Wright, JA; (2007) Functional genomic investigation of the metabolism of Campylobacter jejuni during in vitro growth. Doctoral thesis , UNSPECIFIED.

Abstract

Campylobacter jejuni is an important food-borne pathogen, and a major cause of bacterial gastroenteritis in both developing and developed nations. Despite this, relatively little is known regarding the way in which C. jejuni colonises hosts, causes disease or survives in the environment during transmission between hosts. The publication of the first C. jejuni genome sequence in 2000 highlighted that many of the paradigms from other, better characterised, bacteria such as Escherichia coli and Salmonella enterica do not necessarily hold for Campylobacter spp (Parkhill et al, 2000). This thesis describes the detailed analysis of C. jejuni, during in vitro growth in batch culture. This work was carried out in order to better understand the physiology of this species in this widely used experimental system, to provide a useful data source for the large number of researchers using batch grown C. jejuni, and as a screening process to identify novel and potentially important aspects of C. jejuni physiology. DNA microarray analysis was performed to measure changes in transcript abundance during different phases of the growth cycle, and expression changes were identified by significance and fold-change filtering, and by time course clustering analysis. A subset of the gene expression changes identified by microarray analysis was confirmed by quantitative RT-PCR. A number of phenotypic characteristics of C. jejuni were examined during batch growth, and correlation with gene expression changes was assessed. Microscopy was used in conjunction with bacterial tracking software and fluorescent dyes to monitor motility, respiratory activity and viability throughout the growth cycle. These measures strongly suggested the presence of a two-stage stationary phase, with high levels of motility and respiratory activity until mid-stationary phase, followed by rapid decline of these cellular functions. The utilisation of amino acids, which constitute major carbon sources for C. jejuni, was monitored by analysis of culture supernatants and preferential substrates for growth were identified. Proton nuclear magnetic resonance analysis of culture supernatants was performed and confirmed the depletion of certain amino acids, and identified a pattern of acetate production and assimilation throughout the growth cycle. This trend is characteristic of an „acetate switch‟ mechanism of bacterial metabolism characterised by acetate production during phases of nutrient abundance, followed by assimilation and utilisation of this substrate during nutrient limitation. This switch from production to assimilation occurs in mid-stationary phase. Mutants of the genes putatively involved in the acetate switch were examined. Mutants of the pta and ackA genes, encoding probable phosphate acetyltransferase and acetate kinase enzymes, showed reduced acetate production during growth, and did not exhibit an „acetate switch‟. These mutants appeared to survive less well than the wild-type strain during stationary phase and thereafter. Several genes of unknown function, with potential roles in peptide utilisation were identified using data from the microarray analysis. Two loci, consisting of the Cj0917c gene, and the Cj1580c – Cj1584c putative operon were mutated in C. jejuni by allelic replacement. Growth assays in complex and defined media were performed, in an attempt to identify a putative function. Mutants of Cj0917c showed similar growth characteristics to the wild-type strain, but Cj1580c – Cj1584c deletion mutants exhibited a growth defect in defined media. Assay variability precludes any firm conclusions on the role of this locus, but a putative function in the utilisation of amino acids and di- and tri-peptides is discussed.

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