Date of Award

8-2007

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Steven Ripp, Pamela Small, Stephen Oliver, Steven Wilhelm

Abstract

Construction of two recombinant luxI bacteriophage-based bioluminescent bioreporters was undertaken to develop detection and monitoring systems for Staphylococcus aureus and Salmonella spp. These systems take advantage of the high specificity of bacteriophage for their hosts and the Vibrio fischeri lux operon responsible for quorum sensing bioluminescence. The detection system is composed of two elements, a recombinant phage with luxI which is specific for the target pathogen, and an acylhomoserine lactone (AHL)-inducible bioreporter cell line carrying the reporter lux genes. The goal of this study was to construct Salmonella- and S. aureus-specific recombinant phages which contain luxI.

The luxI expression in S. aureus was controlled by cloning the gene into S. aureus under the control of a Gram-positive promoter and ribosomal binding site (RBS). The same construct was also placed into Escherichia coli and Bacillus subtilis for comparison purposes. Although light was produced by E. coli and B. subtilis, there was no light from S. aureus. The presence of luxI transcript was shown in all strains using real time qRTPCR. Western blotting detected LuxI only in E. coli but not in B. subtilis and S. aureus. After the construct RBS was shown to be effective, the codon utilization of luxI was adapted to S. aureus phage P68 genome to eliminate a possible codon preference problem. Optimization had no effect on S. aureus while it resulted in a significant decrease of light from E. coli and a non-significant increase in B. subtilis. Additional studies are needed to determine reason(s) for luxI expression problem in S. aureus.

Salmonella choleraesuis, which carries luxI under the control of Salmonella phage P22 promoter and a Gram-negative RBS, produced high levels of light. The luxI, under the control of the same promoter and RBS, was cloned into the phage P22 genome using homologous recombination. The recombinant phage P22luxI induced light from the bioreporter only when it was propagated using S. choleraesuis which harbored the homologous recombination plasmid. The bioluminescent bioreporter E. coli OHHLux was shown to produce light in the presence of P22luxI without the S. choleraesuis host. Bioluminescence tests showed P22luxI as the source of the problem. Escherichia coli OHHLux was shown not to be lysed by P22 or P22luxI and not to uptake the P22luxI genome during bioluminescence tests. P22luxI and S. choleraesuis in combination produced AHL by using an AHL bioreporter, Agrobacterium tumefaciens A136. Since A. tumefaciens A136 is not specific enough to use for quantification of AHL, another bioreporter was constructed by placing the E. coli OHHLux plasmid into Klebsiella strains. They produced light with P22luxI without the host like the previous bioreporter. Future studies should focus on constructing a bioreporter strain which does not produce light when mixed with P22luxI without its host. Optimization, and sensitivity and specificity determination studies of the system should be performed using this bioreporter strain.

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