* Plant Science Laboratory, Migal Galilee Technology Center, Kiryat-Shmona, Israel
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
Correspondence: E-mail: rachel{at}migal.org.il.
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Abstract |
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Key Words: amino acid methionine biosynthesis pathway cystathionine -synthase O-acetylhomoserine sulfhydrylase substrate specificity evolution
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Introduction |
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The methionine biosynthesis pathways differ also at the point where threonine and methionine biosynthesis pathways branch. In plants, this branch point occurs at OPH, which is used as a substrate for both threonine and cystathionine biosynthesis (fig. 1C). In bacteria and fungi, these pathways diverge from homoserine (one metabolite upstream of OPH). In these latter groups, OPH is used solely for threonine biosynthesis (fig. 1A and B).
To elucidate the evolutionary processes, which led to different types of methionine biosynthesis in various organisms, we analyzed the substrate specificity of three enzymes that utilize different homoserine-esterified substrates in vivo. We analyzed two CGSs, the first from Escherichia coli that utilizes OSH and the second from Arabidopsis thaliana that utilizes OPH. Both of them take part in the transsulfuration pathway. The third enzyme studied was OAH-sulfhydrylase of Leptospira meyeri, representing the enzyme that utilizes OAH and acts through direct sulfhydrylation. It was found that these three enzymes could utilize OAH and OSH in vivo, although at different levels of efficiency, but only the plant enzyme could utilize OPH. In addition, the two CGSs were revealed to be bifunctional enzymes, which can participate in the direct sulfhydrylation pathway, as well as in their inherent transsulfuration pathway.
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Materials and Methods |
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Bacterial Strains
The bacteria strains used in this study are listed in table 1. The double and triple mutations of E. coli were prepared by P1 transduction (Miller 1972). To prepare a double mutant of metB-thrB (YBB1), the thrB gene was transferred by P1 transduction from Hfr 3000 YA73, after lysogenizing the latter with CAG12093 (carB96:Tn10) (the car gene is closest to the threonine operon in the E. coli genome). This enabled the use of tetracycline selection for subsequent thrB:Tn10 transductions. The thrB:Tn10 was introduced into LE392 to form the YBB1 mutant. The nature of the YBB1 cells was tested on minimal medium, lacking threonine and methionine.
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Two different media were used to grow the E. coli bacterial strains. The first was Luria-Bertani (LB), and the second was the M9 minimal medium (Sambrook, Fritsch, and Maniatis 1989). The latter medium was supplemented with 1 mg/ml thiamine and 40 mg/ml of each amino acid, with the exception of threonine and methionine. For positive control, methionine and threonine were added at a concentration similar to the other amino acids. The antibiotics used were ampicillin, 100 µg/ml; chloramphenicol, 25 µg/ml; and tetracycline, 12.5 µg/ml.
Plasmid Construction
For the complementation tests, three different genes, the MetB gene of E. coli, the AtCGS gene of A. thaliana, and the MetY gene of L. meyeri, were subcloned into the plasmid, pACYC184 (Cmr Tetr) at SphI and SalI sites, canceling the tetracycline resistance of this plasmid. However, the use of these sites enabled the alien genes to be controlled by the promoter of the tetracycline resistance gene. The forward primers contained the Shine-Delgarno sequences, derived from the expression vector pQE (Qiagen), which also included the SphI restriction site, containing the ATG translation-initiation codon. A SalI site was inserted into the reverse primer.
Arabidopsis CGS cDNA was PCR amplified from a flower cDNA library, kindly donated by the Arabidopsis Biological Resource Center (Columbus, Ohio). Fragments of DNA, encoding mature CGS (without its plastid transit peptide), starting with Val-68 (Ravanel et al. 1998), were amplified with the following primers: 5'-AGCATGCAAAGAGGAGAACTATGGT CCGTCAGCTGAGCATTAAAGCC-3' and 5'-ATCAGATGGCTTCGAGAG CTTGA-AGAA-3'. MetB was amplified from E. coli DNA with the primers: 5'-AAAAGAGGAGAACTATGACGCGTAAACAGGCCACC-3' and 5'-ATTACCCCTTGTTTGCAGC CCGG-3'. The MetY of L. meyeri was amplified from the pb12 plasmid, kindly donated by Isabel Saint-Girons, using the following primers: 5'-AGCATGCAAAGAGGAGAACT ATGGTAGGACCATCGGGGGAATC-3' and 5'-ATCAGATATTTTTTAATG CCTCTTC-3'. The amplified fragments were ligated to a pGEMT-cloning vector (Promega), and subsequently into pACYC184, via SphI/SalI sites. The nucleotide sequences of the constructed plasmids were verified by DNA sequencing. MetX of L. meyeri on pET20b+ plasmid was kindly provided by Isabel Saint-Girons. This plasmid possesses gene resistance to ampicillin (Bourhy et al. 1997).
Complementation of E. coli Methionine Auxotrophs
pACYC-184 carrying the three different genes was employed to transform the various E. coli mutants. The transformed bacteria were plated on LB medium and grown overnight at 37°C. Colonies were picked and grown in LB broth to an optical density OD600 of 1.0. One milliliter of the bacteria was pelleted and washed twice with M9 medium containing 0.2% glucose. The bacteria were plated on an M9 plate or grown in M9 liquid medium.
Sequence Analysis
Protein sequences of the organisms listed in table 2 were aligned, using ClustalX (Thompson et al. 1997), a Windows application based on ClustalW (Higgins et al. 1996). Phylogenetic trees were reconstructed by quartet-puzzling maximum likelihood using Tree-Puzzle, version 5.0 (Strimmer and von Haeseler 1996), with 10,000 puzzling steps, applying quartet sampling for substitution process and neighbor-joining tree for rate variation determination. Trees were visualized using the program TreeView (Page 1996).
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Results |
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The Ability of the Enzymes to Use OAH as a Substrate
The ability of the A. thaliana and E. coli CGSs to use OAH was tested by complementing the triple mutant metA, metB, and thrB (YABB1) with the genes encoding each of these enzymes separately. In this mutant, neither OPH nor OSH could be produced, and the mutant also lacked the E. coli CGS, which may utilize OAH as a substrate. OAH that was synthetically synthesized was added to M9 liquid medium (40 mg/ml). OAH-sulfhydrylase of L. meyeri was used as a positive control. As shown in figure 3, although the control plasmid vector did not support mutant growth, the plant and the bacterial CGSs efficiently complemented this mutant, demonstrating that they are able to utilize OAH.
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The Ability of the Enzymes to Use OPH as a Substrate
OPH serves as a substrate for plant CGS enzymes (fig. 1C). Although OPH exists in bacteria in the threonine biosynthesis pathway, the E. coli CGS cannot utilize it for methionine biosynthesis in vivo, as deduced from the methionine auxotrophy of the metA mutant (Michaeli and Ron 1981). To ascertain whether the OAH-sulfhydrylase encoded by the MetY gene can use OPH as a substrate, the thrC-metA (YAC1) double mutant was formed. In this mutant, OSH was not produced, and OPH was expected to accumulate, since it could not be converted into threonine because of a mutation in the thrC gene. The YAC1 mutant was transformed with the recombinant MetY gene, as well as with the Arabidopsis CGS gene, as a positive control. Whereas the AtCGS complemented this mutant, the MetY gene did not (fig. 4A and B). This implies that only the plant enzyme can use OPH for methionine synthesis.
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Evolution of the Transsulfuration and the Direct Sulfhydrylation Pathways
To learn more about the evolutionary history of the methionine biosynthesis pathway, a phylogenic tree of amino acid sequences of CGS, OAH, or OSH sulfhydrylase enzymes of various bacteria, fungi, and plants was constructed, using quartet-puzzling maximum likelihood. The same tree topology was also observed when the neighbor-joining method was applied for tree reconstruction.
Four major groups could be distinguished with high internal branch support values (fig. 6). The first group consisted of CGSs of plants that naturally utilize OPH and cysteine and act through transsulfuration. The second group contained CGSs of bacteria that intrinsically use OSH (Escherichia or Salmonella) or OAH (Bacillus, Helicobacter, or Corynobacterium) through transsulfuration. The third group contained enzymes of bacteria that are active in the direct sulfhydration pathway and use OSH as a substrate. The fourth group included enzymes of bacteria and fungi that function through direct sulfhydration but utilize OAH. These two latter groups appear to share a common ancestor, which most probably functioned though direct sulfhydration.
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Discussion |
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Although, in general, gram-negative bacteria produce OSH, whereas gram-positive bacteria produce OAH, this division is not unambiguous. The results obtained from two different species of Pseudomonas demonstrated, for example, that they use different homoserine-esterified substrates: P. syringae produces OAH and P. aeruginosa produces OSH, although they belong to the same genus (Andersen, Beattie, and Lindow 1998). Phylogenic analysis (fig. 6) could not discriminate between bacterial CGS homologs that use OAH (Bacillus and Helicobacter) and those that utilize OSH (Salmonella and E. coli). However, the two branches of direct sulfhydrylation clustered according to the different homoserine-esterified substrates, OAH or OSH (fig. 6). The plant CGS homologs that utilize OPH were grouped in a specific cluster, which is distant from the other branches of the tree. This may reflect its substrate specificity (OPH) and the fact that the plants enzymes possess N-terminal regulatory region that is absent from bacterial and fungal enzymes (Hacham, Avraham, and Amir 2002).
The ability of the enzymes to use different homoserine-esterified substrates was accompanied by their ability to utilize different sulfur sources. CGS enzymes of Arabidopsis and E. coli, which can utilize several homoserine-esterified substrates, can also substitute cysteine for sulfide as a sulfur source in methionine synthesis and thus acts also as sulfhydrylase. In vitro tests with bacterial and plant enzymes have shown that they can use various sulfur and thiol compounds for methionine synthesis (Kanzaki et al. 1987; Ravanel et al. 1998).
Although the CGS enzymes have the ability to act through transsulfuration as well as through direct sulfhydrylation, recently available genome sequence data indicate that some bacteria and fungi possess separate independent loci for these two different routes. Thus, both CGS and OAH or OSH sulfhydrylase are found in these organisms. This has been described, for example, in Niesseria meningitidis and Corynebacterium glutamicum (Hwang et al. 1999), in Mycobacterium tuberculosis, and in the yeast S. cervicia (Marzluf 1997). However, it is not clear whether these two different pathways are physiologically active or whether they are involved in methionine synthesis in these organisms. Genetic and biochemical evidence shows that in C. glutamicum, the transsulfuration and the direct sulfhydrylation pathways are utilized with almost equal efficiency (Hwang et al. 2002). However, in yeast, it was shown that direct sulfhydrylation is the active route (Marzluf 1997). Complementation tests and activity measurements demonstrated that P. aeruginosa and P. putida are capable of synthesizing methionine by OSH or OAH sulfhydrylase and CGS activities, although the direct sulfhydrylation pathway is strongly favored (Foglino et al. 1995; Vermeij and Kertesz 1999). In both bacteria, the transsulfuration pathway is highly expressed when cysteine is supplied as the sole sulfur source (Vermeij and Kertesz 1999).
The direct sulfhydrylation pathway probably exists in all species of plant, bacteria, and fungi. In some bacteria and fungi, only OAH or OSH sulfhydrylase are present. In other organisms, sulfhydrylase activity is performed by CGS (i.e., CGS has the ability to act also as a sulfhydrylase). This is the case, for example, in plants (Ravanel et al. 1998), in B. subtilis (Auger et al. 2002), and in E. coli (Simon and Hong 1983). The E. coli enzyme can act through transsulfuration and direct sulfhydrylation with nearly the same efficiency, as was demonstrated in this study. In many other bacteria and fungi, the two pathways are separate and the organisms show both CGS and sulfhydrylase activities, performed by independent enzymes.
Based on the phylogenic tree and the results obtained from the complementation tests, we conclude that the ancestral gene encoded an enzyme that had the OAH /OSH sulfhydrylase activities. It is likely that early organisms relied exclusively on the sulfhydrylase activity and that the ancestral protein evolved by gradual mutations, which resulted in increasingly efficient utilization of one of the homoserine-esterified substrates. Thus, although one homoserine-esterified substrate was preferred, the alternative source could still be utilized, albeit less efficiently. In other organisms, the ancestral gene evolved to form the CGS. Although CGS uses cysteine as a sulfur source for homocysteine synthesis, it is also able to utilize sulfide and to act as sulfhydrylase. Thus, the CGS maintains the substrate flexibility of its progenitor. In some cases, the CGS underwent duplication, as suggested for B. subtilis. Two CGS paralogs were found in this bacterium, although the function of one of them (yrhB) remains unclear (Auger et al. 2002). In some bacteria and fungi, which have both CGS and an independent sulfhydrylase enzyme, the ancestral gene had probably been duplicated, and subsequent mutations gradually produced the activities of the present-day enzymes. The duplication is likely to have occurred early in evolution, since these two paralogs (CGS and OAH or OSH sulfhydrylase) now cluster in different branches of the phylogenic tree (fig. 6). The plant CGS homologs may have develop from the bacterial CGS, but their homoserine esterified substrate changed to OPH, although they can still use the other homoserine-esterified substrates as well as sulfide. Nonetheless, direct sulfydrylation apparently works at low efficiency in plants, as shown for Arbidopsis in this study, and by a tobacco mutant that lacks the cystathionine ß-lyase (i.e., lacks the transsulfuration pathway). The latter mutant is auxotrophic to either methionine or homocysteine (Negrutiu et al. 1985).
The evolutionary rationale behind the different organisms' choice of various routes for methionine synthesis, including the sulfur sources and the homoserine-esterified substrates, is probably linked to their interior metabolic network. This may be dependent on their natural habitat specialization, and further study is warranted to clarify this point.
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Acknowledgements |
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Footnotes |
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