Distinct segregation dynamics of the two Vibrio cholerae chromosomes

Abstract

SummaryThe study of prokaryotic chromosome segregation has focused primarily on bacteria with single circular chromosomes. Little is known about segregation in bacteria with multipartite genomes. The human diarrhoeal pathogen Vibrio cholerae has two circular chromosomes of unequal sizes. Using static and time-lapse fluorescence microscopy, we visualized the localization and segregation of the origins of replication of the V. cholerae chromosomes. In all stages of the cell cycle, the two origins localized to distinct subcellular locations. In newborn cells, the origin of chromosome I ( oriCI vc ) was located near the cell pole while the origin of chromosome II ( oriCII vc ) was at the cell centre. Segregation of oriCI vc occurred asymmetrically from a polar position, with one duplicated origin traversing the length of the cell towards the opposite pole and the other remaining relatively fixed. In contrast, oriCII vc segregated later in the cell cycle than oriCI vc and the two duplicated oriCII vc regions repositioned to the new cell centres. DAPI staining of the nucleoid demonstrated that both origin regions were localized to the edge of the visible nucleoid and that oriCI vc foci were often associated with specific nucleoid substructures. The differences in localization and timing of segregation of oriCI vc and oriCII vc suggest that distinct mechanisms govern the segregation of the two V. cholerae chromosomes.

par genes and the pathology of chromosome loss in Vibrio cholerae

“…3 A and C). The subcellular locations of YFP-ParB1 and CFP-ParB2 foci in WT cells are consistent with previous studies of chrI and chrII origin localization (22,23). Thus, YFP-ParBI and CFP-ParB2 mark the subcellular locations of the origin region of each chromosome.…”

“…The BacLight live/dead bacterial viability kit for microscopy (Molecular Probes) was used for the membrane integrity assay. All images were acquired and processed as described previously (22,37).…”

“…In newborn cells, the origin of replication of chrI (oriCI vc ) is found near one of the cell poles, whereas the origin of replication of chrII (oriCII vc ) is found near the cell center (22,23). The origin region of chrI undergoes an asymmetric segregation process: one copy of the duplicated oriCI vc remains at the pole while the other copy traverses the cell to the opposite pole (22)(23)(24). In contrast, the duplicated copies of oriCII vc segregate in a symmetric fashion from the cell center to the quarter positions of the cell, the sites of the new cell centers (22,23).…”

“…The origin region of chrI undergoes an asymmetric segregation process: one copy of the duplicated oriCI vc remains at the pole while the other copy traverses the cell to the opposite pole (22)(23)(24). In contrast, the duplicated copies of oriCII vc segregate in a symmetric fashion from the cell center to the quarter positions of the cell, the sites of the new cell centers (22,23). The pattern of oriCI vc segregation is very similar to that reported for the Caulobacter crescentus chromosome (25), whereas the pattern of oriCII vc segregation is similar to that reported for P1 and the F plasmid (3,26).…”

The bacterial nucleoid: A highly organized and dynamic structure

“…These problems are circumvented by a novel technique, which uses the lac or tet repressor fused to a fluorescent protein to specifically decorate tandem repeats of the cognate repressor binding site inserted at a chromosomal locus of interest Straight et al, 1996;Michaelis et al, 1997;Lau et al, 2003]. Using these two approaches, it was shown that bacteria possess mechanisms to orient the replication origin and terminus towards opposite poles in newborn cells Webb et al, 1997;Niki and Hiraga, 1998;Jensen and Shapiro, 1999;Fogel and Waldor, 2005]. This finding already indicates some degree of order within the nucleoid.…”