Characterization of temperate phage Che12 and construction of a new tool for diagnosis of tuberculosis.
A temperate phage, Che12, able to infect Mycobacterium tuberculosis, was isolated from soil samples taken from tuberculosis sanatorium area in Chennai, India. The plaque morphology of this phage showed varying grades of turbidity on lawns of M. tuberculosis. The temperate nature of Che12 was established by super infection immunity. Phage integration into the host genomic DNA was confirmed by Southern hybridization using Che12 DNA as a probe. PCR amplification and sequencing of a part of the integrated phage genome in a M. tuberculosis lysogen also confirmed the temperate nature of Che12. The morphology of the phage particles was observed by electron microscopy, revealing similarities to other mycobacteriophages like L5, D29 and TM4. A luciferase reporter phage, phAETRC16, was constructed by cloning firefly luciferase gene into Che12. Infection of viable M. tuberculosis cells by phAETRC16 resulted in expression of luciferase leading to sustained light output. Che12, a true temperate phage infecting M. tuberculosis, is thus ideally suited for developing a diagnostic tool facilitating rapid diagnosis of M. tuberculosis.
Kumar
Vanaja
V
Tuberculosis Research Centre (Indian Council of Medical Research), Bacteriology, Mayor V.R. Ramanathan Road, Chetput, Chennai 600 031, India. vanaja_kumar@hotmail.com
Loganathan
Prabakaran
P
Sivaramakrishnan
Gomathi
G
Kriakov
Jordan
J
Dusthakeer
Azger
A
Subramanyam
Balaji
B
Chan
John
J
Jacobs
William R
WR
Paranji Rama
Narayanan
N
D000818:Animals; D004279:DNA, Viral / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D007194:India; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009160:Mycobacteriophages / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000821:virology; D009169:Mycobacterium tuberculosis / Q000821:virology*; D015163:Superinfection / Q000276:immunology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000235:genetics; D010948:Viral Plaque Assay
D004279:DNA, Viral; D049409:Luciferases, Firefly