Blackwell Publishing Journal Backfiles 1879-2005
In Escherichia coli, the 5 kb mdoA locus is involved in the osmotically controlled biosynthesis of periplasmic membrane-derived oligosaccharides (MDOs). The structure of this locus was analysed by in vitro cassette insertion, transposon mutagenesis, and gene-fusion analysis.A ‘neo’ cassette, derived from the neomycin phosphotransferase II region of transposon Tn5, was inserted into mdoA, borne by a multicopy plasmid. This plasmid was shown to complement two previously described mdoA mutations, depending on the orientation of the exogenous gene. Thus, the gene altered by these mutations could be expressed under the control of the exogenous promoter. Moreover, the ‘neo’ cassette inactivated another, uncharacterlzed, mdo gene, because when this insertion was transferred into the chromosome MDO synthesis was abolished.The existence of a second gene was confirmed by complementation analysis with a collection of Tn1000 insertions into mdoA. Two groups were defined, and the two genes are organized into an operon (mdoGH). This conclusion was reached because Tn1000 insertions in the first gene displayed a polar effect on the expression of the second gene.An active gene fusion was obtained on a multicopy plasmid between the beginning of mdoH and lacZ. The hybrid β-galactosidase activity followed the same osmotically controlled response as that described for of MDO synthesis. This regulation was unaffected by the presence, or absence, of MDOs In the periplasm. Finally, the amount of mdoA-specific mRNAs, determined by dot blot hybridization, decreased when the osmolarity of the growth medium increased.
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