Publications by authors named "Dhruba K Chattoraj"

38 Publications

Translation Initiation Control of RNase E-Mediated Decay of Polycistronic mRNA.

Front Mol Biosci 2020 6;7:586413. Epub 2020 Nov 6.

Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, South Korea.

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In , mRNA decay initiates with endonucleolytic cleavage by RNase E. Translating ribosomes impede RNase E cleavage, thus providing stability to mRNA. In transcripts containing multiple cistrons, the translation of each cistron initiates separately. The effect of internal translation initiations on the decay of polycistronic transcripts remains unknown, which we have investigated here using the four-cistron transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates , , and mRNA with fewer but full cistrons. Blocking translation initiation reduced stability, particularly of the mutated cistrons and when they were the 5'-most cistrons. This indicates that, together with translation failure, the location of the cistron is important for its elimination. The instability of the 5'-most cistron did not propagate to the downstream cistrons, possibly due to translation initiation there. Cistron elimination from the 5' end was not always sequential, indicating that RNase E can also directly access a ribosome-free internal cistron. The finding in operon of mRNA decay by cistron elimination appears common in and .
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http://dx.doi.org/10.3389/fmolb.2020.586413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681074PMC
November 2020

Interactions of replication initiator RctB with single- and double-stranded DNA in origin opening of Vibrio cholerae chromosome 2.

Nucleic Acids Res 2020 11;48(19):11016-11029

Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-4260, USA.

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.
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http://dx.doi.org/10.1093/nar/gkaa826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641748PMC
November 2020

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Nucleic Acids Res 2020 01;48(1):200-211

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.
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http://dx.doi.org/10.1093/nar/gkz939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145717PMC
January 2020

Commentary: Functionality of Two Origins of Replication in Strains With a Single Chromosome.

Front Microbiol 2019 19;10:1314. Epub 2019 Jun 19.

Center of Cancer Research (CCR), National Cancer Institute (NCI) and National Institute Health (NIH), Bethesda, MD, United States.

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http://dx.doi.org/10.3389/fmicb.2019.01314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594386PMC
June 2019

A Requirement for Global Transcription Factor Lrp in Licensing Replication of Chromosome 2.

Front Microbiol 2018 10;9:2103. Epub 2018 Sep 10.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.

The human pathogen, , belongs to the 10% of bacteria in which the genome is divided. Each of its two chromosomes, like bacterial chromosomes in general, replicates from a unique origin at fixed times in the cell cycle. Chr1 initiates first, and upon duplication of a site in Chr1, , Chr2 replication initiates. Recent experiments demonstrate that binds the Chr2-specific initiator RctB and promotes its initiator activity by remodeling it. Compared to the well-defined RctB binding sites in the Chr2 origin, is an order of magnitude longer, suggesting that other factors can bind to it. We developed an screen to identify additional -binding proteins and identified the global transcription factor, Lrp, as one such protein. Studies and indicate that Lrp binds to and facilitates RctB binding to Chr2 replication is severely defective in the absence of Lrp, indicative of a critical role of the transcription factor in licensing Chr2 replication. Since Lrp responds to stresses such as nutrient limitation, its interaction with RctB presumably sensitizes Chr2 replication to the physiological state of the cell.
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http://dx.doi.org/10.3389/fmicb.2018.02103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139311PMC
September 2018

Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage.

PLoS Genet 2018 05 24;14(5):e1007426. Epub 2018 May 24.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, crtS, on Chr1. The mechanism by which replication of crtS allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra crtS copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated crtS copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that crtS activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.
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http://dx.doi.org/10.1371/journal.pgen.1007426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991422PMC
May 2018

Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony.

PLoS One 2016 8;11(12):e0166722. Epub 2016 Dec 8.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States of America.

The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites. The altered SeqA binding, however, does not affect initiation synchrony. Synchrony is also unaffected when individual SeqA sites are mutated. An apparent exception was one mutant where the mutation also changed an overlapping DnaA site. In this mutant, the observed asynchrony could be from altered DnaA binding, as selectively mutating this SeqA site did not cause asynchrony. These results reveal robust initiation synchrony against alterations of individual SeqA binding sites. The redundancy apparently ensures SeqA function in controlling replication in E. coli.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166722PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5145175PMC
July 2017

Opening the Strands of Replication Origins-Still an Open Question.

Front Mol Biosci 2016 30;3:62. Epub 2016 Sep 30.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health Bethesda, MD, USA.

The local separation of duplex DNA strands (strand opening) is necessary for initiating basic transactions on DNA such as transcription, replication, and homologous recombination. Strand opening is commonly a stage at which these processes are regulated. Many different mechanisms are used to open the DNA duplex, the details of which are of great current interest. In this review, we focus on a few well-studied cases of DNA replication origin opening in bacteria. In particular, we discuss the opening of origins that support the theta (θ) mode of replication, which is used by all chromosomal origins and many extra-chromosomal elements such as plasmids and phages. Although the details of opening can vary among different origins, a common theme is binding of the initiator to multiple sites at the origin, causing stress that opens an adjacent and intrinsically unstable A+T rich region. The initiator stabilizes the opening by capturing one of the open strands. How the initiator binding energy is harnessed for strand opening remains to be understood.
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http://dx.doi.org/10.3389/fmolb.2016.00062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043065PMC
September 2016

Initiator protein dimerization plays a key role in replication control of Vibrio cholerae chromosome 2.

Nucleic Acids Res 2014 26;42(16):10538-49. Epub 2014 Aug 26.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, Bethesda, MD 20892, USA

RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation. Mutations in rctB that reduce dimer binding increase monomer binding to 12-mers but decrease monomer binding to 39-mers. The mechanism of this paradoxical binding behavior has been unclear. Using deletion and alanine substitution mutants of RctB, we have now localized to a 71 amino acid region residues important for binding to the two kinds of DNA sites and for RctB dimerization. We find that the dimerization domain overlaps with both the DNA binding domains, explaining how changes in the dimerization domain can alter both kinds of DNA binding. Moreover, dimerization-defective mutants could be initiation-defective without apparent DNA binding defect. These results suggest that dimerization might be important for initiation beyond its role in controlling DNA binding. The finding that determinants of crucial initiator functions reside in a small region makes the region an attractive target for anti-V. cholerae drugs.
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http://dx.doi.org/10.1093/nar/gku771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176361PMC
January 2015

Chromosome segregation proteins of Vibrio cholerae as transcription regulators.

mBio 2014 May 6;5(3):e01061-14. Epub 2014 May 6.

ABSTRACT Bacterial ParA and ParB proteins are best known for their contribution to plasmid and chromosome segregation, but they may also contribute to other cell functions. In segregation, ParA interacts with ParB, which binds to parS centromere-analogous sites. In transcription, plasmid Par proteins can serve as repressors by specifically binding to their own promoters and, additionally, in the case of ParB, by spreading from a parS site to nearby promoters. Here, we have asked whether chromosomal Par proteins can likewise control transcription. Analysis of genome-wide ParB1 binding in Vibrio cholerae revealed preferential binding to the three known parS1 sites and limited spreading of ParB1 beyond the parS1 sites. Comparison of wild-type transcriptomes with those of ΔparA1, ΔparB1, and ΔparAB1 mutants revealed that two out of 20 genes (VC0067 and VC0069) covered by ParB1 spreading are repressed by both ParB1 and ParA1. A third gene (VC0076) at the outskirts of the spreading area and a few genes further away were also repressed, particularly the gene for an outer membrane protein, ompU (VC0633). Since ParA1 or ParB1 binding was not evident near VC0076 and ompU genes, the repression may require participation of additional factors. Indeed, both ParA1 and ParB1 proteins were found to interact with several V. cholerae proteins in bacterial and yeast two-hybrid screens. These studies demonstrate that chromosomal Par proteins can repress genes unlinked to parS and can do so without direct binding to the cognate promoter DNA. IMPORTANCE Directed segregation of chromosomes is essential for their maintenance in dividing cells. Many bacteria have genes (par) that were thought to be dedicated to segregation based on analogy to their roles in plasmid maintenance. It is becoming clear that chromosomal par genes are pleiotropic and that they contribute to diverse processes such as DNA replication, cell division, cell growth, and motility. One way to explain the pleiotropy is to suggest that Par proteins serve as or control other transcription factors. We tested this model by determining how Par proteins affect genome-wide transcription activity. We found that genes implicated in drug resistance, stress response, and pathogenesis were repressed by Par. Unexpectedly, the repression did not involve direct Par binding to cognate promoter DNA, indicating that the repression may involve Par interactions with other regulators. This pleiotropy highlights the degree of integration of chromosomal Par proteins into cellular control circuitries.
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http://dx.doi.org/10.1128/mBio.01061-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010829PMC
May 2014

Chromosome I controls chromosome II replication in Vibrio cholerae.

PLoS Genet 2014 Feb 27;10(2):e1004184. Epub 2014 Feb 27.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication.
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http://dx.doi.org/10.1371/journal.pgen.1004184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937223PMC
February 2014

Chromosome segregation in Vibrio cholerae.

J Mol Microbiol Biotechnol 2014 17;24(5-6):360-70. Epub 2015 Feb 17.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Md., USA.

The study of chromosome segregation is currently one of the most exciting research frontiers in cell biology. In this review, we discuss our current knowledge of the chromosome segregation process in Vibrio cholerae, based primarily on findings from fluorescence microscopy experiments. This bacterium is of special interest because of its eukaryotic feature of having a divided genome, a feature shared with 10% of known bacteria. We also discuss how the segregation mechanisms of V. cholerae compare with those in other bacteria, and highlight some of the remaining questions regarding the process of bacterial chromosome segregation.
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http://dx.doi.org/10.1159/000368853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363095PMC
October 2015

Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II.

PLoS Genet 2013 Jun 20;9(6):e1003579. Epub 2013 Jun 20.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.
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http://dx.doi.org/10.1371/journal.pgen.1003579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688505PMC
June 2013

Insensitivity of chromosome I and the cell cycle to blockage of replication and segregation of Vibrio cholerae chromosome II.

mBio 2012 8;3(3). Epub 2012 May 8.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland, USA.

Unlabelled: Vibrio cholerae has two chromosomes (chrI and chrII) whose replication and segregation are under different genetic controls. The region covering the replication origin of chrI resembles that of the Escherichia coli chromosome, and both origins are under control of the highly conserved initiator, DnaA. The origin region of chrII resembles that of plasmids that have iterated initiator-binding sites (iterons) and is under control of the chrII-specific initiator, RctB. Both chrI and chrII encode chromosome-specific orthologs of plasmid partitioning proteins, ParA and ParB. Here, we have interfered with chrII replication, segregation, or both, using extra copies of sites that titrate RctB or ParB. Under these conditions, replication and segregation of chrI remain unaffected for at least 1 cell cycle. In this respect, chrI behaves similarly to the E. coli chromosome when plasmid maintenance is disturbed in the same cell. Apparently, no checkpoint exists to block cell division before the crippled chromosome is lost by a failure to replicate or to segregate. Whether blocking chrI replication can affect chrII replication remains to be tested.

Importance: Chromosome replication, chromosome segregation, and cell division are the three main events of the cell cycle. They occur in an orderly fashion once per cell cycle. How the sequence of events is controlled is only beginning to be answered in bacteria. The finding of bacteria that possess more than one chromosome raises the important question: how are different chromosomes coordinated in their replication and segregation? It appears that in the evolution of the two-chromosome genome of V. cholerae, either the secondary chromosome adapted to the main chromosome to ensure its maintenance or it is maintained independently, as are bacterial plasmids. An understanding of chromosome coordination is expected to bear on the evolutionary process of chromosome acquisition and on the efficacy of possible strategies for selective elimination of a pathogen by targeting a specific chromosome.
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http://dx.doi.org/10.1128/mBio.00067-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350373PMC
August 2012

Replication regulation of Vibrio cholerae chromosome II involves initiator binding to the origin both as monomer and as dimer.

Nucleic Acids Res 2012 Jul 24;40(13):6026-38. Epub 2012 Mar 24.

Laboratory of Biochemistry and Molecular Biology, NCI, 37 Convent Drive, NIH, Bethesda, MD 20892-4260, USA.

The origin region of Vibrio cholerae chromosome II (chrII) resembles plasmid origins that have repeated initiator-binding sites (iterons). Iterons are essential for initiation as well as preventing over-initiation of plasmid replication. In chrII, iterons are also essential for initiation but over-initiation is prevented by sites called 39-mers. Both iterons and 39-mers are binding sites of the chrII specific initiator, RctB. Here, we have isolated RctB mutants that permit over-initiation in the presence of 39-mers. Characterization of two of the mutants showed that both are defective in 39-mer binding, which helps to explain their over-initiation phenotype. In vitro, RctB bound to 39-mers as monomers, and to iterons as both monomers and dimers. Monomer binding to iterons increased in both the mutants, suggesting that monomers are likely to be the initiators. We suggest that dimers might be competitive inhibitors of monomer binding to iterons and thus help control replication negatively. ChrII replication was found to be dependent on chaperones DnaJ and DnaK in vivo. The chaperones preferentially improved dimer binding in vitro, further suggesting the importance of dimer binding in the control of chrII replication.
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http://dx.doi.org/10.1093/nar/gks260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401445PMC
July 2012

Cell size and the initiation of DNA replication in bacteria.

PLoS Genet 2012 1;8(3):e1002549. Epub 2012 Mar 1.

Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America.

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ~30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA.
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http://dx.doi.org/10.1371/journal.pgen.1002549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291569PMC
September 2012

Chromosome dynamics in multichromosome bacteria.

Biochim Biophys Acta 2012 Jul 28;1819(7):826-9. Epub 2012 Jan 28.

Laboratory of Molecular Biology and Biochemistry, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.

On the basis of limited information, bacteria were once assumed to have no more than one chromosome. In the era of genomics, it has become clear that some, like eukaryotes, have more than one chromosome. Multichromosome bacteria provide opportunities to investigate how split genomes emerged, whether the individual chromosomes communicate to coordinate their replication and segregation, and what selective advantages split genomes might provide. Our current knowledge of these topics comes mostly from studies in Vibrio cholerae, which has two chromosomes, chr1 and chr2. Chr1 carries out most of the house-keeping functions and is considered the main chromosome, whereas chr2 appears to have originated from a plasmid and has acquired genes of mostly unknown origin and function. Nevertheless, unlike plasmids, chr2 replicates once and only once per cell cycle, like a bona fide chromosome. The two chromosomes replicate and segregate using separate programs, unlike eukaryotic chromosomes. They terminate replication synchronously, suggesting that there might be communication between them. Replication of the chromosomes is affected by segregation genes but in a chromosome specific fashion, a new development in the field of DNA replication control. The split genome allows genome duplication to complete in less time and with fewer replication forks, which could be beneficial for genome maintenance during rapid growth, which is the norm for V. cholerae in broth cultures and in the human host. In the latter, the expression of chr2 genes increases preferentially. Studies of chromosome maintenance in multichromosomal bacteria, although in their infancy, are already broadening our view of chromosome biology. This article is part of a Special Issue entitled: Chromatin in time and space.
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http://dx.doi.org/10.1016/j.bbagrm.2012.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348396PMC
July 2012

Kurt Nordström (1935-2011).

Plasmid 2012 Mar 10;67(2):73. Epub 2012 Jan 10.

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http://dx.doi.org/10.1016/j.plasmid.2012.01.003DOI Listing
March 2012

A 29-mer site regulates transcription of the initiator gene as well as function of the replication origin of Vibrio cholerae chromosome II.

Plasmid 2012 Mar 9;67(2):102-10. Epub 2012 Jan 9.

Laboratory of Biochemistry and Molecular Biology, NCI, NIH, Bethesda, MD 20892-4260, USA.

The region responsible for replication of Vibrio cholerae chromosome II (chrII) resembles those of plasmids that have repeated initiator binding sites (iterons) and an autorepressed initiator gene. ChrII has additional features: Its iterons require full methylation for initiator (RctB) binding, which makes them inactive for a part of the cell cycle when they are hemi-methylated. RctB also binds to a second kind of site, called 39-mers, in a methylation independent manner. This binding is inhibitory to chrII replication. The site that RctB uses for autorepression has not been identified. Here we show that a 29-mer sequence, similar to the 39-mers, serves as that site, as we find that it binds RctB in vitro and suffices to repress the rctB promoter in vivo. The site is not subject to methylation and is likely to be active throughout the cell cycle. The 29-mer, like the 39-mers, could inhibit RctB-dependent mini-chrII replication in Escherichia coli, possibly by coupling with iterons via RctB bridges, as was seen in vitro. The 29-mer thus appears to play a dual role in regulating chrII replication: one independent of the cell cycle, the other dependent upon iteron methylation, hence responsive to the cell cycle.
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http://dx.doi.org/10.1016/j.plasmid.2011.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319240PMC
March 2012

Transition from a plasmid to a chromosomal mode of replication entails additional regulators.

Proc Natl Acad Sci U S A 2011 Apr 28;108(15):6199-204. Epub 2011 Mar 28.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Plasmid origins of replication are rare in bacterial chromosomes, except in multichromosome bacteria. The replication origin of Vibrio cholerae chromosome II (chrII) closely resembles iteron-bearing plasmid origins. Iterons are repeated initiator binding sites in plasmid origins and participate both in replication initiation and its control. The control is mediated primarily by coupling of iterons via the bound initiators ("handcuffing"), which causes steric hindrance to the origin. The control in chrII must be different, since the timing of its replication is cell cycle-specific, whereas in plasmids it is random. Here we show that chrII uses, in addition to iterons, another kind of initiator binding site, named 39-mers. The 39-mers confer stringent control by increasing handcuffing of iterons, presumably via initiator remodeling. Iterons, although potential inhibitors of replication themselves, restrain the 39-mer-mediated inhibition, possibly by direct coupling ("heterohandcuffing"). We propose that the presumptive transition of a plasmid to a chromosomal mode of control requires additional regulators to increase the stringency of control, and as will be discussed, to gain the capacity to modulate the effectiveness of the regulators at different stages of the cell cycle.
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http://dx.doi.org/10.1073/pnas.1013244108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076835PMC
April 2011

Participation of chromosome segregation protein ParAI of Vibrio cholerae in chromosome replication.

J Bacteriol 2011 Apr 21;193(7):1504-14. Epub 2011 Jan 21.

Lab. of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, 37 Convent Drive, Rm. 6044, NIH, Bethesda, MD 20892-4260, USA.

Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found that deletion of parBI also resulted in a less polar localization of oriI. However, unlike the deletion of parAI, the deletion of parBI increased the oriI number. Replication was normal when both parAI and parBI were deleted, suggesting that ParBI mediates its action through ParAI. Overexpression of ParAI in a parABI-deleted strain also increased the DNA content. The results are similar to those found for Bacillus subtilis, where ParA (Soj) stimulates replication and this activity is repressed by ParB (SpoOJ). As in B. subtilis, the stimulation of replication most likely involves the replication initiator DnaA. Our results indicate that control of chromosomal DNA replication is an additional function of chromosomal par genes conserved across the Gram-positive/Gram-negative divide.
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http://dx.doi.org/10.1128/JB.01067-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067663PMC
April 2011

DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

PLoS Genet 2010 May 6;6(5):e1000939. Epub 2010 May 6.

Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.
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http://dx.doi.org/10.1371/journal.pgen.1000939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865523PMC
May 2010

EMBO Conference on Replication and Segregation of Chromosomes, Geilo, Norway, June 16-20. Replication and segregation of chromosomes in the three domains of life: EMBO conference reports common grounds. Meeting report.

Plasmid 2009 Mar 24;61(2):89-93. Epub 2008 Dec 24.

Laboratories of Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4260, USA.

A meeting of the EMBO Conference Series on Replication and Segregation of Chromosomes was held in Geilo, Norway, 16-20 June, 2008, under a scenic backdrop of high mountains. The meeting focused on the mechanistic details of replication and segregation primarily from well-characterized systems. Because the same basic principles govern chromosome maintenance in all three domains of life, participants encountering parallel processes in distantly-related organisms were stimulated to interact. Another successful aspect of the meeting was the quality of the posters, several of which were chosen for platform presentation and two for special rewards. The organizers Kirsten Skarstad and Erik Boye deserve praise for their skillful organization of the meeting, the highlights of which are discussed below.
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http://dx.doi.org/10.1016/j.plasmid.2008.10.003DOI Listing
March 2009

Selective chromosome amplification in Vibrio cholerae.

Mol Microbiol 2007 Nov 17;66(4):1016-28. Epub 2007 Oct 17.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4260, USA.

Most bacteria have one chromosome but some have more than one, as is common in eukaryotes. How multiple chromosomes are maintained in bacteria remains largely obscure. Here we have examined the behaviour of the two Vibrio cholerae chromosomes as a function of growth rate. At slow growth rates, both chromosomes were maintained at copy numbers of one to two per cell. Increasing the growth rate by nutritional shift-up amplified the origin-proximal DNA of the larger chromosome (chrI) to four copies per cell, but not that of the smaller chrII. The latter was amplified when its specific initiator was supplied in excess or a specific negative regulator was deleted. The growth rate-insensitive behaviour of chrII, whose origin is similar to origins of members of a major class of plasmids, was shared by some but not all of several representative plasmids tested in V. cholerae. Also, unlike plasmid replication, chrII replication is known to be initiated at a specific stage of the cell cycle. Raising chrII copy number decreased growth rate, suggesting that this chromosome might serve as a repository for necessary but potentially deleterious genes.
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http://dx.doi.org/10.1111/j.1365-2958.2007.05973.xDOI Listing
November 2007

Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae.

J Bacteriol 2007 Oct 17;189(20):7450-63. Epub 2007 Aug 17.

Laboratory of Biochemistry and Molecular Biology, NCI, NIH, Bethesda, MD 20892-4260, USA.

MreB is an actin homolog required for the morphogenesis of most rod-shaped bacteria and for other functions, including chromosome segregation. In Caulobacter crescentus and Escherichia coli, the protein seems to play a role in the segregation of sister origins, but its role in Bacillus subtilis chromosome segregation is less clear. To help clarify its role in segregation, we have here studied the protein in Vibrio cholerae, whose chromosome I segregates like the one in C. crescentus and whose chromosome II like the one in E. coli or B. subtilis. The properties of Vibrio MreB were similar to those of its homologs in other bacteria in that it formed dynamic helical filaments, was essential for viability, and was inhibited by the drug A22. Wild-type (WT) cells exposed to A22 became spherical and larger. The nucleoids enlarged correspondingly, and the origin positions for both the chromosomes no longer followed any fixed pattern. However, the sister origins separated, unlike the situation in other bacteria. In mutants isolated as A22 resistant, the nucleoids in some cases appeared compacted even when the cell shape was nearly normal. In these cells, the origins of chromosome I were at the distal edges of the nucleoid but not all the way to the poles where they normally reside. The sister origins of chromosome II also separated less. Thus, it appears that the inhibition or alteration of Vibrio MreB can affect both the nucleoid morphology and origin localization.
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http://dx.doi.org/10.1128/JB.00362-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168437PMC
October 2007

Tryptophanase in sRNA control of the Escherichia coli cell cycle.

Mol Microbiol 2007 Jan 5;63(1):1-3. Epub 2006 Dec 5.

Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4260, USA.

The field of gene regulation underwent a major revolution with the discovery of small non-coding RNAs (sRNAs) and the various roles they play in organisms from bacteria to man. Escherichia coli has more than 60 sRNAs that are transcribed primarily from intergenic regions. They usually target the leader region of mRNAs and prevent their translation. Protein targets are relatively rare. In this issue of Molecular Microbiology, Chant and Summers provide an example of a totally unexpected protein target. They show that dimers of plasmid ColE1 make an sRNA that interacts directly with the enzyme tryptophanase and enhances its affinity for its substrate, tryptophan. A breakdown product, indole, then arrests cell division until the dimers are resolved to monomers. The monomerization helps to prevent plasmid loss. Targeting a catabolic enzyme to buy time for recombination is an amazing example of adaptation, which illustrates the power of a selfish element (a plasmid in this case) to exploit the host cell machinery to its advantage.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05517.xDOI Listing
January 2007

IHF-dependent activation of P1 plasmid origin by dnaA.

Mol Microbiol 2006 Dec;62(6):1739-51

Laboratory of Biochemistry, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4255, USA.

In bacteria, many DNA-protein interactions that initiate transcription, replication and recombination require the mediation of DNA architectural proteins such as IHF and HU. For replication initiation, plasmid P1 requires three origin binding proteins: the architectural protein HU, a plasmid-specific initiator, RepA, and the Escherichia coli chromosomal initiator, DnaA. The two initiators bind in the origin of replication to multiple sites, called iterons and DnaA boxes respectively. We show here that all five known DnaA boxes can be deleted from the plasmid origin provided the origin is extended by about 120 bp. The additional DNA provides an IHF site and most likely a weak DnaA binding site, because replacing the putative site with an authentic DnaA box enhanced plasmid replication in an IHF-dependent manner. IHF most likely brings about interactions between distally bound DnaA and RepA by bending the intervening DNA. The role of IHF in activating P1 origin by allowing DnaA binding to a weak site is reminiscent of the role the protein plays in initiating the host chromosomal replication.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05479.xDOI Listing
December 2006

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

Proc Natl Acad Sci U S A 2006 Aug 27;103(32):12051-6. Epub 2006 Jul 27.

Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4260, USA.

The bacterium Vibrio cholerae has two chromosomes. The origin of replication of chromosome I is similar to that of Escherichia coli. The origin-containing region of chromosome II (oriCII) resembles replicons of plasmids such as P1, except for the presence of an additional gene, rctA [Egan, E. S. & Waldor, M. K. (2003) Cell 114, 521-530]. 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.
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http://dx.doi.org/10.1073/pnas.0605120103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1567695PMC
August 2006

Origin inactivation in bacterial DNA replication control.

Mol Microbiol 2006 Jul;61(1):9-15

Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

Initiation of DNA replication is a highly regulated process in all organisms. Proteins that are required to recruit DNA polymerase - initiator proteins - are often used to regulate the timing or frequency of initiation in the cell cycle by limiting either their own synthesis or availability. Studies of the Escherichia coli chromosome and of bacterial plasmids with iterated initiator binding sites (iterons) have revealed that, in addition to initiator limitation, replication origin inactivation is used to prevent replication that is untimely or excessive. Our recent studies of plasmid P1 revealed that this additional mode of control becomes a requirement when initiator availability is limited only by autoregulation. Thus, although initiator limitation appears to be a well-conserved and central mode of replication control, optimal replication might require additional control mechanisms. This review gives examples of how the multiple mechanisms can act synergistically, antagonistically or be partially redundant to guarantee low frequency events. The lessons learned are likely to help understand many other regulatory systems in the bacterial cell.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05229.xDOI Listing
July 2006

Segregation of the replication terminus of the two Vibrio cholerae chromosomes.

J Bacteriol 2006 Feb;188(3):1060-70

Laboratory of Biochemistry, NIH, Bethesda, MD 20892-4255, USA.

Genome duplication and segregation normally are completed before cell division in all organisms. The temporal relation of duplication and segregation, however, can vary in bacteria. Chromosomal regions can segregate towards opposite poles as they are replicated or can stay cohered for a considerable period before segregation. The bacterium Vibrio cholerae has two differently sized circular chromosomes, chromosome I (chrI) and chrII, of about 3 and 1 Mbp, respectively. The two chromosomes initiate replication synchronously, and the shorter chrII is expected to complete replication earlier than the longer chrI. A question arises as to whether the segregation of chrII also is completed before that of chrI. We fluorescently labeled the terminus regions of chrI and chrII and followed their movements during the bacterial cell cycle. The chrI terminus behaved similarly to that of the Escherichia coli chromosome in that it segregated at the very end of the cell division cycle: cells showed a single fluorescent focus even when the division septum was nearly complete. In contrast, the single focus representing the chrII terminus could divide at the midcell position well before cell septation was conspicuous. There were also cells where the single focus for chrII lingered at midcell until the end of a division cycle, like the terminus of chrI. The single focus in these cells overlapped with the terminus focus for chrI in all cases. It appears that there could be coordination between the two chromosomes through the replication and/or segregation of the terminus region to ensure their segregation to daughter cells.
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http://dx.doi.org/10.1128/JB.188.3.1060-1070.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1347332PMC
February 2006