title: Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius.
abstract: Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure.
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Robert Sobukwe Road, Bellville, Cape Town, South Africa. vanzyllj@gmail.com.
authors.forename: Leonardo Joaquim
authors.initials: LJ
authors.lastname: van Zyl
authors.affiliation: TMO Renewables Limited, 40 Alan Turing Road, The Surrey Research Park, Guildford, Surrey, GU2 7YF, UK.
authors.forename: Mark Paul
authors.initials: MP
authors.lastname: Taylor
authors.affiliation: Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Robert Sobukwe Road, Bellville, Cape Town, South Africa.
authors.forename: Marla
authors.initials: M
authors.lastname: Trindade
mesh_terms: D001435:Bacteriophages / Q000502:physiology*; D005818:Genetic Engineering; D056506:Geobacillus / Q000821:virology*; D009154:Mutation; D010948:Viral Plaque Assay; D053586:Virus Internalization; D014779:Virus Replication
keywords: Bacteriophage; Geobacillus; Immunity; Polysaccharide pyruvyl transferase; Resistance