The Physiological and Molecular Characterization of a Small Colony Variant of Escherichia coli and Its Phenotypic Rescue
The Physiological and Molecular Characterization of a Small Colony Variant of Escherichia coli and Its Phenotypic Rescue
复制标题
大肠杆菌小菌落变种的生理和分子特征及其表型拯救
作者：
Victor Santos;I. Hirshfield
小菌落变种(SCV)可以被定义为自然产生的细菌亚群，其特征是菌落大小减少和独特的生化特性。在过去的二十年里，由于金黄色葡萄球菌的SCV在人类反复感染中的作用，人们对其进行了广泛的研究。然而，关于大肠杆菌的SCV的研究工作很少，这项工作主要集中在定义这些大肠杆菌菌落的生理和形态上，如体积小和生长缓慢。大肠杆菌JW0623在硫辛酸合成酶基因(LIPA)中存在LIPA零突变，是一种硫辛酸营养缺陷体。与亲本BW25113相比，该突变株在LB期生长到对数生长期时，对酸性(pH=3)、过氧化氢、高温和渗透胁迫表现出显著的抗性。利用RT-PCR和实时荧光定量RT-PCR技术，比较了两个菌株中某些基因的表达，试图建立大肠杆菌SCV的分子图谱。这些基因包括糖酵解、TCA循环、电子传递、铁获取、生物膜形成和环丙烷脂肪酸合成。研究还表明，在LB培养基中加入5μg/ml的硫辛酸可以挽救突变株的表型，逆转其缓慢生长、抗性特征和基因表达升高的趋势。这些结果表明，LIPA中的突变导致了一种类似于金黄色葡萄球菌电子传递缺陷的SCV的大肠杆菌SCV。这些菌株是典型的营养缺陷菌，通过将缺失的代谢物添加到富含盐的培养基中而被挽救。基因表达的全球变化是可逆的，取决于营养缺乏症分子是否存在。从进化的角度来看，很明显，它的生存途径是表达赋予抗逆性的基因。
Small colony variants (SCVs) can be defined as a naturally occurring sub-population of bacteria characterized by their reduced colony size and distinct biochemical properties. SCVs of Staphylococcus aureus have been studied extensively over the past two decades due to their role in recurrent human infections. However, little work has been done on SCVs of Escherichia coli, and this work has focused on the physiology and morphology that define these colonies of E. coli, such as small size and slow growth. E. coli strain JW0623, has a null lipA mutation in the lipoic acid synthase gene (lipA), and is a lipoic acid auxotroph. When the mutant was grown in LB medium to log phase, it showed remarkable resistance to acid (pH 3), hydrogen peroxide, heat and osmotic stress compared to its parent BW25113. Using RT-PCR and real time RT-PCR, the expression of certain genes was compared in the two strains in an attempt to create a molecular profile of Escherichia coli SCVs. These include genes involved in glycolysis, TCA cycle, electron transport, iron acquisition, biofilm formation and cyclopropane fatty acid synthesis. It was also demonstrated that the addition of 5 μg/ml of lipoic acid to LB medium allows for the phenotypic rescue of the mutant; reversing its slow growth, its resistance characteristics, and elevated gene expression. These results indicate that the mutation in lipA leads to an E. coli SCV that resembles an electron transport defective SCV of S. aureus These strains are typically auxotrophs, and are phenotypically rescued by adding the missing metabolite to rich medium. There are global shifts in gene expression which are reversible and depend on whether the auxotrophic molecule is absent or present. Looking at the E. coli SCV from an evolutionary point of view, it becomes evident that its path to survival is to express genes that confer stress resistance.