It was recently shown that
Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (
28); (ii) they can move on solid surfaces using different motility mechanisms (
16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (
2,
3); and (iv) they harbor the largest prokaryotic genomes found so far (
15,
27). The large genome might be directly related to their complex life-style and the diverse secondary (
3) and primary (
9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. ) (
22,
23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (
24), which is also the preferred pathway to IV-CoA in the myxobacteria
Myxococcus xanthus and
Stigmatella aurantiaca (Fig. ). However, in
bkd mutants, where no or only residual leucine degradation is possible (
30), the alternative pathway is induced (Fig. ), presumably to ensure the production of iso-fatty acids (iso-FAs) (
5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in
M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (
29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (
26).
Open in a separate windowBiosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in
M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.
Open in a separate windowShort representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δ
bkd); (C) DK5624 (Δ
bkd mvaS::
kan); (D) HB002 (Δ
bkd liuC::
kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In
M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (
4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (
7) and prenylated secondary metabolites like aurachin (
22) or leupyrrins (
6), as well as volatiles like geosmin or germacradienol in
M. xanthus and
S. aurantiaca (
11,
13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in
M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of
M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the
bkd locus as well as a plasmid insertion in the
mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (
5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. ). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (
4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δ
bkd liuC::
kan], HB015 [Δ
bkd MXAN_4265::
kan], DK5624 [Δ
bkd mvaS::
kan], HB019 [Δ
bkd mvaS::
kan mvaS+], and HB020 [Δ
bkd MXAN_4265::
kan mvaS+]) have been published previously (
4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (
26).
M. xanthus HB002 (Δ
bkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. ) and its capability to form fruiting bodies is strongly reduced (Fig. ). The residual amount of iso-FAs results from a second BKD activity in
M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in
bkd mutants (
23,
30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second
bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as
liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (
4). As expected, the FA profile (
4) as well as the developmental phenotype (data not shown) can be complemented (Fig. ) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in
M. xanthus. Unexpectedly, addition of MVL (Fig. ) also partially restored fruiting body formation without restoring the FA profile (Fig. ). Similarly,
M. xanthus HB015 (Δ
bkd MXAN_4265::
kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. ) (
4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. ). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on
mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of
mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (
4). The resulting strain, HB020 (Δ
bkd MXAN_4265::
kan mvaS+), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.
Open in a separate windowFruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δ
bkd liuC::
kan), HB015 (Δ
bkd MXAN_4265::
kan), DK5624 (Δ
bkd mvaS::
kan), HB019 (Δ
bkd mvaS::
kan mvaS+), and HB020 (Δ
bkd MXAN_4265::
kan mvaS+). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in
M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. ). However, genetic complementation with
mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. , bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for
M. xanthus (
8), which has a predatory life-style (
1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (
10,
22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. . No difference in growth was observed between culture with and culture without MVL for HB002 (Δ
bkd liuC::
kan) and HB015 (Δ
bkd MXAN_4265::
kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in
liuC [Fig. ]) or reduced leucine degradation and a mutation in
mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. ). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (
26).As isoprenoids represent probably the most diverse class of natural products (
14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (
7,
11-
13), prenylated secondary metabolites (
6,
22), and carotenoids (
18-
21) are known from myxobacteria in general, and a major volatile compound from
M. xanthus is the terpenoid geosmin (
13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (
4,
5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in
S. aurantiaca (
25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (
17). Nevertheless, the importance of isoprenoids for
M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved.
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