Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis

Chang, Sheng-Yung; Chang, Shing; Gray, O

doi:10.1128/jb.169.9.3952-3962.1987

Cited by 40 publications

(33 citation statements)

References 34 publications

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“…The role of the M-Os in plasmid stability has recently been proposed, as the deletion of certain M-Os results in plasmid segregational instability (3,4,5,8 …”

Section: Discussionmentioning

confidence: 99%

Replication origins of single-stranded-DNA plasmid pUB110

et al. 1989

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The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB10 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymnerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.

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“…The role of the M-Os in plasmid stability has recently been proposed, as the deletion of certain M-Os results in plasmid segregational instability (3,4,5,8 …”

Section: Discussionmentioning

confidence: 99%

Replication origins of single-stranded-DNA plasmid pUB110

et al. 1989

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“…Bacteriophage M13 mplO and mpll and plasmids pUC8, pUC13 (31,37), pACYC184 (7), and pFC54.t (38) have been described previously. Plasmid pDH5413, a derivative of pDH5060 (8), was obtained from Shing Chang, Cetus Corporation, and its restriction map is shown in Fig. la.…”

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confidence: 99%

Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators

Wittman¹,

Wong²

1988

J Bacteriol

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. Microbiol. 76:217-230, 1973). The molecular organization of the genes coding for penicillinase (penP) and its repressor (penI) has recently been determined (T. Himeno, T. Imanaka, and S. Aiba, J. Bacteriol. 168: 1128Bacteriol. 168: -1132Bacteriol. 168: , 1986). These two genes are transcribed divergently from within a 364-nucleotide region separating the coding sequences. We cloned and sequenced the repressor gene (pen1c) from strain 749/C that constitutively produces penicillinase. Gelfand, Cetus Corporation. E. coli MC1000 (6) was kindly provided by Malcolm J. Casadaban, University of Chicago. Bacteriophage M13 mplO and mpll and plasmids pUC8, pUC13 (31, 37), pACYC184 (7), and pFC54.t (38) have been described previously. Plasmid pDH5413, a derivative of pDH5060 (8), was obtained from Shing Chang, Cetus Corporation, and its restriction map is shown in Fig. la. Plasmid pTW75 was constructed from p1201-P156-RBS1 (40) by elimination of the EcoRI site proximal to the ribosomebinding site. The construction of plasmids pHCW-P1, pHCW-P2, pHCW-P3, and pHCW-P4 are described in Fig. lb. Double-stranded phage DNA and plasmid DNA were prepared by the method of Birnboim and Doly (3).Expression of repressor genes and purification of penI. The flanking sequences of the penI+ and penIc genes were modified by oligonucleotide-directed site-specific mutagenesis to simplify subcloning into an E. coli expression vector. A HindlIl restriction site was introduced before the ATG initiation codon, and a BamHI site was introduced after the TGA codon of each of the four penl gene alleles. The penI genes were then subcloned as HindIII-BamHI cassettes into pFC54.t, and the resulting plasmids were used to transform strain DG116. Induction of the PL promoter was performed as described elsewhere (13).To purify the penicillinase repressor, we simplified the protocol for purifying X repressor (21)

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“…Our initial characterization of the plasmid obtained raised some doubt in our minds as to whether we really had pLS14, the plasmid requested, so we have designated it pPOD2000. It may be related to but is not identical to two plasmids from Bacillus amyloliquefaciens which have been partially characterized (Bron et al, 1987;Chang et al, 1987). Plasmid pPOD2000 is maintained at relatively low copy number (approximately five copies per chromosome equivalent) and has no directly selectable markers.…”

Section: Introductionmentioning

confidence: 99%

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance

Gleave

Mountain²,

Thomas³

1990

Journal of General Microbiology

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In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPGlOOO (5.0kb) which carries both a selectable marker (chloramphenicol resistance from Sfuphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonasputidh expressed from a cloned promoter of Bacillus phage SP02) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPGlOOO insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

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Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis

Cited by 40 publications

References 34 publications

Replication origins of single-stranded-DNA plasmid pUB110

Replication origins of single-stranded-DNA plasmid pUB110

Regulation of the penicillinase genes of Bacillus licheniformis: interaction of the pen repressor with its operators

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance

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