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Nucleic Acids Res. 2004; 32(11): 3340–3353.
Published online Jun 23, 2004. doi:  10.1093/nar/gkh659
PMCID: PMC443535

Comparative genomics of the methionine metabolism in Gram-positive bacteria: a variety of regulatory systems


Regulation of the methionine biosynthesis and transport genes in bacteria is rather diverse and involves two RNA-level regulatory systems and at least three DNA-level systems. In particular, the methionine metabolism in Gram-positive bacteria was known to be controlled by the S-box and T-box mechanisms, both acting on the level of premature termination of transcription. Using comparative analysis of genes, operons and regulatory elements, we described the methionine metabolic pathway and the methionine regulons in available genomes of Gram-positive bacteria. A large number of methionine-specific RNA elements were identified. S-boxes were shown to be widely distributed in Bacillales and Clostridia, whereas methionine-specific T-boxes occurred mostly in Lactobacillales. A candidate binding signal (MET-box) for a hypothetical methionine regulator, possibly MtaR, was identified in Streptococcaceae, the only family in the Bacillus/Clostridium group of Gram-positive bacteria having neither S-boxes, nor methionine-specific T-boxes. Positional analysis of methionine-specific regulatory sites complemented by genome context analysis lead to identification of new members of the methionine regulon, both enzymes and transporters, and reconstruction of the methionine metabolism in various bacterial genomes. In particular, we found candidate transporters for methionine (MetT) and methylthioribose (MtnABC), as well as new enzymes forming the S-adenosylmethionine recycling pathway. Methionine biosynthetic enzymes in various bacterial species are quite variable. In particular, Oceanobacillus iheyensis possibly uses a homolog of the betaine–homocysteine methyltransferase bhmT gene from vertebrates to substitute missing bacterial-type methionine synthases.


Sulfur-containing amino acid methionine is synthesized de novo by most microorganisms and plants after the initial steps of inorganic sulfate assimilation and synthesis of cysteine or homocysteine (1). The fact that methionine is the universal N-terminal amino acid of proteins as well as the use of its derivative S-adenosylmethionine (SAM) in a variety of methyltransferase reactions argue for the importance of methionine in the cellular metabolism. There are two alternative pathways of methionine synthesis in microorganisms. The transsulfuration pathway of Escherichia coli involves cystathionine as an intermediate and utilizes cysteine as the sulfur source (2). In contrast, the direct sulfhydrylation pathway found in Saccharomyces cerevisiae (3), Leptospira meyeri (4) and Corynebacterium glutamicum (5) bypasses cystathionine and uses inorganic sulfur instead. While most microorganisms synthesize methionine via either one of these pathways, C.glutamicum utilizes both pathways (6).

Biosynthesis of methionine starts at homoserine, which is the common precursor for amino acids of the aspartate family, isoleucine, threonine and methionine (Figure (Figure1).1). Homoserine is derived from aspartate semialdehyde by the hom gene product. Acylation of homoserine is catalyzed by homoserine acetyltransferase MetB in Bacillus subtilis (7) and by homoserine succinyltransferase MetA in E.coli (8), and these two enzymes are homologous. L.meyeri uses homoserine acetyltransferase MetX not related to the above enzymes (9). In the transsulfuration pathway, homocysteine is formed from O-acetylhomoserine or O-succinylhomoserine and cysteine in two steps, catalyzed by cystathionine γ-synthase MetI and cystathionine β-lyase MetC in B.subtilis (10). In the alternative pathway, O-acetylhomoserine is directly converted to homocysteine by O-acetylhomoserine sulfhydrylase MetY, utilizing sulfide as the sulfur donor (11).

Figure 1
The methionine metabolic pathway in Gram-positive bacteria. The common gene names from B.subtilis, E.coli and Leptospira interrogans are used. Genes whose function was assigned in this study are indicated by asterisks. Different parts of the pathway ...

Unlike the transsulfuration/sulfhydrylation enzymes that are present only in organisms with de novo methionine synthesis, methionine synthase is required by all organisms to ensure regeneration of the methyl group of SAM (1). Two types of methionine synthases can perform this function in E.coli. Reaction catalyzed by B12-dependent protein MetH with coenzyme B12 as a cofactor is more than 100-fold faster than the reaction catalyzed by B12-independent isoenzyme, MetE (8). In enterobacteria, the methyl group of methionine is donated by methyl-tetrahydrofolate (methyl-THF). The latter is formed by reduction of methylene-THF in a reaction catalyzed by the metF gene product. B.subtilis has only the B12-independent methionine synthase (formerly named MetC) and an ortholog of MetF, encoded by yitJ, that contains an extra N-terminal domain highly similar to the homocysteine-binding domain of MetH (12).

E.coli is able to uptake methionine by a high-affinity transport system encoded by the metD locus (8). Recently it was shown that this methionine transporter and its orthologs in various bacteria (yusCBA in B.subtlis, renamed metNPQ) constitute a new family of the ABC superfamily (1316). No other methionine transporters have been identified in bacteria so far.

S-adenosylmethionine synthase, encoded by the metK gene, is responsible for formation of SAM from methionine and ATP. SAM is essential for a large number of methylation processes and is also used for modification of rRNA nucleotides and polyamine synthesis (17). The main product of the transmethylation reaction is S-adenosylhomocysteine (SAH). In E.coli, this molecule seems to be recycled to homocysteine in two steps: a nucleosidase encoded by the pfs gene (mtn in B.subtilis) hydrolyzes SAH into adenine and S-ribosylhomocysteine, which is then cleaved by a specific hydrolase to form homocysteine (18,19). Homocysteine can then be metabolized to synthesize cysteine and methionine. Notably, some organisms are able to synthesize cysteine from homocysteine via the reverse transsulfuration pathway (3,20). The cystathionine β-synthase and γ-lyase activities required for this pathway are possibly encoded by the yrhA and yrhB genes in B.subtilis (12,21). Methylthioadenosine is produced by the polyamine synthesis pathway and then hydrolyzed by a nucleosidase (encoded by mtn in B.subtilis), yielding methylthioribose (19). The latter is efficiently recycled in B.subtilis via the methionine salvage pathway encoded by the mtnKSUVWXYZ gene cluster (2123).

Not methionine, but SAM, a major constituent of the intermediary metabolism, is the regulatory molecule for the methionine biosynthesis both in E.coli and B.subtilis. However, the mechanisms of SAM-dependent regulation differ in these bacteria. SAM-responsive repressor MetJ binds to tandemly repeated MET box sequences (5′-AGACGTCT-3′) and represses transcription of most met genes in E.coli (8). In B.subtilis, these genes are regulated by attenuation of transcription using a highly conserved regulatory leader sequence, the S-box (24,25). SAM directly and specifically binds to this RNA structural element, causing formation of the downstream terminator hairpin and subsequent premature termination of transcription (2628). In the absence of effector molecules, formation of a more energetically favorable antiterminator, which is alternative both to the S-box domain and the terminator structure, leads to transcription read-through. The S-box domain and the terminator hairpin fold independently, while the antiterminator structure consists of several conserved helices of the S-box and a new stem–loop which is alternative to the terminator and the base stem of the S-box. Moreover, Gram-positive bacteria have another control system involving premature transcription termination (T-box), which regulates expression of various aminoacyl-tRNA synthetases and genes involved in the amino acid biosynthesis (25,29,30). The T-box sequence is able to bind uncharged tRNA, promoting formation of the antiterminator. The major role in regulation is played by the T-box ‘specifier codon’, which interacts with the anticodon of an uncharged tRNA. As the position of this regulatory codon in the T-box structure is fixed, one can predict the amino acid specificity of the regulatory signal (31). For example, the presence of a T-box with an isoleucine specifier codon upstream of ileS in Thermoanaerobacter tengcongensis argues against a recently published theory that this gene is mis-annotated and encodes a methionyl-tRNA synthase (23).

Comparative genomics is a powerful technique for reconstruction of metabolic pathways and their DNA- or RNA-level regulation in bacteria (3237). We analyzed the methionine pathway and its regulation in various Gram-positive bacteria. We extended the S-box and methionine-specific T-box regulons, that appear to be widely distributed in bacilli, clostridia and lactobacilli, identified a new regulatory signal for methionine biosynthetic and transport genes in streptococci, reconstructed the likely evolutionary scenario for methionine regulons of Gram-positive bacteria, and described the possible mechanism of dual regulation of the reverse transsulfuration pathway by cysteine and SAM in Clostridium acetobutylicum via RNA regulatory structures and antisense RNA. After reconstruction of the methionine metabolic pathways and regulatory interactions in Gram-positive bacteria, we identified several new enzymes and transporters involved in the methionine metabolism. In particular, we identified a set of candidate genes forming the complete SAM-recycling pathway.


Complete and partial sequences of bacterial genomes were downloaded from GenBank (38). Preliminary sequence data were obtained also from the websites of The Institute for Genomic Research (http://www.tigr.org), University of Oklahoma's Advanced Center for Genome Technology (http://www.genome.ou.edu), the Sanger Centre (http://www.sanger.ac.uk), the DOE Joint Genome Institute (http://www.jgi.doe.gov) and the ERGO Database, Integrated Genomics, Inc. (39).

The conserved secondary structure of the S-box and T-box leaders was derived using the RNAMultAln program (A. A. Mironov, in preparation). This heuristic program simultaneously creates a multiple alignment and a conserved secondary structure for a set of RNA sequences using positional relationship of conserved sequence boxes and paired regions of candidate helices. This program is not based on the energy minimization, but rather uses simple heuristic rules of base pairing and analysis of complementary substitutions. The RNA-PATTERN program (40) was used to search for new S-boxes and methionine-specific T-boxes in bacterial genomes. The input RNA pattern described the RNA secondary structure and the sequence consensus motifs as a set of the following parameters: the number of helices, the length of each helix, the loop lengths and description of the topology of helix pairs. The latter is defined by coordinates of helices. For instance, two helices may be either independent or embedded helices, or else they could form a pseudoknot structure. This definition is similar to an approach implemented in the Palingol algorithm (41). Free energy of the S-box structures were calculated using Zuker's algorithm of free energy minimization (42,43) implemented in the Mfold program (http://bioinfo.math.rpi.edu/~mfold/rna). RNA secondary structure of the antiterminator/antisequestor conformation, which includes parts of the S-box and overlaps with the terminator/sequester hairpin (Figure (Figure7A),7A), was predicted using Mfold with the input including the S-box sequence extended downstream as far as the center of the terminator/sequestor hairpin. This structure is more energetically favorable than S-box in the absence of ligand. To model the effect of ligand binding that stabilizes the S-box, formation of the terminator/sequester hairpins was modeled with input sequence starting immediately downstream of S-box.

Figure 7
Regulation of the methionine biosynthesis and transport genes in bacteria by S-box riboswitch (A), T-box structure (B), unknown transcriptional regulator binding the MET-box operator (C), activator MetR (D) and repressor MetJ (E). The mechanisms of the ...

A simple iterative procedure implemented in the program SignalX was used for construction of the MET-box profile from a set of upstream gene fragments (44). Weak palindromes were selected in each region. Each palindrome was compared to all other palindromes, and the palindromes most similar to the initial one were used to make a profile. The positional nucleotide weights in this profile were defined as

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where N(b, k) was the count of nucleotide b in position k (45). The candidate site score was defined as the sum of the respective positional nucleotide weights:

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where k was the length of the site. Z-score can be used to assess the significance of an individual site.

These profiles are used to scan the set of palindromes again, and the procedure was iterated until convergence. Thus a set of profiles is constructed. The quality of a profile was defined as its information content (46)

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where f(i, k) is the frequency of nucleotide i in position k of sites generating the profile. The best profile is used as the recognition rule.

Each genome was scanned with the profile, and genes with candidate regulatory sites in the upstream regions (in positions −325 to +25 relative to the translation start) were selected. The threshold for the site search was defined as the lowest score observed in the training set.

Protein similarity search was done using the Smith–Waterman algorithm implemented in the GenomeExplorer program (47). Orthologous proteins were initially defined by the best bidirectional hit criterion (48) and if necessary confirmed by construction of phylogenetic trees for the corresponding protein families. The phylogenetic trees of the methionine biosynthesis and transport proteins were constructed by the maximum likelihood method implemented in PHYLIP (49). Multiple sequence alignments were done using CLUSTALX (50). Transmembrane segments were predicted using TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). The COG (48), InterPro (51) and PFAM (52) databases were used to verify the protein functional and structural annotation.

Alignments of S-box and T-box sequences mentioned in this paper, as well as sequences of proteins whose functions were assigned here are available as supplementary Figures Figures1,1, ,22 and and3,3, respectively.

Figure 2
Conserved S-box structure. Capitals indicate invariant positions. Lower case letters indicate strongly conserved positions. Degenerate positions: R = A or G; Y = C or U; D = A, G or U; H = A, C or U; N = any nucleotide. ...
Figure 3
Phylogenetic tree of the substrate-binding components MetQ of bacterial methionine transporters from the MUT family. Proteins are denoted by genome abbreviations (listed in Table Table1).1). Multiple paralogs are numbered. Genes predicted to ...


Reconstruction of methionine regulons

Initially, orthologs of known methionine biosynthetic and transport genes (MET) were identified by similarity search in the genomes of all available Gram-positive bacteria (Table (Table1).1). For further analysis, positional clusters (including possible operons) of the MET genes were also described in this table. The hom gene for homoserine dehydrogenase shared by the methionine and threonine biosynthesis was considered only if it was co-localized or co-regulated with other MET genes.

Table 1.
Methionine biosynthesis and transport genes and methionine-specific regulatory elements in Gram-positive bacteria

S-box regulon

Then, we constructed the pattern of the S-box motif using the training set of 18 S-box leader regions (24), and scanned available genomic sequences using the RNA-PATTERN program. Multiple alignment of 100 S-box leaders from 23 bacterial genomes confirms high degree of conservation of the S-box primary and secondary structure (see supplementary Figure Figure1).1). Similar to other metabolite-binding RNA elements (32), the S-box motif has a set of helices (P2 to P5) closed by a single base stem (P1), and numerous highly conserved regions (Figure (Figure2).2). In fact, the regions of sequence conservation cover most of the S-box sequence, and therefore, may be involved in SAM binding or tertiary interactions. Only two stem–loops at the ends of the fourth and fifth helices are not conserved on the sequence level and have variable length. The end loop of helix P3 (5′-CnGG-3′) possibly forms a pseudoknot structure with the interior loop between helices P4 and P5 (5′-CCnG-3′). This possible pseudoknot interaction is confirmed by several compensatory substitutions and could be required for the formation of stable S-box tertiary structure. For instance, the S-box structure on the Bacillus cereus mtnW gene contains a possible pseudoknot (5′-CGAG-3′; 5′-CTCG-3′) with complementary substitutions in the conserved positions of both arms.

Among Gram-positive bacteria, the S-box motif is widely distributed in the orders Bacillales and Clostridia, but it has not been found in Lactobacillales, including Enterococcus, Streptococcus and Lactococcus species (Table (Table1).1). Positional analysis of genes possessing this regulatory motif has showed that the S-box regulon in Gram-positive bacteria contains most genes of the methionine biosynthesis and transport, as well as the SAM synthase gene metK. The S-box regulon is most extensive in three bacilli, B.subtilis, B.cereus and O.iheyensis, where it contains 11, 16 and 13 regulatory elements, respectively, and includes additional genes for the cysteine biosynthesis and methionine salvage pathways, as well as hypothetical genes. The detailed phylogenetic and positional analysis of the S-box-regulated genes in Gram-positive bacteria is given in the next section.

The S-box motif is not restricted to the Bacillus/Clostridium group of bacteria. In two actinobacteria, Streptomyces coelicolor and Thermobifida fusca, S-box precedes the hypothetical gene SCD95A.26. In addition, single S-box motif was found upstream of the metY-metX operon in the genomes of Chlorobium tepidum, Chloroflexus aurantiacus and Cytophaga hutchisonii, as well as upstream of the metNPQ operon encoding methionine transport system in Petrotoga miotherma. A more relaxed search in (28) identified candidate S-boxes also in the genomes of Fusobacterium nucleatum (upstream of the metK and metN genes), Deinococcus radiodurans (metH and metN), Xanthomonas campestris (metX) and Geobacter sulfurreducens (metB and metX). All other bacterial taxonomic groups with available genomic sequences seem to lack the S-box regulatory system.

Recent experiments (2628) demonstrated that SAM-dependent regulation of the methionine biosynthesis genes of B.subtilis involves attenuation of transcription using formation of alternative secondary structures in the S-box region. Here we tested whether the same regulatory mechanism could operate for all found S-boxes. Downstream of all S-boxes from the Bacillus/Clostridium group of bacteria we identified additional hairpins that are followed by runs of thymidines and therefore are candidate terminators of transcription. In addition, we observed complementary RNA regions that partially overlap both the proposed terminator and the base stem (helix P1) of the S-box. Thus, the same termination-antitermination S-box mechanism possibly operates in all Gram-positive bacteria.

Analysis of the upstream regions of the S-box-regulated genes from two actinobacteria reveals another possible mode of regulation. In this case the S-box motif directly overlaps the ribosome-binding site of the SCD95A.26 gene, possibly acting as a sequestor. We predict that in these bacteria, SAM-stabilized S-box structure directly represses initiation of translation. Similar mode of regulation was previously suggested for other metabolite-responsive riboswitches in actinobacteria (32).

Methionine-specific T-box regulon

Using a training set of experimentally known T-box structures from B.subtilis and other Gram-positive bacteria (31), we constructed a pattern for the T-box motif and scanned bacterial genomes using the RNA-PATTERN program. The pattern was absolutely specific since candidate T-boxes were mostly found upstream of amino acid metabolism-related genes (data not shown). Then we selected 38 T-box sequences with AUG (methionine) specifier codons (see supplementary Figure Figure22 for multiple alignment). Although the T-box system is widely used in bacteria from the Bacillus/Clostridium group (where more than 200T-boxes were identified; A. G. Vitreschak, unpublished results), methionine-specific T-boxes occur only in a limited number of species.

In the genomes of Bacillus halodurans and six clostridia, there is only one methionine-specific T-box that precedes the metS gene encoding methionyl-tRNA synthetase (not included in Table Table1).1). The methionine T-box regulation seems to be extensively used only in the Lactobacillales group (30 Met-T-boxes), where it exclusively controls methionine biosynthesis and transport genes (Table (Table1).1). This phylogenetic distribution is consistent with the absence of the S-box regulatory system in all available genomes of Lactobacillales (see Discussion). Among all other available bacterial genomes, we have found only one additional methionine T-box. It precedes the metICFE-mdh operon in Staphylococcus aureus, a bacterium that mainly uses the S-box system.

Methionine regulation in streptococci

Then we attempted to analyze potential methionine regulons in Streptococcaceae, a large family of Gram-positive bacteria, which have neither S-boxes nor methionine-specific T-boxes. For this aim, we collected upstream regions of all MET genes from Lactococcus lactis and Streptococcus species and applied the signal detection procedure. A highly conserved 17 bp palindromic sequence (named MET-box) with consensus 5′-TATAGTTtnaAACTATA-3′ was identified in all streptococci (Table (Table2).2). To find new members of the candidate regulon, the derived profile for the MET-box signal was used to scan the genomes. In various Streptococcus species, the MET regulon appears to include most methionine biosynthesis and transport genes, as well as several hypothetical genes including yxjH, mdh, fhs and folD (Table (Table1).1). The predicted MET regulon in L.lactis contains only one transcriptional unit, the metEF operon. Given the palindromic structure of the derived signal, typical to binding signals of transcriptional regulators, we propose that the MET-box signal plays a role in transcriptional regulation of the methionine metabolism in Streptococcus species and L.lactis, although the responsible regulatory protein remains to be identified. Interestingly, in a recent study it was shown that the LysR-type transcriptional regulator MtaR is necessary for the efficient methionine uptake in Streptococcus agalactiae (53). MtaR has orthologs in other studied Streptococcus species as well as in L.lactis. Since we have found MET-boxes upstream of the predicted methionine transport operons in S.agalactiae, we suppose that this MET-box is the DNA binding signal of MtaR. Moreover, the MET-box signal is similar to signals recognized by LysR-family regulators in length and palindromic symmetry.

Table 2.
Candidate methionine-specific DNA signals (MET-boxes) in Streptococcaceae

Reconstruction of the methionine pathway

Positional analysis of a large number of regulatory elements (S-, T- and MET-boxes) in Gram-positive bacteria allowed us to identify new genes possibly involved in the methionine metabolism. In addition, we analyzed the candidate MetJ binding sites upstream of some methionine synthesis and transport genes in gamma proteobacteria (data not shown). The detailed analysis of new members of the methionine regulons and reconstruction of the metabolic pathways in various organisms is presented below.

Methionine biosynthesis

The pathway of methionine biosynthesis via the transsulfuration or direct sulfhydrylation route is conserved in most Gram-positive bacteria, but some steps vary. Only complete genomes of F.nucleatum, Clostridium perfringens, Enterococccus faecalis, S.agalactiae and Streptococcus pyogenes, as well as unfinished genomes of several lactobacilli and Streptococcus uberis, lack the de novo synthesis pathway but possess the SAM synthase gene metK (Table (Table1).1). We suggest that this metabolic gap could be filled by methionine-specific transport systems detected in these genomes (metNPQ or metT, see below).

We observed several cases of expansion of the methionine regulon, when the upstream reactions preceding the methionine biosynthetic pathway become methionine regulated. One example is homoserine dehydrogenase (the hom gene product in B.subtilis), which is shared by the threonine and methionine pathways (Figure (Figure1).1). In most Gram-positive bacteria the hom gene is co-localized with the threonine biosynthesis genes thrBC (data not shown). However, in the genomes of two bacilli, three clostridia and Lactobacillus plantarum, we have found a second hom paralog belonging to the methionine regulon, either S-box or T-box (Table (Table1).1). Another example is the cysteine biosynthesis gene cluster (cysH-ylnABCDEF), which is a member of the methionine S-box regulon in B.subtilis and B.cereus, but not in other Bacillales. Interestingly, even in these two related bacteria, the in vitro affinities of candidate cysH S-box motifs to the effector molecule (SAM) vary by two orders of magnitude (28). Furthermore, the expression of the B.subtilis cysH operon is not repressed by methionine in vivo, indicating disfunction of the cysH S-box element (54). Indeed, this S-box contains a unique C→A substitution destabilizing helix 3. Moreover, upstream regions of cysH genes of other closely related genomes (B.halodurans, Bacillus stearothermophilus, O.ihyensis) demonstrate residual similarity to upstream regions of B.subtilis and B.cereus cysH, but cannot fold into the S-box structure (data not shown). In any case, in B.cereus, this functional regulatory interaction seems to be rational since the bacillary transsulfuration pathway of the methionine biosynthesis uses cysteine as a sulfur donor.

The first step of the methionine biosynthesis is catalyzed by one of two non-homologous homoserine O-acetyltransferases, MetB or MetX. The main difference of these two isoenzymes is that, in contrast to MetX from L. meyeri, the MetB enzyme from B.subtilis is feedback inhibited by SAM (7,9). The MetB isoenzyme detected in most Gram-positive bacteria possessing the methionine pathway is replaced by MetX in Staphylococcus, Listeria and T.tengcongensis, whereas the B.cereus genome encodes both proteins (Table (Table1).1). Phylogenetic distribution of the MetB and MetX isoenzymes differs significantly: the former prevails in enterobacteria, firmicutes and cyanobacteria whereas the latter is common in other proteobacteria, actinobacteria, various early branching bacteria and in fungi. Notably, the SAM-inhibited isoenzyme MetB is not methionine regulated in many cases, at least not using S-, T-, or MET-boxes (e.g. in B.subtilis). In contrast, the MetX synthesis is S-box controlled with the exception of B.cereus where both enzymes are present.

Then we have analyzed distribution of the direct sulfhydrylation and transsulfuration pathways of the methionine biosynthesis, which are catalyzed by the MetY and MetI-MetC, respectively. Among Gram-positive bacteria, the former prevails over the latter: twelve species have only MetY, two species have MetI-MetC, whereas seven remaining methionine-producing bacteria possess both pathways (Table (Table1).1). The role of single metI genes (not accompanied by metC) in several genomes containing the metY gene is still not clear. Thus, in contrast to B.subtilis and S.aureus, other methionine-producing firmicutes potentially use the direct sulfhydrylation pathway and, therefore, they do not require cysteine for the methionine synthesis.

Two non-homologous methionine synthases, coenzyme B12-dependent MetH and B12-independent MetE, are known in bacteria. The metE gene was detected in all methionine-producing firmicutes, except clostridia and two bacilli that have the metH gene. B.cereus and B.halodurans have both the B12-dependent methionine synthase MetH, which belongs to the S-box regulon, and the B12-independent isoenzyme MetE, that is likely regulated by coezyme-B12 via the B12-element riboswitch (34). The only exception is O.iheyensis, which has neither metE, nor metH. Analysis of candidate S-box signals allowed us to identify a new member of the methionine regulon in this bacterium, OB0691, which is similar to the betaine–homocysteine methyltransferase BhmT from mammals (55). BhmT catalyzes conversion of homocysteine to methionine, like the bacterial MetE and MetH isoenzymes, but it uses another methyl donor, betaine, instead of methyl-THF. Thus, we predict that O.iheyensis uses a eukaryotic-type methionine synthase and does not require the methylene-THF reductase MetF, which is absent in this bacterium.

B12-dependent methionine synthases from clostridia and Thermotogales lack the C-terminal domain which is involved in reactivation of spontaneously oxidized coenzyme B12, and therefore is required for the catalysis (denoted metH in Table Table1).1). In all these bacteria, except T.tengcongensis, we found a hypothetical gene located immediately upstream of the metH gene (see Thermotoga maritima TM0269 as a representative of this gene family). This gene, named msd, has no homologs in other genomes. However, we identified a conserved sequence motif (hhhThG-28-hEhhh[DE]-//-RxxxGY-32-Pxx[SA][TV]x[GA]hh, where ‘h’ denotes any hydrophobic amino acid), which is common to all full-length MetH proteins and to the Msd proteins. As shown by (56), the RxxxGY motif is critical for binding of SAM to the C-terminal reactivation domain of methionine synthase MetH. Thus, we tentatively assign the missing function of reactivation of the B12-dependent methionine synthase to the product of the msd gene. The three-dimensional structure of the TM0269 protein was recently resolved in Joint Center for Structural Genomics (http://www.jcsg.org/), but this did not lead to assignment of a cellular role for this protein.

The metF gene encoding methylene-THF reductase has been identified in most Gram-positive bacteria that have methionine synthases (MetH or MetE), an exclusion being Clostridium tetani. The MetF proteins from Bacillales, C.acetobutylicum, L.plantarum and two streptococci have an additional N-terminal domain highly similar to the homocysteine-binding domain of MetH (denoted metF in Table Table1).1). Previously, it was proposed that this domain could be involved in positive allosteric regulation of MetF by homocysteine (12).

Recycling of methylene-THF from THF is connected with interconversion of serine and glycine mediated by GlyA, belonging to the methionine regulon in E.coli (57). The glyA gene is present in all Gram-positive bacteria, but never in the methionine regulon. The alternative pathway of methylene-THF recycling, which proceeds in two steps and requires ATP and NADP, is mediated by FolD and Fhs. The corresponding genes are candidate members of the MET-box regulon in Streptococcus pneumoniae, whereas the folD and metF genes form one possible operon in Clostridium difficile (Table (Table1).1). On the other hand, the folD gene is a candidate member of the purine regulon in some γ-proteobacteria (58), that is also rational since methylene-THF is required for the purine biosynthesis. These facts once again demonstrate genome-specific regulon expansions, and, in particular, indicate considerable variability in regulation of the methylene-THF synthesis in bacteria.

Methionine transport

The only known transport system for methionine is the ABC transporter MetNIQ of enteric bacteria, which belongs to the methionine uptake transporter (MUT) family (1315). An ortholog of this system in B.subtilis, encoded by the metNPQ operon, is regulated by the S-box system (12). Recently it was shown that metNPQ encodes an ABC permease transporting methionine sulfoxide, d-, and l-methionine (16). To describe candidate methionine transporters in Gram-positive bacteria, we combined similarity search for the metNPQ orthologs with identification of methionine-specific regulatory sites and with positional analysis of genes.

Candidate methionine transporters metNPQ are widely distributed in Gram-positive bacteria: they are absent only in two clostridia species (Table (Table1).1). In most cases, components of methionine transporters are encoded by clusters of co-localized genes which are preceded by S-boxes (in Bacillales and clostridia), methionine-specific T-boxes (in lactobacilli) or MET-boxes (in streptococci). To analyze the possible origin of a large number of metNPQ paralogs in Gram-positive bacteria, we constructed the phylogenetic tree for the substrate-binding components of MUT-family transporters (Figure (Figure3).3). Though in some cases, e.g. in L.lactis, Campylobacter jejuni, metQ paralogs possibly result from recent genome-specific duplications, in most other cases they have diverged early. Since almost all large branches of the tree contain members of various methionine regulons, including MetJ-regulated orthologs from enterobacteria (15), we believe that the methionine specificity has been retained by all members of the MUT family. However, we cannot exclude the possibility that some members of the MUT family have a more broad specificity.

A new type of candidate methionine transporters, named MetT, was identified in some bacteria from the Bacillus/Clostridium group and γ-proteobacteria. Among Gram-positive bacteria, only B.cereus, S.aureus and three clostridia species have the metT genes, which are regulated by upstream S-boxes in all cases (see Table Table11 and Figure Figure44 for genomic identifiers). Among γ-proteobacteria, only Vibrio and Shewanella species have metT genes, and they are preceded by candidate MetJ sites (data not shown). MetT proteins contain eleven predicted transmembrane segments and are similar to proteins from the NhaC Na+:H+ antiporter superfamily. Existence of likely S-adenosylmethionine-regulated metT in the complete genome of C.perfringens that has no methionine biosynthesis genes suggests that MetT is a methionine transporter.

Figure 4
Phylogenetic tree of the NhaC Na+:H+ antiporter superfamily including predicted methionine-, lysine- and tyrosine-specific transporters. Gene identifiers are shown for annotated complete genome sequences. Genes predicted to be regulated ...

The phylogenetic tree of transporters from the NhaC superfamily consists of four deeply diverged branches (Figure (Figure4).4). The first branch comprises predicted lysine transporters LysW, most of which are preceded by regulatory LYS-elements (35). The second branch contains predicted methionine transporters MetT, preceded by either candidate S-boxes (in Gram-positive bacteria) or MetJ binding sites (in γ-proteobacteria). The third branch includes predicted tyrosine transporters TyrT, most of which are members of the tyrosine T-box regulon (59). The fourth branch includes orthologs of malate : lactate antiporter MleN from B.subtilis (60). Thus analysis of candidate regulatory signals allows us to tentatively assign specificities to the remaining three large sub-groups of transporters from the NhaC superfamily.

Analysis of the methionine-specific regulatory signals allowed us to identify two more hypothetical methionine-related ABC transport systems in Gram-positive bacteria. The first one, named mtsABC, is present in all streptococci, some lactobacilli, and C.perfringens, S.aureus and B.cereus (Table (Table1).1). MtsA (SMU.1935c in Streptococcus mutans) has five predicted transmembrane segments and is not similar to any known protein. The MtsB and MtsC components (SMU.1934c and SMU.1933c) are similar to typical ATP-binding (CbiO) and transmembrane (CbiQ) components of various ABC transporters, respectively. In most cases mtsABC genes are clustered with the hcp gene (SMU.1936c) that encodes a hypothetical cytosolic protein.

The second methionine-related ABC transport system, named tom (for Transporter for Oligopeptides or Methionine), belongs to a large family of oligopeptide ABC transporters. In B.cereus and Listeria monocytogenes, the tom gene clusters (BC0207-BC0208-BC0209-BC0210-BC0211 and LMO2196-LMO2195-LMO2194-LMO2193-LMO2192, respectively) are preceded by S-boxes. Moreover, E.faecalis has a single tomA gene (EF3081) for a substrate-binding component of transporter, which is preceded by a methionine-specific T-box. The available data were insufficient to assign specificities of these two methionine-regulated transporters by the genome context analysis and metabolic reconstruction. One possibility is that they are involved in the uptake of some methionine precursors or oligopeptides.

Methionine salvage and SAM recycling

The methionine salvage genes mtnKSUVWXYZ involved in the methylthioribose utilization in B.subtlis (2123) were identified only in three Bacillus species, always as members of the S-box regulon (Table (Table1).1). In B.cereus, one S-box motif was found upstream of the BC0768-767-766 gene cluster (named mtnABC) encoding a hypothetical ABC transport system. An ortholog of this system exists in the genome of B.stearothermophilus, but not in other studied bacterial genomes. Based on S-box regulatory site, similarity to the ribose ABC transporter and phylogenetic co-occurrence with the mtnKSUSVWXYZ genes, we tentatively assign the methylthioribose specificity to the MtnABC transport system. The absence of MtnABC in B.subtilis, which also can grow on methylthioribose (2,19), suggests existence of other specific transport systems.

In addition to the autotrophic pathway of cysteine biosynthesis, some bacterial species (including B.subtilis) can synthesize this amino acid through the reverse transsulfuration pathway using methionine as a precursor (61). In this pathway, methionine is first converted to homocysteine via the SAM recycling pathway (Figure (Figure1).1). In an attempt to discover missing genes of this pathway, we started with positional analysis of the B.subtilis yrhA and yrhB genes apparently encoding the cystathionine β-synthase and γ-lyase, respectively (12). Orthologs of these genes were found in all Bacillus species, S.aureus and three clostridia and are always clustered forming one candidate operon (Table (Table1).1). Moreover, the yrhAB genes form possible operons with the SAH nucleosidase gene mtn in bacilli, and with orthologs of the B.subtilis luxS gene in O.iheyensis, C.perfringens and Clostridium botulinum. The functional role of the B.subtilis LuxS protein is not known, though its structure has been recently determined (62). The autoinducer-2 production protein LuxS from proteobacteria catalyzes transformation of S-ribosylhomocysteine to homocysteine (63). We assign the previously missing ribosylhomocysteinase function of the SAM recycling pathway to the luxS gene product. Orthologs of the luxS and mtn genes were identified in most Gram-positive bacteria, corroborating the existence of SAM recycling pathways in these organisms.

Two other genes co-localized in positional clusters with the yrhAB genes (yrrT from bacilli and ubiG from clostridia) both contain SAM-binding motifs and are similar to various SAM-dependent methyltranferases (Table (Table1).1). It was known that SAH is synthesized from SAM as a by-product of numerous methylation reactions in the cell (2). Based on co-localization with the yrhAB genes, we propose that the pathway of reverse synthesis of cysteine from methionine could require specific SAM-dependent methylases, and assign this role to YrrT in bacilli and UbiG in clostridia.

The hypothesis that the ubiG-yrhAB operon of C.acetobutylicum is involved in the cysteine synthesis is further supported by observation of a cysteine-specific T-box upstream of this operon, likely mediating repression by cysteine (12). Moreover, a backward-directed S-box motif located immediately downstream of this operon could regulate formation of an antisense transcript for this locus, assuming activation of the ubiG-yrhAB operon by methionine. Thus we predict that genes for the reverse transsulfuration pathway in C.acetobutylicum are apparently expressed only in the conditions of methionine excess and cysteine deficiency (see Figures Figures11 and and5).5). Some other mode of regulation by interference of transcription from the complementary strand also could be involved, but it would not change the main conclusion.

Figure 5
Predicted regulation of the C.acetobutylicum ubiG-yrhBA operon by S-adenosylmethionine-specific S-box and cysteine-specific T-box regulatory signals.

Two highly similar genes of unknown function, yxjH and yxjG, are members of the S-box regulon in B.subtilis (24). They encode proteins with moderate similarity to the C-terminal part of the B12-independent methionine synthase MetE. Mutation analysis has showed that these genes are not required for the de novo methionine synthesis in B.subtlis (12). Orthologs of these genes (denoted yxjH) were identified in various Gram-positive bacteria, several proteobacteria and archaea (see the phylogenetic tree in Figure Figure6).6). Similar to B.subtilis, the yxjH genes in O.iheyensis and L.monocytogenes are S-box regulated. Moreover, most yxjH orthologs from Lactobacillales and Streptococcus species belong to the methionine T-box and MET-box regulons, respectively (Table (Table11).

Figure 6
Phylogenetic tree of bacterial orthologs of the B.subtilis yxjH gene. Proteins are denoted by the genome abbreviations (listed in Table Table1).1). Multiple gene paralogs are numbered. Genes predicted to be regulated by S-boxes, T-boxes and MET-boxes ...

Negative regulation of the B12-independent methionine synthase isozymes by B12-element and vitamin B12 is common in bacteria possessing both B12-dependent and B12-independent isozymes (34). At that, vitamin B12-responsive regulatory elements were detected upstream of the yxjH orthologs in Rhodopseudomonas palustris and Bacteroides fragilis, two bacteria possessing B12-dependent methionine synthases (34). It indicates that these two B12-regulated yxjH orthologs likely function as B12-independent methionine synthases.

Further, based on results of positional analysis and phylogenetic profiling, we suggest that YxjH could function as an alternative methionine synthase enzyme mainly involved in the SAM recycling pathway. Indeed, we identified several cases of co-localization of the yxjH genes with the candidate SAM recycling gene luxS (in Lactobacillus gasseri, Lactobacillus casei and Leuconostoc mesenteroides) and with the methionine biosynthesis genes metICB (in Oenococcus oeni). Notably, the latter genome lacks both methionine synthase genes (metE and metH). In addition, we found methionine-regulated yxjH genes in some species from the order Lactobacillales, which lack genes for the de novo methionine synthesis but possess genes for both methionine transport and SAM recycling. However, our assignment of the methionine synthase role to YxjH contradicts the observation that the metE B.subtilis mutant is a methionine auxotroph, thus suggesting that MetE is the only methionine synthase in this species (12,64). Additional experiments are required to prove this tentative assignment of yxjH to the SAM recycling pathway.

Other candidate members of methionine regulons

Candidate methionine transporter operons in Streptococcus, Oenococcus, Leuconostoc and Oceanobacillus species include a hypothetical gene (hmrA or hmrB) from the M20 family of zinc metallopeptidases. Known members of this family catalyze the release of an N-terminal amino acid, usually neutral or hydrophobic, from a polypeptide (65). Single hmrA gene of B.cereus (BC3176) is a member of the S-box regulon. In addition, methionine regulons of B.cereus, S.aureus, L.mesenteroides and three Streptococcus species contain a putative metal-dependent hydrolase gene, named mdh (genes BC0395, SA0343 and SMU1172 in complete genomes and COG entry 1878). In other Gram-positive bacteria, the mdh genes are not regulated by methionine, at least not by known systems. The role of the hmrA, hmrB and mdh gene products in the methionine metabolism is not clear.

In S.coelicolor and T.fusca, S-box precedes the SCD95A.26 gene encoding a hypothetical pyridoxal-phosphate dependent enzyme (PFAM entry PF00291). Enzymes of this class catalyze various reactions in the metabolism of amino acids (66). SCD95A.26 is a distant homolog of the threonine synthase gene thrC. However, actinobacteria have a proper thrC gene located within the threonine biosynthetic gene cluster. Since the complete genome of S.coelicolor lacks orthologs of known genes for the synthesis of homocysteine, the direct precursor of methionine, we tentatively suggest that SCD95A.26 could be involved in the homocysteine synthesis in actinobacteria.


Methionine biosynthetic and transport genes are regulated by different mechanisms in various microbial species (Figure (Figure7).7). The S-box system in Bacillales and Clostridia orders and the methionine-specific T-box system in Lactobacillales are RNA-dependent regulatory systems that both control transcription termination, although in a different manner. The S-box RNA structure is stabilized by direct binding of an effector molecule, S-adenosylmethionine, whereas the T-box senses lack of amino acid via the presence of uncharged tRNAs. Bacteria from the Streptococcaceae family, lacking both these RNA-dependent systems, are predicted to have a classical DNA-dependent system for regulation of the methionine metabolism, which includes numerous MET-box sites and the MtaR regulatory protein. Regulation of the methionine biosynthesis and transport genes in Gram-negative enterobacterium E.coli is also DNA-dependent and involves two known transcription factors, activator MetR and repressor MetJ, that bind operators absolutely different from MET-box (8). The most intriguing mode of regulation was found for the C.acetobutylicum ubiG-yrhAB operon, which seems to be regulated by both S- and T-box systems (Figure (Figure55).

Inspite of a variety of regulatory systems, the cores of the methionine regulons in Gram-negative and Gram-positive species almost completely coincide. They include most genes required for the de novo methionine synthesis and transport. In contrast, SAM synthase gene metK belongs to the methionine regulons only in bacilli/clostridia and γ-proteobacteria, via the S-box and MetJ regulatory systems, respectively. In lactobacilli and streptococci this gene is not regulated by either the T-box or MET-box systems. One possible explanation for this could be the use of different effector molecules for the methionine regulons in these taxonomic groups. SAM is a known effector of the S-box and MetJ regulatory systems, whereas methionine itself is involved in the regulation by the T-box system via methionine-specific tRNA. Apparently, in the latter case it is not necessary to regulate SAM synthesis by the methionine availability. The effector molecule for the predicted MET-box system in streptococci is not known. In this bacterial group, MET-boxes precede most of the methionine biosynthesis and transport genes but not the metK genes (Table (Table1).1). Using the same biochemical logic we tentatively assign the role of the regulatory molecule in streptococci to methionine itself.

Compared to other riboswitches, S-boxes demonstrate somewhat mosaic phylogenetic distribution. They were observed in two major groups of firmicutes, bacilli and clostridia, and in a number of other taxons. Although riboswitches are subject to frequent horizontal transfer (32), and it cannot be ruled out in this case, especially for isolated proteobacterial genomes (Xanthomonas and Geobacter), the existence of S-boxes in a variety of genomes argues for their ancient origin. In particular, the most parsimonious evolutionary scenario for firmicutes seems to be the following. S-boxes were present in the last common ancestor of bacilli and clostridia, i.e. the last common ancestor of all firmicutes, and it was lost in the Streptococcaceae and Lactobacillales lineages. In streptococci, the role of S-boxes in the regulation of methionine metabolism was assumed by the transcriptional regulator MtaR with the MET-box binding signal, whereas in lactobacilli, the S-box regulon was absorbed by the expanded Met-T-box regulon, which initially included only aminoacyl-tRNA synthetases. One possible example of an early stage of such expansion could be the Met-T-box upstream of the metICFE-mdh operon in S.aureus. On the other hand, degradation of a S-box is exemplified by the case of cysH genes of B.subtilis and closely related genomes.

The comparative analysis of regulation supplemented by genome context and similarity search techniques allowed us to identify several new methionine-related transport systems, assign missing functions of the SAM recycling pathway, and demonstrate variability in the upper part of the methionine pathway in different species. Furthermore, we identified genome-specific extensions of the methionine regulon that involve genes shared with other biochemical pathways. This study not only demonstrates once again the power of comparative genomics in functional annotation of genes, prediction of regulatory interactions and metabolic reconstruction, but also provides one of the first examples where the possible scenario of evolution of several systems regulating one metabolic pathway could be tentatively reconstructed.


Supplementary Material is available at NAR Online.

[Supplementary Material]


We are grateful to Vadim Brodyansky for the MetJ recognition profile and to Alexandra Rachmaninova, Valerie de Crecy-Lagard and Andrei Osterman for discussions. We are grateful to Isabelle Martin-Verstraete for critical reading of the manuscript and many helpful suggestions. This study was partially supported by grants from the Ludwig Institute for Cancer Research (CRDF RBO-1268), the Howard Hughes Medical Institute (55000309) and the Russian Fund of Basic Research (04-04-49361-a).


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