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

不同细菌中协调细胞分裂的机制存在重要差异。我们研究的长期目标是了解非模式生物粪肠球菌（Enterococcus faecalis）和淋病奈瑟菌（Neisseria gonorrhoeae）的细胞分裂机制。Ef和Ng在与细胞分裂有关的蛋白质补体和相互作用方面存在差异。在本提案中，我们将重点研究来自Ng的细胞分裂蛋白FtsI。我们的大部分研究将集中在了解Ef的细胞分裂。我们在Ef中发现了一种新的蛋白MLJD1，它参与细胞分裂。MLJD1与细胞分裂蛋白DivIVAEf相互作用，MLJD1的插入失活导致指示异常细胞分裂的表型。MLJD1的同源物在革兰氏阳性（Gm+）细菌中保守，而在革兰氏阴性（Gm-）细菌中不保守。MLJD1在枯草芽孢杆菌（Bacillus subtilis, Bs）中的同源物CcpN （control catabolite protein of糖异生基因控制分解代谢蛋白）调控糖异生通路中基因的转录。CcpN在细胞分裂中的作用尚不清楚。我们最近发现CcpN与细胞分裂蛋白DivIVABs结合。

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.