Dubytska Lydia, Godfrey Henry P, Cabello Felipe C
Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA.
J Bacteriol. 2006 Mar;188(5):1969-78. doi: 10.1128/JB.188.5.1969-1978.2006.
ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZ(Bbu)). Its gene product (FtsZ(Bbu)) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZ(Bbu) might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZ(Bbu) antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZ(Bbu) could interfere with the function of E. coli FtsZ, ftsZ(Bbu) was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZ(Bbu) generated a filamentous phenotype. This suggested interference of ftsZ(Bbu) with E. coli FtsZ function and confirmed the role of ftsZ(Bbu) in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.
ftsZ对许多微生物的细胞分裂至关重要。在大肠杆菌和枯草芽孢杆菌中，FtsZ在细胞分裂隔膜前沿的环形成中发挥作用。伯氏疏螺旋体基因组中存在ftsZ的同源物（ftsZ(Bbu)）。其基因产物（FtsZ(Bbu)）与其他细菌的FtsZ蛋白高度同源，但其功能尚未确定。由于ftsZ(Bbu)的功能缺失突变体可能是致死性的，因此采用tetR/tetO系统对该基因在伯氏疏螺旋体中进行调控。将四环素响应性修饰的杂交伯氏疏螺旋体启动子控制下的ftsZ(Bbu)反义DNA序列的62个核苷酸克隆到pKFSS1中。将该构建体电穿孔导入携带位于伯氏疏螺旋体flaB启动子控制下的染色体tetR的伯氏疏螺旋体宿主菌株中。经脱水四环素诱导后，反义ftsZ RNA的表达导致丝状伯氏疏螺旋体的产生，这些丝状菌无法分裂，且生长比未诱导的细胞更慢。为了确定FtsZ(Bbu)是否能干扰大肠杆菌FtsZ的功能，从染色体DNA中扩增出ftsZ(Bbu)，并置于四环素调控的杂交启动子控制下。将该构建体导入大肠杆菌并用脱水四环素诱导后，ftsZ(Bbu)的过表达产生了丝状表型。这表明ftsZ(Bbu)对大肠杆菌FtsZ功能有干扰作用，并证实了ftsZ(Bbu)在细胞分裂中的作用。这是关于通过四环素控制反义RNA表达产生等效的伯氏疏螺旋体条件致死突变体的首次报道。
