Novel cell division mechanisms in round bacteria: Enterococcus faecalis and Neisseria gonorrhoeae as model organisms

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Important differences exist in the mechanisms used to coordinate cell division in different bacteria. The long-term objectives of our research are to understand cell division mechanisms in the non-model organisms Enterococcus faecalis (Ef) and Neisseria gonorrhoeae (Ng). Both Ef and Ng differ in the protein complements and interactions implicated in cell division. In the present proposal, we will focus on the cell division protein FtsI from Ng. Most of our research will focus on understanding cell division in Ef. We discovered a new protein in Ef, MLJD1, which is involved in cell division. MLJD1 interacts with the cell division protein DivIVAEf and insertional inactivation of mljd1 causes phenotypes indicative of aberrant cell division. Homologues of MLJD1 are conserved in Gram-positive (Gm+) bacteria but not Gram-negative (Gm-) bacteria. The homologue of MLJD1 in Bacillus subtilis (Bs), named CcpN (control catabolite protein of gluconeogenic genes), regulates the transcription of genes in the gluconeogenic pathway. The role of CcpN in cell division is unknown. We recently discovered that CcpN binds to DivIVABs, a cell division protein. Hypothesis: Because of their high identity, MLJD1 and CcpN have similar cell division and transcriptional regulation functions, which may be unique to Gm+ bacteria. Our short term goals are to: 1) Understand the biological roles of MLJD1Ef and CcpNBs in cell division. mljd1 and ccpN will be inactivated or over-expressed in Ef or Bs and effects on their hosts will be examined and characterized using various microbiological, genetic, biochemical, and microscopic methods. 2) Investigate whether MLJD1 plays a role in a) gluconeogenesis, and b) transcriptional regulation of gluconeogenic genes. These roles will be researched using biochemical assays (gluconeogenesis) and gel retardation or other assays (transcriptional regulation). 3) To investigate the cell division interactome of Ef by Bacterial-two-Hybrid (B2H) or other methods including Tandem Affinity Purification coupled with mass spectrometry. 4) Determine the structure of MLJD1 by nuclear magnetic resonance and/or X-ray crystallography. Lastly, we will continue working on cell division mechanisms in Ng by focussing on FtsINg, a penicillin-binding protein, whose role in cell division is uncharacterized. 5) We will ascertain the role of the N-terminus of FtsINg in cell division, and to understand whether FtsINg N-terminus mutations, that affect cell division, play a role in Ng's susceptibility to -lactam antibiotics (e.g. penicillin) using strategies of gene mutation, protein interaction studies, and microbiological and microscopic methods. The ability of mutated ftsINg to bind penicillin will be determined. Impact: This research will produce novel insights on how Ef and Ng divide and will identify unique targets on MLJD1 and FtsI for antimicrobial development to combat drug resistance.