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. 2006 Aug;188(15):5626-31.
doi: 10.1128/JB.00250-06.

A parA homolog selectively influences positioning of the large chromosome origin in Vibrio cholerae

Djenann Saint-Dic  1 , Brian P Frushour, Jason H Kehrl, Lyn Sue Kahng
Affiliations

A parA homolog selectively influences positioning of the large chromosome origin in Vibrio cholerae

Djenann Saint-Dic et al. J Bacteriol. 2006 Aug.

Abstract

A Vibrio cholerae deletion mutant lacking VS2773, a parA partitioning gene homolog located in a parAB operon on the large chromosome, displays altered positioning of the large chromosome origin. Deletion of a second parA homolog on the large chromosome (VC2061) does not affect its origin positioning. The origin position of the small chromosome is unchanged by either or both of these deletions, suggesting that VC2773 function is specific to the replicon on which it is carried. VC2773 and VC2772 form a parABS system with inverted repeats found near the large chromosome origin.

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Figures

FIG. 1.
FIG. 1.
Positions of V. cholerae chromosome origins in wild-type strain N16961. (A and B) Positioning of the large chromosome origin in cells. (C and D) Positioning of the small chromosome origin. Graphs show positions of signals in cells containing one (red) or two (black) fluorescent foci; they depict the lengths of the cells on the y axis and the positions of the fluorescent foci along the length of the cell on the x axis. Photomicrographs depict the Cy3 probe signals superimposed on phase-contrast images of cell bodies. Bars, 2 μm. (E) Examples of dually labeled cells, where the oriCI probe signal is red (Cy3) and the oriCII probe signal is green (fluorescein).
FIG. 2.
FIG. 2.
Positions of V. cholerae chromosome origins in strains LK626, LK681, and LK712. Graphs show positions of signals in cells containing one (red) or two (black) fluorescent foci. The labeling of axes and scale are the same as those in Fig. 1. (A and B) Positioning of the large chromosome origin in LK626. (C and D) Positioning of the small chromosome origin in LK626. (E and F) LK681 (N16961 with pJK4) labeled with the oriCI probe. (G and H) LK712 (LK626 with “knocked-in” VC2773) labeled with the oriCI probe.
FIG. 3.
FIG. 3.
Isolation of the promoter for the V. cholerae chromosome 1 parAB locus. (A) Northern blot showing transcripts detected using a parB probe in three separate samples of each strain. The positions of RNA markers and their sizes in kb are depicted to the side of the blot. Arrowheads, transcripts; asterisk, background band from rRNA. (B) Beta-galactosidase activities (in Miller units) assayed in bacteria grown in LB at 37°C to an OD600 of 0.4 to 0.6, using the following strains: wild-type parent strain KFV10, KFV10 with a plasmid containing a promoterless lacZ gene (pRKlac290), and KFV10 with pSD19 (pRKlac290 containing the upstream region of parA) (averages of three experiments are shown).
FIG. 4.
FIG. 4.
Stabilization of mini-F plasmids containing putative parS sequences by coresident plasmids carrying the VC2773-VC2772 parAB locus. Experiments were performed multiply, and the graphs are from a representative experiment. Bacteria were grown in M9-CSA with chloramphenicol to maintain the mini-F derivative; at an OD of ∼0.3, they were back diluted into medium lacking chloramphenicol (time zero) and plated at the specified generation times. Colonies were patched onto chloramphenicol to assay for the presence of the mini-F plasmid. The y axes in all panels show the percentages of colonies still retaining the mini-F derivative. (A) Stability of the sop+ mini-F plasmid pDAG114, as well as its unstable derivative pDAG203, in the presence of a coresident pBluescript vector or pSD25 (carrying both parA and parB). (B to D) Stabilities of pDS38, pSD39, and pSD44 (pDAG203 carrying IR1, IR2, and IR3, respectively). Each plasmid was assayed in the presence of empty pBluescript vector, pSD9 (with an in-frame deletion of VC2773, thus carrying parB), pSD21 (with an in-frame deletion of VC2772, thus carrying parA), or pSD25 (carrying both parA and parB). The legend for each graph indicates the par genes supplied in trans.
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