The replication enhancer crtS depends on transcription factor Lrp for modulating binding of initiator RctB to ori2 of Vibrio cholerae.

Replication of Vibrio cholerae chromosome 2 (Chr2) initiates when the Chr1 locus, crtS (Chr2 replication triggering site) duplicates. The site binds the Chr2 initiator, RctB, and the binding increases when crtS is complexed with the transcription factor, Lrp. How Lrp increases the RctB binding and how RctB is subsequently activated for initiation by the crtS-Lrp complex remain unclear. Here we show that Lrp bends crtS DNA and possibly contacts RctB, acts that commonly promote DNA-protein interactions. To understand how the crtS-Lrp complex enhances replication, we isolated Tn-insertion and point mutants of RctB, selecting for retention of initiator activity without crtS. Nearly all mutants (42/44) still responded to crtS for enhancing replication, exclusively in an Lrp-dependent manner. The results suggest that the Lrp-crtS controls either an essential function or more than one function of RctB. Indeed, crtS modulates two kinds of RctB binding to the origin of Chr2, ori2, both of which we find to be Lrp-dependent. Some point mutants of RctB that are optimally modulated for ori2 binding without crtS still remained responsive to crtS and Lrp for replication enhancement. We infer that crtS-Lrp functions as a unit, which has an overarching role, beyond controlling initiator binding to ori2.

Kinetic principles of ParA2-ATP cycling guide dynamic subcellular localizations in Vibrio cholerae.

Dynamic protein gradients are exploited for the spatial organization and segregation of replicated chromosomes. However, mechanisms of protein gradient formation and how that spatially organizes chromosomes remain poorly understood. Here, we have determined the kinetic principles of subcellular localizations of ParA2 ATPase, an essential spatial regulator of chromosome 2 segregation in the multichromosome bacterium, Vibrio cholerae. We found that ParA2 gradients self-organize in V. cholerae cells into dynamic pole-to-pole oscillations. We examined the ParA2 ATPase cycle and ParA2 interactions with ParB2 and DNA. In vitro, ParA2-ATP dimers undergo a rate-limiting conformational switch, catalysed by DNA to achieve DNA-binding competence. This active ParA2 state loads onto DNA cooperatively as higher order oligomers. Our results indicate that the midcell localization of ParB2-parS2 complexes stimulate ATP hydrolysis and ParA2 release from the nucleoid, generating an asymmetric ParA2 gradient with maximal concentration toward the poles. This rapid dissociation coupled with slow nucleotide exchange and conformational switch provides for a temporal lag that allows the redistribution of ParA2 to the opposite pole for nucleoid reattachment. Based on our data, we propose a 'Tug-of-war' model that uses dynamic oscillations of ParA2 to spatially regulate symmetric segregation and positioning of bacterial chromosomes.

The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in E. coli chromosomal replication.

DnaA, the initiator of Escherichia coli chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5'-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in dnaA46ts cells and prevented cell growth. The growth defect was rescued by overexpressing the initiation inhibitor, SeqA, indicating that the growth inhibition resulted from overinitiation. Small deletions within the linker showed another unexpected phenotype: cellular growth without requiring normal levels of anionic membrane lipids, a property found in DnaA mutated in its ATPase domain. The deleted proteins were defective in association with anionic membrane vesicles. These results show that changes in the linker domain can alter DnaA functions similarly to the previously shown changes in the ATPase domain.

The dimerization interface of initiator RctB governs chaperone and enhancer dependence of Vibrio cholerae chromosome 2 replication.

Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA. A structurally homologs interface exists in RctB, the replication initiator of Vibrio cholerae chromosome 2 (Chr2). Chaperones also promote Chr2 replication, although both monomers and dimers of RctB bind to origin, and chaperones increase the binding of both. Here we report how five changes in the dimerization interface of RctB affect the protein. The mutants are variously defective in dimerization, more active as initiator, and except in one case, unresponsive to chaperone (DnaJ). The results indicate that chaperones also reduce RctB dimerization and support the proposal that the paradoxical chaperone-promoted dimer binding likely represents sequential binding of monomers on DNA. RctB is also activated for replication initiation upon binding to a DNA site, crtS, and three of the mutants are also unresponsive to crtS. This suggests that crtS, like chaperones, reduces dimerization, but additional evidence suggests that the remodelling activities function independently. Involvement of two remodelers in reducing dimerization signifies the importance of dimerization in limiting Chr2 replication.

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3'-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

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

In bacteria, mRNA decay is a major mechanism for regulating gene expression. In Escherichia coli, 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 galETKM transcript. We find that RNase E cleaves a few nucleotides (14-36) upstream of the translation initiation site of each cistron, generating decay intermediates galTKM, galKM, and galM 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 gal operon of mRNA decay by cistron elimination appears common in E. coli and Salmonella.

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

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.

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

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.

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

The human pathogen, Vibrio cholerae, 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, crtS, Chr2 replication initiates. Recent in vivo experiments demonstrate that crtS 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, crtS is an order of magnitude longer, suggesting that other factors can bind to it. We developed an in vivo screen to identify additional crtS-binding proteins and identified the global transcription factor, Lrp, as one such protein. Studies in vivo and in vitro indicate that Lrp binds to crtS and facilitates RctB binding to crtS. 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.

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

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.

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

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.

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

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.

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

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.

Chromosome segregation proteins of Vibrio cholerae as transcription regulators.

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.

Chromosome I controls chromosome II replication in Vibrio cholerae.

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.

Chromosome segregation in Vibrio cholerae.

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.

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

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.

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

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.

Cell size and the initiation of DNA replication in bacteria.

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.