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Mycobacterium species have a complex cAMP regulatory network indicated by the high number of adenylate cyclases annotated in their genomes. However the need for a high level of redundancy in adenylate cyclase genes remains unknown. We have used semiquantitiative RT-PCR to examine the expression of the eight Mycobacterium smegmatis cyclases with orthologs in the human pathogen Mycobacterium tuberculosis, where cAMP has recently been shown to be important for virulence. All eight cyclases were transcribed in the cell in all environments tested, and only four demonstrated environmental-mediated changes in transcription. M. smegmatis genes MSMEG_0545 and MSMEG_4279 were upregulated during starvation conditions while MSMEG_0545 and MSMEG_4924 were downregulated in H2O2 and MSMEG_3780 was downregulated in low pH. Promoter fusion constructs containing M. tuberculosis H37Rv promoters showed consistent regulation compared to their M. smegmatis orthologs. Overall our findings indicate that while low levels of transcriptional regulation occur, regulation at the mRNA level does not play a major role in controlling cellular cyclase availability in a given environment.
Figure 1. Regulation of M. smegmatis adenylate cyclase genes under starvation conditions.
Semi-quantitative RT-PCR was used to compare adenylate cyclase mRNA levels between late-log phase cultures incubated for 4 hours in mycomedia (control) or PBS (starvation). (A) A representative depiction of PCR amplified cDNA separated using agarose gel electrophoresis. (B) Data obtained from ImageJ quantifying the band densities observed on the depicted agarose gel comparing the cDNA levels under control (black bars) and starvation (white bars) conditions. (C) Average of three different experiments represented as fold differences of starvation compared to control expression. * indicates statistically significant difference between expression in control and starvation for an individual gene (p<0.05)
Figure 2. Regulation of M. smegmatis adenylate cyclase genes under low pH and oxidative stress
Semi-quantitative RT-PCR was used to compare adenylate cyclase mRNA levels between late-log phase cultures incubated for 4 hours in mycomedia (control, black bars) , mycomedia adjusted to pH 5.5 (low pH, hatched bars), and mycomedia containing H2O2 (H2O2, grey bars). Results are the average of three independent experiments and are expressed in fold difference comparing experimental condition to control. * indicates conditions with statistically significant differences in expression compared to control for an individual gene (p<0.05)
Figure 3. Regulation of M. tuberculosis adenylate cyclase genes.
GFP: promoter fusions containing M. tuberculosis promoters were electroporated into M. smegmatis and used to examine adenylate cyclase expression in late log phase cultures after 4 hour incubation in mycomedia (control, black bar), PBS (starvation, white bar), mycomedia adjusted to pH 5.5 (low pH, hatched bars), and mycomedia containing H2O2 (H2O2, grey bars). Fluorescence was normalized to 106 cells and represented as the average of three independent experiments. * indicates conditions with statistically significant differences in expression compared to control for an individual gene (p<0.05)
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