Browsing by Subject "Bacterial genetics"

Now showing 1 - 4 of 4
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    The cloning and characterisation of an endoglucanase and an endoxylanase from Clostridium acetobutylicum in Escherichia coli
    (1988) Zappe, Harold; Woods, David R; Jones, David T
    Clostridium acetobutylicum P262 is an endospore forming Gram-positive obligate anaerobe which has been used for the industrial production of acetone and butanol. Strains of C. acetobutylicum have been reported to exhibit some activity towards cellulosic and hemicellulosic substrates. The aim of this thesis was to establish a genebank of C. acetobutylicum P262 DNA in Escherichia coli and to isolate and characterise genes encoding enzymes which show activity towards hemicellulose and cellulose.
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    Developmental genetic studies on Thiobacillus ferrooxidans
    (1988) Ramesar, Rajkumar Sewcharan; Rawlings , Douglas E; Woods, David R
    Thiobacillus ferrooxidans is an industrially important bacterium which is used in bioleaching operations. The work reported in this investigation extends current knowledge of the genetics of this organism. Conjugation was attempted as a means for plasmid DNA transfer to T. ferrooxidans. Recombinant T. ferrooxidans plasmids, pDER401 and pDER405, were shown to code for mobilization and replication functions in Escherichia coli and Thiobacillus novellus strains. The plasmids were mobilizable at high frequency by the IncP plasmid, R68.45. Attempts to transfer the T. ferrooxidans recombinant plasmids directly from E. coli to T. ferrooxidans were unsuccessful. In multistage mating experiments, plasmid DNA was transferred from E. coli to T. novellus, and from T. novellus to Thiobacillus intermedius. However, in subsequent matings, plasmid transfer from these thiobacilli to T. ferrooxidans could not be shown. A genomic library of T. ferrooxidans ATCC 33020 was constructed in the plasmid vector, pEcoR251, for the purpose of cloning a recA-like gene from this organism. The library consisted of approximately 1,78 X 10⁴ clones carrying chromosomal DNA fragments of about 3-12 kilobases (kb). The library was successfully screened for functional complementation of E. coli auxotrophic mutants. Clones that conferred resistance to methyl methane sulfonate (MMS), a DNA-damaging agent, were isolated in an E. coli recA⁻ mutant. In an attempt to clone a homologous marker, T. ferrooxidans ATCC 33020 was mutated to rifampicin resistance (Rifʳ) and DNA from the mutant strain was used in the construction of plasmid- and cosmid-based libraries. The plasmid library contained approximately 1,35 X 10⁴ clones with inserts of about 1-13 kb. The cosmid library consisted of approximately 8.2 X 10³ colonies, 4.0 X 10⁴ in vitro packaged cosmids, and an amplified in vivo-packaged cosmid lysate containing approximately 1.82 X 10¹¹ infectious particles, carrying inserts of about 35-55 kb. Complementation of E. coli auxotrophic mutants was observed with the plasmid and cosmid library of the T. ferrooxidans Rifʳ strain. Screening both libraries for a Rifʳ marker was unsuccessful. Three recombinant plasmids, pRSR100, pRSR101, and pRSR102, each containing the functional analogue of the E. coli recA gene, were isolated from the plasmid-based genomic library of T. ferrooxidans ATCC 33020. The plasmid, pRSR100, was used for further characterization of the cloned recA-like gene. pRSR100 complemented defects in DNA repair and homologous recombination in an E. coli recA- strain. Antiserum raised against E. coli RecA⁻ protein reacted with two protein bands with an apparent Mᵣ of approximately 40 000 and 38 000 in extracts of the recA deletion mutant, E. coli JK696, containing pRSR100. A single band with an apparent Mᵣ of approximately 40 000 was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum. The nucleotide sequence of the T. ferrooxidans recA gene has been determined. No SOS box characteristic of LexA- regulated promoters could be identified in the 196-bp region upstream of the coding region. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and P. aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with ATPase and constitutive protease activity have been substituted, the cloned protein has retained these activities. The cloned recA gene was expressed in E. coli from both the λ Pᵣ and lac promoters. However, no expression from the 2.2 kb T. ferrooxidans DNA preceding the gene was evident.
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    Gene cloning studies in two nocardioform bacteria
    (1988) Hill, Russell; Woods, David R
    Nocardioforms are Gram-positive, aerobic actinomycetes and are a metabolically diverse group which produce antibiotics, useful enzymes, are important in the biotransformation of organic compounds and the decomposition of organic wastes and are important medically. A gene cloning vector designated pLR591 was constructed from the broad host range, multicopy Streptomyces plasmid pIJ702 and the positive selection Escherichia coli plasmid pEcoR251. This plasmid has useful features for the construction of actinomycete genomic libraries. Cloning of DNA into the unique Bg1II endonuclease site of pLR591 inactivated the lethal EcoRI gene derived from pEcoR251, thereby selecting for recombinant plasmids containing inserted DNA. The thiostrepton resistance gene derived from pIJ702 was shown to be functional in Streptomyces lividans enabling selection of recombinant pLR591 plasmids containing foreign DNA in S. lividans. The vector pLR591 therefore functions as a positive selection Streptomyces-E. coli shuttle vector facilitating construction of actinomycete genomic libraries in E. coli and subsequent transfer of recombinant plasmids into S. lividans.
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