( 2002 )
Streptomyces spp. contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth.
PMID : 11832503 : DOI : 10.1099/00221287-148-2-391
Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of approximately 108 kDa. The activity of this protein depended on the presence of 5'-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR. The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3-5x10(5) copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2.8x10(4) copies), and decreased markedly at the mid-exponential (4x10(3) copies) and late-exponential phases (1.1x10(3) copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
( 2004 )
Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces: cross-regulation and physiological role in response to oxygen limitation.
PMID : 15522084 : DOI : 10.1111/j.1365-2958.2004.04325.x
Ribonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygen-dependent class I RNR are co-transcribed with nrdS, which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR, which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five- to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS, whereas disruption of cobN, a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS. In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.
( 2010 )
Coevolution of antibiotic production and counter-resistance in soil bacteria.
PMID : 20067498 : DOI : 10.1111/j.1462-2920.2009.02125.x
We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.
( 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
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.
( 2014 )
Isolation and diversity of natural product biosynthetic genes of cultivable bacteria associated with marine sponge Mycale sp. from the coast of Fujian, China.
PMID : 24693980 : DOI : 10.1139/cjm-2013-0785
The marine sponge Mycale sp., a potential source of natural bioactive products, is widely distributed along the coast of Fujian, China. The cultivable bacterial community associated with Mycale sp., the antibacterial activities, and the PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetase) gene diversity of these bacteria were investigated. Phylogenetic analysis of the 16S rRNA gene showed that the 51 isolates from Mycale sp. belonged to Actinobacteria, Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Among them, some bacteria were first isolated from marine sponge. The 20 isolates with antimicrobial activities were primarily clustered within the groups Actinobacteria, Gammaproteobacteria, and Bacillus. Strain HNS054, which showed 99% similarity to Streptomyces labedae, exhibited the strongest antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus MTCC 1430, Bacillus subtilis MTCC 441) and Vibrio species. The screening of natural product biosynthetic genes revealed that 8 Actinobacteria species with antimicrobial activities possessed PKS-KS (ketosynthase) or NRPS-A domains, and the Nocardiopsis species contained a hybrid or mixed PKS-NRPS system. The phylogenetic analysis of the amino acid sequences indicated that the identified KS domains clustered with those from diverse bacterial groups, including Actinobacteria, Alphaproteobacteria, Cyanobacteria, and Firmicutes. Most KS domain sequences had high homology (>80%) to type I KSs, but the KS domain of Nocardiopsis sp. strain HNS048 had 77% similarity to the type II KS domain of Burkholderia gladioli. The NRPS-A domains of the 8 isolates were grouped into the Gammaproteobacteria, Actinobacteria, and Firmicutes groups. The NRPS-A gene of strain HNS052, identified as Nocardiopsis cyriacigeorgica, showed only 54% similarity to Rhodococcus opacus. All results suggested that Mycale sp. harboured diverse bacteria that could contribute to the production of novel bioactive substances in the future.
( 2014 )
Taxonomic evaluation of Streptomyces albus and related species using multilocus sequence analysis and proposals to emend the description of Streptomyces albus and describe Streptomyces pathocidini sp. nov.
PMID : 24277863 : DOI : 10.1099/ijs.0.058107-0 PMC : PMC4851252
In phylogenetic analyses of the genus Streptomyces using 16S rRNA gene sequences, Streptomyces albus subsp. albus NRRL B-1811(T) forms a cluster with five other species having identical or nearly identical 16S rRNA gene sequences. Moreover, the morphological and physiological characteristics of these other species, including Streptomyces almquistii NRRL B-1685(T), Streptomyces flocculus NRRL B-2465(T), Streptomyces gibsonii NRRL B-1335(T) and Streptomyces rangoonensis NRRL B-12378(T) are quite similar. This cluster is of particular taxonomic interest because Streptomyces albus is the type species of the genus Streptomyces. The related strains were subjected to multilocus sequence analysis (MLSA) utilizing partial sequences of the housekeeping genes atpD, gyrB, recA, rpoB and trpB and confirmation of previously reported phenotypic characteristics. The five strains formed a coherent cluster supported by a 100 % bootstrap value in phylogenetic trees generated from sequence alignments prepared by concatenating the sequences of the housekeeping genes, and identical tree topology was observed using various different tree-making algorithms. Moreover, all but one strain, S. flocculus NRRL B-2465(T), exhibited identical sequences for all of the five housekeeping gene loci sequenced, but NRRL B-2465(T) still exhibited an MLSA evolutionary distance of 0.005 from the other strains, a value that is lower than the 0.007 MLSA evolutionary distance threshold proposed for species-level relatedness. These data support a proposal to reclassify S. almquistii, S. flocculus, S. gibsonii and S. rangoonensis as later heterotypic synonyms of S. albus with NRRL B-1811(T) as the type strain. The MLSA sequence database also demonstrated utility for quickly and conclusively confirming that numerous strains within the ARS Culture Collection had been previously misidentified as subspecies of S. albus and that Streptomyces albus subsp. pathocidicus should be redescribed as a novel species, Streptomyces pathocidini sp. nov., with the type strain NRRL B-24287(T).
( 2012 )
Reassessment of the status of Streptomyces setonii and reclassification of Streptomyces fimicarius as a later synonym of Streptomyces setonii and Streptomyces albovinaceus as a later synonym of Streptomyces globisporus based on combined 16S rRNA/gyrB gene sequence analysis.
PMID : 22286909 : DOI : 10.1099/ijs.0.040287-0
The 16S rRNA and gyrB genes of 22 Streptomyces strains belonging to the Streptomyces griseus cluster were sequenced, and their taxonomic positions were re-evaluated. For correct analysis, all of the publicly available sequences of the species were collected and compared with those obtained in this study. Species for which no consensus sequence could be identified were excluded from the phylogenetic analysis. The levels of 16S rRNA gene sequence similarity within the cluster ranged from 98.6 to 100% with a mean value of 99.6 �� 0.3%, and those of the gyrB gene ranged from 93.6 to 99.9% with a mean value of 96.3 �� 1.5%. The observed average nucleotide substitution rate of the gyrB gene was ten times higher than that of the 16S rRNA gene, showing a far higher degree of variation. Strains sharing 99.3% or more gyrB sequence similarity (corresponding to an evolutionary distance of 0.0073) always formed monophyletic groups in both trees. Through the combined analysis of the two genes, clear cases of synonymy could be identified and, according to the priority rule, the assertion of the status of Streptomyces setonii as a distinct species and the reclassification of Streptomyces fimicarius as a later synonym of S. setonii and Streptomyces albovinaceus as a later synonym of Streptomyces globisporus are proposed. Emended descriptions of S. setonii and S. globisporus are provided.
( 1988 )
Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.
PMID : 3045077 : DOI : 10.1128/jb.170.9.3817-3826.1988 PMC : PMC211376
beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.