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.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3 arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3 end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5 and 3 flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophagederived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.

Shigellosis is a public health threat in developed as well as developing countries like “India.” While antibiotic therapy is the mainstay of treatment for shigellosis, current emergence of multidrug-resistant strains of Shigella spp. has posed the problem more challenging. Lytic bacteriophages which destroy antibiotic resistant Shigella spp. have great potential in this context and hence their identification and detailed characterization is necessary. In this study we presented the isolation and a detailed characterization of a novel bacteriophage Sfin-1 , which shows potent lytic activity against multidrug-resistant isolates of Shigella flexneri , Shigella dysenteriae , Shigella sonnei obtained from clinical specimens from shigellosis patients. It is also active against Escherichia coli C. The purified phage is lytic in nature, exhibited absorption within 5–10 min, a latent period of 5–20 min and burst size of ∼28 to ∼146 PFU/cell. The isolated phage shows stability in a broad pH range and survives an hour at 50°C. Genome sequencing and phylogenetic analyses showed that Sfin-1 is a novel bacteriophage, which is very closely related to T1-like phages (89.59% identity with Escherichia virus T1). In silico analysis indicates that Sfin-1 genome consists of double stranded linear DNA of 50,403 bp (GC content of 45.2%) encoding 82 potential coding sequences, several potential promoters and transcriptional terminators. Under electron microscopy, Sfin-1 shows morphology characteristics of the family Siphoviridae with an isometric head (61 nm) and a non-contractile tail (155 nm). This is most likely the first report of a lytic bacteriophage that is active against three of the most virulent multidrug-resistant Shigella species and therefore might have a potential role in phage therapy of patients infected with these organisms.

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