Functional analysis of bacteriophage cf-m3 intergenic region for superinfection immunity regulation

Abstract To understand the immunity determinants of bacteriophage cf, we applied DNA shuffling to mutate the intergenic region (IG) for isolation of possible cf immunity mutants. After superinfection screening, an immunity mutant cf-m3 was obtained with a 106-109 fold greater superinfection ability compared with wild type cf. Nine mutations were characterized, four located within the coding region for a hypothetical repressor, PT gene (ORF165), and five located upstream of the PT coding region. To establish whether the major immunity determinant is the predicted PT protein or in the cis region, phage cf-ls-P and cf-ls-R were generated which showed as low superinfection efficiency as the wild type cf and had no superinfection ability on the cf-lysogen, respectively. This result indicates that rather than superinfection inhibition, the PT protein and the un-transcribed cis element function individually as positive regulators for cf superinfection immunity. Greater superinfection ability depends on the simultaneous presence of both elements. To learn more about the regulation of cf immunity, we characterized in silico the wild-type genome of cf (NCBI NC_001396) and the 61 non-redundant clustered regularly interspaced short palindromic repeats (CRISPR) spacers of Xanthomonas citri identified from 643 CRISPR spacers of 28 strains in the CRISPR database. No homology between CRISPR spacers and the cf genome was found. This indicates that the cf acts through a different immunity mechanism from the CRISPR system. This work yields further insight into the mechanism of cf superinfection immunity.