In a very simplistic way, methylotrophy can be divided into three specific metabolic goals: (i) oxidation (demethylation, dehalogenation) of a primary methylated substrate, producing formaldehyde, methyl- or methylene radical (typically transferred onto tetrahydrofolate; H4F), (ii) oxidation of formaldehyde (methyl- or methylene-H4F) to CO2, and (iii) assimilation of a C1 unit, the latter taking place at the levels of formaldehyde [the ribulose monophosphate (RuMP) cycle], CO2[the Calvin–Benson–Bassham (CBB) cycle], or a combination of methylene-H4F and CO2 (the serine cycle; Fig. 1). Described below are the major and the most well-studied pathways and enzymes that are used for these tasks by methylotrophs, or ‘the parts’ of methylotrophy.
The two well-studied types of methane-oxidizing enzymes, the soluble methane monooxygenase (sMMO) and the membrane-bound methane monooxygenase (pMMO) remain the hallmark enzymes in the aerobic methane oxidation (Hakemian and Rosenzweig, 2007; Trotsenko and Murrell, 2008). The pMMO appears to be the enzyme almost universally widespread among methanotrophs, while the sMMO is only found in some organisms, typically in addition to pMMO, a single exception so far being members of the family Beijerinckiaceae that only possess sMMO (Dedysh et al., 2000; Chen et al., 2010a; Vorobev et al., 2011). The diversity of the pMMO enzymes has been expanded recently to include orthologues that significantly diverge in sequence, and gene clusters encoding these orthologues tend to be present in the genomes of known methanotrophs, along with the ‘canonical’ pMMO gene clusters (Tavormina et al., 2010). In some cases, a function has been determined for a divergent orthologue. For example, a divergent pMMO has been shown to be responsible for high-affinity methane oxidation in Methylocystis sp. strain SC2, with the ‘canonical’ enzyme being responsible for low-affinity methane oxidation (Baani and Liesack, 2008). However, in most cases, functions of the divergent pMMO orthologues remain unknown (Tavormina et al., 2010).
The importance of pMMO originally demonstrated in methanotrophic Proteobacteria continues to hold when it comes to the newly discovered methanotroph phyla, such as aerobic methanotrophic Verrucomicrobia (Methylacidiphilum infernorum; Hou et al., 2008; Op den Camp et al., 2009) and anaerobic methanotrophs belonging to a candidate Phylum NC10 (Candidatus Methylomirabilis oxyfera; Ettwig et al., 2010). Although these recentlydiscovered organisms are unrelated to proteobacterial methanotrophs, they appear to employ typical if evolutionarily diverged pMMOs to metabolize methane. The genomes of verrucomicrobial methanotrophs contain multiple copies of pMMO gene clusters, one of which encodes a divergent enzyme, all copies being expressed (Hou et al., 2008; Op den Camp et al., 2009), while the genome of Candidatus M. oxyfera contains a single pMMO gene cluster. In the latter case, even though the organism grows anaerobically, methane oxidation by pMMO still requires oxygen, which is proposed to be generated by a novel enzyme, NO dismutase, the identity of which remains unknown (Ettwig et al., 2010). Thus, this latter process represents a ‘false’ anaerobic methane oxidation.
True anaerobic methane oxidation so far has only been demonstrated in Archaea, and, based on metagenomic analyses of the communities enriched in ANME type archaea (Knittel and Boetius, 2009), it is proposed to proceed through a reverse methanogenesis pathway (Hallam et al., 2004; Thauer and Shima, 2008; also see discussion by Chistoserdova et al., 2005b). Recent experiments demonstrating methane activation by methyl-CoM reductase from a methanogenic archaeon (Scheller et al., 2010) not only corroborate this hypothesis, but also indicate that all methanogens carry a potential in methane oxidation. While, at the moment, oxidation of methane via reverse methanogenesis is an attribute of Archaea, it is not inconceivable that a similar pathway may be present in yet undiscovered anaerobic bacteria. It has been now well documented that gene complements enabling a partial reverse methanogenesis pathway are present in diverse groups of bacteria (Chistoserdova et al., 2004a; Kalyuzhnaya et al., 2005a), including methylotrophs, in which this pathway serves for formaldehyde oxidation (Chistoserdova et al., 1998; 2005c and see below).
Over decades, much of the work in the field of methylotrophy concentrated on methanol dehydrogenase (MDH), an enzyme catalysing methanol oxidation and another hallmark of methylotrophy (Anthony, 1982; 2004; Williams et al., 2005). A MDH first characterized in Methylobacterium extorquens (Anthony, 1982; 2004) and later detected in most of the model methylotrophs was a heterotetramer consisting of subunits encoded by genes designated mxaFI, contained pyrroloquinoline quinone (PQQ) as cofactor and required a cytochrome c electron acceptor encoded by mxaG that was part of the same gene cluster (Lidstrom et al., 1994; Vuilleumier et al., 2009), along with the accessory genes encoding functions required for inserting Ca++ into the active centre of the enzyme (mxaACKL) and other yet unknown functions (mxaRS) essential for generating an active MDH. However, while the enzyme has been now studied in great detail, it is becoming clear that the classic MDH is not every methylotroph's choice. Indeed, few mxaF genes are found in metagenomic data sets representing important environmental samples. For example, while Methylophilus-like and Roseobacter-like sequences represent some of the most abundant ribotypes in global ocean samples (Rusch et al., 2007), few mxaF homologues were recorded in the same metagenomic data set (L. Chistoserdova, unpubl. obs.). Likewise, analysis of a Lake Washington metagenomic data set specifically enriched in methylotroph sequences via stable isotope probing (Kalyuzhnaya et al., 2008a) revealed the presence of few mxaF genes, suggesting that the distribution of this metabolic module in freshwater environments is also limited.
Recently, it has been demonstrated that the newly discovered methylotrophs belonging to the orders of Burkholderiales and Rhodocyclales encode a different type of MDH, named MDH2 (Kalyuzhnaya et al., 2008b). The properties of the partially purified enzymes responsible for the MDH activity in these species were somewhat different from those exhibited by typical mxaFI-encoded enzymes, and the enzyme appeared to be composed of a single type of subunit. Protein sequences translated from mdh2 genes showed very low similarity to MxaF proteins (less than 35% amino acid identity). However, they were highly similar (up to 80% amino acid identity) to a class of PQQ-linked dehydrogenases that included alcohol dehydrogenases, typically revealing low affinity for methanol (Kalyuzhnaya et al., 2008b).
A number of methylotrophs have now been characterized that are capable of methanol metabolism but contain neither mxaFI nor mdh2 (Giovannoni et al., 2008; Hou et al., 2008; Kalyuzhnaya et al., 2008b). These must possess a different type of MDH. One gene, named xoxF, encoding a polypeptide whose sequence is very similar to the sequence of MxaF (approximately 50% amino acid identity) has been proposed to encode an enzyme replacing the classic MDH (Giovannoni et al., 2008; Hou et al., 2008; Wilson et al., 2008). However, the role of XoxF in methylotrophy has not been unequivocally proven (see discussion below).
Gram-positive methylotrophic bacteria possess PQQ-independent, NAD(P)-binding alcohol dehydrogenases revealing no homology to MDH enzymes from Gram-negative bacteria. These form decameric structures of a single type of subunit and possess broad substrate specificities, similarly to MDHs from Gram-negative bacteria (Bystrykh et al., 1993; Brautaset et al., 2004).
Oxidation of methylamine can be carried out either via direct oxidation to formaldehyde, by methylamine dehydrogenase (MADH; in Gram-negative bacteria) or by methylamine oxidase (in Gram-positive bacteria), or via an indirect pathway that involves transfer of the methyl group onto glutamate, followed by the oxidation of N-methylglutamate (NMG), presumably to methylene-H4F (Chistoserdova et al., 2009; Latypova et al., 2010). Of these known systems, the MADH system is most well studied and represents another hallmark of methylotrophy (Davidson, 2004; Wilmot and Davidson, 2009). So far, all the organisms possessing the MADH enzyme system, encoded by a number of conserved genes (mauBEDAGLMN), have been demonstrated to be bona fide methylotrophs. The mau gene clusters and respective enzymes and accessory proteins are well conserved among Alpha-, Beta- and Gammaproteobacteria, with the exception of the specific electron acceptor that may be represented by an amicyanin (Chistoserdov et al., 1994), an azurin (Gak et al., 1995) or a cytochrome (Kalyuzhnaya et al., 2008a). In some organisms, the mau gene cluster was found to be flanked by IS elements, suggesting that the methylamine oxidation capability may be a subject of lateral gene transfers (Vuilleumier et al., 2009). Indeed, there are a few examples of closely related methylotroph species differing with regard to possessing the mau genes (Kalyuzhnaya et al., 2008a; Vuilleumier et al., 2009), supporting this hypothesis.
While, in terms of its environmental distribution and the role in methylamine-degrading capability, the NMG pathway may be as important if not more important than MADH, the genetic determinants for this pathway have been revealed only recently (Latypova et al., 2010), indentifying three specific enzymes involved in the pathway, γ-glutamylmethylamide synthase, N-methylglutamate synthase and N-methylglutamate dehydrogenase, often encoded by a single cluster of genes (Latypova et al., 2010; Chen et al., 2010b). In some methylotrophs, the NMG pathway is the only pathway for methylamine oxidation (Latypova et al., 2010; Chen et al., 2010b), while in others it is present along with MADH, playing a secondary role (Hendrickson et al., 2010). The NMG pathway is also present in non-methylotrophs, which employ it for utilization of methylamine as a nitrogen source (Chen et al., 2010c). So far, like MADH, this pathway has only been found in Proteobacteria and has also been proposed to be a potential subject of lateral gene transfers (Chen et al., 2010c).
Few methylamine oxidases have been characterized in Gram-positive methylotrophs so far (Hartmann and McIntire, 1997; Wilce et al., 1997). Based on sequence homology, they are closely related to a range of amine oxidases with different substrate specificities, all covalently binding cofactor topaquinone and requiring Cu2+ ions. Such amine oxidases are widespread among Gram-positive and Gram-negative bacteria and eukaryotic organisms. As a single gene is sufficient to encode such an enzyme, genome-based predictions of methylotrophy are difficult based on a single respective gene.
Other primary C1 substrate-oxidizing pathways
A variety of other methylated compounds serve as substrates for methylotrophs, and their metabolism is enabled by a number of well-characterized specific modules. Some of these modules are involved in degradation of important green house gases other than methane, highlighting the role of methylotrophs in global cycling of not only carbon but nitrogen, sulfur and halogenated compounds (Kelly and Murrell, 1999; Schäfer et al., 2007; Trotsenko and Murrell, 2008; Boden et al., 2011). Degradation of monohalogenated methanes such as chloromethane or bromomethane involves two specific methyltransferases, CmuA and CmuB, the former transferring the methyl group of a monohalogenated methane to a corrinoid protein, and the latter further transferring it to H4F (Vannelli et al., 1999; Schäfer et al., 2007). Degradation of dihalogenated methanes is carried out by a specific dehalogenase/glutathione S-transferase (DcmA; Leisinger et al., 1994). Some of the most environmentally important methylated sulfur species are dimethylsulfoniopropionate (DMSP), dimethylsulfide (DMS) and methanesulfonic acid (MSA; Kelly and Murrell, 1999; Yoch, 2002). The latter two are known to promote growth of methylotrophs, while degradation of DMSP has not been described as a bona fide case of methylotrophy so far. Microbial degradation of DMSP is known to proceed via two alternative routes, demethylation (catalysed by DMSP-dependent demethylase DmdA) and cleavage, catalysed by a few different types of DMSP lyases producing DMS (Todd et al., 2011). The demethylation pathway appears to be the predominant pathway in nature, reportedly utilized by over half of the bacterioplankton cells in ocean surface waters (Howard et al., 2011). Some of the bacteria degrading DMSP rely on it as the source of sulfur (Tripp et al., 2008), while others, such as Ruegeria (Silicibacter) pomeroyi, must also be able to utilize it as a carbon source (see below). DMS is degraded by the action of DMS monooxygenase (DmoAB; Boden et al., 2011). The specific module responsible for degradation of MSA is the MSA-specific monooxygenase (MsmABCD; Baxter et al., 2002). Environmentally important methylated nitrogen-containing compounds upstream of methylamine include trimethylamine and its product dimethylamine, the modules for their degradation being respective dehydrogenases (TMAD, DMAD) and respective monooxygenases (TMAM, DMAM; Anthony, 1982; Fig. 1). The TMA/DMA-oxidizing enzymes are not methylotrophy-specific as they are known to function in nitrogen metabolism of diverse bacteria (De Boer et al., 1989; Liffourrena et al., 2010). It is likely that additional, not yet recognized primary oxidation modules supporting methylotrophic metabolism exist, and these are still awaiting to be discovered.
Oxidation of formaldehyde is an important step in methylotrophy, not only in terms of energy generation but also in terms of keeping the intracellular levels of formaldehyde at non-toxic levels. Methylotrophs have a variety of formaldehyde-oxidizing systems from which to chose. These range from a single gene/single enzyme systems [NAD-linked formaldehyde dehydrogenases (FaDH); mycothiol-linked FaDH] to multigene/multienzyme cofactor-linked C1 transfer pathways (Vorholt, 2002; Chistoserdova et al., 2009). One clever way to oxidize formaldehyde is by employing the reactions of the assimilatory RuMP cycle with the addition of one single enzyme, 6-phosphogluconate dehydrogenase (Anthony, 1982). Most well-studied methylotrophs possess more than one means for oxidizing formaldehyde, while in novel and less well-studied methylotrophs, formaldehyde-oxidizing systems are yet to be experimentally defined (Giovannoni et al., 2008; Hou et al., 2008).
It is intriguing to observe that some of the simple and very efficient single enzyme systems such as NAD-linked, glutathione-independent FaDH (exemplified by the well-studied enzyme from Pseudomonas putida; Tanaka et al., 2003) are not found in most methylotrophs. However, they appear to be the major formaldehyde detoxification systems in organisms such as Pseudomonas and non-methylotrophic Burkholderia species that, like methylotrophs, tend to possess multiple formaldehyde oxidation systems (Marx et al., 2004; Roca et al., 2009). Conversely, one of the most elaborate pathways, the one employing tetrahydromethanopterin (H4MPT) as a cofactor and encoded by at least 20 genes, is the most widespread pathway for formaldehyde oxidation in methylotrophs (Chistoserdova et al., 2009). This pathway was first discovered in M. extorquens AM1 (Chistoserdova et al., 1998) and demonstrated to be an indispensable pathway for both formaldehyde oxidation with generation of NAD(P)H and formaldehyde detoxification is this organism (Hagemeier et al., 2000; Marx et al., 2003a). Mutants of M. extorquens with lesions in the genes involved in this pathway are all methylotrophy-negative, and some demonstrate a remarkable threshold for methanol sensitivity (as low as 90 µM while wild-type organism grows at 100 mM; Chistoserdova et al., 2005c). However, in the organisms possessing dissimilatory RuMP cycle, the H4MPT-linked pathway appears to serve an auxiliary function as the cyclic pathway is the major pathway in oxidizing and detoxifying formaldehyde, and mutants with lesions in this pathway can be easily generated (Chistoserdova et al., 2000; Kalyuzhnaya et al., 2005a). Some autotrophic methylotrophs, such as Paracoccus and the closely related Rhodobacter species (the latter use methanol photosynthetically), rely on the glutathione (GSH)-linked formaldehyde oxidation pathway as the major means for both energy generation and formaldehyde detoxification (Ras et al., 1995; Barber and Donohue, 1998), and these species do not contain genes or activities for the H4MPT-linked pathway. With this respect it is interesting to point out that heterologously expressed genes for this pathway were able to complement mutants of M. extorquens AM1 deficient in genes of the H4MPT-linked pathway (Marx et al., 2003a).
Gram-positive methylotrophs, beside the cyclic route, employ mycothiol-dependent FaDHses (Vorholt, 2002). In addition, dye-linked enzymes have been described for some methylotrophs (Klein et al., 1994) but their role in methylotrophy remains poorly understood.
A C1 transfer pathway linked to tetrahydrofolate (H4F), analogous to the H4MPT-linked pathway, exists in many methylotrophs (and across all life). Typically, this pathway involves FolD, a bifunctional enzyme possessing methylene-H4F dehydrogenase and methenyl-H4F cyclohydrolase activities. Most methylotrophs for which genome sequences are available possess this gene (see Table 1), and in methylotrophs that grow on compounds whose primary degradation involves demethylation reactions, such as chloromethane-degrading M. extorquens (formerly chloromethanicum) CM4, FolD appears to be specifically involved in dissimilation of these compounds, along with respective methyltransferases (CmuAB), methyl-H4F reductase (MetF) and formyl-H4F hydrolase (PurU; Vannelli et al., 1999).
Table 1. Examples of occurrence of methylotrophy metabolic modules in major functional groups.
However, some methylotrophs, for example M. extorquens AM1 and Methylococcus capsulatus, do not posses FolD. Instead, they posses two alternative enzymes, MtdA and Fch, responsible for methylene-H4F dehydrogenase and methenyl-H4F cyclohydrolase activities respectively. These enzymes have been characterized in M. extorquens AM1, and, based on enzyme kinetics (Vorholt et al., 1998; Pomper et al., 1999), mutant phenotypes (Chistoserdova and Lidstrom, 1994; Marx and Lidstrom, 2004) and metabolite flux analysis (Marx et al., 2005; Crowther et al., 2008), they appear to be involved in a pathway that acts in a reductive rather than oxidative direction in this organism, i.e. this pathway enables transfer of C1 units into the serine cycle using H4F as an adduct (Crowther et al., 2008). The latter study thus challenged the long-standing dogma of spontaneous reaction between formaldehyde and H4F being of physiological significance. The difference in physiological roles of MtdA and FolD is highlighted by mutant complementation experiments: while MtdA could act as a functional substitute for FolD (Studer et al., 2002), FolD could not complement a lesion in MtdA (Marx and Lidstrom, 2004).
Intriguingly, while not revealing sequence homology with FolD, MtdA is homologous to MtdB, and is also active with H4MPT (Chistoserdova et al., 1998; Vorholt et al., 1998; Hagemeier et al., 2000; Vorholt, 2002). The significance of this activity remains unclear as MtdA cannot substitute for MtdB in the H4MPT-linked oxidative pathway (Chistoserdova et al., 1998; L. Chistoserdova, unpublished), but it suggests that this enzyme may have an additional, not yet identified function.
To complicate the matters further, a novel class of Mtd enzymes, named MtdC, have been recently described. These are so far found in representatives of the division Planctomycetes and in a novel deeply divergent phylum of uncultivated bacteria from Lake Washington (Vorholt et al., 2005), which I will term here Phylum LW for convenience (Supporting information, Fig. S1). These enzymes had high affinities for methylene-H4MPT and NADP but low affinities for methylene H4F or NAD, distinguishing them from MtdA and MtdB enzymes (Supporting information, Table S1). Accordingly, it has been demonstrated that MtdC cannot functionally substitute for either MtdA or MtdB (Vorholt et al., 2005). Thus, while phylogenetically more related to MtdA, MtdC must fulfil a function more similar to the function of MtdB, as part of the H4MPT-linked pathway for formaldehyde oxidation/detoxification (Vorholt et al., 2005).
Analysis of the recently available genomic sequences suggests that the diversity of Mtd enzymes must extend beyond the Mtd enzymes described above. While the divergent Mtd protein in the newly described genome of NC10 phylum methylotroph Candidatus M. oxyfera is likely a divergent MtdB (Supporting information, Fig. S1), the divergent Mtd proteins in the genomes of Methylophaga thiooxidans, Nitrosococcus halophilus and Anaerobaculum hydrogeniformans may represent new classes and may express a different set of substrate specificities, based on their phylogenetic positions (Supporting information, Fig. S1). It is noteworthy that so far genes encoding Fch enzymes have only been found in organisms possessing MtdA but not MtdC, suggesting that the novel Mtd enzymes are likely employed in the dissimilatory rather than assimilatory pathways.
Understanding the specific functions of different variants of H4F-utilizing C1 transfer pathways is further complicated by the use of two alternative enzymes for transferring C1 units between the levels of formyl-H4F and formate: formyl-H4F ligase (FtfL) being a reversible enzyme (Marx et al., 2003b) and formyl-H4F hydrolase (PurU) being a non-reversible enzyme (Nagy et al., 1995). Thus, in the organisms possessing FtfL the pathway can in principle operate in both directions while in the organisms possessing only PurU the pathway can only operate in the oxidative direction. It is remarkable that the organisms utilizing the MtdA/Fch variant of the pathway always employ FtfL, while organisms utilizing the FolD variant can employ either FtfL or PurU or both (Table 1). Few methylotrophs, exemplified by Methylibium petroleiphilum and M. extorquens CM4 encode both variants of the pathway (Vannelli et al., 1999; Studer et al., 2002; Kane et al., 2007). In the latter, PurU was demonstrated to specifically operate in degradation of chloromethane, and FtfL cannot functionally substitute for PurU (Vannelli, 1999; Studer et al., 2002). However, from the experimental data available, it is not clear whether FtfL is simply not present at sufficient levels under these growth conditions or whether it can only operate in the reductive direction in this organism.
Methylotrophs, like other organisms, tend to encode multiple formate dehydrogenases (FDH). For example, M. extorquens species possess four different FDH enzymes, which include a tungsten-containing FDH (FDH1; Laukel et al., 2003), a predicted molybdenum-containing FDH (FDH2; Chistoserdova et al., 2004b), a predicted cytochrome-linked FDH that is likely periplasmic (FDH3; Chistoserdova et al., 2004b), and a novel type of FDH (FDH4; Chistoserdova et al., 2007a), and all of these appear to be functional. However, when each of the enzymes was disrupted in a wild-type background, mutants in only one of them, FDH4, revealed a phenotype: these mutants accumulated formate in the late stationary phase of growth on methanol (Chistoserdova et al., 2007a). If an intact FDH4 was present, the three other enzymes appeared redundant during growth on methanol. However, the situation was different when the organism grew on formate. FDH4 alone could not sustain the growth, and at least one of the other FDH enzymes was needed (Chistoserdova et al., 2004b). FDH4 in this organism also appeared to be somehow involved in acid stress response, but the mechanism of this involvement remains unknown (Chistoserdova et al., 2007a).
In organisms oxidizing formaldehyde via the RuMP cycle, the formate oxidation step is expected to be less critical for methylotrophy, and, accordingly, activities of FDH enzymes are typically measured at very low levels (Anthony, 1982). Relative contributions of alternative FDHses to methylotrophy were recently investigated in a RuMP cycle methylotroph, Methylobacillus flagellatus that possesses FDH enzymes homologous to FDH2 and FDH4 of M. extorquens (Hendrickson et al., 2010). It was concluded from this study that the formate oxidation step is essential even when the dissimilatory RuMP cycle is functionally present, as no mutants could be generated simultaneously lacking both FDHses, and FDH4 appeared to play a more important function in fitness of this organism compared with FDH2 (Hendrickson et al., 2010).
Overall, based on the available genomic data, FDHses are not conserved among methylotroph groups, but rather these are randomly distributed. For example, close homologues of the FHD2 enzymes described above are widespread across methylotrophic and non-methylotrophic Proteobacteria, while non-proteobacterial methylotrophs encode FDH enzymes homologous to the ones of other representatives of respective (or related) phyla (Hou et al., 2008; Ettwig et al., 2010).
The RuMP cycle
The RuMP cycle functions in adding a molecule of formaldehyde onto a C5 sugar (RuMP), thus producing a familiar currency of universally widespread cellular metabolism, glucose 6-phosphate, part of which is then converted into C3 molecules and used for cell biosyntheses, another part is oxidized generating NAD(P)H, and the remaining part is used to regenerate the acceptor molecule (Anthony, 1982). Only two specific enzymes, hexulosephosphate synthase (HPS) and hexulosephosphate isomerase (HPI), are needed to enable the RuMP cycle, in addition to the enzymes of glycolysis, pentosephosphate cycle and Entner–Doudoroff pathways. High activities of HPS and HPI are typically signatures of methylotrophy via the RuMP cycle (Anthony, 1982; Lidstrom, 2006). However, the presence of the hps/hpi gene pair is not always a sign of methylotrophy capability as the same cycle is utilized by some non-methylotrophs, serving as an additional means for formaldehyde detoxification (Yasueda et al., 1999). Indeed, homologues of hps/hpi are found in the genomes of a variety of non-methylotrophic species, including Bacillus, Escherichia, etc. and also in some Archaea (Kato et al., 2006).
The serine cycle. In the serine cycle, methylene-H4F and CO2 are assimilated through a series of reactions involving amino acids and organis acids, to result in C3 and C4 metabolites for building biomass and glyoxylate that serves as the acceptor for methylene-H4F (Anthony, 1982; Chistoserdova et al., 2009; Peyraud et al., 2009). The cycle, while specific to methylotrophy, borrows a number of genes/enzymes from other common metabolic pathways and some of these (such as serine hydroxymethyltransferase, enolase and malate dehydrogenase) have dual functions in methylotrophs, being involved in pathways with different metabolic goals. Key enzymes that are typically measured to indicate the operation of the serine cycle are hydroxypyruvate reductase and serine glyoxylate aminotransferase. Serine cycle genes are typically found in clusters, and are subjects of coordinated regulation (Kalyuzhnaya and Lidstrom, 2003; 2005). Such clusters have been analysed in a number of methylotrophs and potential methylotrophs (Vuilleumier et al., 2009), and their structure and content suggest a complex history. For example, the structure of the serine gene cluster first characterized in M. extorquens AM1 (Chistoserdova et al., 2003) is highly conserved in some alphaproteobacterial methylotrophs, and these clusters are remarkably syntenic with the cluster in a betaproteobacterial methylotroph, M. petroleiphilum (Vuilleumier et al., 2009). However, the genome of R. pomeroyi, also an Alphaproteobacterium, contains a cluster that shows no synteny to the M. extorquens cluster (Vuilleumier et al., 2009). In addition, some of the genes in the cluster reveal very low levels of homology to their counterparts in other Alphaproteobacteria, suggesting independent evolution of the serine cycle in different bacterial lineages. Gene clustering is also different in Hyphomicrobium denitrificans, and some of the genes (such as that encoding phosphoenolpyruvate carboxylase, PPC, HPR) show little homology with other PPC and HPR genes.
Genomes of gammaproteobacterial methanotrophs also harbour most of the serine cycle genes, but few of them are clustered (Ward et al., 2004). However, the gene for one key enzyme of the serine cycle, the PPC, is missing from the genomes. Thus the cycle might be incomplete in these bacteria, or a modification of the cycle using an alternative enzyme is operational. The proteomic analysis of M. capsulatus revealed that most of the serine cycle genes are expressed during growth on methane (Kao et al., 2004).
Glyoxylate regeneration. The assimilation of C1 units via the serine cycle requires regeneration of glyoxylate from acetyl-CoA. It has been recognized early on that, while this task can be carried out by the classic glyoxylate shunt, many serine cycle methylotrophs employ instead an alternative glyoxylate regeneration pathway (Anthony, 1982) that remained a puzzle for over three decades. This pathway has been recently resolved by a combination of efforts from different laboratories, and it is now known as ethylmalonyl-CoA pathway (EMCP; Erb et al., 2007; 2008; Chistoserdova et al., 2009; Peyraud et al., 2009). The pathway shares reactions and enzymes with the serine cycle (malate thiokinase, malyl-CoA lyase), the tricarboxylic acid cycle (succinate dehydrogenase, fumarase), the polyhydroxybutyrate cycle (beta-ketothiolase, acetoacetyl-CoA reductase) and with other metabolic pathways (ethylmalonyl-CoA mutase, propionyl-CoA carboxylase), in addition to the specific reactions such as ethylmalonyl-CoA mutase (Erb et al., 2008) and crotonyl-CoA reductase/carboxylase (Erb et al., 2007). Respectively, genes for the latter enzymes serve as markers for probing genomes for the presence of EMCP. The EMCP is not specific to methylotrophy. It is also utilized for metabolism of C2 compounds by both facultative methylotrophs, such as M. extorquens (Okubo et al., 2010), and non-methylotrophic bacteria such as Rhodobacter and Streptomyces (Alber, 2010). In the latter case, genes for the EMCP are present in the genomes but not the genes for the serine cycle. It is interesting to point out that the EMCP functions as a cycle during growth on C1 compounds (regenerating a molecule of glyoxylate per each molecule of methylene-H4F and each molecule of CO2 assimilated; Chistoserdova et al., 2009), while during growth on C2 compounds, it functions as a liner pathway (Erb et al., 2010; Okubo et al., 2010).
Some methylotrophs, however, opt to use the glyoxylate shunt instead of the EMCP. For example, the genomes of M. petroleiphilum and Methylocella silvestris, while lacking genes for the key enzymes of the EMCP, contain the genes for the glyoxylate shunt (Kane et al., 2007; Chen et al., 2010a) and the genome of H. denitrificans encodes both pathways. In M. capsulatus, neither the EMCP nor the glyoxylate shunt appears to be encoded, consistent with the hypothesis of this pathway fulfilling an auxiliary metabolic task.
The CBB cycle. A number of methylotrophs possess genes and enzymes of the CBB cycle, some as the only means for C1 assimilation and others in addition to other C1 assimilatory cycles. In the latter case, the contribution of the CBB cycle to methylotrophy remains poorly understood (Chistoserdova et al., 2005b). The three major groups that rely on the CBB for methylotrophy are (i) the alphaproteobacterial autotrophs (such as Paracoccus denitrificans and Xanthobacter autotrophicus; Baker et al., 1998), (ii) methanotrophic Verrucomicropbia (M. infernorum; Op den Camp et al., 2009) and (iii) methanotrophs belonging to the NC10 phylum (Ettwig et al., 2010). Based on sequence comparisons, the key CBB genes (cbbLS) may be subjects of lateral transfers. For example, two cbb gene clusters are found in the genome of M. petroleiphilum, one more related to the clusters in other Betaproteobacteria, the other more related to the clusters in Alphaproteobacteria (Kane et al., 2007). While analysis of the genome of the NC10 representative Candidatus M. oxyfera revealed that most of the C1 metabolism genes significantly diverge from the genes found in other groups of methylotrophs, the cbb operon showed high homology with the operons from Proteobacteria (Ettwig et al., 2010). The cbb genes of M. infernorum are most closely related to the genes in Chloroflexi and Actinobacteria.