The SigF regulon in Mycobacterium smegmatis reveals roles in adaptation to stationary phase, heat, and oxidative stress

Abstract

SigF is an alternative sigma factor that is highly conserved among species of the genus Mycobacterium. In this study we identified the SigF regulon in Mycobacterium smegmatis using whole-genome microarray and promoter consensus analyses. In total, 64 genes in exponential phase and 124 genes in stationary phase are SigF dependent (P < 0.01, >2-fold expression change). Our experimental data reveal the SigF-dependent promoter consensus GTTT-N((15-17))-GGGTA for M. smegmatis, and we propose 130 potential genes under direct control of SigF, of which more than 50% exhibited reduced expression in a Delta sigF strain. We previously reported an increased susceptibility of the Delta sigF strain to heat and oxidative stress, and our expression data indicate a molecular basis for these phenotypes. We observed SigF-dependent expression of several genes purportedly involved in oxidative stress defense, namely, a heme-containing catalase, a manganese-containing catalase, a superoxide dismutase, the starvation-induced DNA-protecting protein MsDps1, and the biosynthesis genes for the carotenoid isorenieratene. Our data suggest that SigF regulates the biosynthesis of the thermoprotectant trehalose, as well as an uptake system for osmoregulatory compounds, and this may explain the increased heat susceptibility of the Delta sigF strain. We identified the regulatory proteins SigH3, PhoP, WhiB1, and WhiB4 as possible genes under direct control of SigF and propose four novel anti-sigma factor antagonists that could be involved in the posttranslational regulation of SigF in M. smegmatis. This study emphasizes the importance of this sigma factor for stationary-phase adaptation and stress response in mycobacteria.

FIG. 1.

Validation of microarray data with real-time PCR. Gene expression ratios (ΔsigF strain/wild type) of the six genes MSMEG_1804 (sigF), MSMEG_2927 (opuCB), MSMEG_6213 (mcat), MSMEG_6232 (katA), MSMEG_6467 (dps1), and MSMEG_6515 (treS) were determined by both microarray analysis (solid bars) and quantitative real-time PCR (open bars) for stationary-phase (μ = 0.07 ± 0.01) LBT batch cultures of M. smegmatis mc²155 wild-type and ΔsigF strains. Relative gene expression ratios were tested for significance (**, P ≤ 0.01; *, P ≤ 0.05). Results are shown as means ± standard deviations for four (solid bars) or three (open bars) biological replicates.

FIG. 2.

Identification of the SigF promoter consensus. Visually identified promoter motifs upstream of 49 SigF-regulated genes (from microarray analysis) were used for a genome-wide virtual footprint analysis. (A) Promoter spacing variation between the −10 and −35 elements in the training set. (B) Number of promoters sorted into categories according to their distance to the start codon. (C) Derived SigF promoter consensus determined using the WebLogo tool.

FIG. 3.

SigF-dependent pigmentation of bacterial colonies. M. smegmatis mc²155 wild-type (WT) and ΔsigF deletion strains were grown on LBT agar under standard fluorescent light at 37°C for 5 days.