Characterization of a Mycobacterium smegmatis uvrA mutant impaired in dormancy induced by hypoxia and low carbon concentration

Abstract

Background: The aerobic fast-growing Mycobacterium smegmatis, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of M. smegmatis dormancy, by identifying and characterizing genes involved in this behavior.

Results: In a library generated by transposon mutagenesis, we searched for M. smegmatis mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the uvrA gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the uvrA gene. The restoration of the uvrA activity in M. smegmatis by complementation with the uvrA gene of M. tuberculosis, confirmed that i) uvrA inactivation was indeed responsible for the inability of M. smegmatis cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective uvrA genes of M. tuberculosis and M. smegmatis are true orthologs. The rate of survival of wild type, uvrA mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively.

Conclusions: Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.

Figure 1

Effect of nutrient limitation on M. smegmatis growth. (A) M. smegmatis wild type and (B) S1 strains were grown in M9 minimal medium supplemented with glucose at the final concentration of 0.4% (wt, white square; S1, black square); 0.2% (wt, white circle; S1, black circle) or 0.01% (wt, white triangle; S1, black triangle). The growth rate was monitored for 35 hours by measuring OD_600nm. For each strain the data reported in graph represent the mean of three independent experiments.

Figure 2

Screening of M. smegmatis mutant library. A) (Left panel) M. smegmatis wild type and ppk mutant were grown in M9 minimal medium supplemented with glucose 0.2% until OD_600nm = 1.0, serially diluted up to 10^-5 and transferred by using a metal replicator on agar plates. (Right panel) After incubation at 37°C for 4-5 days for aerobic cultures, or incubation for 2 weeks in an AnaeroGen gas pack system at 37°C followed by incubation under aerobic condition at 37°C for 4-5 days, plates were compared. B) Individual screening of 6 selected mutants. Each clone was grown in M9 minimal medium supplemented with glucose 0,2% until OD_600nm = 1.0, serially diluted up to 10^-5 and transferred by using a metal replicator on agar plates. Clones 1, 3 and 6 were considered as moderately affected clones. Clone 2 was considered as severely affected. ND = Non Diluted culture

Figure 3

Effect of hypoxia and low carbon content on M. smegmatis dormancy. M. smegmatis wild type, S1 (uvrA::tn611), S1-uvrA-Ms and S1-uvrA-Tb strains were grown in M9 minimal medium supplemented with glucose 0.2% until OD_600nm = 1.0. Bacterial cultures were then serially diluted up to 10^-5 and transferred to agar plates. After incubation at 37°C for 4-5 days for aerobic cultures, or 2 weeks for anaerobic cultures in an AnaeroGen gas pack system at 37°C followed by incubation under aerobic condition at 37°C for 4-5 days, plates were compared. ND = Non Diluted culture

Figure 4

UV irradiation assay. A) M. smegmatis wild type, S1 (uvrA::tn611), S1-uvrA-Ms and S1-uvrA-Tb strains were streaked from left to right on LB plates. Plates were either exposed or not to UV radiation (0, 15, 30 and 45 seconds). B) M. smegmatis wild type, S1, S1-uvrA-Ms and S1- uvrA-Tb cells in exponential phase were harvested and resuspended in PBS (see Methods for details). Aliquots were exposed to different UV doses (0, 2, 4 and 6 mJ/cm²). The percentage of survival of each strain was determined and represented as the mean value of three independent experiments.

Figure 5

Effect of hydrogen peroxide on cell growth. M. smegmatis cells of wild type, S1, S1-uvrA-Ms and S1- uvrA-Tb strains were grown in LBT with (A) or without (B) 5 mM H₂O₂ and OD_600nm determined every 3 hours. For each strain the data reported in graph is the mean of three independent experiments.