Chromosome I controls chromosome II replication in Vibrio cholerae

Abstract

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.

Figure 1. The chrII initiator, RctB, binds to sites outside of the chrII origin.

ChIP-chip profiles in the WT V. cholerae strain, N16961 (CVC209) in regions where the binding was considered above background. The background was assessed from RctB ChIP signals from ΔrctB strain, MCH1 (CVC2099). Regions were selected where the average difference in binding between the two strains was at least 2 in three independent experiments. (The average difference over the entire genome was −0.08±0.86.) The origin region of chrII, where RctB has known binding sites, is shown in (A), regions outside of the origin in chrII are shown in (B), and a region in chrI is shown in (C). chrII-1 to -12 and chrI-1 to -10 refer to fragments from the peak regions of (B) and (C) profiles, respectively, that were tested further for activity.

Figure 2. The newly identified chrII sites inhibit mini-chrII replication in E. coli and bind purified RctB.

(A) The activity of the chrII origin, oriII, was tested using a three-plasmid system in E. coli, where one plasmid carried oriII, poriII (pTVC35), another supplied RctB under arabinose control (pTVC11) and the third was either the empty vector (vector; pTVC243) or carried one of the new sites. As positive controls we used plasmids carried an iteron (piteron; pBH127) or a 39-mer (p39-mer; pTVC222). RctB was supplied at low (light gray bar) and high (dark gray bar) concentrations, using arabinose at 0.002% and 0.2%, respectively. The copy numbers of poriII were normalized to the copy number of poriII (called 1) when the third plasmid was the empty vector (pTVC243) and arabinose was at 0.002%. The mean values and standard deviations are from three independent experiments. (B) Binding of purified RctB to the new sites was tested by EMSA. Percent binding ([intensity of the retarded band/combined intensities of retarded and free bands]×100) at 2 nM (+) or 20 nM (++) are shown by light and dark gray bars, respectively. The error bars are from three independent measurements of band intensities from the same gel.

Figure 3. The newly identified chrI site enhances mini-chrII replication, improves cell growth and binds purified RctB.

(A) oriII activity was tested as in Figure 2A. The copy numbers with the empty vector are shown by dotted lines for reference purposes. (B) Growth of E. coli cells harboring three plasmids: poriII (pTVC35), prctB (pTVC11) and either an empty vector (pTVC243; solid lines) or the same vector carrying the chrI-4 fragment (pchrI-4 = pBJH170; dotted lines). The growth was tested in LB under antibiotic selection for all three plasmids, and OD₆₀₀ was monitored using the Synergy HT plate reader (BioTek, USA). Freshly transformed colonies from plates containing 0.2% arabinose were suspended in LB and cultivated at 37°C. (Upon saturation of growth, the cultures were diluted and they grew with their characteristic lag periods, indicating that the lag is unlikely to be due to accumulation of mutants.) The amount of RctB was varied either by omitting the inducer (−ara; gray lines) or by adding the inducer at 0.2% (+ara; black lines). (C) RctB binding to the chrI-4 sequence was tested by DNase I footprinting in vitro. The chrI-4 sequence was present in a supercoiled plasmid (pBJH170), and the DNase I treatment was done either in the absence or in the presence of 20 nM RctB. The sites of DNase I cleavage were mapped by primer extension. The extension products are shown only for the first 50 nt of the minimal 70 bp site (as in chrI-9) within which changes (shaded in blue) in the presence of RctB were significant. The DNase I level was adjusted so that there is one nick in the region of interest, making it unlikely that the plasmid was supercoiled during the DNase I probing. It remains possible that only the initial binding of RctB requires supercoiling but once bound the complexes remain stable enough to reveal the footprint in relaxed plasmids.

Figure 4. The chrI site modulates DNA binding of RctB and enhances poriII activity in ΔdnaKJ host.

(A) The effect of chrI-4 (present in pBJH170) on oriII activity was tested as in Figure 2A, except that four different the poriII plasmids were used (from top pTVC210, pTVC25, pTVC31 and pTVC524), containing different numbers of regulatory iterons and 39-mer sites (arrowheads and squares, respectively). Copy numbers were determined at high RctB concentration (0.2% arabinose) only. At low RctB concentration the copy number 39-mer carrying plasmids, pTVC210 and pTVC25, were too low to be measured reliably. (B) Non-essentiality of the 39-mer binding activity of RctB in the enhancer function. The copy numbers were measured as in (A) except that poriII was pTVC210 and RctB was a 39-mer binding defective mutant (RctBΔC157) . (C) In vivo binding of RctB to iterons and a 39-mer in the presence of empty vector (pKT25) or the same vector carrying chrI-4 or chrI-6 (pBJH188 or pBJH243, respectively). The vector and pchrI-6 were used as negative controls. The chrII origin contains two promoters, P_rctA and P_rctB, with overlapping iterons and a 39-mer, respectively. RctB binding to these sites represses the promoters. Activities of the promoters (in pTVC126 and pTVC500, respectively) were determined at three concentrations of arabinose at 0% (white bars), 0.002% (gray bars) and 0.2% (black bars). The error bars are from three independent measurements. (D) The effect of chrI-4 on the growth of ΔdnaKJ cells (BR4392) carrying poriII (pTVC31), prctB (pTVC11) and either the empty vector (pTVC243) (solid lines) or the same vector containing chrI-4 (pBJH170) (dotted lines). The cells were initially grown in LB containing antibiotics to select all three plasmids and 0.2% arabinose. At time zero, the cultures were diluted 1000× with fresh medium containing antibiotics and either no arabinose (gray lines) or 0.002% arabinose (black line).

Figure 5. The newly identified RctB binding sites affect V. cholerae growth and chromosomal replication.

(A) Growth was monitored by colony size of WT V. cholerae cells (CVC209) transformed with either an empty vector (pBR322 derivative, pTVC243) or the same vector containing chrII-10 (pTVC350) (first two panels), or an empty vector of lower copy number (pACYC177) and the same vector containing chrI-4 (pBJH188) (last two panels). (B) Chromosomal replication was tested by measuring relative frequencies of four markers: oriI, oriII, terI and terII, in cells with pchrII-10 (blue bars) or pchrI-4 (red bars). The relative frequencies were then expressed after dividing the corresponding relative frequencies in cells with empty vectors. Plasmids used were as in (A). Relative marker frequencies were also determined for the ΔchrII-10 mutant (CVC2565) and ΔchrI-4 mutant (CVC2542), and expressed after dividing with the corresponding ratios from the otherwise isogenic WT cell (CVC1121) (light blue and red bars). Data are averages from three independent experiments. (C) Localization of oriI and oriII in WT (CVC2553) and ΔchrI-4 mutant (CVC2554) strains. oriI and oriII were marked by inserting P1parS and pMTparS, and detected by GFP-P1ParB and mCherry-pMTParB, respectively. Plots show focus positions in cells with one focus (dark green for oriI and dark orange for oriII) and two foci (light green for oriI or light orange for oriII). Focus positions were measured from the pole from which the nearest focus was closer than the nearest focus from the opposite pole. The pole used to measure focus positions is placed on the abscissa and the other (distal) pole is shown as black circles. 500 cells were analyzed in each experiment.