The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.Natural products and their derivatives provide the basis for medicines targeting a wide range of human diseases. The Gram-negative myxobacteria, members of the d-subgroup of proteobacteria, are an important source of novel classes of secondary metabolites 1 . Of these, the genus Sorangium is particularly valuable, as 46% of metabolites isolated from myxobacteria 1 , including the potent antitumor compound epothilone 2 , derive from this group. The majority of myxobacterial metabolites are polyketides, nonribosomal polypeptides or hybrids of the two structures, many of which are synthesized on gigantic molecular assembly lines composed of polyketide synthase (PKS) and nonribosomal polypeptide synthetase (NRPS) multienzymes 3 . Sorangium strains exhibit additional characteristic features, including 'social behavior' , cell movement by gliding, biofilm formation and morphological differentiation culminating in complex multicellular structures called fruiting bodies 4 . Three myxobacterial suborders are known 5 and the availability of the genome sequence of Myxococcus xanthus (Cystobacterineae) 6 enables comparative analysis with the Sorangium cellulosum (Sorangiineae) genome to illuminate the basis for several important behavioral and metabolic differences. These include the ability of Sorangium strains to degrade complex plant materials (Fig. 1). S. cellulosum So ce56, an obligate aerobe, was established previously as a model Sorangium strain 7 by virtue of its favorable growth characteristics and ability to differentiate reproducibly under laboratory conditions. It synthesizes the cytotoxic chivosazoles 7 and the catecholate-type siderophores myxochelins 8 . Comparison of the complete genome sequence of strain S. cellulosum
The biosynthetic mta gene cluster responsible for myxothiazol formation from the fruiting body forming myxobacterium Stigmatella aurantiaca DW4/3-1 was sequenced and analyzed. Myxothiazol, an inhibitor of the electron transport via the bc 1 -complex of the respiratory chain, is biosynthesized by a unique combination of several polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS), which are activated by the 4-phosphopantetheinyl transferase MtaA. Genomic replacement of a fragment of mtaB and insertion of a kanamycin resistance gene into mtaA both impaired myxothiazol synthesis. Genes mtaC and mtaD encode the enzymes for bis-thiazol(ine) formation and chain extension on one pure NRPS (MtaC) and on a unique combination of PKS and NRPS (MtaD). The genes mtaE and mtaF encode PKSs including peptide fragments with homology to methyltransferases. These methyltransferase modules are assumed to be necessary for the formation of the proposed methoxy-and -methoxy-acrylate intermediates of myxothiazol biosynthesis. The last gene of the cluster, mtaG, again resembles a NRPS and provides insight into the mechanism of the formation of the terminal amide of myxothiazol. The carbon backbone of an amino acid added to the myxothiazolacid is assumed to be removed via an unprecedented module with homology to monooxygenases within MtaG.Myxobacteria are Gram-negative soil bacteria that are assigned to the two suborders Cystobacterineae and Sorangineae. Both belong to the ␦-group of the Proteobacteria (1). They are distinguished from most other bacteria by their ability to glide in swarms, to feed cooperatively, and to form fruiting bodies upon starvation (2, 3). In addition, they have been shown to produce a wide variety of secondary metabolites with unique structures and biological activities (for reviews, see Refs. 4 and 5). These include the electron transport inhibitors myxothiazol (6), stigmatellin (7), and myxalamids (5, 8) produced by different strains of Stigmatella aurantiaca (Cystobacterineae) and the epothilones produced by Sorangium cellulosum (Sorangineae) (9) (structures are given in Fig. 1). Due to their antitumor activity, epothilones have attracted great attention (10 -12). Myxothiazol as well as epothilones contain a thiazole ring that is formed by the incorporation of cysteine into the polyketide backbone (13). Thiazoline and thiazolidine structures of bacitracin in Bacillus licheniformis (14) and the bacterial siderophores yersiniabactin and mycobactin have recently been shown to be biosynthesized by a NRPS 1 or a combined PKS/NRPS in Yersinia pestis and Mycobacterium tuberculosis (15)(16)(17) . 22)). No such combinations have been published so far for the formation of a thiazole coupled to a polyketide structure. In addition to the bis-thiazole moiety, myxothiazol has some unique features: the unusual leucine derived starter unit 3-methyl-butyryl-CoA (13) and the linear polyketide backbone, which includes a -methoxy-acrylate and a terminal amide structure.Little is known about the biochemistry o...
Rolling circle amplification (RCA) is an elegant biochemical method by which long single-stranded DNA molecules with a repeating sequence motif can be readily synthesized. In RCA, small circular single-stranded oligonucleotides serve as templates for the polymerization of the complementary strand. A DNA polymerase with an efficient strand displacement activity can copy the circular template without stopping. This results in a long DNA strand with periodic sequence. We here demonstrate that this method, using DNA recognition and biotin-streptavidin binding, provides a simple procedure for DNA-directed nanoscale organization of matter. As an example, a 74 nucleotide (nt) long circular DNA molecule is amplified into a sequence-periodic single strand with a length up to several micrometers. Hybridization of this long periodic DNA template to the biotinylated complement of the sequence motif results in a long DNA duplex with a periodic arrangement of biotin binding sites. On this duplex, streptavidin-coated particles can be organized into one-dimensional arrays. The resulting DNA constructs are characterized by gel electrophoresis and atomic force microscopy.
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