Elucidating the functional role of Mycobacterium smegmatis recX in stress response

Abstract

The RecX protein has attracted considerable interest because the recX mutants exhibit multiple phenotypes associated with RecA functions. To further our understanding of the functional relationship between recA and recX, the effect of different stress treatments on their expression profiles, cell yield and viability were investigated. A significant correlation was found between the expression of Mycobacterium smegmatis recA and recX genes at different stages of growth, and in response to different stress treatments albeit recX exhibiting lower transcript and protein abundance at the mid-log and stationary phases of the bacterial growth cycle. To ascertain their roles in vivo, a targeted deletion of the recX and recArecX was performed in M. smegmatis. The growth kinetics of these mutant strains and their sensitivity patterns to different stress treatments were assessed relative to the wild-type strain. The deletion of recA affected normal cell growth and survival, while recX deletion showed no significant effect. Interestingly, deletion of both recX and recA genes results in a phenotype that is intermediate between the phenotypes of the ΔrecA mutant and the wild-type strain. Collectively, these results reveal a previously unrecognized role for M. smegmatis recX and support the notion that it may regulate a subset of the yet unknown genes involved in normal cell growth and DNA-damage repair.

Figure 1

(A,B) Relative levels of expression of M. smegmatis mc²155 recA and recX genes at the early log, mid-log and stationary phases under aerobic and hypoxic growth conditions. (C) Relative levels of expression of sigH2 and sigH7 genes at the early log, mid-log and stationary phases. The signal intensities were determined as described in the Methods section. The histograms represent the mean values of three independent experiments. The expression levels were determined and normalised to 16S ribosomal RNA expression and induction ratios calculated relative to the amount of transcript in the aerobic early log phase culture. The error bars represent the standard error of the mean calculated from 3 independent replicates.

Figure 2

(A) Expression levels of M. smegmatis mc²155 RecA and RecX proteins under aerobic and hypoxic growth conditions. (B) The quantification of relative changes in RecA protein levels under aerobic and hypoxic growth conditions. (C) The quantification of relative changes in RecX levels under aerobic and hypoxic growth conditions. The histograms represent the mean values determined by quantification of Western blots from three independent experiments. The loading control GroEL was used for normalization of RecA and RecX expression levels. The error bars represent the standard error of the mean calculated from 3 independent replicates. Significant differences are indicated by **p < 0.01, and *p < 0.05. ns, not significant.

Figure 3

Isolation of M. smegmatis mc²155 ΔrecX and ΔrecA ΔrecX mutant strains. (A) physical map of the M. smegmatis recA recX, ΔrecAΔrecX and ΔrecX regions. The horizontal lines with arrowheads on both ends represent the size of the genomic DNA fragment between NdeI and SalI restriction sites corresponding to the wild-type, ΔrecA ΔrecX and ΔrecX strains. The lightning bolt symbols indicate recognition sites for the indicated restriction enzymes. The little black boxes (adjacent to the NdeI recognition site) indicate hybridization probes used in Southern blotting experiments. (B) Southern blot analysis of genomic DNA from the wild-type and ΔrecX strains. Lane 1 and 4, molecular weight markers; 2, genomic DNA from the wild-type strain; 3, genomic DNA of the ΔrecX strain. (C) Southern blot analysis of genomic DNA of the wild-type and ΔrecAΔrecX strains. Lane 1, molecular weight markers; 2, genomic DNA of the wild-type strain; 3, genomic DNA of the ΔrecAΔrecX double mutant strain.

Figure 4

Insertion of a hygromycin resistance gene into M. smegmatis mc²155 recA-recX locus exerts no polar effect. The precA and precX denote pVV16-vector bearing one functional copy of either recA or recX gene under the control of the Hsp60 constitutive promoter. EV denotes pVV16 empty vector. precA and precX indicate plasmids bearing wild-type copies of M. smegmatis recA and recX genes respectively. These have been transformed into the indicated wild-type and mutant strains. Complementation of M. smegmatis mc²155ΔrecA ΔrecX and ΔrecX mutant strains for aerobic growth (panel A) and sensitivity against UV irradiation (panel B) with plasmids bearing wild-type alleles of M. smegmatis recA or recX. (C), quantitative real-time PCR analysis of genes around the recA-recX locus of M. smegmatis mc²155 wild-type and mutant strains. The error bars represent the standard error of the mean calculated from 3 independent replicates. Significant differences are indicated by ns, not significant.

Figure 5

The kinetics of growth of M. smegmatis mc²155 wild-type, ΔrecA and ΔrecA ΔrecX and ΔrecX strains under normal growth conditions. Each data point is the mean of three independent experiments and the error bars represent standard deviations of the mean values.

Figure 6

Effect of DNA-damaging agents on the survival of M. smegmatis mc²155 wild-type and ΔrecA, ΔrecA ΔrecX and ΔrecX strains. The plates were exposed to: (A) five J/m² UV light; (B) 0.01% MMS; (C) 1 mM H₂O₂ and (D) 5 μM ciprofloxacin. Control represents the growth of strains without any DNA damaging agent. The results are representative data (n = 3) from three independent experiments.

Figure 7

Survival of exponential-phase cultures exposed to a broad range of increasing concentrations of DNA damaging agents. The survival of M. smegmatis wild-type, ΔrecA, ΔrecA ΔrecX and ΔrecX strains was investigated by determining the number of CFUs. (A) The survival of cells treated with (A), MMS; (B) H₂O₂; (C) UV radiation and (D) ciprofloxacin. The error bars represent the standard error of the mean calculated from 3 independent replicates. Significant differences are indicated by *p < 0.05; **p < 0.01 and ***p < 0.001. ns, not significant.

Figure 8

UV radiation induces the expression of recA and recX in M. smegmatis. (A) kinetics of accumulation of recA mRNA in control and following exposure to UV radiation; (B) kinetics of accumulation of recX mRNA in control and following exposure to UV radiation. (C) Representative Western blots showing the kinetics of accumulation of RecA and RecX proteins in control and UV-induced M. smegmatis cells. (D,E) Quantifications of Western blot data shown in panel C. The error bars represent the standard error of the mean calculated from 3 independent replicates.