3-Hydroxy-3-Methylglutaryl-Coenzyme A (CoA) Synthase Is Involved in Biosynthesis of Isovaleryl-CoA in the Myxobacterium Myxococcus xanthus during Fruiting Body Formation

Abstract

Isovaleryl-coenzyme A (IV-CoA) is the starting unit for some secondary metabolites and iso-odd fatty acids in several bacteria. According to textbook biochemistry, IV-CoA is derived from leucine degradation, but recently an alternative pathway that branches from the well-known mevalonate-dependent isoprenoid biosynthesis has been described for myxobacteria. A double mutant was constructed in Myxococcus xanthus by deletion of genes involved in leucine degradation and disruption of mvaS encoding the 3-hydroxy-3-methylglutarylcoenzyme A synthase. A dramatic decrease of IV-CoA-derived iso-odd fatty acids was observed for the mutant, confirming mvaS to be involved in the alternative pathway. Additional quantitative real-time reverse transcription-PCR experiments indicated that mvaS is transcriptionally regulated by isovalerate. Furthermore, feeding studies employing an intermediate specific for the alternative pathway revealed that this pathway is induced during fruiting body formation, which presumably increases the amount of IV-CoA available when leucine is limited.

Novel Iso-branched Ether Lipids as Specific Markers of Developmental Sporulation in the Myxobacterium Myxococcus xanthus

“…This pathway is so far unique to myxobacteria and is highly induced in bkd mutants. Recently, we were able to show that this pathway is also induced during fruiting body formation and might function as an alternative source for isovaleryl-CoA under these leucine limiting conditions (27). Furthermore, we could show that the decrease in iso-FA formation is even more severe in mutants where both pathways to isovaleryl-CoA are disrupted (27).…”

“…General-CTT medium (28), TPM agar (11), glycerol- (29) and starvation buffer-induced sporulation (30), large scale fruiting body formation (27), and GC-MS-based fatty acid analysis of total cellular lipids (27) have all been published. M. xanthus strains used in this study are listed in Table 1.…”

The unique DKxanthene secondary metabolite family from the myxobacterium Myxococcus xanthus is required for developmental sporulation

“…Four of them were identified as being part of a complex hybrid PKS/NRPS biosynthetic gene cluster Ϸ47 kb in size. The involvement of this gene cluster in the DKX biosynthesis was confirmed by targeted gene inactivation via plasmid insertion according to published procedures (22). This gave rise to mutants M. xanthus PM1284 and M. xanthus PM1297 (both DKX Ϫ ) carrying plasmid integrations in two separate genes of the DKX biosynthetic gene cluster [dkxA (MXAN 4305) encoding a NRPS adenylation domain and dkxO (MXAN 4290) encoding a thioesterase, respectively].…”

“…Briefly, internal fragments of genes dkxA (encoding a NRPS adenylation domain) and dkxO (encoding a thioesterase) were amplified by PCR using primers pcrart1284 -1 (5Ј-GCGTCCGAGCTCCACAAC-3Ј) and pcrart1284 -2 (5Ј-GCCAATGGGCACCGGCTC-3Ј) for dkxA and primers pcrart1297-1 (5Ј-CAGGCGTCGC-CTCGTCTC-3Ј) and pcrart1297-2 (5Ј-GACGGCCTGGAC-CACCTG-3Ј) for dkxO. The PCR constructs were cloned into plasmid pCR2.1-TOPO (Invitrogen), and the resulting plasmids were purified from E. coli TOP10 (Invitrogen) and introduced into M. xanthus DK1050 by electroporation as described (22), leading to kanamycin-resistant DKxanthene negative mutants PM1284 (dkxA::kan; DKX Ϫ ) and PM1297 (dkxO::kan; DKX Ϫ ), respectively.…”

“…Identification of the DKxanthene biosynthesis gene cluster in DK1050 was accomplished by transposon mutagenesis using the transposon pMycoMar (21) and by targeted gene disruption experiments using standard procedures (22). Briefly, internal fragments of genes dkxA (encoding a NRPS adenylation domain) and dkxO (encoding a thioesterase) were amplified by PCR using primers pcrart1284 -1 (5Ј-GCGTCCGAGCTCCACAAC-3Ј) and pcrart1284 -2 (5Ј-GCCAATGGGCACCGGCTC-3Ј) for dkxA and primers pcrart1297-1 (5Ј-CAGGCGTCGC-CTCGTCTC-3Ј) and pcrart1297-2 (5Ј-GACGGCCTGGAC-CACCTG-3Ј) for dkxO.…”