Investigating the key components of the chitinase secretion system in Serratia marcescens

Abstract

The opportunistic bacterial pathogen Serratia marcescens secretes a ‘chitinolytic machinery’ comprising ChiA, ChiB, ChiC, and Cbp21, that is essential for the efficient utilisation of the extracellular polysaccharide chitin. Secretion of these proteins appears to be a two-step process in S. marcescens with initial export to the bacterial periplasm being followed by a final secretion step across the outer membrane. Successful secretion of the chitinolytic machinery ultimately depends on the products of a four-gene operon, chiWXYZ, which resembles a phage lysis cassette. At the outset of this project the structure, function and interrelationships between the ChiWXYZ proteins was largely unknown and thus a major aim of this work was to provide mechanistic insight into this system. This project therefore details the characterisation of key molecular events in the secretion process. The chiW and chiX genes encode putative holin and endolysin proteins, respectively, and these are essential for secretion of chitinases. Using X-ray crystallography, the structure of full-length ChiX was resolved to 1.34 Å, revealing structural similarities to the lysostaphin-type (LAS) family of peptidases. Purified ChiX was shown to possess L-Ala D-Glu endopeptidase activity, establishing the peptidoglycan cross-link in the periplasm as its substrate. ChiX has no obvious signal peptide, however experiments outlined here provided evidence that the holin ChiW was responsible for ChiX passage to the periplasm. Finally, proteomic data sets for strains overproducing a central regulator of the system, ChiR, were analysed in an attempt to discover hitherto unknown components or substrates of the chitinase secretion machinery.

Date of Award	2016
Original language	English
Awarding Institution	University of Dundee
Sponsors	Medical Research Council
Supervisor	Frank Sargent (Supervisor)