Functional analysis of an intergenic non-coding sequence within mce1 operon of M.tuberculosis

<p>Abstract</p> <p>Background</p> <p>The <it>mce </it>operons play an important role in the entry of <it>M. tuberculosis </it>into macrophages and non-phagocytic cells. Their non-redundant function as well as complex regulation is implied by the phenotype of <it>mce </it>mutants. Recently, <it>mce1 </it>operon was found to extend over 13 genes, <it>fadD5 </it>(Rv0166) being the first gene of the operon. The presence of a non-coding sequence of 200 base pairs between Rv0166 and Rv0167 is peculiar to <it>mce1 </it>among the four <it>mce </it>operons of <it>M.tuberculosis</it>. We have examined the function of this region.</p> <p>Results</p> <p>We predicted putative promoter activity of the 200 base pairs of non-coding, intergenic region between Rv0166 and Rv0167 <it>in silico </it>using MEME software and designate it as intergenic promoter, IGPr. We demonstrate both promoter activity and a putative negative regulatory function of this fragment by reporter assays carried out in the surrogate host <it>M.smegmatis</it>. We find that the repressive elements not only control the native promoter but also repress a heterologous promoter of <it>M.smegmatis</it>. The higher activity of the intergenic promoter in a clinical isolate in comparison with the wild type sequence from <it>M.tuberculosis </it>H37Rv could be correlated with a point mutation within the negative element. We have mapped two transcription start sites for <it>mce1 </it>operon both of which are utilized in <it>M.tuberculosis </it>H37Rv as well as the clinical isolate VPCI591. Our studies show that the promoter activity in the non-coding region is relevant not only in reporter gene expression but also in the expression of <it>mce1 </it>operon in <it>M. tuberculosis </it>cells grown in synthetic medium.</p> <p>Conclusion</p> <p>The <it>mce </it>operon of <it>M.tuberculosis </it>H37Rv potentially can be transcribed from two promoters P1 and P2, former mapping upstream of Rv0166 and the latter in the non-coding intergenic region between Rv0166 and Rv0167. The transcription initiation from P1 results in a transcript with Rv0166 while that from P2 will be without it. The sequences between the translation start site of Rv0167 and the promoter P2 have a negative regulatory role, as point mutation within the sequence leads to enhanced activity of P2 as well as a heterologous promoter from <it>M.smegmatis</it>. The mutation detected in the clinical isolate VPCI591 therefore behaves like a gain-of-function mutation.</p

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains

<p>Abstract</p> <p>Background</p> <p>The presence of four mammalian cell entry (<it>mce</it>) operons in <it>Mycobacterium tuberculosis </it>suggests the essentiality of the functions of the genes in these operons. The differential expression of the four <it>mce </it>operons in different phases of <it>in vitro </it>growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome.</p> <p>Here we investigate the extent of polymorphism in eight genes in the <it>mce1 </it>and <it>mce4 </it>operons of <it>M. tuberculosis </it>from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform.</p> <p>Results</p> <p>We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of <it>mce1 </it>and <it>mce4 </it>operons revealed that <it>yrbE1A </it>[<it>Rv0167</it>] was most polymorphic in <it>mce1 </it>operon while <it>yrbE4A </it>[<it>Rv3501c</it>] and <it>lprN </it>[<it>Rv3495c</it>] had the highest number of SNPs in the <it>mce4 </it>operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to <it>M. tuberculosis </it>using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation.</p> <p>Conclusions</p> <p>Our results show that SNPs in the coding sequences of <it>mce1 </it>and <it>mce4 </it>operons in clinical isolates can be significantly high. Moreover, <it>mce4 </it>operon is significantly more polymorphic than <it>mce1 </it>operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in <it>mce1 </it>operon and synonymous substitutions are more in <it>mce4 </it>operon. <it>In silico </it>modeling predict that nonsynonymous SNP at <it>mce1A </it>[<it>Rv0169</it>], a virulence gene could play a pivotal role in causing functional changes in <it>M. tuberculosis </it>that may reflect upon the biology of the bacteria.</p

High yield synthesis of intrinsic, doped and composites of nano-zinc oxide using novel combinatorial method

A novel synthesis of the production of luminescent zinc oxide (ZnO), either in its intrinsic, metal, nonmetal-doped or composite forms with high yield has been developed by parallel iterative techniques, within a combinatorial library prepared by the reduction of nitroarenes. The reduction of nitroarenes by aluminium/zinc dusts in alkaline medium (pH 10 +/- 2) forms azoxy compounds, whereas in acidic medium (pH 4.9 +/- 0.2) forms phenyl hydroxylamine and zinc/aluminium dust gets oxidised into respective hydroxide. Here, we demonstrate the reduction of nitroarenes at neutral pH (7.0 +/- 0.2), which forms intrinsic as well as doped ZnO at 50 +/- 5 degrees C using zinc dust alone or mixtures of salts of several transition and non-transition metals in presence of 1:10 ratio of solvent and water. Interestingly, it is observed that the photoluminescence emission could be tuned in a wide range from 390 to 615 nm useful for many display related devices