Chromosome 1 licenses chromosome 2 replication in <i>Vibrio cholerae</i> by doubling the <i>crtS</i> gene dosage

<div><p>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 <i>Vibrio cholerae</i>, 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, <i>crtS</i>, on Chr1. The mechanism by which replication of <i>crtS</i> allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra <i>crtS</i> copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated <i>crtS</i> 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 <i>crtS</i> 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.</p></div

p<i>crtS</i> allows Chr2 replication in Chr1 replication-blocked cells.

<p>(A) Time-lapse microscopy of a cell with p<i>crtS</i> (CVC3028) grown in the presence of 0.2% arabinose. Under this condition, cells with two or more <i>ori2</i> foci can be seen in cells with one <i>ori1</i> focus (panel 4), indicating Chr2 replication in the absence of Chr1 replication. Scale bar, 2 μm. (B) Histogram of <i>ori2</i> foci number per cell (x-axis) as percentages of cells that have one <i>ori1</i> focus (y-axis). Note that the percentage of cells with two or more <i>ori2</i> foci (yellow + blue bars) increases from 7.9 ± 0.9% (Σn = 498 cells) in vector carrying cells (CVC3145) to 28 ± 2.1% (Σn = 590 cells) in p<i>crtS</i> carrying cells. Data represents mean ± SEM of percentages calculated from three biological replicates with at least 100 cells in each replicate. Statistical significance was calculated using a Student’s <i>t</i>-test.</p

Cessation of Chr2 replication upon excision of <i>crtS</i>.

<p>(A) Scheme to excise <i>crtS</i> by flanking it with <i>attB</i> and <i>attP</i> sites and inducing the ΦC31 integrase gene from the <i>ptet</i> promoter with the inducer, aTc. Without induction, <i>ptet</i> is repressed by the product of <i>tetR</i>. The <i>nat</i> marker allows to monitor the frequency of excision. (B) Time-lapse microscopy of cells (CVC3082) without the integrase induction (panels 1–5), the same cells with induction (panels 6–10), and when the induction was in p<i>crtS</i>-carrying cells (CVC3137, panels 11–15). Chr2 replicates normally in the absence of the inducer, as seen by the increase from one to four <i>ori2</i> (red foci) per cell in about two generations. Induction of the integrase shows the inability of Chr2 to replicate even as cells continue to grow and divide, since only one <i>ori2</i> focus is visible after two generations. <i>ori2</i> foci duplication is seen upon integrase induction when p<i>crtS</i> was present. Scale bars, 2 μm. (C) Histogram of cells with different numbers of <i>ori2</i> foci in cells with or without p<i>crtS</i> after 150 min of integrase induction. Upon induction, the number of cells with no <i>ori2</i> focus increased significantly. The presence of p<i>crtS</i> partially complements the Chr2 replication defect upon excision of the chromosomal copy of <i>crtS</i>, as seen by the reduction in cells with no <i>ori2</i> focus from 49 ± 2.3% in WT cells to 28 ± 2.3% in p<i>crtS</i>-carrying cells. Total cells (Σn) counted for: WT -aTc = 1340, WT +aTc = 1250; p<i>crtS</i> -aTc = 1097, p<i>crtS</i> + aTc = 1173.</p

Blocking replication of Chr1 blocks Chr2 replication.

<p>(A) Scheme for blocking Chr1 replication by induction of <i>ter</i>-binding Tus protein from <i>pbad</i> promoter. (B) Time-lapse microscopy of <i>V</i>. <i>cholerae</i> (CVC3022) showing <i>ori1</i> (green) and <i>ori2</i> (red) foci when cells were grown either without inducer (panels 1–4) or with 0.2% inducer (arabinose) to induce Tus for blocking Chr1 replication (panels 5–12). Without inducer, cells were born with two <i>ori1</i> foci and one <i>ori2</i> focus (panel 1). Upon cell growth, the <i>ori2</i> focus duplicates (panel 2), followed by duplication of <i>ori1</i> before cell division (panels 3,4). With Tus induction, many cells show one <i>ori1</i> focus, indicating a block of Chr1 replication (panel 5). In most of these cells, the <i>ori2</i> focus remains single, indicating that Chr2 replication is blocked (panels 6–8). In a few cells, the <i>ori2</i> focus was seen to divide even as the <i>ori1</i> focus remained single, (panels 9–12). Scale bars, 2 μm. (C) Marker frequency analysis by qPCR. Markers analyzed were <i>ori1</i>, <i>ter1</i>, <i>ori2</i> and <i>ter2</i> of <i>V</i>. <i>cholerae</i> grown in LB in the absence (black) or presence (grey) of 0.2% arabinose for 150 min. The induction reduced the <i>ori1/ter1</i> and <i>ori2/ter2</i> values. Data represents mean ± standard error of mean (SEM) from three biological replicates, each performed in triplicate. Statistical significance was calculated using a Student’s <i>t</i>-test. (D) Histogram of <i>ori2</i> foci number per cell (x-axis) as percentages in cells that have one <i>ori1</i> focus (y-axis). The cells with no visible <i>ori2</i> focus (grey bars, ~17%) are possibly due to weak tdTomato fluorescence. Data represents mean ± SEM of percentages calculated from three biological replicates each with at least 100 cells (Σn = total cells counted = 522). (<i>E</i>) Mean length of cells with one <i>ori1</i> focus, two <i>ori2</i> foci and one or two <i>ori2</i> foci. Note that the cells with two <i>ori2</i> foci are longer, revealing that with accumulation of mass Chr2 can overcome its dependence on Chr1 replication. Error bars denote SEM.</p

p<i>crtS</i> promotes growth of cells dependent on RctB function.

<p>Growth curve of <i>E</i>. <i>coli</i> cells after transformation with three plasmids: One is p<i>rctB</i> (circles, pTVC14) or p<i>rctB</i>L156R (diamonds, pJJ263); the second is p<i>crtS</i> (filled-in symbols, pBJH188) or the corresponding empty vector (empty symbols, pACYC177), and the third is mini-Chr2 (pJJ114). The results show that the DnaK-interaction defective mutant, RctBL156R, is able to support growth of <i>E</i>. <i>coli</i> dependent on mini-Chr2 replication, when provided with p<i>crtS</i>. Representative growth curves are shown from two biological replicates each performed in triplicates.</p

Model for Chr2 replication licensing by remodeled RctB.

<p>The triggering of Chr2 replication initiation requires two copies of <i>crtS</i>. The two copies are normally produced by passage of the Chr1 replication fork across the <i>crtS</i> site (left diagram) or, can also be provided in the absence of Chr1 replication by inserting an extra copy of <i>crtS</i> (right diagram). This suggests that the product of replication but not the passage of the fork <i>per se</i> is obligatory for <i>crtS</i> function. In our experiments, the requirement for a second copy of <i>crtS</i> could be obviated by increasing RctB supply (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007426#pgen.1007426.g004" target="_blank">Fig 4</a>). This, together with the findings that to date all revertants of Δ<i>crtS</i> map in <i>rctB</i> and that <i>crtS</i> alters the DNA binding activities of RctB, lead us to propose that <i>crtS</i> activates RctB. The two <i>crtS</i> copies are apparently required to activate RctB sufficiently for initiation.</p

Extra copies of <i>crtS</i> in Chr1 promote Chr2 replication in Chr1 replication-blocked cells.

<p>(A) Histograms of <i>ori2</i> foci per cell in different strains when Chr1 replication was not blocked. The circles above the histograms show the location of <i>crtS</i> copies (stars) in Chr1 and their distance in Mb from <i>ori1</i> (tick mark at 12 o’clock). In these experiments, only <i>ori2</i> was fluorescently marked. The number of cells with ≥2 <i>ori2</i> foci increases in strains with two <i>crtS</i> copies (CVC3061 (ii) and CVC3093 (iii)), and increases even more in cells with three <i>crtS</i> copies (CVC3151 (iv)). Total cells (Σn) counted in (ii) to (iv) were 1233, 1609 and 1329, respectively. Deletion of the native <i>crtS</i> returns the distribution to that of WT cells (CVC3112 (v); Σn = 1346). (B) Time-lapse microscopy of cells with extra copies of <i>crtS</i> in Chr1 when its replication was blocked (CVC3056, panels 1–4; CVC3092, panels 5–8; and CVC3150, panels 9–12). In the schematics, the block to Chr1 replication is shown by crossed circles. In these cells, <i>ori2</i> foci duplicated frequently even as <i>ori1</i> focus remained unduplicated. Scale bars, 2 μm. (C) Histograms of <i>ori2</i> foci number per cell at 150 min after addition of 0.2% arabinose. Percentage of cells with one <i>ori1</i> focus and ≥2 <i>ori2</i> foci increases from 13 ± 0.15% (Σn = 522) in cells with one <i>crtS</i> (CVC3022) to 37 ± 5.1% (Σn = 726) in cells with an extra <i>crtS</i> 10 kb upstream (CVC3056), to 42 ± 5.6% (Σn = 516) in cells with an extra <i>crtS</i> 900 kb downstream (CVC3092), and to 41 ± 1.6% (Σn = 469) in cells with two extra <i>crtS</i> (CVC3151). The increase from two to three <i>crtS</i> carrying cells is not considered significant (<i>p</i>-value = 0.79). Data in (A) and (C) represent mean ± SEM of percentages calculated from three biological replicates with at least 100 cells in each replicate. Statistical significance was calculated using a Student’s <i>t</i>-test.</p

Excess RctB requires the unreplicated <i>crtS</i> site to promote Chr2 replication.

<p>Histogram of <i>ori2</i> foci numbers in cells with either the vector (CVC3169), or p<i>rctB</i>LOW (CVC3167) or p<i>rctB</i>HIGH (CVC3168), after integrase and Tus induction to excise <i>crtS</i> and block Chr1 replication. The number of cells with two <i>ori2</i> foci does not increase significantly in the presence of the vector vs. p<i>rctB</i>LOW (9.1 ± 0.5%, Σn = 544 vs. 12 ± 2.1%, Σn = 456), and increases about 2-fold to 17 ± 2.1% (Σn = 551) in the presence of p<i>rctB</i>HIGH. Note that when <i>crtS</i> was not excised the increases were higher, about 2- and 5-fold in the presence of p<i>rctB</i>LOW and p<i>rctB</i>HIGH, respectively (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007426#pgen.1007426.g004" target="_blank">Fig 4B</a>). Data represents mean ± SEM of percentages calculated from three biological replicates with at least 100 cells in each replicate. Statistical significance was calculated using a Student’s <i>t</i>-test.</p

Increasing RctB concentration promotes Chr2 replication in Chr1 replication-blocked cells.

<p>(A) Time-lapse microscopy of Chr1 replication-blocked cells with additional RctB at low (CVC3052) or high (CVC3125) levels supplied from plasmid p<i>rctB</i>LOW or p<i>rctB</i>HIGH. Increasing RctB using either plasmid allowed Chr2 replication independent of Chr1 replication. Scale bars, 2 μm. (B) Histogram of <i>ori2</i> foci number per cell in the presence of low or high RctB, 150 min after addition of 0.2% arabinose. Percentage of cells with one <i>ori1</i> focus and ≥2 <i>ori2</i> foci (yellow + blue bar) increased from 7.9 ± 0.9% (Σn = 498) in cells with vector (CVC3145) to 22 ± 1.9% (Σn = 622) in cells with p<i>rctB</i>LOW (CVC3052) and to 46 ± 3.5% (Σn = 516) in cells with p<i>rctB</i>HIGH (CVC3125). Data represent mean ± SEM of percentages calculated from three biological replicates with at least 100 cells in each replicate. Statistical significance was calculated using a Student’s <i>t</i>-test.</p