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

在不同细菌中用于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（Ef）和淋病奈瑟菌（Ng）的细胞分裂机制。Ef和Ng两者在涉及细胞分裂的蛋白质互补和相互作用方面不同。在本提案中，我们将重点关注来自Ng的细胞分裂蛋白FtsI。我们的大部分研究将集中在理解Ef中的细胞分裂。我们在Ef中发现了一个新的蛋白质MLJD 1，它参与细胞分裂。MLJD 1与细胞分裂蛋白DivIVAEf相互作用，mljd 1的插入失活导致指示异常细胞分裂的表型。MLJD 1的同源物在革兰氏阳性（Gm+）细菌中是保守的，但在革兰氏阴性（Gm-）细菌中不是。枯草芽孢杆菌（Bacillussubtilis，Bs）中MLJD 1的同源物CcpN（controlcataboliteproteinoftumeogenicgenes）调控着产芽孢途径中基因的转录。CcpN在细胞分裂中的作用尚不清楚。我们最近发现CcpN与DivIVAB结合，DivIVAB是一种细胞分裂蛋白。** 假设：MLJD 1与CcpN具有高度的同源性，具有相似的细胞分裂和转录调控功能，这可能是Gm+细菌所独有的。** 我们的短期目标是：1）了解MLJD 1 Ef和CcpNB在细胞分裂中的生物学作用。mljd 1和ccpN将在Ef或Bs中失活或过表达，并将使用各种微生物学、遗传学、生物化学和显微镜方法检查和表征对其宿主的影响。2)研究MLJD 1是否在a）胚胎发生和B）胚胎发生基因的转录调控中发挥作用。这些作用将使用生物化学测定（胚胎发生）和凝胶阻滞或其他测定（转录调控）进行研究。3)采用细菌双杂交（B2 H）或串联亲和纯化结合质谱等方法研究Ef的细胞分裂相互作用组。4)通过核磁共振和/或X射线晶体学确定MLJD 1的结构。最后，我们将继续研究Ng中的细胞分裂机制，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)我们将确定FtsINg的N-末端在细胞分裂中的作用，并了解是否FtsINg N-末端突变，影响细胞分裂，发挥作用，在吴的敏感性-内酰胺类抗生素（如青霉素）使用基因突变的策略，蛋白质相互作用的研究，以及微生物和显微镜的方法。将测定突变的ftsINg结合青霉素的能力。* 影响：这项研究将产生关于Ef和Ng如何划分的新见解，并将确定MLJD 1和FtsI的独特靶标，用于抗菌剂开发以对抗耐药性。

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.*****