Construction and application of flaA sigma-28 promoter fusions to the virulence and ecology of Campylobacter jejuni

Allen, Kevin James
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University of Guelph

To facilitate studies in both ecology and virulence associated processes of 'C. jejuni, flaA' [sigma]28 promoter fusions to the 'luxAB' and 'luxCDABE' genes of ' Xenorhabdus luminescens' and to the 'gfp' gene of ' Aequorea victoria' were constructed and placed in plasmids pRY' luxAB', pRY'luxCDABE' and pRY'gfpuv' respectively. Using pRY'luxCDABE', it was shown that the ' flaA' [sigma]28 promoter responds to various environmental and chemotactic stimuli. pH, bovine bile, deoxycholic acid, L-fucose and osmolarity all were found to up-regulate the 'flaA' promoter while viscosity acted to down-regulate it. Chemotactic stimulators were found to up-regulate the 'flaA' promoter. Amino acids (L-glutamate, L-aspartate and L-proline) and organic acids (citric acid, fumaric acid, [alpha]-ketoglutarate and succinic acid) were found to up-regulate 'flaA' promoter activity. The presence of D-glucose and D-sucrose at high concentrations resulted in up-regulation of 'flaA' promoter indicating that osmolarity is responsible for this. As a result, it is likely that the signal used to stimulate chemotaxis is also used to modulate 'flaA' promoter activity. As well, 'lux'+ 'C. jejuni ' ATCC 33291 was used to assess eggshell ecology. The 'lux '+ phenotype was effective at visualizing eggshell colonisation but was not able to predict eggshell penetration by ' C. jejuni' ATCC 33291. The phenotype was able to detect both slight cracks and flaws in eggshells. Using SEM, penetration of the inner membrane was observed in cracked eggs. This molecular-based approach, combined with SEM, indicates that vertical transmission may be possible in eggs.

Campylobacter jejuni, fiaA [sigma]28 promoter, Amino acids, Organic acids, Eggshells