Cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional regulators: kstR and kstR2

Abstract

Mycobacterium tuberculosis is able to use a variety of carbon sources in vivo and current knowledge suggests that cholesterol is used as a carbon source during infection. The catabolized cholesterol is used both as an energy source (ATP generation) and as a source of precursor molecules for the synthesis of complex methyl-branched fatty acids. In previous studies, we described a TetR-type transcriptional repressor, kstR, that controls the expression of a number of genes involved in cholesterol catabolism. In this study, we describe a second TetR-type repressor, which we call kstR2. We knocked this gene out in Mycobacterium smegmatis and used microarrays and quantitative RT-PCR to examine the effects on gene expression. We identified a palindromic regulatory motif for KstR2, showed that this motif is present in three promoter regions in mycobacteria and rhodococcus, and demonstrated binding of purified KstR2 to the motif. Using a combination of motif location analysis, gene expression analysis and the examination of gene conservation, we suggest that kstR2 controls the expression of a 15 gene regulon. Like kstR, kstR2 and the kstR2 regulon are highly conserved among the actinomycetes and studies in rhodococcus suggest a role for these genes in cholesterol catabolism. The functional significance of the regulon and implications for the control of cholesterol utilization are discussed.

Fig. 1.

The putative kstR2 regulon in M. smegmatis. (a) Representation of the M. smegmatis genome from MSMEG_5893 to MSMEG_6043. The directions of the arrows represent gene direction and the arrows filled in black are genes controlled by kstR (Kendall et al., 2007). (b) The magnified region corresponds to MSMEG_5999 to MSMEG_6017 and the bottom two lines of arrows correspond to the M. tuberculosis and R. jostii RHA1 genomes. Orthologous genes are indicated by the grey shaded areas. Although MSMEG_6010 and Rv3558 have a conserved context (neighbouring genes are conserved) there is no homology between these two genes and they are not considered to be orthologues. The locations of the KstR2 motif are indicated by asterisks and the sizes of the intergenic regions in the M. smegmatis genome are shown. Genes indicated by thickly outlined arrows in the rhodococcal genome have been shown previously to be induced by cholesterol (Van der Geize et al., 2007). Genes indicated by arrows filled in black in the M. smegmatis genome are those in the kstR2 regulon.

Fig. 2.

Sequence logo of the KstR2 binding motif. Sequence logos (Crooks et al., 2004) show the relative frequency of each base at each position of the motif. The y-axis shows the information content.

Fig. 3.

Binding of purified KstR2_Mtb to 30 bp probes containing the motif. (a) EMSA of purified KstR2_Mtb to 30 bp probes from the three intergenic regions (Rv3549c–Rv3550, Rv3557c–Rv3558 and Rv3560c–Rv3561). Lanes: 1, Rv3549c–Rv3550; 2, Rv3557c–Rv3558; 3, Rv3560c–Rv3561. +, with protein; −, without protein. (b) Specific binding of purified KstR2_Mtb to a 30 bp probe from the Rv3557c–Rv3558 intergenic region. Lanes: 1, labelled probe with protein; 2, labelled probe with protein and with 150-fold excess unlabelled probe; 3, labelled probe with protein and with an excess of poly[d(I-C)].

Fig. 4.

Expression levels of MSMEG_6001 and MSMEG_6038 in a wild-type, ΔkstR2_Msm mutant and ΔkstR/ΔkstR2_Msm mutant background. The expression levels were measured in mid-exponential-phase aerated cultures using RTq-PCR as described in Methods. The results are expressed relative to sigA which was not significantly different (unpaired Student's t-test; P>0.05) in the mutants compared with the wild-type. Levels of sigA expression for the wild-type, ΔkstR2_Msm and ΔkstR/ΔkstR2_Msm were 90 495 (±10 458), 89 635 (±4929) and 75 180 (±3818) copies (μl cDNA)⁻¹, respectively. Significantly upregulated (de-repressed) genes in the mutants compared with the wild-type are marked with asterisks (unpaired Student's t-test; P<0.05). Error bars represent ±1 sd. Black bars, MSMEG_6001 (a KstR2-regulated gene); white bars, MSMEG_6038 (a KstR-regulated gene).