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

Abstract

Fis protein is shown here to bind to 10 sites in the ␥ origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the ␥ origin for the plasmid-encoded 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 -protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up variants cannot drive replication of R6K ␥-origin plasmids carrying the bla gene encoding resistance to penicillin (Pen r ) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cm r ). In contrast, R6K replication driven by wild-type is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up , 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 ␥ origin, suggesting that excess transcription from the bla gene may inactivate replication driven by 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.The small, abundant DNA-binding proteins Fis integration host factor (IHF), and HU (for reviews, see references 14, 24, 25, and 69) have roles in a variety of DNA transactions, including replication of many replicons. Fis binds and bends oriC, the origin of replication of the chromosome (22, 30), and is involved in chromosome replication (22). IHF also binds oriC (21, 63), and both HU and IHF assist DnaA in unwinding the origin (37) and participate in replication of oriC minichromosomes (21,42). IHF also has roles in the replication and stable maintenance of phage f1 (33, 34) and plasmids pSC101 (3-5, 27, 74, 75) and R6K (11-13, 15, 17, 45).Plasmid R6K encodes resistance to penicillin (Pen r ) and streptomycin (49) and contains three origins of replication: ␣, ␥, and ␤ (Fig. 1). Each origin requires in cis the 277-bp core segment and an additional segment unique to that origin (47,48,(70)(71)(72). The ␥ origin, for example, includes the core and the adjacent enhancer (for a recent review, see reference 16). The enhancer contains DnaA box 1 (79, 80) and a small segment, stb, involved in stable maintenance of ␥-origin-containing plasmids (81). We subdivided the core into three distinct regions (16): (i) the AT-rich region, bound by and IHF (11,13,15,17,19,45,52); (ii) seven 22-bp direct repeats (DRs) bound by (19,23,28,60); and (iii) a multiprotein-binding region interacting with D...

Effects of Fis on Escherichia coli gene expression during different growth stages

“…Subsequently, it was found to be engaged in other site-specific DNA recombination processes (Ball & Johnson, 1991;Thompson et al, 1987;Weinreich & Reznikoff, 1992), to alter DNA supercoiling Skoko et al, 2005;Travers et al, 2001), to influence the initiation of DNA replication (Filutowicz et al, 1992;Messer et al, 1991;Ryan et al, 2004;Wold et al, 1996;Wu et al, 1996), and to regulate a growing number of genes directly or indirectly (Ball et al, 1992;Bosch et al, 1990;Gonzalez-Gil et al, 1996;Green et al, 1996;Jacobson & Fuchs, 1998;Keane & Dorman, 2003;Pease et al, 2002;Ross et al, 1990;Shin et al, 2003;Xu & Johnson, 1995a, c). Prominent among these regulated genes are the rRNA and various tRNA genes, which are directly stimulated by Fis Hirvonen et al, 2001;Nilsson & Emilsson, 1994;Ross et al, 1990).…”

Binding Modes of the Initiator and Inhibitor Forms of the Replication Protein π to the γ ori Iteron of Plasmid R6K

“…Construction of plasmids pFW25 (23) and pRK1 (20) was previously described. pFL731 is a derivative of pUC9 in which a single iteron is cloned into the HincII site.…”

Long CTG·CAG Repeats from Myotonic Dystrophy Are Preferred Sites for Intermolecular Recombination

“…Nevertheless, we describe the recombination frequencies as "apparent frequencies." Accordingly, we used a pFW25 vector that contains the R6K ␥ origin (␥ ori) of replication (34). The ␥ ori is a unidirectional origin, which can function only in the presence of the protein encoded by the pir gene (45).…”

“…The CTG⅐CAG-containing sequences were subcloned into pBR322 and pFW25 (34) as follows. Fragments containing (CTG⅐CAG) n were prepared by digesting the pUC19NotI derivatives with either NotI or EcoRI/HindIII (New England Biolabs, Inc.).…”