Multipartite Regulation of<i>rctB</i>, the Replication Initiator Gene of<i>Vibrio cholerae</i>Chromosome II

Pal, Debasish; Venkova-Canova, Tatiana; Srivastava, Preeti; Chattoraj, Dhruba K.

doi:10.1128/jb.187.21.7167-7175.2005

Cited by 67 publications

(59 citation statements)

References 32 publications

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“…30) by cloning origin fragments (black lines) into vector pGP704 and supplying RctB from an arabinose-inducible source of RctB (pTVC11) (the system is shown schematically on the bottom left corner). The arabinose concentration was 0.002%, which produced RctB at a level approaching that of V. cholerae (31). For pTVC20 and pTVC25, the inducer concentration also was 0.2%.…”

Section: Resultsmentioning

confidence: 84%

“…Similar results were obtained with a different oriCII plasmid, pTVC36, which lacks the rctB leader region proposed to be a target for rctA acting as an antisense RNA (ref. 31; data not shown). The V. cholerae sequence present in pTVC36 is identical to those present in pTVC33 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This increased plasmid yield as found in ref. 31, making quantification of plasmid bands more reliable. The copy numbers were normalized relative to that of pTVC22, also determined at two different arabinose concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…The 11-mer itself matches the consensus 12-mer sequence in eight positions. RctB-binding sites for autoregulation also show only six͞seven matches to the 12-mer (31). A search of RctB repeats outside of ig2 by using the program RSAT (http:͞͞ embnet.ccg.unam.mx͞rsa-tools) identified 36 sites in chrI and 23 sites in chrII, when only up to three mismatches to the 12-mer was allowed (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, RctB controls its own titration through rctA transcription. RctB also represses its own transcription (autorepression) (28,31). These control steps seem suited to adjust for both upward and downward fluctuations in RctB availability and contribute to initiator homeostasis (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae

Messerschmidt

Kemter

Schindler

et al. 2014

Biotechnology Journal

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Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications.

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Modular Assembly of Synthetic Secondary Chromosomes

Zumkeller,

Schindler,

Felder

et al. 2024

Methods in Molecular Biology

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Multipartite Regulation ofrctB, the Replication Initiator Gene ofVibrio choleraeChromosome II

Cited by 67 publications

References 32 publications

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae

Modular Assembly of Synthetic Secondary Chromosomes

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