Genetics and Cell Morphology Analyses of the Actinomyces oris srtA Mutant.
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.
Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, 6431 Fannin Street, R226/MSE, Houston, TX, 77030, USA. chenggang.wu@uth.tmc.edu.
Chenggang
C
Wu
Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX, USA.
Melissa Elizabeth
ME
Reardon-Robinson
Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, 6431 Fannin Street, R224/MSE, Houston, TX, 77030, USA. ton-that.hung@uth.tmc.edu.
Hung
H
Ton-That
D000190:Actinomyces / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D019881:Aminoacyltransferases / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004251:DNA Transposable Elements; D017353:Gene Deletion; D020043:Genes, Essential; D006360:Heat-Shock Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D006360:Heat-Shock Proteins; C090745:global stress protein A, bacteria; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
Actinomyces oris; Allelic exchange; Electron microscopy; Glycosylation; Sortase; Tn5 transposon mutagenesis