Dataset Information

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

ABSTRACT: pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

SUBMITTER: Alegre MT

PROVIDER: S-EPMC201981 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

Alegre M T MT Cournoyer B B Mesas J M JM Guérineau M M Normand P P Pernodet J L JL

Applied and environmental microbiology 19941201 12

pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-te ...[more]

PMID: 7811067

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Project description:Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by non-ribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster (BGC) in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-utilizing enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here, we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize.SignificanceThe discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, for example of their evolution, activity, and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Project description:Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145.The high coverage and specificity (detection of three nucleotide differences) of the new microarrays used has highlighted the macroscopic genomic differences between two S. lividans strains and S. coelicolor. In a series of case studies we have validated the microarray and have identified subtle changes in genomic structure which occur in the Asp-activating adenylation domains of CDA non-ribosomal peptide synthetase genes which provides evidence of gene shuffling between these domains. We also identify single nucleotide sequence inter-species differences which exist in the actinorhodin biosynthetic gene cluster. As the glyoxylate bypass is non-functional in both S. lividans strains due to the absence of the gene encoding isocitrate lyase it is likely that the ethylmalonyl-CoA pathway functions as the alternative mechanism for the assimilation of C2 compounds.This study provides evidence for widespread genetic recombination, rather than it being focussed at 'hotspots', suggesting that the previously proposed 'archipelago model' of genomic differences between S. coelicolor and S. lividans is unduly simplistic. The two S. lividans strains investigated differ considerably in genetic complement, with TK24 lacking 175 more genes than its wild-type parent when compared to S. coelicolor. Additionally, we confirm the presence of bldB in S. lividans and deduce that S. lividans 66 and TK24, both deficient in the glyoxylate bypass, possess an alternative metabolic mechanism for the assimilation of C2 compounds. Given that streptomycetes generally display high genetic instability it is envisaged that these high-density arrays will find application for rapid assessment of genome content (particularly amplifications/deletions) in mutational studies of S. coelicolor and related species.

Dataset Information

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

Publications

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

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