ABSTRACT

Regulation is a key concept for understanding the dynamics of metabolism in bacteria. This report outlines a simple laboratory experiment aimed at studying a key form of regulation in bacterial metabolism, exemplifying how microbes switch the flux of carbon towards alternative metabolic fates as a function of nutrient availability and according to their physiological status. The experiment involves the use of a Sinorhizobium meliloti wild-type strain and its isogenic derivative carrying a null mutation in the gene encoding the synthase that generates the storage polymer polyhydroxybutyrate (PHB). These strains serve to characterise the effects of growth phase and carbon availability at the end of balanced growth on the extent of PHB accumulation by rhizobia. The experimental set-up can be completed in a main 4-h laboratory session, followed by an additional 1-h session after an initial incubation for subsequent bacterial-colony counting (48 h), and requires equipment usually available at any university. The protocol described here is performed by the undergraduates of our biochemistry course. We consider this exercise to be a useful complementary tool for spotlighting the concept of metabolic regulation in bacteria and for promoting scientific thinking along with the development of students’ wet-bench laboratory skills.

摘要

调控是了解细菌代谢动力学的关键概念。本报告概述了一个简单的实验室实验，旨在研究细菌代谢中一种关键的调节形式，举例说明微生物如何根据营养可用性和生理状态将碳通量转换为替代代谢命运。该实验涉及使用苜蓿中华根瘤菌野生型菌株及其同基因衍生物在编码产生储存聚合物聚羟基丁酸酯（PHB）的合酶的基因中携带无效突变。这些菌株用于表征生长阶段和平衡生长结束时的碳有效性对根瘤菌 PHB 积累程度的影响。实验设置可以在 4 小时的主要实验室会话中完成，然后在初始孵育后额外的 1 小时会话用于随后的细菌菌落计数（48 小时），并且需要通常在任何大学都可用的设备。此处描述的协议由我们生物化学课程的本科生执行。