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19 Sorangium cellulosum
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In 1937 Imshenetski and Solntseva isolated a new species of cellulose-degrading myxobacteria, which they called Sorangium cellulosum. 16S rRNA gene sequencing of nine isolates of Sorangium and their comparison with Polyangium cellulosum as the reference strain proved a close phylogenetic relationship (evolutionary distance, less than 3% on the nucleotide level). The use of single antibiotics or better combinations of several antibiotics that act on different targets may be helpful, because Sorangium species usually turn out to be multiresistant. Cells of the suborder “Cystobacterineae” on the one hand and “Sorangiineae” and “Nannocystineae” on the other hand can easily be distinguished because they differ in cell morphology, as can be detected using phase-contrast microscopy. Strains of Byssophaga cruenta, characterized by their intense blood-red color, and the very common S. cellulosum strains are the only myxobacteria which degrade crystalline cellulose and can use it as the sole carbon source. At the Gesellschaft für Biotechnologische Forschung (GBF) an isolation and screening program was initiated in the late 1970s to evaluate the potential of the different genera of myxobacteria as producers of secondary metabolites. A ddc gene product, which was previously found only in eukaryotes, has also been identified in Sorangium strains. As discussed in this chapter, the fascinating microorganisms of the genus Sorangium attract more and more attention, because they undergo a complex life cycle, possess the largest bacterial genomes known to date, and show a high potential as producers of biotechnologically important natural products.
Raster electron microscopic pictures of fruiting Sorangium. (a) Vegetative swarm colony. (b) Sporangioles on the agar surface, some of which are broken. (c) Myxospores of Sorangium. The surface structure is the result of drying. Pictures by K. Gerth and H. Lünsdorf.
Dependence of the generation time of Sorangium strains on incubation temperature. So ce26 is a mesothermophilic isolate. An increase of the temperature from 30 to 40°C results in an increase of the generation time from 11 to 19 h. GT-46 and GT-41 are moderately thermophilic Sorangium strains. The generation time decreases with an increase in temperature. At 42°C the temperature optimum is reached with a generation time of 6.5 h.
Myxobacterial producers of novel secondary metabolites. With 47% of total production, Sorangium strains are the most outstanding producers of novel metabolites.
Frequency of some selected metabolites derived from S. cellulosum strains. The data are given as numbers of producer strains from 1,700 screened isolates. From Gerth et al. (2003) with the permission of Elsevier, B.V.
A survey of “novel” metabolites from Sorangium. Typical linear and macrocyclic polyketides are presented. Some of them are likely to be biosynthesized by combinations of peptide synthetases and polyketide synthetases, e.g., eliamid. Socein is one of the rare polypeptides active against fungi and yeasts.
(a) Structures of myxobacterial secondary metabolites with a benzoic acid moiety. (b) Benzoyl-CoA biosynthesis in S. cellulosum So ce26 (soraphen producer). Mutants E4 and E5 are nonproducer mutants. Mutant E4 excretes traces of cinnamic acid and high concentrations of phenyl propionic acid into the culture supernantant. Mutant E5 recovers the ability of soraphen production in the presence of these compounds. From Gerth et al. (2003) with the permission of Elsevier, B.V.
Transposon mutagenesis in S. cellulosum. (a) mariner-based transposon. IR, inverted repeats, P aphII , promoter of the aphII gene; Ω, transcription terminator of the hygromycin resistance gene (HygR); oriR6Kγ, conditional origin of replication. (b) Transposon region when integrated into the chromosome. (c) Transposon recovery, consisting of ligation of chromosomal DNA from mutants after restriction with an enzyme which does not cut inside the transposed element (e.g., MluI). Using Primer 1 and Primer 2 the flanking chromosomal regions can be sequenced from the recovered plasmid. (d) Analysis of mutants, using a bioassay (e.g., comparison of nonproducers of chivosazol obtained by transposon mutagenesis with the wild type, which shows an inhibition zone on the S. cerevisiae indicator plate) (Kopp et al., 2004).
Identification of the chivosazol biosynthetic gene cluster by inactivation of PKS genes using plasmid integration by homologous recombination. (a) Inactivation plasmid containing the selection marker HygR and a homologous region obtained by PCR using degenerate PKS primers. (b) Biosynthetic gene cluster; localization of mutations is marked. (c) HPLC chromatogram of culture extracts of S. cellulosum So ce56 wild type (wt) and chivosazol-negative mutants (Mutant1 and Mutant2). (d) Bioassay for chivosazol production using Hansenula anomala.