Preponderance of Fis-binding Sites in the R6K Gamma Origin and the Curious Effect of the Penicillin Resistance Marker on Replication of This Origin in the Absence of Fis

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1996 Aug 1

PMID 8759862

Citations 7

Authors

F Wu

J Wu

J Ehley

M Filutowicz

Affiliations

Soon will be listed here.

Abstract

Fis protein is shown here to bind to 10 sites in the gamma origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase. However, the requirement of Fis for R6K replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up pi variants cannot drive replication of R6K gamma-origin plasmids carrying the bla gene encoding resistance to penicillin (Penr) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cmr). In contrast, R6K replication driven by wild-type pi is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up pi, Fis deficiency, or drug markers) prevents R6K replication. The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin. Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact. However, replication is restored by insertion of transcriptional terminators on either side of the gamma origin, suggesting that excess transcription from the bla gene may inactivate replication driven by pi copy-up mutants in the absence of Fis. This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.

Citing Articles

Sequence-specific interactions of Rep proteins with ssDNA in the AT-rich region of the plasmid replication origin.

Wegrzyn K, Fuentes-Perez M, Bury K, Rajewska M, Moreno-Herrero F, Konieczny I Nucleic Acids Res. 2014; 42(12):7807-18.

PMID: 24838560 PMC: 4081077. DOI: 10.1093/nar/gku453.

Plasmid R6K replication control.

Rakowski S, Filutowicz M Plasmid. 2013; 69(3):231-42.

PMID: 23474464 PMC: 3691012. DOI: 10.1016/j.plasmid.2013.02.003.

Structure-based functional analysis of the replication protein of plasmid R6K: key amino acids at the pi/DNA interface.

Kunnimalaiyaan S, Rakowski S, Filutowicz M J Bacteriol. 2007; 189(13):4953-6.

PMID: 17449630 PMC: 1913429. DOI: 10.1128/JB.00109-07.

Mechanism of origin activation by monomers of R6K-encoded pi protein.

Bowers L, Kruger R, Filutowicz M J Mol Biol. 2007; 368(4):928-38.

PMID: 17383678 PMC: 2001305. DOI: 10.1016/j.jmb.2007.02.074.

Dimers of pi protein bind the A+T-rich region of the R6K gamma origin near the leading-strand synthesis start sites: regulatory implications.

Kruger R, Filutowicz M J Bacteriol. 2000; 182(9):2461-7.

PMID: 10762246 PMC: 111308. DOI: 10.1128/JB.182.9.2461-2467.2000.

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