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

在不同细菌中，协调细胞分裂的机制存在着重要的差异。我们研究的长期目标是了解非模式生物粪肠球菌(Ef)和淋球菌(Ng)的细胞分裂机制。Ef和Ng在细胞分裂所涉及的蛋白质互补和相互作用方面有所不同。在目前的提案中，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中的细胞分裂。我们在EF中发现了一种新的蛋白质MLJD1，它参与细胞分裂。MLJD1与细胞分裂蛋白DivIVAEf相互作用，mljd1的插入失活导致细胞分裂异常的表型。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。枯草芽孢杆菌MLJD1的同源物CcpN(控制糖异生基因的分解代谢蛋白)调控糖异生途径中的基因转录。CcpN在细胞分裂中的作用尚不清楚。我们最近发现CcpN与DivIVAbs结合，DivIVAbs是一种细胞分裂蛋白。假设：由于高度同源性，MLJD1和CcpN具有相似的细胞分裂和转录调节功能，这可能是GM+细菌所独有的。我们的短期目标是：1)了解MLJD1Ef和CcpNBs在细胞分裂中的生物学作用。Mljd1和ccpN将在EF或BS中失活或过度表达，并将使用各种微生物学、遗传学、生化和显微方法来检测和表征其对宿主的影响。2)研究MLJD1是否在a)糖异生和b)糖异生基因的转录调控中发挥作用。将使用生化分析(糖异生作用)和凝胶抑制或其他分析(转录调控)来研究这些作用。3)用细菌双杂交(B2H)或串联亲和纯化结合质谱仪等方法研究EF的细胞分裂互动组。4)通过核磁共振和/或X射线结晶学确定MLJD1的结构。最后，我们将继续研究Ng的细胞分裂机制，重点是FtsINg，一种青霉素结合蛋白，其在细胞分裂中的作用尚不清楚。5)我们将利用基因突变、蛋白质相互作用研究、微生物学和显微镜等方法，确定FtsINg的N-末端在细胞分裂中的作用，并了解影响细胞分裂的FtsINg N-末端突变是否在Ng对β-内酰胺类抗生素(如青霉素)的易感性中起作用。突变的ftsINg与青霉素结合的能力将被测定。影响：这项研究将对EF和Ng如何划分产生新的见解，并将确定MLJD1和FTSI上用于抗菌素开发以对抗耐药性的独特靶点。

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.