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 ChIPchip 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

Control of rep Gene Expression in Plasmid pGA1 from Corynebacterium glutamicum

The cryptic multicopy plasmid pGA1 (4,826 bp) from Corynebacterium glutamicum LP-6 belongs to the fifth group of rolling-circle-replicating plasmids. A determinant, which negatively controls pGA1 replication, was localized in the leader region of the rep gene coding for the initiator of plasmid replication. This region, when cloned into the compatible vector pEC6, was found to cause decrease of segregational stability of the pGA1 derivative pKG48. A promoter and a single transcriptional start site were found in the rep leader region in orientation opposite to the rep gene. These results suggest that a small countertranscribed RNA (ctRNA) (ca. 89 nucleotides in length), which might inhibit translation of pGA1 rep gene, is formed. Analysis of predicted secondary structure of the pGA1-encoded ctRNA revealed features common with the known ctRNAs in bacteria. Inactivation of the promoter P-ctRNA caused a dramatic increase of copies of the respective plasmid, which proved a negative role of the ctRNA in control of pGA1 copy number. A region between the promoters Prep and P-ctRNA with a potential to form secondary structures on both ctRNA and rep mRNA was found to cause low activity of the rep promoter even when promoter P-ctRNA was deleted. Thus, the sequence within the rep leader region itself seems to act, in addition to the ctRNA, as a second regulatory element of a novel type, negatively influencing expression of the pGA1 rep gene

Multipartite Regulation of rctB, the Replication Initiator Gene of Vibrio cholerae Chromosome II

Replication initiator proteins in bacteria not only allow DNA replication but also often regulate the rate of replication initiation as well. The regulation is mediated by limiting the synthesis or availability of initiator proteins. The applicability of this principle is demonstrated here for RctB, the replication initiator for the smaller of the two chromosomes of Vibrio cholerae. A strong promoter for the rctB gene named rctBp was identified and found to be autoregulated in Escherichia coli. Promoter activity was lower in V. cholerae than in E. coli, and a part of this reduction is likely to be due to autorepression. Sequences upstream of rctBp, implicated earlier in replication control, enhanced the repression. The action of the upstream sequences required that they be present in cis, implying long-range interactions in the control of the promoter activity. A second gene specific for chromosome II replication, rctA, reduced rctB translation, most likely by antisense RNA control. Finally, optimal rctBp activity was found to be dependent on Dam. Increasing RctB in trans increased the copy number of a miniplasmid carrying oriCII(VC), implying that RctB can be rate limiting for chromosome II replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis

ParB2 can silence P<i>rctA</i> and P<i>rctB</i> in the presence of <i>parS2-B</i> in <i>E. coli</i>.

<p>The top map shows the origin region as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003579#pgen-1003579-g001" target="_blank">Figure 1</a>. The lines below the map show the regions of the origin that were cloned upstream of a promoter-less <i>lacZ</i> gene and tested for promoter activity in <i>E. coli</i>. Fragments marked 1A–3A are present in pTVC122-124, respectively, and are orientated to record P<i>rctA</i> activity. The fragments marked 1B–3B are present in pTVC210-11 and pAS1, respectively, and are oppositely oriented to record P<i>rctB</i> activity. The white bars represent <i>β</i>-galactosidase activities obtained in the presence of an empty vector (pACYC184), while the grey bars indicate the activities in the presence of pTVC236 that supplied ParB2 at about 14-fold the physiological level. The copy number of <i>lacZ</i>-carrying plasmids was about 60 per cell, assuming there are four <i>oriC</i> copies in newborn <i>E. coli</i> cells in LB. The activities shown are mean values from three cultures inoculated with independent single colonies.</p

ParB2 spreads into the replication origin of <i>V. cholerae</i> chromosome II (chrII).

<p>The origin region has three functional units: <i>incII</i>, <i>oriII</i> and <i>rctB</i>. <i>incII</i> is required for controlling replication, <i>oriII</i> for initiating replication, and <i>rctB</i> for supplying the chrII-specific replication initiator protein, RctB. RctB binds to two kinds of site: the 11- and 12-mers (arrow heads) and 39-mers (black rectangles). The origin also has binding sites for DnaA and IHF, and the two promoters P<i>rctA</i> and P<i>rctB</i>. The <i>rctA</i> locus has a 39-mer and a binding site for ParB2, <i>parS2-B</i>. The bottom panel shows binding of ParB2 (grey profile) and RctB (black profile) in the origin and flanking regions, determined by ChIP-chip using specific antibodies (denoted as α) against the two proteins. The dashed line represents the average signal for ParB2 (1.1±1.1) over the entire genome. The corresponding value for RctB is 1.1±0.9. <i>parS2-A</i> and <i>parS2-C</i> are the nearest neighbors of <i>parS2-B</i>. The error bars here and elsewhere represent one standard deviation.</p

ParB2 and RctB bind simultaneously to <i>rctA</i>, but competitively to the central 39-mer.

<p>The fragment in the top panel contained the entire <i>rctA</i>, which has <i>parS2-B</i> and a 39-mer for binding ParB2 and RctB, respectively. The fragment in the bottom panel contained only the 39-mer central to <i>incII</i>. The fragments were PCR amplified from pTVC291 and pTVC222, respectively. (The <i>rctA</i> fragment is identical to the fragment 1 of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003579#pgen-1003579-g004" target="_blank">Figure 4</a> but the 39-mer fragment does not have the natural flanks of the fragment 5 of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003579#pgen-1003579-g004" target="_blank">Figure 4</a>). The fragments (2 nM each) were subjected to EMSA with purified RctB and ParB2, each at two concentrations: 3 nM (+) and 30 nM (++) for RctB, and 0.7 µM (+) and 1.4 µM (++) for ParB2. Arrows indicate the bands representing single or simultaneous binding.</p

Increase in copy number of <i>oriII</i> plasmids by ParB2 in the absence of <i>parS2-B</i> in <i>E. coli</i>.

<p>The copy number was measured in <i>E. coli</i> by supplying RctB from Pbad-<i>rctB</i> present in pTVC499, and ParB2 from P<i>lac-parB2</i> present in pTVC501 (cartoon on the top). Lines below the origin map indicate the extent of origin DNA present in different plasmids. The copy-numbers were from cultures containing 0.002% arabinose that supplied a near-physiological level of RctB, and either no IPTG (− ParB2) or 100 µM IPTG (+ ParB2) that supplied ParB2 at about 10-fold the physiological level (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003579#pgen.1003579.s002" target="_blank">Figure S2</a>). The copy numbers are the mean values from three cultures inoculated with independent single colonies. The copy number 1 corresponds to four copies of <i>oriII</i> is per cell. % increase (last column) = 100×[Copy # (+ParB2)−Copy # (−ParB2)]/Copy # (−ParB2).</p