Biology of a Novel Mycobacteriophage, SWU1, Isolated from Chinese Soil as Revealed by Genomic Characteristics

Abstract

bMycobacteriophage SWU1 is a newly isolated phage from a soil sample collected at Gongping village, Pingchang County, Sichuan Province, China, using Mycobacterium smegmatis mc 2 155 as a host. Plaques of SWU1 appear as a unique bull's-eye on an M. smegmatis lawn. In this paper, we report the complete genome sequence of SWU1 and some major findings from the analysis result. Bacteriophages represent unprecedented bioresources and an indispensable quarry for novel genetic tools. This holds true for mycobacteriophages, a magic toolkit for Mycobacterium molecular manipulation and tuberculosis control (7-10, 13, 14). However, most mycobacteriophages are isolated from limited regions, and many prophages and the CRISPR spacers within mycobacterial genomes fail to find matching phages (4), suggesting that more work on the isolation and description of mycobacteriophages remains necessary.Using Mycobacterium smegmatis mc 2 155 as the host organism, a novel mycobacteriophage (SWU1) was isolated from a soil sample collected at Sichuan Province, China. Plaques of SWU1 appear a characteristic bull's-eye with a halo, which is thought an indicator for the appearance of phage-associated depolymerases. In order to understand the formation mechanism of halo, we sequenced and analyzed the genome of SWU1.We used phenol to break up SWU1 particles and isolate the DNA. Purified phage DNA was sheared with a HydroShear DNAfragmenting system (Gene Machines). The 1.5-to-3-kb fragmented DNAs were purified, repaired, and cloned into PUC118 to create a shotgun library. The resulting ligation mixture was used to transform Escherichia coli DH10B. Recovered plasmid was sequenced on an ABI 3730XL DNA Sequencer (Applied Biosystem) by GBI in China (approximately 4.8ϫ coverage). Predicted genes were subjected to database searches using Glimmer software (3). The putative functions of the open reading frames (ORFs) were determined by BLASTP searches (1). The use of a previously constructed L5 codon usage table assisted the identification of SWU1 predicted genes (6).The SWU1 genome was found to be 52,474 bp in length with 94 putative protein-coding genes and 3 tRNA genes with a GϩC content of 62.4%. SWU1 belongs to cluster A2 using the previously stated criteria (5). Among those genes, 91 have sequence similarity to other genes and 22 have been assigned functions, although 3 fail to match any genes. Many of the genes (genes 6, 13, 14, 16, 17, 23, 26, 27, and 28) are presumably involved in phage structure and assembly. Several genes (genes 44, 49, and 50) might encode proteins involved in phage DNA metabolism. Genes 10, 11, and 12 encode LysA, Holin, and LysB, which are components of the lytic cassette of SWU1. The phage integrase (gp33), the phage attachment site (bp 25,583 to 25,625), and the excisionase (gp36) form an integration cassette. We note that gp45 (encoding a single-stranded DNA [ssDNA] annealing protein, SSAP), gp60 (a recombination endonuclease VII), and gp72 (a RecB family exonuclease) are functional genes of homologous recombination. ...

The Global Reciprocal Reprogramming between Mycobacteriophage SWU1 and Mycobacterium Reveals the Molecular Strategy of Subversion and Promotion of Phage Infection

“…M. smegmatis mc 2 155 and mycobacteriophage SWU1 were used for profiling the global gene expression dynamics of the host response to phage infection. M. smegmatis mc 2 155 and SWU1 were grown as described previously (Fan et al, 2012 ; Chen et al, 2013 ).…”

“…Most mycobacteriophages are isolated from America. We have isolated and characterized the first mycobacteriophage SWU1, a lytic phage, from China soil (Fan et al, 2012 ). In this study, we used RNA-seq and functional assay to characterize the reciprocal reprogramming between SWU1 and Mycobacterium , with special aim to unveil how mycobacteriophages usurp the biomolecular machinery of their mycobacterial host.…”

Genomic and proteomic features of mycobacteriophage SWU1 isolated from China soil

“…Genome announcement of SWU1 had been published (Fan et al, 2012). The GenBank accession number for SWU1 is JF946695.…”

Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics

“…The E. coli DH5α used for molecular cloning was grown in the Luria-Broth (LB) medium at 37°C. Mycobacteriophage SWU1 (GenBank ID: JF946695) from Sichuan Province, China, was isolated by using M. smegmatis mc 2 155 as the host (Fan et al 2012;Fan et al 2015). Antibiotics (Sigma-Aldrich) were added at the following indicated concentrations: ampicillin, 100 μg/ml; kanamycin, 50 μg/ml for E. coli and 20 μg/ml for M. smegmatis mc 2 155.…”