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• W2065164437 abstract "The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of ≈200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning. The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of ≈200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning. Bacterial plasmids have been used extensively as model systems in the study of DNA segregation. This is because plasmids encode centromere-like loci, also called partitioning (par) loci, that ensure stable propagation of their replicons (1Gerdes K. Møller-Jensen J. Ebersbach G. Kruse T. Nordstrom K. Cell. 2004; 116: 359-366Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Hayes F. Barilla D. Trends Biochem. Sci. 2006; 31: 247-250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) Plasmidborne par loci invariably consist of two proteins encoded by a bicistronic operon and one or more cis-acting centromere regions where the proteins act. The first gene in the operon (called parA, parF, or parM) encodes an ATPase. The second gene (called parB, parG, or parR) encodes an adaptor protein that binds to its cognate centromere and thereby forms the “partition complex” that, in turn, is recognized by the ATPase. Based on the ATPase, all par loci are divided into two types: Type I loci, which encode Walker box ATPases related to the MinD family, and Type II loci, which encode actin-like ATPases (3Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (704) Google Scholar, 4Koonin E.V. J. Mol. Biol. 1993; 229: 1165-1174Crossref PubMed Scopus (260) Google Scholar, 5Gerdes K. Møller-Jensen J. Jensen R.B. Mol. Microbiol. 2000; 37: 455-466Crossref PubMed Scopus (356) Google Scholar). Based on gene sizes and arrangement, Type I loci are subdivided in Type Ia and Ib. Type Ib ATPases are generally smaller than those of Type Ia, which include ParA of plasmid P1 and SopA of plasmid F. The Type Ib ATPases lack the DNA-binding helix-turn-helix (HTH) domain found in the N-terminal part of the longer Type Ia ATPases (5Gerdes K. Møller-Jensen J. Jensen R.B. Mol. Microbiol. 2000; 37: 455-466Crossref PubMed Scopus (356) Google Scholar, 6Hayes F. Mol. Microbiol. 2000; 37: 528-541Crossref PubMed Scopus (75) Google Scholar). Thus, contrary to the Type Ia ATPases, the Type Ib ATPases do not themselves specifically bind DNA. The molecular mechanism specified by Type II loci is well understood (1Gerdes K. Møller-Jensen J. Ebersbach G. Kruse T. Nordstrom K. Cell. 2004; 116: 359-366Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 7Møller-Jensen J. Borch J. Dam M. Jensen R.B. Roepstorff P. Gerdes K. Mol. Cell. 2003; 12: 1477-1487Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 8Garner E.C. Campbell C.S. Mullins R.D. Science. 2004; 306: 1021-1025Crossref PubMed Scopus (217) Google Scholar). By contrast, the molecular mechanism behind the common and more efficient Type I loci has been more difficult to understand (2Hayes F. Barilla D. Trends Biochem. Sci. 2006; 31: 247-250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 9Ebersbach G. Gerdes K. Annu. Rev. Genet. 2005; 39: 453-479Crossref PubMed Scopus (198) Google Scholar). The Escherichia coli virulence plasmid, pB171, has two par loci designated par1 (Type II) and par2 (Type I) with a peculiar genetic arrangement. The oppositely oriented par1 and par2 loci share a common cis-acting region, parC1, of ≈200 bp only (see Fig. 1, A and B) (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar). parC1 contains 17 6-bp direct repeats (called B1 to B17) organized in two clusters. As described previously (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar), parC1 expresses both par1- and par2-specific incompatibility, indicating that the full-length parC1 fragment contains centromere-like sites for both par loci. The parC2 region downstream of parB also expresses par2-specific incompatibility and contains 18 6-bp direct repeats (B18–B35) that are related to the B repeats in parC1. In contrast, parC2 does not express par1-specific incompatibility (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar). Based on sequence similarity, the B repeats could be divided into two subclasses (I and II) (see Fig. 1, B and C) (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar). Individual repeats belonging to the two subclasses of B repeats are interspersed among each other in parC1 and parC2. In analogy with other Type I par loci (2Hayes F. Barilla D. Trends Biochem. Sci. 2006; 31: 247-250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 9Ebersbach G. Gerdes K. Annu. Rev. Genet. 2005; 39: 453-479Crossref PubMed Scopus (198) Google Scholar), the genetic organization of parC1 and parC2 suggests that ParB binds to the B repeats. parC1 also harbors the P1 and P2 promoters that transcribe par1 and par2 (Fig. 1, A and B) (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar). ParA of par2 is a Walker box ATPase, which forms filaments that oscillate in spiral-shaped structures over the nucleoid (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar, 11Ebersbach G. Gerdes K. Mol. Microbiol. 2004; 52: 385-398Crossref PubMed Scopus (123) Google Scholar). In the presence of par2, plasmids localize to midcell and/or cell quarters, a pattern that is dependent on ParA spiral formation and oscillation (11Ebersbach G. Gerdes K. Mol. Microbiol. 2004; 52: 385-398Crossref PubMed Scopus (123) Google Scholar). Recently, we proposed a working model for how an oscillating and filament-forming protein can localize plasmids primarily at midcell and quarter-cell positions (12Ebersbach G. Ringgaard S. Møller-Jensen J. Wang Q. Sherratt D.J. Gerdes K. Mol. Microbiol. 2006; 61: 1428-1442Crossref PubMed Scopus (98) Google Scholar). The unusual genetic arrangement of the double par locus of pB171 stimulated an analysis of parC1 and parC2. We show here that both ParB and ParR bind to parC1 and that ParB binds to parC2. ParB recognizes the 6-bp direct repeats located within parC1 and parC2. Interestingly ParR recognizes two 10-bp direct repeats located in parC1 just upstream of parM. The ability of ParB and ParR to bind to parC1 is consistent with their transcriptional regulation of the two par operons, i.e. ParR regulates the par1 operon and parB the par2 operon. Surprisingly, however, ParB represses the par1 operon as efficiently as ParR, suggesting that par2 is epistatic to par1. This is, to our knowledge, the first example of cross-talk regulation between two different par loci. Bacterial Strains and Plasmids—Plasmids are listed in Table 1. The following E. coli K-12 strains were used: MC1000, F- Δ(ara-leu) Δlac rpsL15 (13Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1753) Google Scholar); Top10, F- mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG; CH1182, hubA16::aphA; CH1192, hubA16::aphA himA82::TetR (not Δ(galR-lys)); MG1655, bkg Δ(lav-arg) Δ(galR-lys) galP211 (both CH1182 and CH1192 are MG1655 derivatives).TABLE 1Plasmids used and constructedPlasmidRepliconaR1ts indicates the temperature-sensitive runaway replication system (28)Relevant genotype/descriptionbNumbers in brackets are parC1 coordinates according to Ebersbach and Gerdes (10)ResistancecCm and Amp denote the cat and bla genes, respectivelyPrimers used in PCRRef./sourcepRBJ200R1tslacZYA+AmpLaboratory collectionpOU254R1tsLacZYA+ par from R1AmpLaboratory collectionpMG25pUCCloning vector, PA1/O4/O3AmpLaboratory collectionpBAD33pACYCPBADCmRef. 27Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3978) Google ScholarpGE107pACYCPBAD::parRCmB171-57 + B171-58This workpGE121pUCPA1/O4/O3::His6-parRAmpB171-22 + B171-23This workpGE2R1tslacZYA+ par2+AmpRef. 10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google ScholarpGE207pACYCPBAD::parBCmB171-55 + B171-56This workpGE223pUCPA1/O4/O3::His6-parBAmpB171-20 + B171-21This workpGE3112R1tslacZYA+, parC1+ (–192 to +30)AmpB171-47 + B171-48This workpGE3115R1tslacZYA+, parC1 (–192 to –34)AmpB171-47 + B171-50This workpGE3116R1tsAmpB171-47 + B171-51This workpGE3117R1tsAmpB171-47 + B171-52This workpGE3118R1tslacZYA+, parC1 (–192 to –97)AmpB171-47 + B171-53This workpGE3119R1tslacZYA+, parC1 (–192 to –125)AmpB171-47 + B171-54This workpGE3211R1tslacZYA+, parC1 (–192 to +30)AmpB171-39 + B171-40This workpGE3212R1tslacZYA+, parC1 (–125 to +30)AmpB171-39 + B171-41This workpGE3213R1tslacZYA+, parC1 (–97 to +30)AmpB171-39 + B171-42This workpGE3214R1tslacZYA+, parC1 (–83 to +30)AmpB171-39 + B171-43This workpGE3215R1tslacZYA+, parC1 (–62 to +30)AmpB171-39 + B171-44This workpGE3216R1tslacZYA+, parC1 (–35 to +30)AmpB171-39 + B171-45This workpGE3217R1tslacZYA+, parC1 (–4 to +30)AmpB171-39 + B171-46This worka R1ts indicates the temperature-sensitive runaway replication system (28Larsen J.E. Gerdes K. Light J. Molin S. Gene. 1984; 28: 45-54Crossref PubMed Scopus (145) Google Scholar)b Numbers in brackets are parC1 coordinates according to Ebersbach and Gerdes (10Ebersbach G. Gerdes K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15078-15083Crossref PubMed Scopus (117) Google Scholar)c Cm and Amp denote the cat and bla genes, respectively Open table in a new tab Construction of Plasmids Containing parC1-lacZ Fusions— Thirteen mini-R1 derivatives containing parC1 fragments with varying numbers of repeats cloned in front of lacZ in the transcriptional fusion vector pRBJ200 were constructed (see Table 1). In the pGE3100 series the P1 promoter reads into lacZ, and in the pGE3200 series, the P2 promoter reads into lacZ. The different truncated parC1 fragments were generated by PCR amplification using plasmid pB171 as the template (the names of the PCR primers used for the construction of the individual plasmids are shown in Table 1, and oligonucleotide sequences are given in Tables 2, 3, 4). All upstream primers contained an XhoI site, and the downstream primers contained a BamHI site. PCR fragments digested with BamHI and XhoI were cloned into the same sites of pRBJ200. A StuI-BamHI fragment of each pGE3000 series plasmid (harboring the cloned promoter fragment and the lacZYA genes) was subsequently cloned into the StuI-BamHI of plasmid pOU254, resulting in the plasmids used in the LacZ assays. The plasmids were transformed into the E. coli strain MC1000.TABLE 2Primers for construction of truncated ParR binding site in EMSADirection of truncationacw, clockwise; ccw, counterclockwisePrimer namePrimer sequencepar2, cwB171-995′-TATACGTTCATCTATAGCCCCTT-3′B171-1005′-ATCTATAGCCCCTTAAATACTCAA-3′B171-1015′-CCTTAAATACTCAATCTGAGTAAA-3′B171-1025′-TCAATCTGAGTAAATCACTGTTTC-3′B171-1035′-TAAATCACTGTTTCTGGTTGCAAG-3′B171-1045′-TTCTGGTTGCAAGGTAATACTCA-3′B171-1055′-CAAGGTAATACTCAATTTGAGCAC-3′B171-1175′-CTCAATTTGAGCACTATCATAGTT-3′B171-1065′-GCACTATCATAGTTATGAAATCAC-3′par2, ccwB171-PE25′-GATCTCCGTTTAACAGGC-3′par1, ccwB171-1075′-TTTCATAACTATGATAGTGCTCAA-3′B171-1085′-ATGATAGTGCTCAAATTGAGTATT-3′B171-1095′-TCAAATTGAGTATTACCTTGCAAC-3′B171-1105′-TATTACCTTGCAACCAGAAACAGT-3′B171-1115′-CAACCAGAAACAGTGATTTACTCA-3′B171-1125′-CAGTGATTTACTCAGATTGAGTAT-3′B171-1135′-CTCAGATTGAGTATTTAAGGGGCT-3′B171-1145′-GTATTTAAGGGGCTATAGATGAAC-3′B171-1155′-GGCTATAGATGAACGTATACTGCG-3′par2, cwB171-1165′-TCCAGTTCATAGTTAAATGTCTGG-3′a cw, clockwise; ccw, counterclockwise Open table in a new tab TABLE 3Primers for construction of truncated ParB binding site in EMSADirection of truncationacw, clockwise; ccw, counterclockwisePrimer namePrimer sequencepar2, cwB171-405′-CCCCCGGATCCGACGTCGCCCCTTAAATACTCAATCTGAG-3′B171-415′-CCCCCGGATCCGACGTCTATCATAGTTATGAAATCAC-3′B171-835′-TAAATCACTGTTTCTGGTTGCAAG-3′B171-425′-CCCCCGGATCCGACGTCAGTATTATTTATTACATCACATG-3′B171-445′-CCCCCGGATCCGACGTCTTTCATACTTCATAAATATGAATAC-3′B171-455′-CCCCCGGATCCGACGTCATTGACACCTCAGGTTACCGTGTC-3′B171-465′-CCCCCGGATCCGACGTCCATCATACTTCACACATCACA-3′par2, ccwB171-395′-CCCCCCTCGAGCTGCAGGCAACCTCATAAATGTGATGTGTG-3′par1, ccwB171-495′-CCCCCGGATCCGACGTCGATATTTTGACACGGTAACCTGAGG-3′B171-505′-CCCCCGGATCCGACGTCATATGTATTCATATTTATGAAGTATG-3′B171-515′-CCCCCGGATCCGACGTCATATGAAGTGTGTCATGTGATG-3′B171-525′-CCCCCGGATCCGACGTCGTAATAAATAATACTTGTGTTTTG-3′B171-535′-CCCCCGGATCCGACGTCTTGTGTTTTGTGATTTCATAAC-3′B171-545′-CCCCCGGATCCGACGTCAGTGCTCAAATTGAGTATTACC-3′par2, cwB171-405′-CCCCCGGATCCGACGTCGCCCCTTAAATACTCAATCTGAG-3′a cw, clockwise; ccw, counterclockwise Open table in a new tab TABLE 4Additional oligonucleotides used for PCRPrimer namePrimer sequenceB171-105′-CCCCCCTGCAGGGATCCGCAATACTTAAAAACACTAACG-3′B171-115′-CCCCCGACGTCCTCGAGCAATGCTGAAAAACAGACACGC-3′B171-195′-CCCCCGTCGACTCACCATGGGCCAAATACCTC-3′B171-205′-CCCCGGATCCTAAGGATTTATAAATGCATCACCATCACCATCACGTGAAGAAACCCAGCCAGCAAGC-3′B171-215′-CCCCCGTCGACTTATTACGTTAGTGTTTTTAAGTATTGC-3′B171-225′-CCCCGGATCCTAAGGAGGATTTATAAATGCATCACCATCACCATCACGACGATGAACGGAAAAGGAAAAAAT-3′B171-235′-CCCCCGTCGACCCCATTGCTTCATTCTGATATATCAG-3′B171-435′-CCCCCGGATCCGACGTCCATCACATGACACACTTCATATTTC-3′B171-475′-CCCCCCTCGAGCTGCAGGCCCCTTAAATACTCAATCTGAG-3′B171-485′-CCCCCGGATCCGACGTCGCAACCTCATAAATGTGATGTGTG-3′B171-555′-CCCCCGGATCCGAGCTCATAAGGAGTTTTATAAATGGTGAAGAAACC-3′B171-565′-CCCCCCTCGAGGGTACCAAGCTTGCGGCCGCTTATTACGTTAGTGTTTTTAAGTATTGC-3′B171-575′-CCCCCGGATCCGAGCTCATAAGGAGTTTTATAAGTGGACGATGAACGGAAAAGG-3′B171-585′-CCCCCCTCGAGGGTACCAAGCTTTCAGAATAATTTTTTCATTTTAAGACGC-3′B171-595′-CCCCCGGATCCGAGCTCATGAGGTTGCAATGATTACTGTAGTTGG-3′B171-605′-CCCCCGGATCCGAGCTCGCGGCCGCTTAAGGGGCTATAGATGAACGTATAC-3′B171-685′-CCCCCGCGGCCGCGAGCAGGCAAGCTATTTACCG-3′B171-695′-CCCCCGCGGCCGCCCTGGTTAGTTCAACATAGC-3′B171-925′-CCCCGGATCCTAAGGAGGATTTATAAATGCATCACCATCACCATCACGCAGAGGCTCTGGCTCAGCGCCTTG-3′B171-935′-CCCCGGATCCTAAGGAGGATTTATAAATGCATCACCATCACCATCACCCAGAAAAACAATGTCGGACGACAA-3′B171-SP75′-GACGCAATGCGTAACCTTAACCCGG-3′B171-SP105′-CCGGTGCCGTTGCTCATGCAATCG-3′ParM-up5′-CCCCCGGTACCTTAAGGGGCTATAGATGAACGTATACTGC-3′ParM-down5′-CCCCCTCTAGAGGATCCTTACTCCTCTTTGAAAGCCGCGATAGC-3′171SR145′-GGGGCAGCTGGCGAAAGGGGGATGTGCTGC-3′171SR165′-GGGGCAGCTGAATTTCACACAGGAAACAGCTA-3′ Open table in a new tab Construction of Plasmids Expressing ParR (pGE207) or ParB (pGE107)—DNA fragments containing parR and parB were PCR-amplified using pB171 as template. The primers used are listed in Table 1, and the DNA sequences of the oligonucleotides are given in Tables 2, 3, 4. A Shine-Dalgarno sequence (from parMR of R1, included in the upstream primer) was cloned in front of the parR and parB open reading frames. Upstream primers harbored a SacI site and downstream primers a HindIII site. These sites were subsequently used to clone the PCR fragments into the corresponding sites located downstream of the arabinose-inducible PBAD promoter of vector pBAD33. The pBAD33 derivatives were transformed into strains of MC1000 already harboring the pOU254 derivatives. The growth rate of almost all strains (except control strains containing the pBAD33 vector) decreased slightly upon arabinose addition, especially strains expressing ParB. Construction of Plasmids Used for Overexpression of His6-ParR and His6-ParB—ParR and ParB were amplified from plasmid pB171 by using PCR with the following DNA primers: parB, B171-20 + B171-21; parR, B171-22 + B171-23 (sequences are given in Table 4). Upstream primers contained His6 codons inserted between the start codon and the second codon of the ParR and ParB reading frames, respectively. The PCR products were digested with restriction enzymes BamHI and XhoI and cloned into the BamHI and SalI sites located downstream of the IPTG-inducible PA1/O4/O3 promoter of plasmid pMG25 thereby creating plasmids pGE121 (PA1/O4/O3::his6-parR) and pGE223 (PA1/O4/O3::his6-parB). DNA sequences of all PCR-amplified constructs were verified by DNA sequencing by using the CEQ2000 sequencing system provided by Beckman. Protein Purification—E. coli strain Top10 harboring either pGE223 (PA1/O4/O3::his6-parB) or pGE121 (PA1/O4/O3::his6-parR) was grown in LB medium at 37 °C to an A450 of 0.8 before expression of recombinant protein was induced by the addition of isopropyl 1-thio-β-d-galactopyranoside to a final concentration of 1 mm. Induction was continued for 4 h. All of the following steps of the purification procedure were performed on ice or in a 4 °C cold room. Harvested cells were resuspended in 4 ml of lysis buffer/200 ml of culture. Egg white lysozyme was added to resuspended cells at a final concentration of 1 mg/ml and incubated for 30 min. Cells were then sonicated three times for 20 s. The lysate was cleared by centrifugation at 10,000 rpm for 30 min. The cleared lysate from 1 liter of culture was mixed with 1.0 ml of nickel-nitrilotriacetic acid-agarose matrix (Qiagen) and incubated with rotation for 1 h for binding of the histidines in the His6 tag to the nickel ions. The mixture was then allowed to settle in a column. The column was washed twice with 4 ml of wash buffer. Flow-through was collected for further analysis. Recombinant proteins were eluted four times with 0.5 ml of elution buffer. Each elution fraction was stored separately. 5 μl of all samples were analyzed by SDS-PAGE (4% stacking gel, 12.5% separation gel) followed by Coomassie Brilliant Blue staining of the gel. Lysis buffer consisted of 50 mm Na2PO4, 300 mm NaCl, 30 mm imidazole, pH 8.0; wash buffer consisted of 50 mm Na2PO4, 300 mm NaCl, 40 mm imidazole, pH 8.0; elution buffer consisted of 50 mm Na2PO4, 300 mm NaCl, 250 mm imidazole, pH 8.0. LacZ Assay—Cells of strain MC1000 harboring one mini-R1 plasmid (pOU254 derivative) and one expression plasmid (pBAD33 derivative) were grown at 35 °C in AB medium supplemented with 0.5% glycerol, 0.1% casamino acids, 0.1 μg/ml thiamin, and antibiotics (50 μg/ml chloramphenicol and 30 μg/ml ampicillin). The generation time in this medium was about 50 min. Overnight cultures were diluted 50-fold into fresh medium with antibiotics and grown to an A450 of ∼0.04–0.05. Then, 0.2% arabinose was added, and the cultures were grown for 3 h to an A450 of ∼0.4. Samples were collected for β-galactosidase activity measurements carried out according to Miller (14Miller J.F. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). The stability of the R1 plasmids containing the parC1-lacZ fusions were tested by plating on LA plates containing selection only for the pBAD33 derivatives in addition to 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). The R1 plasmids were found to be stable during the time course of the experiment. Prolonged growth of the cultures in the presence of arabinose resulted in reduced plasmid stability especially in strains expressing ParB. Electrophoretic Mobility Shift Assay—DNA oligonucleotides were 32P-end-labeled in a kinase reaction. 20 pmol of oligonucleotide was mixed with 1.5 μl of 10 × polynucleotide kinase (PNK) buffer (1 m Tris-HCl, 100 mm MgCl2, 70 mm DTT, pH 8.0), 2 μl of 10 μCi/μl [γ-32P] ATP (3000 Ci/mmol), 1 unit of PNK buffer, of PNK, and H2O to a total volume of 15 μl. The reaction mixture was then incubated for 30 min at 37 °C followed by 10 min at 70 °C. End-labeled oligonucleotides were then used in a PCR reaction with an additional oligonucleotide designed to obtain the DNA fragment of interest. Oligonucleotides used in construction of truncated parC1 fragments for the ParB electrophoretic mobility shift assay (EMSA) 2The abbreviations used are: EMSA, electrophoretic mobility shift assay; HMW, high molecular weight; DTT, dithiothreitol; IHF, integration host factor; TBE, Tris borate-EDTA. are: fragment a, B171-39 + B171-40, 256 bp; fragment b, B171-39 + B171-83, 216 bp; fragment c, B171-39 + B171-41, 189 bp; fragment d, B171-39 + B171-42, 161 bp; fragment e, B171-39 + B171-44, 126 bp; fragment f, B171-39 + B171-45, 99 bp; fragment g, B171-39 + B171-46, 69 bp; fragment h, B171-40 + B171-49, 223 bp; fragment i, B171-40 + B171-50, 193 bp; fragment j, B171-40 + B171-51, 165 bp; fragment k, B171-40 + B171-52, 144 bp; fragment l, B171-40 + B171-53, 130 bp; fragment m, B171-40 + B171-54, 102 bp (oligonucleotide sequences are listed in Table 3). Primers used in construction of DNA fragments containing ihf1 and ihf2 are: ihf1, B171-SP7 + B171-SP10, 194 bp; ihf2, B171-10 + B171-11, 222 bp. The sequences of these oligonucleotides are listed in Table 4. Primers used in construction of DNA fragments containing P1-parS are: P1-parS, B171-68 + B171-69. Primers used in construction of DNA fragments containing pUC19 DNA are: 171SR14 + 171SR16, 199 bp (oligonucleotide sequences are listed in Table 4). Oligonucleotides used in construction of truncated parC1 fragments for the ParR EMSA are: fragment a, B171-PE2 + B171-99, 355 bp; fragment b, B171-PE2 + B171-100, 345 bp; fragment c, B171-PE2 + B171-101, 335 bp; fragment d, B171-PE2 + B171-102, 325 bp; fragment e, B171-PE2 + B171-103, 315 bp; fragment f, B171-PE2 + B171-104, 305 bp; fragment g, B171-PE2 + B171-105, 295 bp; fragment h, B171-PE2 + B171-117, 285 bp; fragment i, B171-PE2 + B171-106, 275 bp; fragment j, B171-116 + B171-107, 239 bp; fragment k, B171-116 + B171-108, 229 bp; fragment l, B171-116 + B171-109, 219 bp; fragment m, B171-116 + B171-110, 209 bp; fragment n, B171-116 + B171-111, 199 bp; fragment o, B171-116 + B171-112, 189 bp; fragment p, B171-116 + B171-113, 179 bp; fragment q, B171-116 + B171-114, 169 bp; fragment r, B171-116 + B171-115, 159 bp (see Table 2). The standard reaction mixture (20 μl) for the ParB/ParR experiments contained 10 mm Tris-base (pH 7.5), 50 mm KCl, 1 mm MgCl2, 0.5 mm DTT, and 0.1 μg/μl sonicated salmon sperm DNA. In EMSA experiments performed with integration host factor (IHF), the standard reaction mixture (20 μl) contained 25 mm Tris-base (pH 7.5), 50 mm KCl, 50 mm NaCl, 1 mm MgCl2, 0.5 mm DTT, 1 mg/ml bovine serum albumin, and 0.1 μg/μl sonicated salmon sperm DNA. The standard reaction mixture was mixed on ice followed by the addition of 32P-end-labeled DNA fragments. Protein concentrations are given in the figure legends. We used a concentration of 5 nm 32P-labeled DNA fragments throughout. The reactions were then incubated for 20 min at ambient temperature. After incubation, glycerol was added to a concentration of 5%, and reactions were analyzed by electrophoresis on a 0.5× TBE (pH 7.5) 5% polyacrylamide gel in 1× TBE running buffer (0.89 m Tris-base, 0.89 m boric acid, 0.02 m EDTA, pH 8.3) at 200 V for 11/2 h. The polyacrylamide gels were prerun at 200 V for 1 h, and the TBE running buffer was changed once every hour. Gels were dried on paper and exposed to a phosphor screen over night for imaging on a phosphorimaging device. In determination of the Hill coefficient, the amount (log10 (X/(1 - X))) of bound parC1 (X) was plotted as a function of log10 to the respective concentrations of ParB and ParR. Generation of Biotinylated Double-stranded parC1 Fragments for Surface Plasmon Resonance (SPR)—Biotinylated parC1-containing DNA fragments were produced by PCR using a 5′-end-biotinylated B171-39 clockwise primer and B171-40 counterclockwise primer (Table 3) with pGE2 as template DNA. B171-39 was biotinylated. The PCR product was purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences) as recommended by the manufacturer. SPR Measurements—SPR was carried out using a Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) with streptavidin-coated SA sensor chips (Biacore AB). Biacore measures the binding of molecules to ligands immobilized on sensor chips as real-time changes in the optical SPR phenomenon that is caused by changes in the refractive index near the sensor chip. The refractive index changes are linearly dependent on mass binding to the sensor chip, and hence the amount of analyte that binds the immobilized ligand can be calculated. Because the injections of analyte supply constant concentrations, the binding kinetics is monitored. When the flow is changed to buffer without analyte, the dissociation kinetics is monitored, as bound analyte leaves the immobilized ligand. For the binding studies, 150 mm KCl, 4 mm MgCl2, 1 mm DTT, 0.005% Tween 20, 20 mm HEPES, pH 7.5, was used as running buffer. 200 resonance units of the biotinylated parC1 PCR product was captured on flow cell 2 by injecting the cell with running buffer supplied with 0.5 m NaCl, whereas flow cell 1 was left blank for reference subtraction. Analysis of ParB binding to immobilized parC1 was conducted as follows. A continuous flow of running buffer at 10 μl/min over the two flow cells created a stable base line. ParB diluted to the specified concentration in running buffer was then injected over the two flow cells. To release the ParB complex from the immobilized DNA, the flow was increased to 40 μl/min, and two 15-s pulses of 6 m guanidinium hydrochloride were injected over both flow cells leaving the immobilized DNA ready for another binding cycle. The SPR response is dependent on mass bound to the sensor chip. Thus the relative numbers of ParB binding per immobilized parC1 were calculated according to n=Ranalyte×MrligandRligand×Mranalyte(Eq. 1) where Ranalyte is the SPR response contributed by the analyte (i.e. ParB) binding to the immobilized ligand (parC1 or ParB captured by parC1), Rligand is the response contributed by the immobilized ligand, and Mr ligand and Mr analyte are the molecular weights of the ligand and analyte, respectively. Calculation of the Number of ParB Molecules per Iteron in parC1— This calculation is as follows: R(ParB) = 450; R(parC1) = 200; Mr(His6-ParB) = 10,882.5 g/mol; Mr(parC1) = 165,472.4 g/mol. n=R(parB)×Mr(parC1)R(parC1)×Mr(His6-ParB)=450×165,472.4200×10,882.5=34.2≈34(Eq. 2) Thus, at 100 nm ParB, 34 molecules of ParB binds to parC1, corresponding to two ParB molecules per direct repeat in parC1; this suggests that one repeat binds one ParB dimer. R denotes response units in the SPR experiments. In Vitro Transcription Reactions— PCR fragments for P1 and P2 transcription were obtained using the primers B171-SP3/B171-39 and B171-40/B171-SP9 resulting in fragments of 1174 bp and 1114 bp, respectively (Tables 3 and 4). The standard reaction mixture contained 4 μl of 5 × transcription buffer (Promega), 2 μl of 100 mm DTT, 0.8 μl of RNA-guard, 4 μl of NTPmix (2.5 mm ATP, GTP, UTP), 2.4 μl of 100 μm CTP, 1 μl of template DNA (stock concentration, 20 ng/μl), 1 μl of [α-32P]CTP (10 mCi/μl), and 1 unit of DNA polymerase in a total volume of 25 μl. Where indicated protein was added to a concentration of 1 μm for ParB and 10 μm for ParR. Samples were incubated for 60 min at 37 °C followed by the addition of 1 μl of DNase (1 unit) and were then incubated an additional 15 min at 37 °C. Reactions were stopped by the addition of formamide loading buffer and boiled for 10 min at 99 °C followed by denaturing PAGE on a 5% polyacrylamide gel. The gel was dried on paper and exposed to a 32P-sensitive screen overnight for imaging on a PhosphorImager. Oligonucleotides Used for PCR—These primer sequences are listed in Tables 2, 3, 4. The Divergent P1 and P2 Promoters Are Negatively Coupled— As described above, parC1 contains the B1–B17 repeats located in two clusters that are potential operators for ParB binding (Fig. 1B). Further inspection of the parC1 region revealed two 10-bp direct repeats, R1 and R2, separated by" @default.
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• W2065164437 title "Regulatory Cross-talk in the Double par Locus of Plasmid pB171" @default.
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