Characterization of a Mycobacterium smegmatis uvrA mutant impaired in dormancy induced by hypoxia and low carbon concentration.
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.
Dipartimento di Biologia Strutturale e Funzionale, Università Federico II di Napoli, Via Cinthia 4, 80126 Napoli, Italy. angelina.cordone@unina.it
Angela
A
Cordone
Bianca
B
Audrain
Immacolata
I
Calabrese
Daniel
D
Euphrasie
Jean-Marc
JM
Reyrat
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002244:Carbon; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D016296:Mutagenesis; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*
D001426:Bacterial Proteins; D002244:Carbon; D004706:Endodeoxyribonucleases; D010100:Oxygen