L. Van Zyl1, M. P. Taylor1, M. I. Trindade1
1Institution for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Cape Town, 7535
Geobacillus thermoglucosidasius is a promising “platform” organism to use in the production of a range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. We have previously described a novel phage (GVE3) that infects G. thermoglucosidasius and was isolated from stuck fermentations. Here we describe the generation of two G. thermoglucosidasius strains resistant to the phage GVE3. One solution was the over-expression of the phage “immunity” protein encoded by GVE3. To identify spontaneous GVE3 resistant mutants, the lysogen background had to be reduced. Overexpression of a suspected cI-like regulator protein reduced the number of lysogens leading to identification of natural GVE3 resistant mutants. Next generation sequencing of one mutant showed an amber mutation in the polysaccharide pyruvyl transferase (csaB). A double crossover knockout of csaB confirmed the phenotype and resulted in phage resistance. Both solutions prevented lytic phage infection as judged by the absence of phage DNA inside the host, post infection and an inability to form plaques when using the recombinant strains as host. Several other target proteins were also expressed to determine if they could effect phage resistance including a potential anti-holin, HTH-like regulator as well as inactivation of yueB, however none of these resulted in a phage resistance phenotype.