Isolation of a Minireplicon of the Virulence Plasmid pXO2 of Bacillus anthracis and Characterization of the Plasmid-Encoded RepS Replication Protein
- 1Department of Molecular Genetics and Biochemistry and Graduate Program in Molecular Virology and Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
- 2Department of Microbiology and Molecular Genetics, The University of Texas—Houston Health Science Center, Houston, Texas 77030
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FIG. 1.Alignment of the RepS protein of pXO2, Rep63A protein of plasmid pAW63, and RepE protein of pAMβ1 as well as the origins of replication of these three plasmids (1, 7, 27, 34, 45). The alignment was done using the ClustalW program, and the shaded letters indicate identical amino acids or nucleotides. Gaps introduced to maximize alignment are indicated by hyphens. Nucleotide coordinates of the pXO2 origin of replication are indicated in parentheses.
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FIG. 2.Schematic representation of the construction of plasmid pBSCmrepS containing the pXO2 minireplicon. The numbers in parentheses correspond to the nucleotide coordinates of pC194 or pXO2. The direction of transcription of the various genes is indicated by the arrows.
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FIG. 3.Restriction analysis of the pBSCmrepS plasmid isolated from E. coli, B. anthracis, B. cereus, and B. subtilis. Plasmid was digested with BamHI (B) or EcoRI (E). L lanes contain size markers (in kilobases).
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FIG. 4.Purification of the RepS protein of pXO2. Lanes: U, lysates from uninduced E. coli cells; I, lysates from IPTG-induced cells overexpressing the MBP-RepS protein; P, purified MBP-RepS; M, protein molecular mass standards (in kilodaltons).
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FIG. 5.Binding of the RepS protein to ds origin DNA (A) or to ss DNA (B). The indicated amounts of the MBP-RepS or MBP protein were incubated with 5′-end-labeled probes, and the DNA-protein complexes were resolved by electrophoresis on native 6% polyacrylamide gels. The probes used were as follows: a 60-bp region containing the putative ori of pXO2 (ds-ori; oligonucleotide a in Table 1); ss-ori, 60-nt bottom strand of the ori; ss-ns, a 53-nt nonspecific ss oligonucleotide. The positions of free probe (P) and RepS-DNA complex (C) are shown to the left of the gels.
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FIG. 6.Competition EMSA experiments using the 60-bp ds ori (oligonucleotide a) as the probe. 32P-labeled ds ori DNA was incubated in the presence or absence of molar (M) excesses of ds oligonucleotide competitors a, b, and c (A) or ds oligonucleotides d, e, f, ss ori, and nonspecific ss oligonucleotide (B). The “wild-type” ori consists of 60 bp (nt 1 to 60). Positions with a minus sign refer to sequences upstream of position 1 of the ori. Numbers higher than 60 indicate sequences present downstream of the ori. The positions of free probe (P) and RepS-DNA complex (C) are shown to the left of the gels.
- American Society for Microbiology
