Additional analysis demonstrated that manipulation of the level of expression of setB caused defects in chromosome dynamics. Particularly striking are the effects that occur when SetB is overexpressed in wild type cells (Figure 15).
Overproduction of SetB caused dramatic effects on chromosome segregation; essentially no cell had a normal appearance. In general, the cells were elongated with filamentation becoming obvious at the latter part of the growth phase, and the nucleoids appearing either stretched out or fragmented (Figure 15). The chromosomes often appeared as pairs, where one pair seemed to have been pulled away from the other pair (Figure 15b-3). In many instances, it looked as if the chromosomes comprising a pair had been stretched out parallel to each other (Figure 15b-7). Nucleoid fragmentation was the other predominate phenotype, which became more apparent later in the growth phase. Fragmented nucleoids appeared to have been pulled apart, with debris strewn throughout the longitudinal cross-section of the cell (Figures 15b-5 and 15d-1). Our current hypothesis is that SetB, either directly or indirectly, supports the establishment of a connection between the DNA and a force-generating apparatus in the cell. In this context, overproduction of SpcA could result in the application of either too much force or misapplied force to the sister chromosomes, causing them to decondense and, eventually, break apart.
Our proposal that SetB was involved in mediating the application of a force to the daughter chromosomes has gained credence from studies that have indicated that MreB, the bacterial actin ancestor, appears to be involved in chromosome segregation. Ken Geddes' group showed that overproduction of mutant MreB proteins that cannot hydrolyze ATP (and thus cannot form actin-like filaments in the cell) causes a par phenotype. In addition, ParM, a MreB-family protein encoded by the P1 plasmid, is required for plasmid segregation — a process that has been proposed to occur via attachment of the sister plasmid circles to ParM filaments that grow outward from the cell center. We have demonstrated, using 2-hybrid analysis, that SetB interacts with MreB. This represents the first report of a MreB-interacting protein.
