Gonococcal MinD affects cell division in Neisseria gonorrhoeae and Escherichia coli and exhibits a novel self-interaction

The Min proteins are involved in determining cell division sites in bacteri a and have been studied extensively in rod-shaped bacteria. We have recentl y shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this opero n. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibit ed both grossly abnormal cell division and morphology as well as altered ce ll viability. Western blot analysis verified the absence of MinD from N. go norrhoeae (MinD(Ng)) in this mutant. Hence, MinD(Ng) is required for mainta ining proper cell division and growth in N. gonorrhoeae. Immunoblotting of soluble and insoluble gonococcaI cell fractions revealed that MinD(Ng) is b oth cytosolic and associated with the insoluble membrane fraction. The join t overexpression of MinC(Ng) and MinD(Ng) from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed wit h plasmids encoding either MinC(Ng) or MinD(Ng) alone did not display notic eable morphological changes. These studies suggest that MinD(Ng) is involve d in inhibiting gonococcal cell division, likely in conjunction with MinC(N g). The alignment of MinD sequences from various bacteria showed that the p roteins are highly conserved and share several regions of identity, includi ng a conserved ATP-binding cassette. The overexpression of MinD(Ng) in wild -type Escherichia coli led to cell filamentation, while overexpression in a n E. coli minD mutant restored a wild-type morphology to the majority of ce lls; therefore, gonococcaI MinD is functional across species. Yeast two-hyb rid studies and gel-filtration and sedimentation equilibrium analyses of pu rified His-tagged MinD(Ng) revealed a novel MinD(Ng) self-interaction. We h ave also shown by yeast two-hybrid analysis that MinD from E. coli interact s with itself and with MinD(Ng). These results indicate that MinD(Ng) is re quired for maintaining proper cell division and growth in N. gonorrhoeae an d suggests that the self-interaction of MinD may be important for cell divi sion site selection across species.