Highly Iterated Palindrome 1 (HIP1) sequence improves Synechococcus sp. PCC 7002 transformation efficiencies in a homology- and methylation-dependent manner

The ability to precisely control cyanobacterial metabolism first requires the ability to efficiently deliver engineered DNA constructs. Here, we investigate how natural transformation efficiencies in Synechococcus sp. PCC 7002 can be greatly improved by leveraging the native and abundant cyanobacterial Highly Iterated Palindrome 1 (HIP1) sequence. While including at least one homologous HIP1 site within the homology arms of an integrating plasmid increased integration efficiency by up to 7-fold, methylation of those sites by HIP1 methyltransferase (encoded by slr0214 from Synechococcus sp. PCC 6803) boosted this to greater than a 100-fold improvement overall. Non-homologous HIP1 sites also improved transformation efficiencies of both integrating and replicating episomal plasmids (by up to 60- and 9-fold, respectively), but only when methylated. The collective data further reveal that HIP1 does not function as part of a native restriction enzyme system in PCC 7002, but rather may improve transformation efficiency via two complementary mechanisms: i) altering DNA binding/uptake/processing prior to homologous recombination, and ii) increasing the efficiency of homologous recombination in a manner reminiscent of a crossover hotspot instigator (Chi) site. Future studies are needed, however, to more clearly elucidate the specific role of HIP1 during natural transformation of cyanobacteria.