Development of a Quantitative Assay for Mycobacterial Endogenous Arabinase and Ensuing Studies of Arabinase Levels and Arabinan Metabolism in Mycobacterium smegmatis

摘要

Treatment of either Mycobacterium tuberculosis or M. smegmatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [¹⁴C]glucose, followed by a “chase” with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.