII; P, PstI; Xb, XbaI. c8, c9, and s8 indicate the vectors pUC18, pUC19, and pSU21, respectively. ND, not done. (B) Cloning of the L. meyeri metX and metY genes under the control of the plac promoter (small arrows).

High similarity of MetY to O-acetylhomoserine sulfhydrylases. The determination of the sequence of a 2.8-kb PstI-XbaI insert of pb13s8 (Fig. 2A) demonstrated an open reading frame of 1,304 nucleotides encoding a 443-amino-acid protein. Two putative start codons, separated by 21 nucleotides, were found, and the first one was chosen conservatively. The amino acid sequence deduced from metY and analyzed by the BLAST program showed the following sequence identities: 55% with Aspergillus nidulans O-acetylhomoserine sulfhydrylase (28), 50% with yeast O-acetylhomoserine sulfhydrylase (Met17 or Met25), and 40.5% with P. aeruginosa O-succinylhomoserine sulfhydrylase (MetZ) (10, 19). Amino acid sequence alignments of these proteins indicated a high overall similarity (Fig. 3). E. coli cystathionine -synthase (MetB) (7) was less similar to MetY (30% identity). In this respect, the deduced MetY amino acid sequence showed a stronger similarity to the sequences of fungal transsulfuration enzymes than to those of the corresponding bacterial enzymes, MetB and MetZ, which, as already mentioned (10, 12), had a gap of about 40 amino acids in their middle portion, unlike the sequences of the yeast enzymes (see Fig. 3 for MetZ).

View larger version (65K): [in this window] [in a new window]   FIG. 3.   Alignment of L. meyeri serovar semaranga metY (MetYLm) deduced amino acid sequence with the O-acetylhomoserine sulfhydrylases of A. nidulans (OAHSAn; accession no. U19394) and S. cerevisiae (MT17Sc; accession no. P06106) and the O-succinyl homoserine sulfhydrylase of P. aeruginosa (MetZPa; accession no. U10904). Identical residues are represented by bold characters, and similar residues are indicated by dots. A hyphen indicates a gap. The alignment was performed by use of Clustal V. The proposed lysine (K) of the pyridoxal phosphate binding site is indicated by # above the sequences.

Evidence for direct sulfhydrylation in L. meyeri. E. coli metB mutants carrying the pb12 plasmid (metX metY) grew very slowly (generation time, about 24 h) and stopped growing at an optical density at 600 nm (OD₆₀₀) of 0.2 (Table 2). This slow growth could be due to a weak expression of the L. meyeri methionine genes in E. coli. To improve expression, L. meyeri metX and metY genes were cloned under the control of the lac promoter. pxc8 (metX) was obtained by cloning the AccI-HindIII fragment from pb12 into pUC18, and pyc9 (metY) was obtained by deletion of the PstI-Cfr101 insert of pb13c9 (Fig. 2B). However, the growth of metA and metB E. coli strains (AB1932 and WA802) harboring pxc8 and pyc9, respectively, was not improved.

MetY, an enzyme with O-acetylhomoserine sulfhydrylase activity. MetY was overproduced by cloning the metY gene under the control of a very strong and inducible T7 promoter. The molecular mass of the overexpressed MetY protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown) was 45 kDa, in agreement with that calculated from the sequence. Crude cellular extracts of E. coli BL21 (D23) transformed with pET20b⁺ bearing metY exhibited, after induction, two very high activities, O-acetylhomoserine sulfhydrylase (15 µmol of homocysteine/min/mg of protein) and O-acetylserine sulfhydrylase (13 µmol of cysteine/min/mg of protein). A control was made with crude extracts of the same strain transformed with the pET20b⁺ vector alone. No acetylhomoserine sulfhydrylase activity was found, whereas O-acetylserine sulfhydrylase activity was observed at a level similar to that found in the strain bearing metY. However, no cystathionine -synthase activity was found. The O-acetylhomoserine sulfhydrylase activity was specific for the acetyl substrate; succinylhomoserine was not used as a substrate.

	DISCUSSION

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The L. meyeri metY gene was cloned by functional complementation of an E. coli metB mutant. However, the results suggest that cystathionine is not an intermediary metabolite of methionine synthesis in L. meyeri. The data are consistent with the enzymatic activity exhibited in vitro by MetY. Indeed, MetY protein can utilize sulfide for reaction with O-acetylhomoserine to yield homocysteine (Fig. 1B, step 4). In contrast to E. coli MetB protein (Fig. 1A, step 2), MetY protein does not use cysteine as a substrate, indicating that MetY protein is devoid of cystathionine -synthase activity. It is clear that complementation of E. coli metB mutants by the L. meyeri metY gene, however poor, became possible by marginal use of E. coli substrates. In fact, two substrates used by MetY protein differ from those used in E. coli: the homoserine derivative is an O-acetyl derivative, and the source of sulfur is not cysteine. A previous study from our laboratory has also shown that the L. meyeri metX product, O-acetylhomoserine transferase, the enzyme catalyzing the step upstream of MetY (2), does not transfer the acetyl group of acetyl coenzyme A to serine, showing its exclusive specificity for homoserine.

In S. cerevisiae, the Met17 (or Met25) enzyme exhibits both O-acetylserine sulfhydrylase and O-acetylhomoserine sulfhydrylase activities in vitro (32). Unfortunately, it was not possible to determine if L. meyeri MetY had an O-acetylserine sulfhydrylase activity in vitro since the latter activity was also present in E. coli extracts (see Results). The synthesis of cysteine by MetY was thus experimentally tested in vivo by functional complementation of the double E. coli cysK cysM mutant (these two genes specify two isoenzymes with O-acetylserine sulfhydrylase activity) (16). However, no complementation was found (data not shown). This could suggest that L. meyeri MetY is indeed similar to S. cerevisiae Met17 (or Met25), which was found ultimately to behave in vivo only as an O-acetylhomoserine sulfhydrylase (5), and that MetY may also have evolved from the same common ancestor of the -family of the transsulfuration enzymes (24).

At this point, the key goal is to determine the physiological role of the O-acetylhomoserine sulfhydrylase activity of L. meyeri MetY and whether it represents a major or alternative pathway. The organization of the two genes metX and metY in an operon (2) suggests the participation of both genes in the methionine pathway. Since the complementation of an E. coli metB mutant by the L. meyeri metY gene was effective, we rather expected to isolate an L. meyeri gene encoding cystathionine -synthase. However, the L. meyeri inserts from the seven recombinant cosmids able to complement both metA and metB E. coli mutants overlapped, indicating that they originated from the same region of the chromosome. We thus propose that the transsulfuration pathway via cystathionine does not exist in L. meyeri; this is in contrast to the situation found for fungi, which have both operating pathways for methionine biosynthesis (transsulfuration and sulfhydrylation). The proposed pathway for methionine biosynthesis for L. meyeri is shown in Fig. 4. With regard to metabolic regulation, we have reported that MetX, the first enzyme of the L. meyeri pathway, is not feedback inhibited (2). The concentration of methionine and S-adenosylmethionine giving 67 and 75% inhibition of O-acetylhomoserine sulfhydrylase (MetY), respectively, was 10 mM. The methionine or S-adenosylmethionine inhibition of O-acetylhomoserine sulfhydrylase seems not to be physiologically significant. Further studies are needed to examine regulation at the level of repression by methionine or its metabolites.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Proposed pathway for methionine biosynthesis in L. meyeri. MetX, O-acetylhomoserine transferase; MetY, O-acetylhomoserine sulfhydrylase.

It was of interest to compare the methionine biosynthetic pathway in a pathogenic species of Leptospira to that in the saprophytic L. meyeri species. A 525-bp DNA fragment from Leptospira interrogans serovar icterohaemorrhagiae strain Verdun was amplified by PCR assay with appropriate oligonucleotides chosen within metY (data not shown). The deduced amino acid sequence of the amplified product (Fig. 3, amino acids 110 to 285) was 86% identical to the amino acid sequence of MetY. Interestingly, the large insertion of 40 amino acids characteristic of MetY protein (Fig. 3, amino acids 234 to 276) and of yeast enzymes was found in the corresponding amino acid sequence of this pathogenic species. These results could suggest that the direct sulfhydrylation pathway for methionine is also operating in a pathogenic species of Leptospira.