Molecular Genetics in Rhodopseudomonas Sphaeroides: Transformation, Plasmids, and Gene Cloning
Fornari, Chester Stephen
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https://hdl.handle.net/2142/71163
Description
Title
Molecular Genetics in Rhodopseudomonas Sphaeroides: Transformation, Plasmids, and Gene Cloning
Author(s)
Fornari, Chester Stephen
Issue Date
1983
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Abstract
A Tris-dependent transformation system was developed for R. sphaeroides. Washing the cells with 0.5M Tris-buffer, pH 7.2, at 0(DEGREES)C prior to exposure to DNA was found to be necessary for inducing a competent state for DNA uptake. Polyethylene glycol (PEG) and Ca('2+) were also determined to be necessary components of this system and were added at the time of DNA addition to Tris-treated cells. The transformation method was most effective with plasmid DNA in the CCC configuration and frequencies as high as 1 x 10('-5) transformants per viable cell were achieved using RSF1010 DNA, although other plasmids, such as pRK290 and RP4 were capable of transforming Tris-treated cells. A versatile cloning vector, pUI81, was constructed from RSF1010 and a pBR322 derivative, pSL25 and shown to be effective in transforming Tris-treated R. sphaeroides.
The indigenous, cryptic plasmids of R. sphaeroides 2.4.1 were structurally analyzed. Ten strains were examined for plasmid content and the molecular relationships among them by agarose gel electrophoresis and filter hybridizations. It was found that all strains contained at least one high molecular weight plasmid, and some had as many as six. The sizes in kilobases (kb) ranged from 42 to 150 and the total plasmid content in some strains accounted for as much as 18% of the genome. All of the larger (> 42 kb) plasmids among the various strains were found to be homologous with each other, although the homology was restricted primarily to the largest (114 kb) plasmid of strain 2.4.1. The 2.4.1. 42 kb plasmid was analyzed in detail and a partial restriction enzyme map was determined. Methods for the high yield preparation of pure plasmid DNA from the various strains were also developed and a procedure for fractionating bulk plasmid DNA was also devised.
The genes for the ribulose-1,5,-bisphosphate carboxylase and the nitrogenase enzyme complex of R. sphaeroides 2.4.1, as well as for Rs. rubrum, R. palustris and R. capsulata, were identified and located by probing chromosomal digests with the nif genes from K. pneumoniae and the carboxylose gene from R. rubrum. The 2.4.1 carboxylase gene on a 2800 bp EcoRI-Bam HI fragment was subsequently cloned out of low melting point agarose onto pBR322 and transformed into E. coli strain SF8.
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