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Genomic Characterization of Novel Genes in the Novel Mycobacterium Phage Butters
Author(s) -
Juarez Michelle C.,
Grajales Melanie,
Mageeney Catherine M.,
Kenna Margaret A.,
Ware Vassie C.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1076.2
Subject(s) - lytic cycle , temperateness , biology , lysogenic cycle , gene , mutant , bacteriophage , genome , siphoviridae , genetics , mycobacterium smegmatis , phagemid , microbiology and biotechnology , escherichia coli , virus , mycobacterium tuberculosis , tuberculosis , pathology , medicine
The SEA‐PHAGES program at Lehigh University is a collaborative undergraduate research enterprise focused on isolating and characterizing phages that infect Mycobacterium smegmatis to gain a better understanding of phage genome structure, gene function, and phage biology in general. One specific long term objective of the research program is to characterize phage gene products that function in lytic infection and to evaluate their potential as biocontrol agents to target pathogenic bacteria. Among the phages isolated at Lehigh is Mycobacterium phage Butters – a temperate N cluster phage with one of the smallest known mycobacteriophage genomes (41,491bp) among the >6000 mycobacteriophages isolated and characterized thus far ( www.phagesdb.org ). Among 66 annotated and primarily uncharacterized genes in Butters, two ( gp30, gp31 ) are “orphams” with no known mycobacteriophage family members and one is N‐specific ( gp57 ). To test the hypothesis that specific genes are essential for phage infection, gene deletions were made using a B acterial R ecombineering E lectroporated D NA (BRED) technique to determine if mutant phage could be recovered after gene targeting. Here we focus on gp30 and gp57 . Initial identification of mutant plaques was based on notable changes in plaque size phenotype and the presence of deletion‐sized PCR products. Mutant plaques retained the characteristic turbidity of wildtype plaques, indicating little, if any, perturbation of the lysogenic cycle for this temperate phage and a likely requirement for gp30 and gp57 in lytic growth. In most instances, mutant phage plaques (recovered after mixed BRED infection with wildtype phage DNA) could not be individually propagated after multiple plating rounds, suggesting that an essential component (required for bacterial infection) is missing in the population of mutant phages. We report progress in characterizing mutant phages by: 1) analysis of lysogens recovered from mutant plaques that harbor lytic‐incompetent phages, 2) cloning and sequencing of deletion‐sized PCR products recovered from mutant prophage genomes, and 3) plating mixed phage populations on gene complementation strains to recover mutant phages. Support or Funding Information Lehigh University, Department of Biological Sciences; Howard Hughes Medical Institute Science Education Alliance