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Study on the Antibiotic Biosynthetic Gene Related Linear Plasmid of Streptomyces Roseoflavus Men-myco-93-63

Author: SunWeiMing
Tutor: GuoWei；LiuDaQun
School: Agricultural University of Hebei
Course: Biochemistry and Molecular Biology
Keywords: linear plasmid Antibiotics NRPSs PKSs
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 78
Quote: 1
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Abstract


The soil bacteria Streptomyces roseoflavus Men-myco-93-63, isolated from the soil in which patato scab naturally declined, contains a large number of liposoluble antibiotics.According to the property of‘the linear chromosomes and plasmids of Stremptomyces sp. have proteins covalently bound to the 5’ends of the DNA’, a novel method was established to detect the type and configuration of the Streptomyces sp. plasmids by improving the preparation of agarose embedded block. The type and configuration of two plasmids were determined efficiently from S. roseoflavus Men-myco-93-63 and verified by the two-dimensional electrophoresis. The speculated sizes of these plasmids were 47 kb and 53 kb, respectively.To make it clear whether the biosynthetic genes for antibiotic are located on the plasmid, S. roseoflavus Men-myco-93-63 was treated to cure the linear plasmid with different methods, such as freeze-thaw, SDS/ heat cycle cultivation and protoplast respectively. Some derivative strains (P1, P2, P3, P4, P6, P7 and SH7, SH9) were obtained from the former two methods, which lost theis capacity of producing spores. On the basis of the plasmid profile, these strains carry plasmid pSR53 but loss plasmid pSR47. The bioassay suggested that these mutants did not display inhibitory activity to Verticillium dahliae V41 any more, but unchang or decrease a little to S. scabies H2. From the above results it was inferred that the biosynthesis antibiotics-related genes of inhibiting V. dahliae V41 and regulating spore formation were located on the linear plasmid pSR47. However, the biosynthesis antibiotics-related genes of inhibiting S. scabies H2 were located in the chromosome or linear plasmid pSR53. At the same time, the linear plasmid pSR53 may harbor housekeeping genes of the host bacteria because it was very difficult to be cured. A special-shaped strain SH10 obtained in this study, which was bare in morphological characteristics at first on the PDA medium, could produce yellow soluble pigment and in the course of subculture return to its wild-type. Thus, we suppose that the chromosomal DNA or plasmid pSR53of S. roseoflavus Men-myco-93-63 harbors adsA–like genes from S. griseus, which regulates the morphological differentiation.The method of CTAB preparation of genomic DNA was improved on the basis of the principle of CTAB precipitating the majority of carbohydrates and lipids at high salt concentration in this study and large genomic DNA fragments and linear plasmid DNA were got to construct cosmid library. An assumption of the linear plasmid associated with the 5’terminal protein co-precipitating with the denatured protein after boiling and separating with chromosome was ued to successfully extracted a little of the two linear plasmids of S. roseoflavus Men-myco-93-63.In order to sequence of the pSR47 DNA, the telomere of pSR47 was cloned and the pSR47 DNA library was constructed. 66 of contigs were obrained and they were 131 428bp in length. The partial sequences showed some similarity to the biosynthesis-related genes for antibiotics identified before, such as pSA31, pSA321 and pSA32. The further bioinformatic analysis showed that the antibiotics biosynthetic key enzyme NRPSs/PKSs genes, ssgA, whiB-like and FtsK/SpoIIIE family regulatory genes in morphological differentiation, cytochrome P450 genes, cytochrome metabolism related to genes, a operon of histidine kinase signal transduction pathway and some genes which have founctions of replication, repairing, integration and conjugative were identified.

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