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.
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.
Wu
F
F
Department of Bacteriology, University of Wisconsin-Madison 53706, USA.
Wu
J
J
Ehley
J
J
Filutowicz
M
M
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D016384:Consensus Sequence; D018983:DNA Footprinting; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D005819:Genetic Markers; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D010403:Penicillin Resistance / Q000235:genetics*; D010957:Plasmids; D011815:R Factors; D018741:Replication Origin; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; D005819:Genetic Markers; D034982:Integration Host Factors; D015534:Trans-Activators; C440502:integration host factor, E coli; C045264:replication initiator protein; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D004265:DNA Helicases