Campylobacterjejuni, a Gram-negative enteric pathogen, is the leading cause of bacterial gastroenteritis in the developed world. A C. jejuni strain 8 1-176 transposon library was used to screen for mutants over-producing a calcofluor white (CFW)-reactive polymer implicated in biofilm formation. This identified two lipooligosaccharide (LOS) core mutants: one defective for a two-domain glycosyltransferase (lgtF), and the other defective in a heptosyltransferase (waaF). To determine if other LOS core mutants displayed a similar phenotype, and to explore other biological outcomes of step-wise LOS truncations on C. jejuni stress resistance and pathogenesis, mutant strains defective for GaiT and CstII were also constructed. Silver stain and mass spectrometry analyses confirmed the sequential truncation of sialic acid (ΔcstII), galactose (ΔgalT), two glucoses (ΔlgtF), and heptose II (ΔwaaF). While the ΔlgtF and ΔwaaF mutants exhibited enhanced biofilm formation and ΔlgtF displayed increased sensitivity to complement killing, no effect for these phenotypes and only modest alterations in CFW reactivity were seen with partial outer core truncations. Deletion of LgtF had no effect on mouse colonization in vivo, or on invasion and intracellular survival in epithelial cells in vitro. In contrast, the ΔwaaF mutant exhibited a significant defect in intracellular survival in vitro. Interestingly, the mutants exhibited stepwise increases in susceptibility to the antimicrobial peptide LL-37, with /waaF and ΔlgtF being more susceptible and ΔgalT and ΔstII being more resistant than wild type. In contrast, all of the mutants were highly susceptible to polymyxin B. This is the first report of C. jejuni susceptibility to LL-37 and of LOS affecting polymyxin B resistance. Each of these appears to be independent of overt effects on outer membrane protein expression, membrane stability, or surface hydrophobicity. Together, our data indicate that the length and specific moieties of the LOS play important roles in C. jejuni biology, and suggest a dynamic interplay of the LOS with other stress resistance factors.

Thesis (M. Phil.)--University of Queensland. / Includes bibliographical references.

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Campylobacterjejuni, a Gram-negative enteric pathogen, is the leading cause of bacterial gastroenteritis in the developed world. A C. jejuni strain 8 1-176 transposon library was used to screen for mutants over-producing a calcofluor white (CFW)-reactive polymer implicated in biofilm formation. This identified two lipooligosaccharide (LOS) core mutants: one defective for a two-domain glycosyltransferase (lgtF), and the other defective in a heptosyltransferase (waaF). To determine if other LOS core mutants displayed a similar phenotype, and to explore other biological outcomes of step-wise LOS truncations on C. jejuni stress resistance and pathogenesis, mutant strains defective for GaiT and CstII were also constructed. Silver stain and mass spectrometry analyses confirmed the sequential truncation of sialic acid (ΔcstII), galactose (ΔgalT), two glucoses (ΔlgtF), and heptose II (ΔwaaF). While the ΔlgtF and ΔwaaF mutants exhibited enhanced biofilm formation and ΔlgtF displayed increased sensitivity to complement killing, no effect for these phenotypes and only modest alterations in CFW reactivity were seen with partial outer core truncations. Deletion of LgtF had no effect on mouse colonization in vivo, or on invasion and intracellular survival in epithelial cells in vitro. In contrast, the ΔwaaF mutant exhibited a significant defect in intracellular survival in vitro. Interestingly, the mutants exhibited stepwise increases in susceptibility to the antimicrobial peptide LL-37, with /waaF and ΔlgtF being more susceptible and ΔgalT and ΔstII being more resistant than wild type. In contrast, all of the mutants were highly susceptible to polymyxin B. This is the first report of C. jejuni susceptibility to LL-37 and of LOS affecting polymyxin B resistance. Each of these appears to be independent of overt effects on outer membrane protein expression, membrane stability, or surface hydrophobicity. Together, our data indicate that the length and specific moieties of the LOS play important roles in C. jejuni biology, and suggest a dynamic interplay of the LOS with other stress resistance factors.

Lignocellulosics (LC) are the most abundant and under-utilised renewable resource of energy in the world. The present study is concerned with finding alternative uses to LC as an animal feed using environmentally friendly technologies. The major biological constraint for using LC is related to the low accessibility of cell wall polysaccharides to both cell-free and microbial enzymes. This can only be overcome by some type of processing. This study emphasises the use of physical (steam) and biological (enzymic) treatments. Results have shown that both low (LT) and high temperature steam treatment (HT) are efficient methods for solubilising hemicellulosic sugars and depolymerising lignin. However, HT leads to higher losses of both sugar and dry matter. Higher improvement in fibre bio-availability was obtained with HT and yet, this effect was more evident from enzymic hydrolysis data compared to rumen fermentation. LT can be used for upgrading LC since exogenous chemicals are added. HT showed to be an attractive alternative for producing animal feed and substrate for enzymic saccharification without requiring chemicals. The effects of steam treatment on fibre physical structure were particularly important. Greater effect was noticed under HT in combination with rapid decompression. It was suggested that rapid decompression should be avoided in the context of animal feeding. Experiments on toxic compounds indicated that furans have negligible toxicity to rumen micro-organisms. Phenolic compounds, however, are potentially toxic and can affect the pattern of rumen fermentation, gas production and adhesion of bacteria to substrate.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 197-227). Also available via the Internet.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 41-49). Also available via the Internet.