Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant.
Bacterial Cell Wall Homeostasis
Methods in molecular biology, January 2016
Chenggang Wu, Melissa Elizabeth Reardon-Robinson, Hung Ton-That
Sortase is a cysteine-transpeptidase that anchors LPXTG-containing proteins on the Gram-positive bacterial cell wall. Previously, sortase was considered to be an important factor for bacterial pathogenesis and fitness, but not cell growth. However, the Actinomyces oris sortase is essential for cell viability, due to its coupling to a glycosylation pathway. In this chapter, we describe the methods to generate conditional srtA deletion mutants and identify srtA suppressors by Tn5 transposon mutagenesis. We also provide procedures for analyzing cell morphology of this mutant by thin-section electron microscopy. These techniques can be applied for analyses of other essential genes in A. oris.
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