Characterization of ori and parS-like functions in secondary genome replicons in Deinococcus radiodurans

Abstract

The mechanisms underlying multipartite genome maintenance and its functional significance in extraordinary radioresistance of Deinococcus radiodurans are not well understood. The sequences upstream to parAB operons in chrII (cisII) and MP (cisMP) could stabilize an otherwise, non-replicative colE1 plasmid, in D. radiodurans. DnaA and cognate ParB proteins bound specifically with cisII and cisMP elements. The ΔcisII and ΔcisMP cells showed the reduced copy number of cognate replicons and radioresistance as compared with wild type. Fluorescent reporter–operator system inserted in chrI, chrII, and MP in wild type and cisII mutants showed the presence of all three replicons in wild-type cells. Although chrI was present in all the ΔcisII and ΔcisMP cells, nearly half of these cells had chrII and MP, respectively, and the other half had the reduced number of foci representing these replications. These results suggested that cisII and cisMP elements contain both origin of replication and parS-like functions and the secondary genome replicons (chrII and MP) are maintained independent of chrI and have roles in radioresistance of D. radiodurans.

Genome Editing by CRISPR/Cas12 Recognizing AT-Rich PAMs in Shewanella oneidensis MR-1

“…Therefore, it is necessary for the CRISPR/Cas9 system to find at least one available PAM site within a distance of 20 bp near the target for precise editing, regardless of whether genomic insertion, knockout, or replacement is conducted (Figure a, showing the case of precise replacement). We cannot ensure that a GG sequence will appear within every 20 bp, especially in regions rich in AT sequences, such as some cis -elements and “ ori ”-like sequences. − If there were three PAM options for us to choose from, NGG, TTTV, and ATTN, it would be undoubtedly easier to edit the genome of S. oneidensis MR-1. The application scenarios of the CRISPR/Cas12 system in genome editing were illustrated by taking the construction of an in situ C-terminal fusion of cytochrome protein OmcA, one of the terminal reductases in the EET pathway, as an example (Figure b).…”