Regulatory mechanism of septum formation in bacterial cells

研究了大肠杆菌中隔膜形成的调控机制，重点研究了青霉素结合蛋白（PBP） 3，这是一种分离所必需的肽聚糖合成酶。分析了细胞分裂/包膜生物合成基因簇的转录，包括编码PBP - 3的ftsI基因，PBP - 3的分子内结构域参与与其他细胞分裂成分的相互作用，C端处理前体PBP - 3的蛋白酶Prc的生理功能，以及突变导致不规则细胞分裂/分离的envC基因。在ftsI上游1.9 kb处发现了一个转录启动子，该启动子对包括ftsI在内的9个基因的表达至关重要。对未发现突变等位基因的基因进行零表型检测。活性位点改变的突变体PBP 3的显性负作用被认为是由于其与其他细胞分裂成分的相互作用。基因内抑制突变逆转了这种作用，并与野生型活性位点结合，产生了新的PBP 3突变体，这些突变体与其他分裂组分的相互作用似乎有缺陷。具有缺陷Prc或产生成熟PBP - 3作为主要基因产物的细胞正常生长。c端处理对于分离似乎是不必要的。抑制prc缺陷菌株对渗透/热胁迫敏感性的突变在ftsI中不存在。对抑制基因spr进行了定位和克隆。spr突变在prc^+背景下引起渗透/热胁迫敏感性。克隆了PBP - 7多拷贝敏感性抑制基因，并对其进行了分析。Spr蛋白可能参与肽聚糖代谢。克隆了envC基因，测定了其野生型和突变型等位基因的核苷酸序列。染色体envC的破坏引起细胞形态不规则，但不致命。

Regulatory mechanism of septum formation in Escherichiacoli was investigated with the emphasis on penicilin-binding protein (PBP) 3, a peptidoglycan-synthesizing enzyme essential for septation. Transcription of cell division/envelope biosynthesis gene cluster including the ftsI gene that encodes PBP 3, an intramolecular domain of PBP3 involved in interaction with other cell division components, physiological function of protease Prc that processes precursor PBP 3 in the C terminus, and the envC gene whose mutation causes irregular cell division/separation were analyzed.1. A transcriptional promoter essential for expression of 9 genes including ftsI was identified at 1.9 kb upstream of ftsI.The genes in the cluster with no known mutant alleles were examined for their null phenotype.2. Dominant negative effect by a mutant PBP 3 with the altered active site is supposed to be due to its interaction with other cell division components. Intragenic suppressor mutations that reversed the effect were isolated and combined with the wild-type active site to give novel PBP 3 mutants that seemed defective in interaction with other division components.3. Cells with defective Prc or producing mature PBP 3 as a primary gene product grew normally. C-terminal Processing seemed nonessential for septation.4. Mutations that suppressed the sensitivity of a Prc-defective strain to osmotic/thermal stresses were not in ftsI.The suppressor gene spr was mapped and cloned.5. The spr mutation caused osmotic/thermal stress sensitivity in the prc^+ background. The gene for PBP 7 was cloned as a multicopy suppressor of the sensitivity and analyzed. Protein Spr was likely to be involved in peptidoglycan metabolism.6. The envC gene was cloned and its nucleotide sequence was determined for wild-type and mutant alleles. Disruption of the chromosomal envC caused irregular cell morphology but not lethality.