Browsing by Author "Rawlings, Doug"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Open Access
    Genetic studies on the region downstream of the unc operon of Thiobacillus ferroxidans
    (1996) Oppon, Joseph Cruick-Shank; Rawlings, Doug
    A Tn7-like element was found in a region downstream of a cosmid (p818.1) isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. A probe made from the Tn7-like element hybridized to restriction fragments of identical size from both cosmid p818.1 and T ferrooxidans chromosomal DNA. The same probe hybridized to restricted chromosomal DNA from two other T ferrooxidans strains (ATCC 23270 and 19859). There were no positive signals when an attempt was made to hybridize the probe to chromosomal DNA from two Thiobacillus thiooxidans strains (ATCC 19733 and DSM504) and a Leptospirillum ferrooxidans strain DSM 2705. A 3.5 kb BamHI-BamHI fragment was subcloned from p818.1 downstream the T ferrooxidans unc operon and sequenced in both directions. One partial open reading frame (ORFl) and two complete open reading frames (ORF2 and ORF3) were found. On the basis of high homology to previously published sequences, ORFI was found to be the C-terminus of the T ferrooxidans glmU gene encoding the enzyme GlcNAc I-P uridyltransferase (EC 1.7.7.23). The ORF2 was identified as the T ferrooxidans glmS gene encoding the amidotransferase, glucosamine synthetase (EC 2.6.1.16). The third open reading frame (ORF3) was found to have very good amino acid sequence homology to TnsA of transposon Tn7. Inverted repeats very similar to the imperfect inverted repeat sequences of Tn7 were found upstream of ORF3. The cloned T ferrooxidans glmS gene was successfully used to complement an E.coli glmS mutant CGSC 5392 when placed behind a vector promoter, but was otherwise not expressed in E.coli.
  • Thumbnail Image
    Item
    Open Access
    An investigation into the partition functions of the broad-host-range Plasmid pTF-FC2
    (1997) Smith, Anthony S G; Rawlings, Doug
    The broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2 is stably inherited over many generations despite a low copy number. The pas genes which lie between the repB primase and the repA helicase encode a proteic plasmid stabilisation system and are capable of stabilising the unstable heterologous R1 replicon present on p0U82. The deletion of the pas genes has been shown not to change the copy number of mutant plasmids. This suggested that the pas genes are not involved in replication and function as a stabilisation cassette. The pasA gene encodes an antidote, the pasB gene a toxin which exerts a bacteriocidal effect in an E. coli host and the pasC gene a protein which moderates the toxic effect of PasB. The PasC is unique in proteic plasmid stabilisation systems and reduces PasB toxicity only in the presence of PasA. PasA is able to repress the pas promoter and the addition of PasB increases this repression. PasC has been shown not to effect the pas promoter by itself. In the presence of PasA and PasB, PasC reduces the ability of PasA and PasB to repress the pas promoter. PasC is thought to stabilise the interaction of PasA and PasB and in doing so reduces their ability to function as repressors of the pas operon. The IncQ plasmid RSF1010 which has similarity to pTF-FC2 has two genes in a position analogous to the pasA, pasB and pasC genes. These genes have been found to be unable to function as a plasmid stabilisation system.