Construction of Bacteriophage Luminal DiameterX174 Mutants with Maximum Genome Sizes

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.