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In many bacteria, LuxS functions as a quorum-sensing molecule synthase. However, it also has a second, more central metabolic function in the activated methyl cycle (AMC), which generates the S-adenosylmethionine required by methyltransferases and recycles the product via methionine. Helicobacter pylori lacks an enzyme catalyzing homocysteine-to-methionine conversion, rendering the AMC incomplete and thus making any metabolic role of H. pylori LuxS (LuxSHp) unclear. Interestingly, luxSHp is located next to genes annotated as cysKHp and metBHp, involved in other bacteria in cysteine and methionine metabolism. We showed that isogenic strains carrying mutations in luxSHp, cysKHp, and metBHp could not grow without added cysteine (whereas the wild type could), suggesting roles in cysteine synthesis. Growth of the ΔluxSHp mutant was restored by homocysteine or cystathionine and growth of the ΔcysKHp mutant by cystathionine only. The ΔmetBHp mutant had an absolute requirement for cysteine. Metabolite analyses showed that S-ribosylhomocysteine accumulated in the ΔluxSHp mutant, homocysteine in the ΔcysKHp mutant, and cystathionine in the ΔmetBHp mutant. This suggests that S-ribosylhomocysteine is converted by LuxSHp to homocysteine (as in the classic AMC) and thence by CysKHp to cystathionine and by MetBHp to cysteine. In silico analysis suggested that cysK-metB-luxS were acquired by H. pylori from a Gram-positive source. We conclude that cysK-metB-luxS encode the capacity to generate cysteine from products of the incomplete AMC of H. pylori in a process of reverse transsulfuration. We recommend that the misnamed genes cysKHp and metBHp be renamed mccA (methionine-to-cysteine-conversion gene A) and mccB, respectively.
Helicobacter pylori is a Gram-negative bacterium which causes peptic ulceration, gastric adenocarcinoma, and gastric lymphoma (2). All H. pylori strains possess a homologue of luxS, best known as a quorum-sensing molecule synthase. luxS homologues have been found in around half of all bacterial genomes sequenced to date (38, 42, 44). Some of the molecules formed in the reaction catalyzed by LuxS are collectively termed autoinducer 2 (AI-2) and have been shown to act as signaling molecules. They have been described as mediating a variety of effects in different bacteria, including on virulence. In H. pylori, disruption of luxS has been shown to increase biofilm formation (6) and to reduce in vivo fitness and infectivity (20, 29). Several motility-associated luxSHp phenotypes have also been reported, including loss of growth phase-dependent flaA regulation (24), reduced motility on soft agar plates, and reduced transcription of the flagellar regulator flhA (29, 30).
In most bacteria, the enzyme encoded by luxS has another (possibly primary and sometimes sole) function: it is an integral metabolic component of the activated methyl cycle (AMC). The AMC is a key metabolic pathway that generates S-adenosylmethionine (SAM) as an intermediate product (the full cycle is shown in Fig. Fig.1).1). SAM bears a methyl group with a relatively high transfer potential and is used by numerous methyltransferases to carry out cellular processes, including nucleic acid and protein methylation and detoxification of reactive metabolites (11). The product of the methyltransferase reaction is S-adenosylhomocysteine (SAH), and in the complete AMC, SAM is regenerated from SAH via homocysteine and methionine, ready for another round of methylation/transmethylation (35, 44, 47, 49). The role of LuxS in the AMC is to catalyze cleavage of S-ribosylhomocysteine (SRH) to yield homocysteine and a by-product, 4,5-dihydroxy-2,3-pentanedione (DPD) (Fig. (Fig.1)1) (35, 44, 47, 49). DPD is the precursor of the family of related, interconverting molecules collectively termed “AI-2” (5, 44).
H. pylori is unusual among bacteria in not having a complete AMC. The final step in the classical AMC, the conversion of homocysteine to methionine can be carried out by either of two N5-methyltetrahydrofolate-dependent methyltransferases, MetE or MetH. However, all H. pylori genomes studied to date lack metE and metH homologues (4). This raises the question of whether the only function of LuxS in H. pylori is as a quorum-sensing molecule synthase or whether LuxS in H. pylori has another, as yet undescribed, metabolic role.
In searching for a possible metabolic role for LuxSHp, we formed the hypothesis that it is part of a de novo cysteine biosynthesis pathway that uses methionine as a reduced sulfur source. Our reasoning for this was based on three observations. First, H. pylori has an absolute requirement for methionine (26, 27, 31), in agreement with its lack of metE and metH. Second, H. pylori can synthesize cysteine without apparently making use of oxidized sulfur compounds, such as sulfate: genomic studies show that homologues to the genes which encode the components required for uptake of sulfate and conversion to sulfide are absent (10). Third, all complete H. pylori genomes available to date contain homologues of two genes, annotated as cysK and metB, located immediately upstream of luxS. In other bacteria, these are involved in the generation of cysteine and interconversion of cysteine and methionine, respectively. Recently the metB gene from H. pylori strain SS1 was purified and characterized, but its physiological role was not established (18).
When we pondered the fate of homocysteine, which is not converted to methionine in H. pylori due to a lack of MetE and MetH, coupled with the observation that the gene responsible for its production, luxS, is linked with two other genes predicted to be involved in methionine and cysteine metabolism, we hypothesized that H. pylori is able to utilize homocysteine by converting it to cysteine via the reverse transsulfuration pathway (RTSP), involving the associated genes metB and cysK. We thus set out to address this hypothesis and more specifically to define the roles of the cysK, metB, and luxS homologues in the putative H. pylori RTSP.
The strains and plasmids used in this study are shown in Table Table1.1. Escherichia coli strains DH5α and DS941 (37, 51) were used in cloning/subcloning experiments. E. coli was routinely propagated in Luria-Bertani (LB) broth or on LB agar plates at 37°C under normal atmospheric conditions. Vibrio harveyi was grown in either LB or AB medium (12) at 30°C, also under normal atmospheric conditions. H. pylori strains were routinely passaged on horse blood agar (Oxoid) every 2 to 3 days and were grown in a MACS VA500 microaerobic workstation (Don Whitley Scientific) using a humidified atmosphere consisting of 6% O2, 3% H2, 5% CO2, and 86% N2. Antibiotic selection was carried out at 100 μg ml−1 with ampicillin (or carbenicillin) and 50 μg ml−1 for kanamycin.
For H. pylori metabolic supplementation experiments, the complete chemically defined medium (cCDM) of Reynolds and Penn was formulated as previously described (31), with variations as indicated in the text. The concentrations of methionine and cysteine used were 505 μM and 1.0 mM, respectively. For the variations of CDM lacking cysteine (see Results for description of use), different chemicals were added: homocysteine (at 1.0 mM), cystathionine (at 1.0 mM), or potassium sulfide (a range of concentrations up to 1.0 mM were utilized). All chemicals were of high purity (cell culture tested) and were obtained from Sigma-Aldrich Co. CDM batch culture comparative growth studies were carried out as described previously (9).
AI-2 measurements were performed as described previously (3, 47). Twenty-four-hour culture supernatant samples, corresponding to mid-late log phase, were tested. AI-2 activity was expressed as fold change in induced bioluminescence from the reporter strain compared with that for the negative control. Negative control samples consisted of media which had not been inoculated but otherwise were treated as test cultures. Positive controls were carried out using in vitro-synthesized AI-2, prepared as described previously (8).
Recombinant DNA techniques were carried out using standard methods (34). PCR amplifications were performed using either standard DNA polymerase (GoTaq; Promega, Madison, WI) or proofreading DNA polymerase (Novagen KOD; Merck Chemicals Ltd., United Kingdom). Restriction digests were performed in accordance with the manufacturer's recommendations (Promega, Madison, WI). E. coli genomic and plasmid DNA was isolated using the Qiagen range of preparatory kits (Qiagen, United Kingdom). H. pylori genomic DNA was purified as described previously (22). DNA sequencing was conducted using standard fluorescent dye terminator chemistries, and analysis was performed using the Applied Biosystems 3730 DNA analyzer system (Geneservice, Cambridge, United Kingdom, and Applied Biosystems Inc., Foster City, CA.). Results were analyzed using the BioEdit software suite (13).
All mutants were made following an insertion-deletion strategy. The H. pylori ΔluxS mutant strains were made by transforming J99 and NCTC11637 with pGEMT::luxS::aphA3 (a generous gift from Leo Smeets and Theo Verboom).
The cysK and metB loci were mutagenized as follows. Fragments containing the open reading frames (ORFs) and approximately 400 to 500 bp on either side were amplified from J99 genomic DNA using the following primer pairs: cysK (forward, 5′-CACCATTGACAAATCCTTCC-3′; reverse, 5′-TTTGGTGTTGGGCTTGATAG-3′) and metB (forward, 5′-CCTGATAATCCCGCAGCCTACTA-3′; reverse, 5′-ACCCCCACTTCAGACCACTCAG-3′). These products were then cloned into pGEM-T Easy (Promega, Madison, WI). Inverse PCR (iPCR) products were generated from these using the following primer pairs, which were designed so that resulting clones would contain a deletion of a large part of the open reading frame of each target gene, preventing gain of function recombination/excision events: metB (5′ iPCR, 5′-CGTGAATTCCGGCTAAACCAG-3′ [EcoRI site underlined]; 3′ iPCR, 5′-TGAACAGGATCCGTTAGAAGATT-3′ [BamHI site underlined]) and cysK (5′iPCR, 5′-GCTGTTTTTCTGTGCTGAATTCTT-3′ [EcoRI site underlined]; 3′iPCR, 5′-GTGGATCCGAGGGTTCTATTTTGA-3′ [BamHI site underlined]). These were prepared for cloning by restriction digestion and purified. The apolar aphA3 cassette, conferring kanamycin resistance, was isolated from pMWA2 (46) by restriction digestion with EcoRI/BamHI and ligated into the prepared iPCR products. Positive clones were selected on the basis of kanamycin resistance and confirmed by PCR and DNA sequencing. The resultant vector constructs are nonreplicative in H. pylori and were used as suicide vectors to transform J99 and NCTC11637.
E. coli was transformed with plasmid DNA by electroporation using a Bio-Rad Gene Pulser with pulse controller at a voltage of 1.8 kV, with a resistance of 200 Ω at 25-μF capacitance. H. pylori was transformed by natural transformation using established protocols (22). Recombinant H. pylori strains were recovered by selection on Columbia agar base containing kanamycin, egg yolk emulsion (5% [vol/vol]), and triphenyl tetrazolium chloride at a working concentration of 40 μg ml−1. Inclusion of this indicator made it easier to see the small recombinant colonies.
The following protocol was developed for sampling H. pylori cultures prior to metabolite analysis. Strains were grown in complete CDM for 24 h. All subsequent manipulations were performed on ice or in chilled equipment, and all plastic ware was prechilled prior to use. Three ml of culture at an optical density (OD) at 600 nm (OD600) of 1.0 was taken as a standard sampling volume (variances from this OD were corrected for by using larger or smaller volumes). This was quenched in 5 ml ice-cold phosphate-buffered saline (PBS). Cells were recovered by centrifugation and washed with a further 5 ml ice-cold PBS. The cells were again recovered by centrifugation, and cell pellets were stored at −80°C. Metabolite analysis was performed as described previously (15, 36).
Primary amino acid sequences were aligned using the ClustalW algorithm using BioEdit (13). Accession numbers of individual protein sequences used are as follows: H. pylori, LuxS, NP222818; MetB, NP222819; CysK, NP222820; RecA, NP222862; Helicobacter acinonychis, LuxS, YP665186; MetB, YP665187; CysK, YP665188; RecA, YP664174; Helicobacter hepaticus, LuxS, NP859707; MetB, NP859593; CysK, NP860380; RecA, NP860164; Helicobacter bilis, LuxS, ZP04580164; MetB, ZP04581342; CysK, ZP04581369; RecA, ZP04580683; Helicobacter canadensis, LuxS, ZP03656033; MetB, ZP03656080; CysK, ZP03656337; RecA, ZP03655680; Helicobacter pullorum, LuxS, ZP04808703; MetB, ZP03661228; CysK, ZP03660206; RecA, ZP04808545; Helicobacter winghamensis, LuxS, ZP04583537; MetB, ZP04583291; CysK, ZP04582871; RecA, ZP04583642; Helicobacter cinadei, LuxS, ZP03657497; MetB, ZP03658434; CysK, ZP03658433;RecA, ZP03659220; Wolinella succinogenes, LuxS, NP908224; MetB, NP907217; CysK, NP907372; RecA, NP907645; Campylobacter jejuni, LuxS, YP002344589; MetB, ZP01699839; CysK, YP002344310; RecA, YP179815; Enterococcus faecium, LuxS, ZP00604876; MetB, ZP00604875; CysK, ZP00604874; RecA, ZP00603401; Staphylococcus aureus, LuxS, BAB43220; MetB, NP370984; CysK, BAB56621; RecA, NP371809; E. coli, LuxS, NP417172; MetB, NP418374; CysK, NP416909; RecA, YP853918; and Bacillus subtilis, LuxS, NP390945; MccB, NP390603; MccA, NP390604; and RecA, ZP03591416.
These alignments were used to derive phylogenies based on neighbor-joining methods using the Mega4 suite of programs (40). Bootstrap values were generated based on 1.000 replicates of each calculation (consensus bootstrap trees had identical topologies).
To determine the functions of the putative homologues of MetB, CysK, and LuxS in H. pylori (MetBHp, CysKHp, and LuxSHp), we first constructed a panel of mutant strains deficient in each one of these gene products in two different H. pylori strain backgrounds (J99 and 11637). Each mutant was created using a deletion/insertion approach, using the aphA3 cassette (kanamycin resistance) as a selectable marker (19). The general approach to this mutagenesis strategy is shown in Fig. Fig.2A,2A, and details are described in Materials and Methods. Mutants were confirmed on the basis of PCR and DNA sequencing.
To avoid polar effects of disrupting either cysKHp or metBHp, we used a variant of aphA3 which lacks both the promoter and terminator elements and which we have used successfully in previous studies (46). Since luxS comprises the last of the genes in this operon, we were able to use the V. harveyi AI-2 assay (3, 47) to demonstrate that disruption of either cysKHp or metBHp did not block expression of downstream genes (Fig. (Fig.2B).2B). As predicted, insertion of aphA3 into the luxS gene abrogated AI-2 production completely (Fig. (Fig.2B2B).
In order to determine the effects of mutagenesis of cysKHp, metBHp, and luxSHp, we composed various chemically defined media (CDM) based on the complete CDM (cCDM) of Reynolds and Penn (31). This is a versatile system which allows precise control of the composition of the medium. cCDM contained both of the sulfur-containing amino acids, methionine and cysteine. We also made 4 variants of this medium, all of which lacked cysteine. One had no additional source of sulfur (we refer to this hereinafter as CDM), while the others contained either homocysteine (CDM+HC), cystathionine (CDM+CTT), or potassium sulfide (CDM+S) (see Materials and Methods for concentrations). Using these media with the wild type and the ΔcysKHp, ΔmetBHp, or ΔluxSHp mutants of both H. pylori strain 11637 and J99, we found that all strains grew well in cCDM (Fig. 3A to D for 11637l; J99 not shown). Upon omission of cysteine (CDM), the wild-type strain still grew, although less well than in the complete medium (Fig. (Fig.3A).3A). In contrast, all three mutants were auxotrophic for cysteine, with no growth detected (Fig. 3B to D). These observations confirmed that wild-type H. pylori is able to grow in the absence of cysteine and so possesses a cysteine de novo biosynthesis pathway. They also revealed that cysKHp, metBHp, and luxSHp are all part of this pathway.
We next examined which steps in our proposed H. pylori reverse transsulfuration pathway (RTSP) (Fig. (Fig.1)1) were catalyzed by LuxSHp, CysKHp, and MetBHp. These experiments gave the same results for mutants constructed in the 11637 and J99 backgrounds; the 11637 results are presented in Fig. 3B to D.
For the ΔluxSHp mutant, adding homocysteine to the CDM lacking cysteine improved growth of the ΔluxSHp strain, and CTT restored growth to levels seen with cCDM (CDM with cysteine) (Fig. (Fig.3B).3B). This implies that, as expected, LuxSHp is required for the conversion of SRH to homocysteine.
The ΔcysKHp mutant grew in cCDM (containing cysteine) and in CDM lacking cysteine but containing CTT (CDM+CTT) but not in CDM lacking cysteine (CDM) or CDM lacking cysteine but containing HC (CDM+HC) (Fig. (Fig.3C),3C), suggesting that CysKHp catalyzes the conversion of HC to CTT.
Finally, the ΔmetBHp mutant could grow only in cCDM, indicating an absolute requirement for cysteine (Fig. (Fig.3D).3D). This would be consistent with MetBHp catalyzing the final step from cystathionine to cysteine, although further experimentation is needed to confirm this (see below).
In contrast to results for the wild type, none of the mutants could grow in CDM lacking cysteine but containing potassium sulfide (CDM+S) (data not shown). Thus, it seems that CysKHp is not acting like CysK homologues in other bacteria (which use sulfide to generate cysteine), in agreement with the reported absence of the assimilatory sulfate reduction pathway in H. pylori (10).
Taking these observations together, we propose that H. pylori generates cysteine from methionine via components of the AMC, followed by reverse transsulfuration, with LuxSHp converting SRH to homocysteine and CysKHp converting homocysteine to cystathionine, which is finally converted to cysteine by MetBHp.
To investigate the individual steps in the proposed cysteine biosynthetic pathway and specifically to establish the likely substrates for LuxSHp, CysKHp, and MetBHp, we performed metabolite analyses of the wild-type and mutant strains grown in cCDM (Fig. (Fig.4).4). Specifically, we measured the relative levels of the AMC metabolites SAM, SAH, SRH, and HC, plus CTT. Although methionine and cysteine were also measured during this analysis, both amino acids were present in excess in the growth medium (cCDM). As such, their relative levels were not seen to vary significantly between the strains (data not shown).
For the ΔluxSHp mutant, the first important finding was that SRH was detectable in the cell extract whereas for the wild type it was undetectable (Fig. (Fig.4).4). The other main differences between the metabolite profiles of the ΔluxSHp mutant and the wild type were that HC and CTT were either undetectable or virtually undetectable in the mutant (0% and 0.4% of wild-type levels, respectively). This confirms that HC and CTT cannot be synthesized in the absence of LuxSHp. Furthermore, the accumulation of SRH confirms this to be the in vivo substrate of LuxSHp.
For the ΔcysKHp mutant, most strikingly, the levels of HC were 11- to 12-fold greater than those in the wild type, suggesting that this metabolite was accumulating in this strain. Like the ΔluxSHp mutant, the ΔcysKHp mutant produced minimal levels of CTT. The latter observation confirms that CysKHp is needed for the formation of CTT. The accumulation of HC suggests that this is the substrate for the CysKHp reaction. As an interesting aside, we found approximately 2-fold more SAH in cell extracts from the ΔcysKHp mutant than in those from the wild type. This is discussed below.
Finally, in the ΔmetBHp mutant (unlike the case for all other mutants), CTT was present and was at around 5-fold excess relative to the level in the wild type, indicating that this metabolite was accumulating in this strain. This suggests that CTT is the substrate for the MetBHp reaction. Interestingly, HC was also found to accumulate, although at a lesser level (2- to 3-fold), and, as for the ΔcysKHp mutant, we saw around 2-fold more SAH than in the wild-type extract. These findings may indicate a weak feedback inhibition effect of CTT on CysKHp and of homocysteine on PfsHp, the enzyme catalyzing the reaction immediately prior to the LuxSHp reaction in the AMC. Allosteric inhibition of metabolic enzymes by a downstream metabolite(s) is recognized as a common means of regulation of enzyme activity in prokaryotes; thus, these observations are unsurprising.
Having demonstrated the metabolic role of the cysK-metB-luxS gene cluster in H. pylori, we finally turned our attention to its presence in other bacteria and to its likely origin.
Analysis of the available H. pylori genomes showed that in all cases luxS is located downstream of the two other genes we have examined, annotated as cysK and metB. cysKHp, metBHp, and luxSHp lie in the same coding orientation. We found this same arrangement of genes in Helicobacter acinonychis (regarded as the most closely related Helicobacter species to H. pylori (7). However, in all other helicobacters and the related Campylobacter jejuni and Wolinella succinogenes, this cluster was absent, and homologues of cysK, metB, and luxS were found in diverse genomic locations and were not adjacent to one another.
Protein BLAST searches of CysKHp, MetBHp, and LuxSHp revealed that their closest known homologues are found in Enterococcus faecium, a Gram-positive organism (68%, 74%, and 80% amino acid identities, respectively). Importantly, the E. faecium genes exist as a cluster, with the genes lying in the same order as in H. pylori/H. acinonychis. We conducted phylogenetic analyses using multiple amino acid sequence alignments of LuxS, CysK, and MetB from H. pylori, Helicobacter hepaticus, Helicobacter cinaedi, Helicobacter bilis, Helicobacter pullorum, Helicobacter canadensis, Helicobacter winghamensis, Helicobacter acinonychis, W. succinogenes, C. jejuni, and E. faecium. We also included candidate Gram-negative sequences (E. coli) and two Gram-positive sequences, LuxS, MetB, and CysK from Staphylococcus aureus and LuxS, MccA/YrhA, and MccB/YrhB from Bacillus subtilis. We reconstructed phylogenies using sequences derived from the housekeeping gene product, RecA, from all 14 organisms. Phylogenetic trees were constructed using neighbor joining methods and are shown in Fig. Fig.55.
We found that with the housekeeping gene product RecA, as expected, the helicobacters all occupy the same cluster, which also contains the W. succinogenes sequence. Also as expected, the C. jejuni sequence associates with this cluster but is found to be isolated. The Gram-positive representatives group together, and the E. coli sequence forms an independent outgroup. We repeated this analysis with a further 7 housekeeping gene products (encoded by aroE, atpA, efp, engA, gapA, gyrA, and mutY), which all gave qualitatively similar results, with all of the helicobacter sequences clustering together and the Gram-positive sequences forming separate groups (data not shown). Conversely, with CysK, MetB, and LuxS, the H. pylori and H. acinonychis sequences grouped more closely with the Gram-positive sequences than with those of the other Helicobacter species or species shown to be more closely related to Helicobacter species by other phylogenetic methods (7, 14).
These data suggest that in H. pylori, the three-gene cluster cysK-metB-luxS exists as a conserved syntenic locus that was acquired by horizontal gene transfer from a Gram-positive, enterococcal bacterium after the division of H. pylori/H. acinonychis from the other Helicobacter species but before their division from each other.
LuxS is now generally understood to carry out at least two functions, one in metabolism and one in cell-cell signaling (3, 8, 32, 44, 48). Bacterial signaling systems based on LuxS-generated AI-2 are known to regulate gene expression in several members of the Enterobacteriales (23, 50), notably Vibrio species. Many other bacteria also possess luxS homologues and produce AI-2 but appear to lack an AI-2 sensory apparatus (i.e., LuxP/Q and the Lsr system, respectively) (32), in agreement with suggestions that in most species luxS fulfils primarily a metabolic role in the AMC (32, 47-49).
In this context, H. pylori presents an interesting case: although lacking homologues for the identified AI-2 receptors, the bacterium is known to respond to exogenously added, synthetic AI-2 (30), pointing toward a role of LuxS in cell-cell signaling. On the other hand, Lee and coworkers (2006) proposed that the reduced in vivo fitness observed for an H. pylori SS1ΔluxS mutant was caused by metabolic disturbances, possibly by a disruption of the metabolic flux through the AMC or other LuxS-dependent pathways (20). Interestingly, previous physiological work (26, 27, 31) and genome analyses have revealed that the AMC in H. pylori is incomplete, since the organism is a methionine auxotroph that lacks the genes required for methylation of homocysteine (metE or metH) (10, 28, 41). This made us question whether LuxS in H. pylori had any metabolic roles apart from generating AI-2 and if so, what these roles could be.
Intriguingly, in all currently sequenced H. pylori strains, luxS is part of a three-gene cluster that in addition to luxS contains two other genes with putative functions in methionine and cysteine metabolism (Fig. (Fig.2A).2A). These genes, currently annotated as metB and cysK, until now have been proposed to encode cystathionine γ-synthase and cysteine synthase, respectively (10). The gene cluster (cysK, metB, and luxS) is not only conserved among H. pylori strains and H. acinonychis but also is present in several Gram-positive bacteria (49), suggesting that metBHp and cysKHp are not evolutionary relics of now-inactive metabolic pathways but still fulfil a metabolic function linked to that of luxSHp.
Our hypothesis that the cysK-metB-luxS cluster is responsible for cysteine formation was based on several considerations. First, all H. pylori strains studied by Reynolds and Penn (1994) and the vast majority of strains characterized by Nedenskov (1994) were able to grow without cysteine (27, 31). Since the organism is not capable of using sulfate as a sulfur source (10), a hitherto-unidentified pathway must exist that makes use of the reduced sulfur present in methionine to generate cysteine. Second, the metabolic fate of the homocysteine generated in the LuxS reaction is unclear. High concentrations of homocysteine are toxic in some organisms (33, 43). Third, only two enzymatic steps are required to convert homocysteine to cysteine in the so-called reverse transsulfuration pathway (RTSP) present in some bacteria (for example, in B. subtilis and Pseudomonas putida), where part of the AMC, including LuxS, is known to contribute to methionine-to-cysteine conversion by generating the required homocysteine (16, 45). In addition, the genes in the B. subtilis cluster that show similarity to cysKHp and metBHp have recently been shown to encode functional cystathionine β-synthase and cystathionine γ-lyase, respectively, and have therefore been renamed MccA and MccB (methionine-to-cysteine-conversion genes) (16).
The results of our study are straightforward and in full agreement with the above hypothesis. First, we were able to demonstrate that all genes of the cysK-metB-luxS cluster in H. pylori (in both 11637 and J99) are required for cysteine prototrophy: mutation of any one of these genes rendered the respective strain auxotrophic for cysteine. Chemical complementation restored the growth of all mutants and also indicated a sequential order for the involved enzymatic activities, which is LuxS-CysK-MetB. Our results suggested that the true physiological function of the cysKHp gene product lies in the conversion of homocysteine to cystathionine; the latter is then used by MetBHp to generate cysteine. This interpretation was further corroborated and extended by metabolite profiling. In full agreement with the proposed metabolic functions, the metabolite pools of ΔluxSHp mutants showed accumulation of SRH and depletion of both homocysteine and cystathionine, whereas ΔcysKHp mutants accumulated homocysteine and were depleted for cystathionine. ΔmetBHp mutants were the only strains that accumulated cystathionine. Thus, it appears that an RTSP is operational in H. pylori.
To unequivocally establish the nature of the reactions that link homocysteine with cysteine in H. pylori will require purification and detailed characterization of CysKHp and MetBHp. The latter enzyme has already been studied, but not with respect to its physiological function. Kong et al. (18) cloned the respective gene from H. pylori SS1 and purified the recombinant protein. However, the enzymatic activity of MetBHp was measured only using an unphysiological side reaction that is observed for some enzymes in vitro in the presence of O-acetyl-serine and in the absence of other substrates (18). Thus, the true physiological role of MetBHp remained unclear. With regard to CysKHp, a similar enzyme from Lactobacillus casei, also annotated CysK, has recently been proposed to act as a cystathionine β-synthase, converting homocysteine to cystathionine (17). CysKHp has up to now been thought to act as a cysteine synthase that uses sulfide to generate cysteine (10). However, our finding that sulfide cannot support growth of H. pylori in the absence of cysteine, at least under our conditions, suggests that CysKHp is solely dedicated to homocysteine conversion. Thus, CysKHp appears to be a cystathionine β-synthase, and MetBHp appears to be a cystathionine γ-lyase. We therefore suggest that the genes encoding these proteins be renamed. Specifically, we suggest that cysKHp be renamed mccA (methionine-to-cysteine-conversion gene A) and that metBHp be renamed mccB.
The relevance of the established cysteine biosynthesis pathway for the lifestyle, fitness, and virulence of H. pylori is not clear. The reduced in vivo fitness observed for the H. pylori SS1 luxS mutant (20) may indeed be linked to its inability to convert methionine to cysteine, although a role of AI-2 cannot be excluded. H. pylori and other members of this genus have lost the ability to reduce sulfate (10; our unpublished data). However, it can be assumed that they are well adapted to their specific habitats, which therefore must provide sufficient reduced sulfur sources for these organisms to thrive. Presumably, the conversion of homocysteine to cysteine removes a potentially toxic metabolite and at the same time reduces the (energetically costly) need for cysteine uptake. On the other hand, a similar effect would have been achieved had the organism maintained a functional AMC.
Whatever the selective advantage gained, our genome comparisons revealed that the entire cysK-metB-luxS cluster was obtained by horizontal gene transfer from a Gram-positive bacterium closely related to E. faecium. Several previous analyses already established the Gram-positive origin of luxSHp but did not consider the remaining genes of the cluster (21). Interestingly, similar clusters exist in a number of Gram-positive species (49), for instance, Oceanobacillus iheyensis, Clostridium perfringens, and Clostridium botulinum. Intriguingly, in some Bacillus species, notably B. subtilis, Bacillus cereus, and Bacillus anthracis, similar clusters contain the pfs gene instead of luxS. It thus appears that during the course of evolution, the common ancestor of H. pylori and H. acinonychis has lost its native luxS gene and instead acquired a Gram-positive homologue together with the ability to generate cysteine.
Finally, we would point out that in the absence of exogenous cysteine, growth of H. pylori will be limited by its capacity to generate this compound from homocysteine. Homocysteine availability, in turn, is determined by the number of methylation reactions carried out by the cells (i.e., the number of SAM molecules that can be converted to SAH and then further to homocysteine via the action of Pfs and LuxS; see Fig. Fig.1).1). Under these conditions, generation of the quorum-sensing products comprising AI-2, at least in theory, is directly proportional to growth of the population, and the resulting AI-2 concentration should thus provide a very accurate measure of population density. It is now important to determine whether the other phenotypes associated with luxS mutagenesis in H. pylori are dependent on the homocysteine biosynthesis role of LuxS or its role as the AI-2 synthase.
We thank Leo Smeets (Reinier de Graaf Hospital, Delft, The Netherlands), Theo Verboom (VU University Medical Centre, The Netherlands), and Bonnie Bassler (Princeton University) for kindly providing strains for this study. We also thank Paul Williams (Nottingham University) for his support and discussion of this project and Catherine Ortori (Nottingham University) for technical assistance.
This project was generously supported by a studentship awarded to F.S. by Overseas Research Students Awards Scheme (ORSAS) and Nottingham University, by project grant support from Cancer Research UK, and by funds provided by the Institute of Infection, Immunity and Inflammation (University of Nottingham), the Biotechnology and Biological Sciences Research Council, the Medical Research Council (program grant G9219778), and the National Institute of Health Research through its funding of the Nottingham Digestive Diseases Centre Biomedical Research Unit.
Published ahead of print on 8 January 2010.