Gonococcal MinD Affects Cell Division in Neisseria gonorrhoeae and Escherichia coli and Exhibits a Novel Self-Interaction

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Abstract
The Min proteins are involved in determining cell division sites in bacteria and have been studied extensively in rod-shaped bacteria. We have recently shown that the gram-negative coccus Neisseria gonorrhoeae contains a min operon, and the present study investigates the role of minD from this operon. A gonococcal minD insertional mutant, CJSD1, was constructed and exhibited both grossly abnormal cell division and morphology as well as altered cell viability. Western blot analysis verified the absence of MinD from N. gonorrhoeae (MinD Ng ) in this mutant. Hence, MinD Ng is required for maintaining proper cell division and growth in N. gonorrhoeae . Immunoblotting of soluble and insoluble gonococcal cell fractions revealed that MinD Ng is both cytosolic and associated with the insoluble membrane fraction. The joint overexpression of MinC Ng and MinD Ng from a shuttle vector resulted in a significant enlargement of gonococcal cells, while cells transformed with plasmids encoding either MinC Ng or MinD Ng alone did not display noticeable morphological changes. These studies suggest that MinD Ng is involved in inhibiting gonococcal cell division, likely in conjunction with MinC Ng . The alignment of MinD sequences from various bacteria showed that the proteins are highly conserved and share several regions of identity, including a conserved ATP-binding cassette. The overexpression of MinD Ng in wild-type Escherichia coli led to cell filamentation, while overexpression in an E. coli minD mutant restored a wild-type morphology to the majority of cells; therefore, gonococcal MinD is functional across species. Yeast two-hybrid studies and gel-filtration and sedimentation equilibrium analyses of purified His-tagged MinD Ng revealed a novel MinD Ng self-interaction. We have also shown by yeast two-hybrid analysis that MinD from E. coli interacts with itself and with MinD Ng . These results indicate that MinD Ng is required for maintaining proper cell division and growth in N. gonorrhoeae and suggests that the self-interaction of MinD may be important for cell division site selection across species.