( 2003 )
Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution.
PMID : 12761202 : DOI : 10.1093/jb/mvg002
Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) BIOSCI: Biotechnol. Biochem. 65, 1627-1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.
( 2004 )
Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene (rpoB) sequences.
PMID : 15023980 : DOI : 10.1099/ijs.0.02941-0
The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100 % similarity within the same species and 90.2-99.3 % similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.
( 2011 )
Genetic screening strategy for rapid access to polyether ionophore producers and products in actinomycetes.
PMID : 21421776 : DOI : 10.1128/AEM.02915-10 PMC : PMC3126441
Polyether ionophores are a unique class of polyketides with broad-spectrum activity and outstanding potency for the control of drug-resistant bacteria and parasites, and they are produced exclusively by actinomycetes. A special epoxidase gene encoding a critical tailoring enzyme involved in the biosynthesis of these compounds has been found in all five of the complete gene clusters of polyether ionophores published so far. To detect potential producer strains of these antibiotics, a pair of degenerate primers was designed according to the conserved regions of the five known polyether epoxidases. A total of 44 putative polyether epoxidase gene-positive strains were obtained by the PCR-based screening of 1,068 actinomycetes isolated from eight different habitats and 236 reference strains encompassing eight major families of Actinomycetales. The isolates spanned a wide taxonomic diversity based on 16S rRNA gene analysis, and actinomycetes isolated from acidic soils seemed to be a promising source of polyether ionophores. Four genera were detected to contain putative polyether epoxidases, including Micromonospora, which has not previously been reported to produce polyether ionophores. The designed primers also detected putative epoxidase genes from diverse known producer strains that produce polyether ionophores unrelated to the five published gene clusters. Moreover, phylogenetic and chemical analyses showed a strong correlation between the sequence of polyether epoxidases and the structure of encoded polyethers. Thirteen positive isolates were proven to be polyether ionophore producers as expected, and two new analogues were found. These results demonstrate the feasibility of using this epoxidase gene screening strategy to aid the rapid identification of known products and the discovery of unknown polyethers in actinomycetes.
( 2007 )
Cloning of the gene cluster responsible for biosynthesis of KS-505a (longestin), a unique tetraterpenoid.
PMID : 18071268 : DOI : 10.1271/bbb.70477
KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid.
( 2008 )
A multilocus phylogeny of the Streptomyces griseus 16S rRNA gene clade: use of multilocus sequence analysis for streptomycete systematics.
PMID : 18175701 : DOI : 10.1099/ijs.0.65224-0 DOI : 10.1099/ijs.0.65224-0
Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of Streptomyces have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the Streptomyces griseus 16S rRNA gene clade is the most challenging and least defined group within the genus Streptomyces in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, atpD, gyrB, recA, rpoB, trpB and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within Streptomyces. A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the S. griseus 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other Streptomyces clades for refining the systematic framework of this genus.