Constitutive transcription pMER610
Mini Mu-lac fusions
Springer Online Journal Archives 1860-2000
Abstract The transpositional phage MudI 1734 lacZ was used to construct transcriptional fusions within the plasmid pMJ611, which contains the cloned tellurite resistance (TeR) determinant of the IncHI-2 plasmid pMER610. A series of 70 MudI insertions, in both orientations, causing loss of tellurite resistance in pMJ611, mapped within a 4.3 kb region which included the genes terA-terD and a 0.4 kb region upstream of the site previously reported as the 5′ limit of the TeR determinant. Expression of β-galactosidase from these transcriptional fusions, including those involving the 5′ upstream region, occurred only from inserts transcribed in the direction terA-terD, confirming the transcriptional orientation of the TeR determinant deduced from DNA sequence analysis. Sixteen of the tellurite-sensitive MudI fusions, distributed over the entire determinant and in both orientations, showed the same pattern of expression when transferred by conjugation and homologous recombination to pMER610, except that the β-galactosidase levels were consistently 2- to 3-fold higher in the parent plasmid. Northern analysis with a DNA probe spanning the TeR determinant identified five transcripts of 4.8, 4.0, 2.7, 1.5 and 1.0 kb synthesised by pMER610. Further hybridisations with DNA probes defining sub-sections of the TeR determinant, together with DNA sequence analysis, suggested the presence of three transcriptional start sites, at approximately 0.9 and 0.1 kb upstream of terA, and near the junction between terC and terD. Three transcriptional termination sites, located within terA, near the terC-terD junction and at the 3′ end of terE are also indicated. Both the expression of β-galactosidase from the MudI fusions and the synthesis of ter gene transcripts are constitutive and were not affected by prior exposure of cultures to sub-toxic levels of tellurite. Further DNA sequence analysis reveals that the extensive homology between terD and terE extends to a section of terA.
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