Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation

Abstract

There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.

Figure 1. Interactions between RctB, rctA, ParB2 and parS2 control oriCII-based replication.

A) Schematic of oriCII region of V. cholerae. DNA fragments used as EMSA probes in Figure 3 are shown by dotted lines and the DNA fragment used in the transcription reporter assay in Figure 7 is shown by the double line. Native parS2-B and mutated parS2X sequences are also shown. Numbers correspond to genomic sequence data (NC_002506). B) Overexpression of RctB and ParB2 enable oriCII-based replication with origin fragments that include rctA and parS2. Self-ligated DNA fragments containing either oriCII-rctB [a], rctA-oriCII-rctB [b], or rctA(parS2X)-oriCII-rctB [c] were introduced into DH5α cells harboring control vector (pGZ119EH) (open bars), or rctB (pYB285) (closed bars) or parB2 (pYB273) (gray bars) expression vectors. Mean and standard deviation of 5 independent experiments are shown. *No transformants obtained after overnight incubation in ≥3 experiments.

Figure 2. Map of locations of mutations that enabled replication of pYB292.

Sites of amino acid substitutions are shown by the pins and deletions are shown as bars. More detailed information is presented in Table S1.

Figure 3. Binding of RctB and/or ParB2 to DNA fragments containing rctA and parS2.

A) Binding of wild type or mutant RctB or ParB2 proteins to indicated DNA fragments. The amount of protein used in each lane was 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 31, 100, 316, 1000 ng, from left to right. B) Binding of RctB and ParB2 proteins to rctA. RctB and ParB2 were premixed and then added to the reaction tube. Amounts of proteins (ng) are indicated and the dilution series from left to right was 0.03, 0.3, 3, 30 and 300 ng. Note that similar banding patterns were observed in experiments where either RctB or ParB2 was added prior to the other protein (see Figure S1).

Figure 4. Protection of rctA from DNase I digestion in the presence of RctB or RctB and ParB2.

The DNase I protection assay was performed with 0, 10, 20, 40, 80, 160, 320 and 640 ng RctB bound to a 5′-³²P-labeled DNA probe containing rctA (including parS2-B, indicated at the side of the gel), in the absence or presence of ParB2 (100 ng). M denotes the G+A chemical sequencing ladder. The bracket indicates the ParB2 footprint. The arrowheads indicate nucleotides protected by RctB in multiple independent experiments. The arrows indicate hypersensitive sites.

Figure 5. Titration of ParB2 by multicopy parS2 prevents oriCII replication.

A) Establishment of an oriCII plasmid containing parAB2-rctA-oriCII-rctB (pYB404) without overexpression of RctB or ParB2. B) pYB404 and pYB289 (oriCII-rctB) were introduced into DH5α cells harboring plasmids containing either parS2 (closed bar), parS2X (gray bar), control (open bar), or two copies of parS2 (hatched bar) with various replication origins (indicated on the right, with reported copy numbers shown in parentheses). Means and standard deviations from 3 independent experiments are shown.

Figure 6. Overinitiation of oriCII can influence cell viability.

A) and B) Plating efficiency of indicated strains on indicated media containing chrolamphenicol. C) and D) Phase-contrast images of YBB2003 (ΔrctA/vector) (C) and YBB2004 (ΔrctA/rctB ⁺) (D) after 4 hr of induction by IPTG. bar = 2 µm.

Figure 7. Transcriptional control of parAB2 promoter by RctB and ParB2.

β-galactosidase activities of parAB2 promoter with or without an intact parS2 were measured in the presence of a plasmid expressing RctB (pYB284) (black bars) or ParB2 (pSM922) (gray bars), as well as a vector control (pBAD33) (open bars). Averages and standard deviations are shown and * represents significant (p value<0.01) difference between the indicated groups.