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Title: Secretion of the chitinolytic machinery in Serratia marcescens
Author: Hamilton, Jaeger
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2013
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There are six known secretion systems in Gram negative bacteria, referred to as Type 1 to Type 6 respectively, which are dedicated to moving substrate across the outer membrane. Secretion systems are broadly separated into those that move their substrate across the cell envelope in a single translocation event (one-step systems), and those that are dependent on the Sec or Tat machineries for export to the periplasm (two-step systems). Serratia marcescens is an important opportunistic human pathogen and has gathered a lot of interest due to its repertoire of secreted proteins. These include the haem-scavenging protein HasA, which is secreted by a Type 1 secretion system, and the cytotoxic haemolysin ShlA, which is secreted as part of a two-partner Type 5 secretion system. Serratia marcescens also encodes a Type 6 secretion system, which is known to translocate at least six effector molecules directly into other bacterial target cells. Serratia marcescens is a model organism in terms of its ability to degrade the quite intractable polymer chitin, for which it produces three chitinase enzymes ChiA, ChiB, ChiC and a chitin-binding protein Cbp21, which hydrolyse the ß-1,4 link in the chitin chain and promote binding of chitinase to the chitin substrate respectively. These chitinolytic enzymes are utilised by S. marcescens for both basic physiology and also in pathogenesis. In this work, genetic, biochemical and proteomic approaches identified, for the first time, genes that are essential for the secretion of all three chitinases as well as Cbp21. A genetic screen identified genes encoding a holin-like membrane protein (ChiW) and a putative L-alanyl-D-glutamate endopeptidase (ChiX). Subsequent quantitative proteomics experiments and biochemical analyses established that ChiW and ChiX were required for secretion of the entire chitinolytic machinery. Chitinase secretion was observed to be blocked at a late stage in the mutant strains as normally secreted enzymes were found to accumulate in the periplasm, thus implicating ChiW and ChiX in a novel outer membrane protein translocation process. It is proposed that the bacterial genome-encoded holin-like protein and endopeptidase identified represent a putative secretion system utilised by Gram-negative bacteria. In addition to this, genes encoding the chitinolytic machinery and the putative secretion apparatus were shown to be bimodally regulated and co-ordinately expressed.
Supervisor: Sargent, Frank Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Bacterial membrane biology ; Secretion system ; Chitinase chitin chitinolytic phenotype