Characterization of a Mycobacterium smegmatis uvrAmutant impaired in dormancy induced by hypoxia and low carbon concentration
- Angela Cordone1, 2Email author,
- Bianca Audrain2, 3,
- Immacolata Calabrese1,
- Daniel Euphrasie2 and
- Jean-Marc Reyrat^2
© Cordone et al; licensee BioMed Central Ltd. 2011
Received: 16 June 2011
Accepted: 18 October 2011
Published: 18 October 2011
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.
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.
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.
Mycobacterium tuberculosis, the etiological agent of tuberculosis, has the ability to enter human macrophages and survive inside them in a 'latent' or 'non-proliferating' form for a long period of time. This behavior is termed dormancy or latency. During their lifetime, latent bacilli can reactivate giving rise to active tuberculosis, the transmissible form of the disease [1–3].
The molecular mechanism allowing dormancy is not fully understood due the lack of experimental systems that can closely mimic human latent infections . In the granuloma, dormancy is hypothesized to occur in response to low oxygen, stress and lack of nutrients .
Experimental evidences suggest that, within the granuloma, the in vivo environment where dormant mycobacteria persist, the oxygen concentration is the limiting factor for bacterial growth and the condition that induces dormancy. Therefore, during the last few years, various experimental models using microaerobiosis or anaerobiosis, have been developed to reproduce dormancy in vitro [4–6]. There is also evidence that tubercle bacilli suffer nutrient deprivation in lung lesions . Conditions of nutrient limitation have been used to investigate the ability of M. tuberculosis to persist in a non-growing state for long periods of time [7–9]. Importantly, dormancy is a common behavior to both pathogenic and non-pathogenic mycobacteria, in vitro [4, 10, 11], allowing the study of pathogenic species by using non-pathogens as model.
M. smegmatis is a fast growing non pathogenic mycobacterium frequently used as a model system to study its pathogenic counterpart M. tuberculosis. M. smegmatis becomes dormant in low oxygen concentration conditions  and remains viable for over 650 days when it suffers carbon, nitrogen and phosphorous-starvation . Based on these observations, we decided to use low oxygen and limiting nutrient conditions to develop an in vitro system. Then, we used such system to screen a library of M. smegmatis generated by insertion mutagenesis and look for mutants defective in dormancy . This strategy allowed the isolation of two mutants with insertions mapping in the uvrA gene. The UvrA protein belongs to the nucleotide excision repair system (NER) and is highly conserved among mycobacteria. NER counteracts the deleterious effects of DNA lesions acting as an endonuclease enzyme complex including four Uvr proteins: UvrA, UvrB, UvrC, and UvrD. UvrA, togheter with UvrB, plays a key role in the recognition of DNA damaged sites . UvrC, together with UvrB, perform a single strand incision at both sides of the damaged site and the DNA fragment is removed by the action of the UvrD helicase.
While this DNA-repair system has been largely analyzed in E. coli , it remains poorly characterized in mycobacteria. It has been recently reported that the M. smegmatis genome is predicted to encode two additional UvrA proteins, named UvrA2 and UvrA-like protein, whose function are still unknown .
Here we report that the M. smegmatis UvrA protein is essential for the mycobacterial dormancy behavior and survival in hostile growth conditions, such as low oxygen and carbon content, also observed in the granuloma. Our results, together with recent analyses [16–19], suggest that the NER system plays a key role in M. smegmatis dormancy.
M. smegmatisdormancy is induced under conditions of low oxygen and low carbon availability
Library screening and isolation of M. smegmatismutants with impaired dormancy behavior upon hypoxia and low carbon availability
Ten thousand clones of a transposon library containing more than 20,000 mutants and covering the majority of the M. smegmatis gene pool  were screened as described above to isolate mutants unable to survive a prolonged exposure to low oxygen tension and low carbon availability. The screening allowed us to isolate a total of 278 insertion mutants unable to survive these conditions. Each clone was serially diluted to further confirm the observed phenotype (see a 6-clone sample plate in Figure 2B). During individual screening, 21 clones sensitive to hypoxia and low carbon availability were isolated and divided in two groups: the first group included 8 clones that were completely unable to survive and, therefore, defined as severely affected (S); the second group included the remaining 13 clones that were only partially affected and, therefore, defined as moderately affected (M) (Figure 2B). Most likely, these mutants are unable to either enter or exit the dormant state.
Genes disrupted in M and S mutants identified ( LM)-PCR
M. Tuberculosisortholog(% identity)*d
Mycocerosic Acid synthase
Manganese containing catalase
Riesce(2Fe-2S) domain protein
ABC transporter ATP-binding protein
Short-chain dehydrogenase/reductase SDR
Cell division protein
Adenylate and Guanilate cyclase domain protein
UvrA exinuclease, ABC, A subunit
UvrA exinuclease, ABC, A subunit
UvrA is important for mycobacterial dormancy and survival upon hypoxia
As shown in Table 1, a BLAST search performed using uvrA of M. smegmatis as a query showed that this gene is highly conserved in M. tuberculosis. The orthology between the M. smegmatis and M. tuberculosis UvrA proteins was verified by using the M. tuberculosis uvrA gene to complement the M. smegmatis uvrA deficient strain (Figure 3). The reintroduction of the M. tuberculosis uvrA wt gene (here defined as S1-uvrA-Tb) was able to restore the wt phenotype in the M. smegmatis mutated strain. Our results demonstrate that UvrA is essential for M. smegmatis to enter or exit dormancy upon hypoxia. Moreover, we proved that the M. smegmatis and M. tuberculosis gene products are true orthologs.
UvrA deficiency does not influence M. smegmatisgrowth under nutrient limiting conditions
In addition to hypoxia, nutrient starvation is also supposed to affect cell growth. To check whether the NER deficiency had an effect on cell growth in nutrient limitation, we monitored the growth rate of the uvrA mutant and the complemented strains in minimal medium supplemented with the following final glucose concentration: 0.4%; 0.2% or 0.01% (w/v). As shown in Figure 1B, uvrA mutant cells grown in 0.2% glucose entered stationary phase at a lower optical density (OD600nm≈1.1) in compared to cells of the same strains grown in higher (0.4%) glucose concentration. Moreover, both wt and uvrA cell growth arrested at the limiting glucose concentrations (0.01%). Taken together these results indicate that M. smegmatis growth rate is limited by the amount of carbon available and also that absence of UvrA does not affect M. smegmatis growth under nutrient-limited conditions.
The mycobacterial NER system is involved in the protection from UV-induced damage of DNA
The UvrA NER system contributes to repair DNA oxidative damages
It is hypothesized that inside the granuloma, dormant bacilli are continuously exposed to reactive oxygen species (ROS) and Reactive Nitrogen Intermediates (RNI) [23–27], lipo-soluble molecules that can enter the mycobacterial waxy cell wall, thus causing DNA damages.
In silico analysis of mycobacterial genomes  has shown the presence of genes encoding enzymes involved in different DNA repair system such as Nucleotide Excision Repair (NER), Base Excition Repair (BER), Recombinational Repair, Non-Homologous End-joining repair and SOS repair. Surprisingly, even if mycobacteria lack the mutSL-based post-replicative mismatch repair system , their mutation rate is similar to those of other bacteria . A recent analysis provided evidence that the mycobacterial NER system is able to repair a wider range of DNA damages than the corresponding E. coli system, highlighting its involvement in mismatch recognition and suggesting a crucial role of the NER system in preserving the mycobacterial genome integrity [16, 19]. Although mycobacterial DNA repair systems are still not well characterized , it is possible that their functions are important for survival of tubercle bacilli during latency. Latent mycobacteria, in fact, are continuously exposed to the action of compounds such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Intermediates (RNI) that induce DNA damage [24–27]. The deleterious effects of these intermediates, is probably counteracted by the synergic action of highly efficient and functional DNA repair systems. Oxidative stress results in different types of non-bulky DNA damages such as formation of abasic sites, single and double-stranded breaks, or production of oxidized bases converting guanine to 7,8-dihydro-8-oxoguanine. Although Base Excision Repair (BER) is the main pathway for the removal of this kind of lesion [32–34], we hypothesized that during dormancy the BER system is overwhelmed by extensive DNA damages and that mycobacterial genome integrity might be preserved by a synergic action of different DNA repair systems among which NER. Earlier studies have shown that a M. tuberculosis NER-deficient strain mutated in uvrB, is markedly attenuated for survival in mice and that UvrB protein is required for resistance of M. tuberculosis to both ROS and RNI species in vivo . It has also been recently reported that a M. smegmatis uvrB mutant is sensitive to stress factors such as hypoxia, a condition under which bacteria are not proliferating thus they can accumulate DNA damage over time .
In this study we used hypoxia and low carbon availability as a model for dormant state to screen a library of M. smegmatis insertional mutants. This strategy led to the isolation of two strains mutated in the uvrA gene and unable to survive such condition. We showed that the M. smegmatis UvrA protein is essential to survive the in vitro dormancy condition of growth. Moreover, we demonstrated that the UvrA protein is needed for cell to neutralize both UV light- and oxyradicals-induced damages.
According to these data, it is possible to hypothesize that the uvrA mutant is not able to survive the in vitro dormancy conditions because of sudden oxygen increase following the opening of the jars. The oxidative burst created is probably neutralized by the synergic action of functional DNA repair systems, which maintain the genome integrity. A deficiency in one of the DNA repair systems during this step may result in the accumulation inside the mycobacterial genome of mutations which are not counteracted by the action of the remaining repair systems, resulting in failure of cells to reactivate.
A future analysis of the M. tuberculosis uvrA knock-out mutants using human macrophages and mouse infection as an in vitro and in vivo dormancy model systems will give more insight into mycobacterial survival during latency and will help to better clarify the importance of M. tuberculosis NER system during latency.
In this report we describe the isolation and subsequent analysis of a M. smegmatis strain mutated in the uvrA gene under different stress conditions.
We demonstrate that M. smegmatis UvrA deficient strain is more sensitive to hypoxia, UV radiation and oxidative stress than wild type and that the use of M. smegmatis own gene or the corresponding M. tuberculosis homologous gene, fully restore the wild type ability to resist these factors.
Based on our data, we can conclude that UvrA protein, and thus the NER system, is an importatnt player for adaptation of M. smegmatis to various stress conditions. Further analysis are needed to better clarify the role of NER system in the complex phenomenon of mycobacterial dormancy.
Bacterial strains, media and growth conditions
Mycobacterium smegmatis mc2155  is the parental of all the recombinant strains described below. E. coli DH5α strain (supE44 ΔlacU169 [φ80ΔlacZM15] hsdR17recA1)  was used for all cloning experiments.
M. smegmatis mc2155 and derivatives were grown in LB medium containing 0,05% Tween 80 (LBT).
For nutrient limitation experiments, M. smegmatis mc2155 and derivatives were grown in M9 containing 1 mM Mg2SO4 and supplemented with glucose at the following final concentrations: 0.4%; 0.2% or 0.01% (w/v).
Escherichia coli strains were grown in LB medium. When required, antibiotics were added to the medium at the following final concentrations: ampicillin 100 μg/ml, kanamycin 25 μg/ml. Hygromicin was used at 200 μg/ml for E. coli and 50 μg/ml for M. smegmatis.
In vitrodormancy assay
M. smegmatis transposon insertion mutants  were thawed and printed by using a metal replicator in 96 well plates in M9 medium containing 1 mM Mg2SO4 and 0.2% glucose at 37°C in standing condition until OD600nm = 1.0. After incubation time, wild type and mutant strains were serially diluted 1:10 up to 10-5 and spotted on M9 agar plates containing glucose. Control plates were incubated in normal atmosphere (20% O2) for 4-5 days at 37°C, whereas experimental plates were transferred to anoxic jar (Oxoid) for 2 weeks at 37°C. Hypoxia was generated using AnaeroGen gas pack system (Oxoid) inside jars and anaerobiosis (O2 <1%) was checked by using methylene blue as indicator. Plates were finally removed from the anoxic jar and incubated in normal atmosphere to enable growth of the surviving bacteria.
Sequence (5' - 3')a
Position of annealing b
ctag tctaga gacgtgtccggtgtaggtgt
ctag tctaga atgacctggtggatcgactg
ctag tctaga cgatgccttgaggatcgtg
ctag tctaga gaagatcgaaacccgatacg
Ligation-mediated PCR (LM-PCR)
Transposon insertions were mapped by using LM-PCR as previously reported . LM-PCR reactions were done using SalI and BamHI enzymes (Roche). PCR products were separated by 1.5% agarose gel and the fragments were purified using QIAquick gel extraction kit (Qiagen). The purified fragments were used as templates in sequencing reactions together with oligonucleotide F or G .
UV irradiation assay
M. smegmatis strains were grown in LBT medium up to exponential phase (OD600nm = 0.4-0.6). Samples from these cultures were streaked on LB agar plates. Plates were exposed to UV light during 0, 15, 30 and 45 seconds and then incubated at 37°C for 3-4 days. The percentage of survival of these strains after UV irradiation was also determined; exponential phase cultures of all strains were harvested and pellets were re-suspended in 2 mL of 1× PBS. 200 μL were exposed to UV intensities of 0, 2, 4 and 6 mJ/cm2 (as measured with a VLX 3W dosimeter). Viable counts of the cultures were determined by plating serial dilution on LB plates with appropriate antibiotics after 4 days at 37°C.
Hydrogen peroxide assay
M. smegmatis strains, were grown in triplicate in LBT medium up to stationary phase (OD600 = 1.5). Cultures were serially diluted 1:100 in LBT supplemented with 0 and 5 mM H2O2 freshly prepared, placed in the microtiter well plates and incubated in a Bioscreen C kinetic growth reader at 37°C with constant shaking. Growth was monitored as OD600nm at 3 h intervals for 48 h.
We would like to express a special acknowledgement to Dr. Jean-Marc Reyrat, a great microbiologist and a great person who loved life and his work, who unfortunately passed away before drafting the manuscript. We will never forget him. We thank L. Di Iorio for technical assistance. We acknowledge Ivan Matic for allowing us to use the VLX 3W dosimeter. We thank Ezio Ricca, Maurilio De Felice, Mario Varcamonti and Riccardo Manganelli for critical reading of the manuscript and suggestions. We are grateful to Emilia MF Mauriello for english revision of the manuscript. This work was supported by European Community's Sixth Framework Programme (Project number: LSHP-CT-2006-037566) to JMR
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