Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage
[['Nearly 10% of bacteria from diverse phyla distribute their genetic information into more than one chromosome [1] .', 'The presence of multipartite genomes in bacteria provides a challenge to understand the mechanisms that determine timely replication and segregation of all chromosomes of a genome prior to cell division .', 'Failures in these processes can cause genome instability , slowed cell growth and cell death [2] .', 'Chromosome dynamics in bacteria with multipartite genomes is well studied in the human pathogen Vibrio cholerae .', 'All members of the Vibrio genus possess two circular , unequal-sized chromosomes , Chr1 and Chr2 .', 'The size of Chr1 is fairly conserved ( 3 . 0 to 3 . 4 Mb ) whereas there is greater diversity in the size of Chr2 ( 0 . 8 to 2 . 4 Mb ) [3] .', 'The replication of the two chromosomes is regulated differently [4] .', 'The regulation of Chr1 is similar to that of the well-studied Escherichia coli chromosome and is mediated through the universal initiator of bacterial replication , DnaA [5] .', 'Chr2 replication is regulated by its specific initiator RctB , which is highly conserved in the Vibrionaceae family [4 , 6] .', 'The regulation of Chr2 replication is reminiscent of the copy number control of some plasmids [7 , 8] .', 'The salient feature of Chr2 replication origin is the presence of an array of tandem repeats of 12-mers where the initiator RctB binds .', 'This feature of the origin is common in members of the iteron family of plasmids [5] .', 'Additionally , the central region of RctB is structurally homologous to plasmid initiators [9 , 10] .', 'The Chr2 replicon , however , has many additional features that distinguish it from its presumed plasmid progenitor .', 'For example , in addition to 12-mers , RctB binds to a second kind of site , 39-mers , which inhibits initiation [11] .', 'Another mechanism to inhibit initiation is built-in to the 12-mers in the form of adenine methylation sequences ( GATC ) , which become hemi-methylated following replication initiation and allow binding of the SeqA protein [5 , 12] .', 'Inhibition of replication initiation upon SeqA binding also occurs in the origins of the E . coli chromosome and Chr1 .', 'Thus , although seemingly plasmid-derived , the complexity of Chr2 replication control is comparable to those of other well-studied bacterial chromosomes , which is apparently required to maintain once-per-cell-cycle replication [8] .', 'Replication of Chr2 initiates after Chr1 [13] .', 'The lag in initiation time is such that both chromosomes terminate replication at approximately the same time .', 'The basis of the termination synchrony is due to a novel replication timing mechanism .', 'A unique RctB-binding site , crtS ( Chr2 replication triggering site ) , was discovered in Chr1 within a 153-bp locus [14] .', 'Chr2 replication initiation occurs after the Chr1 replication fork moves past the crtS site [15] .', 'This appears to be a conserved feature in most Vibrio species [16] .', 'The genomic location of crtS is such that it appropriately defers Chr2 replication for termination synchrony .', 'Moving crtS to ectopic sites changes the timing of Chr2 replication in accordance with the time at which the site is duplicated , which indicates that crtS locus needs to be replicated for Chr2 replication initiation [15] .', 'Thus far , this is the only genetic scheme known for coordinating replication timing of individual chromosomes in bacteria with multipartite genomes .', 'Supplying crtS on a multicopy plasmid increases replication of mini-Chr2 ( plasmids with Chr2 replication origin , ori2 ) in E . coli when RctB is provided , although some mini-Chr2 replication is seen in the absence of crtS as well [14] .', 'Increasing crtS copy number in Chr1 also increases Chr2 replication in V . cholerae [15] .', 'These results indicate that crtS function is limiting for Chr2 replication [14 , 15] .', 'This is further supported by the fact that deletion of crtS leads to chromosomal fusion or selection of suppressor mutations [15] .', 'The role of crtS thus appears crucial for Chr2 replication in V . cholerae .', 'The only clue as to how crtS might function comes from the in vivo finding that in its presence , RctB binding to 12-mers increases and to 39-mers decreases , both of which are favorable for initiation [14] .', 'These results suggest that crtS might remodel RctB into a more proficient initiator , which is further supported by the ability of crtS to partially substitute the DnaK and DnaJ chaperones required for RctB function .', 'Overall , these results indicate that crtS replication is required to trigger Chr2 replication and the mechanism of triggering is through remodeling of RctB .', 'However , how replication helps crtS function remains an open question .', 'Replication can serve by doubling the site dosage and the cumulative activity of two sites could suffice to remodel enough RctB to trigger Chr2 replication initiation .', 'It is also possible that the passage of the replication fork across crtS changes the topology of the site to make it more active .', 'Replication could cause crtS to be transiently hemi-methylated or single-stranded , or could clear out possible bound inhibitory factors such that the site can interact with RctB appropriately .', 'To address some of these possible roles of replication in crtS function , here we devised an approach to conditionally block Chr1 replication in V . cholerae and investigated the consequences towards triggering Chr2 replication initiation .', 'We conclude that the crtS locus has significant activity without replication , and doubling the crtS gene dosage , which is normally accomplished by Chr1 replication , suffices to activate Chr2 replication .', 'The product but not the process of replication thus appears obligatory for crtS function .', 'The unreplicated crtS locus appears to promote the initiator function of RctB , as in replication-permissive conditions .', 'Our finding that crtS has activity without replication is expected to simplify mechanistic studies on how crtS triggers Chr2 replication initiation .'], ['In V . cholerae , blocking of Chr2 replication initiation does not prevent Chr1 replication or cell division , indicating that there is no checkpoint to ensure completion of genome duplication before cell division [17] .', 'Here , we blocked Chr1 replication and tested its effect on the replication of Chr2 .', 'In the earlier study , Chr2 replication was blocked by adding an excess of 39-mer sites that specifically inhibited Chr2 replication initiation .', 'Because there is no known Chr1-specific inhibitor of initiation , we utilized the Tus-ter replication fork barrier system of E . coli to conditionally block Chr1 replication [18] .', 'Two E . coli ter sites were inserted on each flank of the Chr1 origin of replication , ori1 , and the ter-binding Tus protein of E . coli was supplied under control of the arabinose-inducible promoter , pbad ( Fig 1A ) .', 'By observing origin proximal fluorescent markers , we could simultaneously visualize Chr1 and Chr2 replication by microscopy .', 'The ori1 region was marked by GFP- P1ParB fluorescent protein bound to a P1parS site inserted at +135 kb from the origin .', 'The ori2 region was marked by tdTomato-pMTParB fluorescent protein bound to a pMTparS site inserted at +40 kb from the origin [17] .', 'The genes encoding the proteins were chromosomally inserted and expressed under control of the IPTG-inducible promoter , ptac .', 'Time-lapse microscopy showed that under our growth conditions , V . cholerae cells were born with two ori1 foci , one near each cell pole , and a single ori2 focus near the cell center ( Fig 1B , panel 1 ) .', 'Subsequently , the ori2 focus duplicated and occupied quarter-cell positions , while the two ori1 foci duplicated prior to completion of cell division ( panels 2–4 ) .', 'This scenario is indicative of multi-fork replication of Chr1 that results when the time to complete duplication of the chromosome exceeds the cell generation time [19 , 20] .', 'Following Tus induction to block Chr1 replication , cells contained only 1 ori1 focus ( panels 5–8 ) .', 'The focus did not duplicate as the cells lengthened for the duration of the time-lapse experiment ( equivalent to at least two generations in liquid medium under replication-permissive conditions ) .', 'Although the cells continued to lengthen , they failed to divide , indicative of blockage of Chr1 replication [21] .', 'The localization of ori1 was also not polar , unlike under normal growth ( S1 Fig ) .', 'The block in Chr1 replication was confirmed by qPCR where the ori1/ter1 ratio was seen to reduce from 2 . 8 to 1 . 4 at 150 min after Tus induction ( Fig 1C ) .', 'In sum , the appearance of cells with one ori1 focus instead of two , elongation of cells without division for more than two generations , reduction in ori1/ter1 ratio and loss of polar localization of ori1 are all consistent with a block to elongation of Chr1 replication forks .', 'In newborn cells that showed the continued presence of a single ori1 focus , the ori2 focus also remained single during the time lapse experiment , indicating that Chr2 replication initiation depends on the prior replication of Chr1 ( Fig 1B , panels 5–8 ) .', 'We were able to reverse the Chr1-replication block by removing the inducer of Tus expression ( arabinose ) using a microfluidic chamber ( S1 Movie; S2 Fig ) .', 'Upon removal of the inducer , ori1 foci duplicated within ~30 min , followed by duplication of the ori2 focus and resumption of cell division .', 'The removal of arabinose also caused a significant decrease in Tus protein level , explaining the resumption of ori1 duplication ( S3A Fig ) .', 'These results demonstrate that our approach to block Chr1 replication arrests the progression of the cell cycle ( Chr2 replication and cell division ) in a reversible fashion .', 'Although it is possible that the cell division block could be responsible for blocking Chr2 replication , our results discussed later argue against this possibility .', 'We restricted our analysis of ori2 foci only to cells that showed a single ori1 focus , which accounted for ~50% of all cells .', 'While majority of these cells had one ori2 focus , some cells exhibited duplication of ori2 foci within the duration of the time-lapse ( Fig 1B , panels 9–12 ) .', 'On a population basis , by counting foci in cells 150 min after the induction of Chr1 block , 13% of one ori1 cells were seen to have two or more ori2 foci ( Fig 1D ) , indicating that Chr2 has some propensity to initiate replication independent of Chr1 replication .', 'Cells with one ori1 and two ori2 foci were longer than cells with one ori1 and one ori2 foci: 4 . 0 ± 0 . 3 μm as compared to 3 . 0 ± 0 . 2 μm ( mean ± standard error , Fig 1E ) , suggesting that given enough accumulation of mass , Chr2 can replicate in the absence of Chr1 replication .', 'This is unlikely to be due to increase of RctB concentration over time , which remains essentially unchanged in Chr1 replication-blocked cells ( S3B Fig ) .', 'Importantly , the results provide the initial indication that unreplicated crtS has activity , as we show later .', 'An earlier study established that replication of the crtS locus on Chr1 is required for Chr2 replication [15] .', 'We also find that the 153-bp crtS in a plasmid ( pcrtS ) increases Chr2 replication ( S4A Fig ) [14] .', 'To test whether the crtS is the only locus of Chr1 whose replication was required for licensing Chr2 replication , we supplied pcrtS , in cells where Chr1 replication was blocked by the Tus-ter complex .', 'In Chr1-replication blocked cells with pcrtS , the number of cells with single ori1 focus and two or more ori2 foci increased 3-fold as compared to the empty vector ( from 8% to 28% , counted after 150 min of Tus induction ) ( Fig 2 ) .', 'Under the growth condition used for microscopy , relative to ori1 the copy number of pcrtS was approximated by qPCR to be 2 . 5 ± 1 . 3 under Chr1-replication permissive growth and 4 . 5 ± 1 . 6 under Chr1-replication blocked growth .', 'The presence of pcrtS had no effect on Chr1 replication under Tus-ter block , as the distribution of ori1 focus was similar whether the cells had pcrtS or the empty vector ( S5 Fig ) .', 'The 153 bp crtS locus thus appears sufficient to license Chr2 replication without requiring replication of any other Chr1 locus .', 'Our results so far are consistent with the view that crtS needs to be newly replicated for timely licensing of Chr2 replication .', 'How does replication help ?', 'To test whether simply doubling the crtS copy number , that normally occurs as the replication fork crosses the site , could suffice for initiating Chr2 replication , an extra copy of crtS was inserted in Chr1 either 10 kb upstream of the native locus at 0 . 80 Mb or 900 kb downstream of the native locus at 1 . 84 Mb ( hereafter called two-crtS cells ) .', 'We also constructed a strain containing three copies of crtS that included both the ectopic copies in addition to the native copy .', 'The strains with extra crtS copies were constructed with or without the Tus-ter block in Chr1 .', 'The cells without the Tus-ter block grew normally , as seen by their growth curves and average cell length ( S6 Fig ) .', 'We also deleted the native crtS locus from the strain where the added crtS copy was 10 kb upstream .', 'Its growth rate , average cell length and number of ori2 foci were similar to those of isogenic cells containing crtS at the native locus only , indicating that the added crtS functions normally at the new location .', 'In two-crtS cells without the Tus-ter block , a larger percentage of cells contained two or more ori2 foci ( ~60% in two-crtS cells vs . ~30% in one-crtS cells ) , indicating that Chr2 initiated earlier in the cell cycle than it did in one-crtS cells ( Fig 3A ) .', 'This was true whether the second crtS copy was close or far from the native site .', 'The average length of cells with two ori2 foci was also shorter in the presence of extra copies of crtS , indicating earlier initiation of Chr2 replication in the cell cycle ( S6C Fig ) .', 'The presence of three crtS copies further increased Chr2 replication such that nearly 80% of cells contained two or more ori2 foci .', 'These results are consistent with findings in an earlier study that showed that crtS activity is limiting for Chr2 replication under normal growth [15] .', 'We next blocked replication of Chr1 by inducing Tus expression .', 'Cells with one ori1 focus showed two ori2 foci in nearly 40% of two-crtS cells ( Fig 3B and 3C ) , which is a 3-fold higher frequency than that was seen in one-crtS cells .', 'Since Chr2 replication initiated more efficiently with two unreplicated copies of crtS , it would suggest that a role of replication is to double the crtS gene dosage and the act of replication may not be required for crtS function .', 'In other words , an unreplicated copy of crtS has activity towards initiating Chr2 replication .', 'The initiation was also similarly efficient whether the two crtS sites were near or far apart , suggesting that the sites probably do not interact with each other for function .', 'Since when Chr1 replication is not blocked , cells with more than one crtS copy also have more ori2 foci , a concern can be that the increased number of cells with two ori2 foci when Chr1 replication is blocked is a mere reflection of the ori2 copy number in the replication-permissive mother cell rather than the consequence of new Chr2 initiation events in the absence of Chr1 replication .', 'To address this concern , individual cells were followed by time-lapse microscopy at 20 min intervals beginning at 30 min after the addition of the inducer to block Chr1 replication ( S7 Fig ) .', 'Cells had either one , two or three crtS copies , as in Fig 3C .', 'Cells were followed from the time a mother cell with two ori1 foci divided into daughters with one ori1 focus .', 'Most of these cells were born with one ori2 focus .', 'During the course of time-lapse spanning ~ 150 min , the ori2 focus duplicated earlier and in more cells when they had more than one copy of crtS .', 'Only in about 4–9% of the newborn cells with multiple crtS copies , there were two ori2 foci and one ori1 focus .', 'These ori2 foci could have been inherited from the mother cell or be the product of duplication within the first 20 min interval of the time-lapse .', 'These results indicate that in cells with two or more ori2 foci scored at the end of time-lapse , the foci were mostly products of new duplication rather than inheritance from the mother ( Fig 3C ) .', 'Increasing the number of crtS copies from two to three did not significantly enhance the number of cells with two or more ori2 ( Fig 3C , S7 Fig ) .', 'In the absence of Chr1 replication , other factors necessary for replication probably become limiting for Chr2 initiation .', 'We note that RctB levels tend to increase with the number of crtS copies ( S8 Fig ) .', 'This is possibly due to increased Chr2 replication and consequently higher dosage of Chr2-encoded rctB gene .', 'A block of Chr1 replication in two-crtS cells also blocked cell division as it did in one-crtS cells .', 'Since in replication-blocked two-crtS cells Chr2 initiated efficiently , it is unlikely that the cell division block could be preventing Chr2 initiation ( Fig 1B , panels 5–8 ) .', 'The replication licensing activity of crtS is most probably mediated through RctB , so far the only factor known to bind crtS [14] .', 'Moreover , increasing the supply of RctB increases Chr2 replication , suggesting that the initiator is normally limiting for replication [4 , 6] .', 'These considerations led us to ask whether increasing RctB can overcome the requirement of Chr1 replication for Chr2 replication .', 'RctB was supplied using plasmids that expressed the initiator constitutively .', 'One plasmid produced RctB at near physiological level ( prctBLOW ) and another at nearly a 9-fold higher level ( prctBHIGH ) , as quantified by Western blotting ( S9A Fig ) .', 'As expected , increasing rctB expression increased Chr2 replication in cells with prctBHIGH when under Chr1 replication-permissive condition ( S9B Fig ) .', 'When Chr1 replication was blocked , a roughly 2-fold increase was observed in the number of cells with one ori1 and two ori2 foci in the presence of prctBLOW as compared to the empty vector ( from 8% to 22% , 150 min after Tus induction ) ( Fig 4A and 4B ) .', 'In the presence of prctBHIGH , the increase in the number of such cells was about 6-fold , accounting for roughly 50% of cells with one ori1 focus ( Fig 4B ) .', 'Thus , increasing the supply of RctB can overcome the dependence on Chr1 replication .', 'We believe that although normally two crtS copies are required , one crtS copy becomes adequate when the concentration of its interacting partner ( RctB ) is increased—a mass action effect .', 'This rescue , we show later , depends on the presence of the solo unreplicated crtS copy , consistent with RctB being the target of crtS .', 'RctB binding to crtS has been detected by DNase I footprinting and in cross-linking experiments [14] .', 'The interaction of RctB with crtS likely remodels the protein and increases its initiator function , since in the presence of the site , RctB binding to 12- and 39-mers in vivo changes in a manner that should favor initiation .', 'RctB is known to require remodeling to function since it depends on molecular chaperones , DnaK and DnaJ , for binding to both 12- and 39-mers and for maintaining a mini-Chr2 in E . coli [22] .', 'The replication defect of mini-Chr2 in ΔdnaKJ cells could be partially overcome by providing pcrtS [14] .', 'These studies suggest that crtS has chaperone-like remodeling activity on RctB .', 'To further probe the chaperone-like function of crtS , we tested an RctB mutant ( RctBL156R ) that is significantly defective in interactions with DnaK [9] .', 'This mutant binds to 12-mers poorly and does not support mini-Chr2 replication even in WT ( dnaKJ+ ) E . coli .', 'Cells dependent on mini-Chr2 replication grew when RctBL156R was the initiator , provided pcrtS was also present ( Fig 5 ) .', 'In comparison to the previous study using a host bearing a dnaK mutation , which can have pleiotropic effects , the present results obtained in the WT ( dnaK+ ) background using a RctB mutant that is specifically defective in interactions with DnaK provide stronger support for the remodeling activity of crtS .', 'Since RctBL156R retains some capacity to interact with DnaK , crtS could be helping RctB-DNA interaction , rather than substituting for the DnaK function .', 'In either case , the results are consistent with a remodeling function of crtS .', 'This is further supported by the observation that pcrtS enhanced the growth of E . coli dependent on mini-Chr2 replication when the mini-Chr2 origin , rather than the initiator RctB , was defective due to insertion of 5 bp within it [11] ( S10 Fig ) .', 'Deletion of crtS is detrimental for cell viability [15] .', 'Survivors are found where either Chr1 and Chr2 have fused or mutations are present .', 'The suppressor mutations so far have been found only in rctB or in its promoter .', 'In view of this finding , we sequenced the whole genome of an apparently viable ΔcrtS strain constructed in our earlier study [14] .', 'The sequencing indeed revealed a mutation in rctB ( S11A Fig ) .', 'Thus , the presence of a compensatory mutation or genomic rearrangements seems necessary for viability of ΔcrtS strains .', 'The RctB mutant was still responsive to crtS , since the initiator activity of the mutant increased in the presence of crtS ( S11B Fig ) .', 'This implies that the mutant is gained in functions not controlled by crtS .', 'To study the consequences of crtS deletion on a shorter time scale , we developed a method to excise crtS and study its effect on Chr2 replication in real-time .', 'Native crtS was flanked with the attB and attP attachment sites of ΦC31 phage and the cognate integrase gene was placed under the control of the ptet promoter [23 , 24] ( Fig 6A ) .', 'Upon induction of the integrase by anhydrotetracycline ( aTc ) , cells continued to divide but duplication of the ori2 foci ceased , giving rise to daughter cells that lacked any ori2 focus ( Fig 6B , S12 Fig and S2 Movie ) .', 'The continuation of cell division confirms a prior observation that the cell cycle is insensitive to block in Chr2 replication [17] .', 'On a population basis , after about two generations of adding aTc , ~50% of cells did not show an ori2 focus .', 'The proportion of cells without ori2 reduced to ~30% in the presence of pcrtS ( Fig 6C ) , implying that the presence of the plasmid helped Chr2 replication .', 'The inability of Chr2 to replicate upon induction of crtS excision indicates that for licensing crtS needs to be replicated anew in each cell cycle .', 'The ability to follow the consequences of crtS excision in real-time allowed us to test whether a single unreplicated copy of crtS has activity towards RctB .', 'Since crtS is crucial for Chr2 replication under normal conditions , an unreplicated crtS copy might still be required for Chr2 replication that is promoted by supplying excess RctB in Chr1 replication-blocked cells ( Fig 4 ) .', 'To test for the crtS dependence , we excised the site using the ΦC31 system as before .', 'One hour after inducing crtS excision , we induced Tus expression to block Chr1 replication .', 'When crtS was not excised , cells containing duplicated ori2 were 2-fold more in the presence of prctBLOW and 5-fold more in the presence of prctBHIGH compared to cells containing the empty vector ( Fig 4B ) .', 'In contrast , when crtS was excised , the percentage of cells with duplicated ori2 was similar in cells containing the empty vector or prctBLOW ( 9% vs . 12% ) and only 2-fold more in cells containing prctBHIGH ( 9% vs . 17% ) ( Fig 7 ) .', 'The residual replication of Chr2 upon crtS deletion could be due to some spontaneous remodeling .', 'These results are consistent with the view that unreplicated crtS possesses significant activity towards promoting RctB function , which becomes particularly crucial near the physiological concentrations of the initiator .'], ['There are several ways the passage of a replication fork can influence the activity of a site .', 'Simply doubling the copy number of a site can increase the cumulative activity that may suffice for function .', 'Indeed , our present data are consistent with this possibility since two unreplicated copies of crtS promoted Chr2 replication more effectively than one unreplicated copy ( Fig 3 ) .', 'Our finding that the act of replication is not essential also makes the following models less likely , though not mutually exclusive .', '1 . Replication changes crtS topology: The passage of a replication fork can drastically change DNA topology in its wake .', 'An approaching fork introduces positive supercoils , whereas after passage of the fork , the DNA gets relaxed or negatively supercoiled [26] .', 'The template strand , copied by lagging strand synthesis , also briefly remains single-stranded at the growing fork .', 'DNA topology-sensitive DNA-protein interactions thus can be profoundly altered during and immediately after the passage of the fork .', 'Our results indicate that the above DNA topology changes are not obligatory for crtS function .', '2 . Replication allows sandwiching of the target: It is conceivable that crtS function may require at least two copies of the site , e . g . , if the function involves sandwiching the target .', 'If interactions between sisters were a requirement , it occurred in our replication-blocked experiments without hemi-catenation [26] , and without hemi-methylation and SeqA binding [27] , common in newly replicated sister chromosomes , and with nearly equal facility when two unreplicated copies were near or far from each other ( Fig 3 ) .', '3 . Replication clears crtS of bound factors: It is possible that the passage of the fork dissociates factors that may bind and inhibit crtS function [28] .', 'We note that Chr2 does not replicate in all cells in the absence of Chr1 replication , even when provided with two crtS copies .', 'A likely reason could be blocking of Chr1-replication .', 'Since most , if not all , required factors for replication are synthesized from Chr1 , they may have been inadequately produced when cells had a single copy of Chr1 .', 'This is indicated when crtS copies were increased from two to three .', 'Cells with 2 or more ori2 increased from 61% to 81% under replication permissive condition ( Fig 3A ) , but remain unchanged at ~40% under replication blocked condition , despite the extra crtS ( Fig 3C ) .', 'Unless the cause for Chr2 not replicating in all cells is better understood , whether replication does more than just double the crtS copy number , such as change DNA topology as discussed earlier , cannot be ruled out from the present results .', 'It is possible that unreplicated crtS could be an inhibitor of Chr2 replication , for example , by titrating an activator of Chr2 replication or by coupling with ori2 , as in plasmid handcuffing [29] .', 'In these scenarios , inhibitory activity is expected to increase in the presence of additional unreplicated crtS copies .', 'Our finding that unreplicated crtS copies instead increase Chr2 replication makes these scenarios unlikely .', 'Moreover , should crtS be functioning only as an inhibitor in the absence of replication , deletion of the site should suffice to activate Chr2 replication , but it does not .', 'In sum , crtS appears to serve only a positive role in ori2 firing .', 'A single copy of crtS has significant but insufficient activity .', 'Doubling its copy number , normally afforded by replication , appears to raise the activity above the threshold required for initiating replication ( Fig 8 ) .', 'We showed earlier that RctB binding characteristics change in the presence of crtS [14] and provide new evidence here that the site relieves the chaperone requirement of RctB activity to some extent and allows the initiator to function with a defective origin ( Fig 5 and S10 Fig ) .', 'These results are consistent with crtS being a remodeler of RctB that promotes its initiator function , analogous to the role played by chaperones [9] .', 'There is precedence for DNA-binding mediated remodeling of initiator proteins and modulation of their function by chaperones [30–36] .', 'In our experiments , excess RctB could relieve the requirement for crtS replication , but the presence of an unreplicated crtS copy was still helpful ( Figs 4 vs . 7 ) .', 'Even though two crtS copies are required , presumably at the increased RctB concentration , there was enough interactions with an unreplicated copy of crtS to yield sufficient remodeled RctB for initiating Chr2 replication .', 'There is not sufficient structural information available on RctB at present for a meaningful discussion on the molecular nature of the remodeling .', 'How does a cell ensure once per cell cycle replication of Chr2 ?', 'By linking the replication of Chr2 to that of Chr1 via crtS the cell ensures that a trigger is received by Chr2 every time Chr1 is replicated .', 'Given that Chr1 initiation is tightly regulated , this is a fail proof mechanism for licensing Chr2 replication .', 'Our results suggest that doubling the crtS copy number could serve as the sensor for Chr2 initiation , by affecting the binding preference of RctB .', 'However , the nature of the block that prevents Chr2 replication in the absence of crtS duplication is not known ( Fig 1 ) , making it difficult to speculate how the block is overcome efficiently upon crtS duplication ( Fig 3 ) .', 'The Chr2 initiator RctB is limiting for Chr2 replication and indications are strong that crtS helps to overcome this rate-limiting component .', 'The negative regulatory forces then prevent further replication in the same cell cycle to ensure only one round of replication per cell cycle .', 'In our experiments , pcrtS failed to fully complement excision of the chromosomal crtS , although the site was present in similar copy number when plasmid-borne ( Figs 2B vs . 3C ) .', 'What compromises crtS activity when plasmid-borne is not clear but pcrtS is known to reduce colony size [14] .', 'We report here further evidence for stress to cells by pcrtS ( S4B Fig ) .', 'In the classical N16961 El Tor background of V . cholerae , colonies are smaller and display a rugose morphology in the presence of pcrtS but not the vector .', 'Although our microscopy strains do not show the rugose morphology ( expected because they are hapR+ [37] ) , their colony sizes are still smaller .', 'How pcrtS affects global cell physiology remains unknown .', 'Moreover , for unknown reasons , plasmids in general have reduced copy number and are unstable in V . cholerae [6] .', 'The instability of pcrtS and the stress caused by it are confounding factors in comparing crtS activity from the chromosome and the plasmid .', 'Further studies on why plasmid copy number is lower in V . cholerae and how pcrtS stresses cells will be required before the importance of location for crtS function can be understood .', 'Origin licensing is a key mechanism for limiting initiation of eukaryotic DNA replication to once per cell cycle [38] .', 'Although initiation occurs in S-phase , the helicases are loaded in the G1-phase and they cannot be loaded later in S-phase .', 'A helicase-loaded origin in G1-phase is considered licensed to initiate replication , although the actual initiation awaits activation of the helicases in S-phase by kinases CDK and DDK .', 'It is clear that crtS participation is critical for origin firing of Chr2 , but the exact step ( s ) of the initiation reaction in which crtS participation is required remains unclear .', 'The initiators of the iteron family of plasmids believed to be the progenitor of Chr2 are known to facilitate helicase loading in addition to DNA binding [39 , 40] .', 'A crtS-remodeled initiator could also be more proficient in helicase loading , in which case it would have a role beyond formation of the initial initiator-origin complex .', 'In phage λ , loading and activation of the helicase occur at separate stages , the latter requiring molecular chaperones [41] .', 'A role of the chaperone-mimic crtS in loading/activation of the helicase therefore cannot be ruled out .', 'In that case , the crtS-mediated licensing could be 
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['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 .']
['The timing of DNA replication initiation is controlled in all growing cells .', 'The conventional wisdom is that in bacteria such as E . coli , initiation occurs when sufficient active form of the initiator protein accumulates .', 'The same scenario possibly applies to V . cholerae Chr1 , whose replication in turn determines the timing of replication of its second chromosome , Chr2 .', 'This is because Chr2 replication initiation depends on the replication of a site , crtS , in Chr1 .', 'Here we show that the crtS site has significant activity towards triggering Chr2 replication without itself being replicated .', 'We propose that the doubling of crtS copy number , normally afforded by replication , allows accumulation of enough active Chr2-specific initiator to trigger initiation .', 'The study provides a novel example of how the timing of a gene function can be controlled by the timing of its duplication .']
['bacteriology', 'medicine and health sciences', 'pathology and laboratory medicine', 'chemical compounds', 'cell cycle and cell division', 'pathogens', 'cell processes', 'vibrio', 'microbiology', 'light microscopy', 'carbohydrates', 'organic compounds', 'plasmid construction', 'vibrio cholerae', 'dna replication', 'microscopy', 'bacterial genetics', 'dna construction', 'dna', 'molecular biology techniques', 'microbial genetics', 'bacteria', 'bacterial pathogens', 'microbial genomics', 'research and analysis methods', 'bacterial genomics', 'medical microbiology', 'fluorescence microscopy', 'microbial pathogens', 'chemistry', 'molecular biology', 'genetic loci', 'biochemistry', 'arabinose', 'cell biology', 'nucleic acids', 'organic chemistry', 'genetics', 'monosaccharides', 'biology and life sciences', 'physical sciences', 'genomics', 'organisms']