Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38

• We present the complete sequence of pColE3.
• pColE3 is most closely related to a plasmid from enterohemorrhagic E. coli.
• We used Phred DNA quality scores to infer the presence of single-stranded DNA.
• We used DNA staining dyes to demonstrate the presence of single-stranded DNA that coexists with double-stranded DNA.
• We demonstrate single-stranded DNA in other colicins but not in pColE1.
• Single-stranded DNA colicins may represent a new subclass of rolling-circle replication plasmids.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage.