Identification of key structural determinants of the IntI1 integron integrase that influence attC × attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse

Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system.

International audienceThe Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player

Beta-galactosidase activity in <i>M. smegmatis</i> expressing <i>lacZ</i> under the control of the promoter regions of the <i>M. tuberculosis</i> H37Rv <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> genes.

<p>The <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> promoter-<i>lacZ</i> fusions carried by the pJEM15 plasmid were introduced into <i>M. smegmatis</i>. The positive control is <i>M. smegmatis</i> transformed with pJEM31(PAN) containing <i>lacZ</i> under the control of a mobile genetic element normalized against <i>M. smegmatis</i> transformed with pJEM15 empty vector. The results shown are the mean of two independent experiments.</p

DNase I footprinting assay of the <i>pks2</i> and <i>msl3</i> promoter regions with PhoP-P.

<p>Radiolabeled PCR fragments corresponding to (A) pks2 (−136 to +62), (B) msl3 (−312 to −79), (C) fadD21(−194 to +7) and (D) lipF (−639 to −450) were used as DNA targets. Various amounts of PhoP-P were used with pks2 and msl3 (lane 1: 0; lane 2: 1.8 pmol; lane 3: 3.6 pmol; lane 4: 7.2 pmol and lane 5: 14.4 pmol) and were incubated with 0.2 pmol of DNA before DNase 1 digestion. For fadD21 and lipF different amount of PhoP-P were used ((lane 1: 0; lane 2: 1.8 pmol; lane 3: 18 pmol; lane 4: 180 pmol and lane 5: 900 pmol) and were incubated with 1 pmol of DNA before DNase I digestion. Lane 6: A+G Maxam and Gilbert reaction. Protected regions are indicated by a line with colored regions. The blue, orange and red segments correspond to the DR1, DR2 and DR3 sites, respectively.</p