title: Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5.
abstract: The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.
authors.affiliation: Department of Microbiology, Bose Institute, Kolkata, India.
authors.forename: Bidisha
authors.initials: B
authors.lastname: Bhattacharya
authors.forename: Nabanita
authors.initials: N
authors.lastname: Giri
authors.forename: Mahasweta
authors.initials: M
authors.lastname: Mitra
authors.forename: Sujoy K Das
authors.initials: SK
authors.lastname: Gupta
mesh_terms: D003001:Cloning, Molecular; D005415:Flavins / Q000378:metabolism; D008242:Lysogeny; D009160:Mycobacteriophages / Q000201:enzymology* / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000378:metabolism / Q000821:virology; D009711:Nucleotides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005415:Flavins; D009711:Nucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D012264:Ribonucleotide Reductases; D013940:Thymidylate Synthase