title: Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38.
abstract: 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.
authors.affiliation: Biology Department, California State University Northridge, Northridge, CA 91330-8303, United States.
authors.forename: Magali
authors.initials: M
authors.lastname: Morales
authors.affiliation: Biology Department, California State University Northridge, Northridge, CA 91330-8303, United States.
authors.forename: Hedieh
authors.initials: H
authors.lastname: Attai
authors.affiliation: Ellington High School, Ellington, CT 06029, United States.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Troy
authors.affiliation: Biology Department, California State University Northridge, Northridge, CA 91330-8303, United States; Interdisciplinary Research Institute for the Sciences (IRIS), California State University Northridge, Northridge, CA 91330-8303, United States. Electronic address: david.bermudes@csun.edu.
authors.forename: David
authors.initials: D
authors.lastname: Bermudes
mesh_terms: D000165:Acridine Orange / Q000378:metabolism; D003086:Bacteriocin Plasmids / Q000235:genetics*; D001483:Base Sequence; D004396:Coloring Agents / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D018628:Gene Dosage; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
chemical_list: D004396:Coloring Agents; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D000165:Acridine Orange
keywords: Acridine orange metachromatic staining; Colicin; Phred quality scores; Picogreen; Rolling-circle replication; Single-stranded DNA