Date of Graduation

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Young Min Kwon

Committee Member

Billy M. Hargis

Second Committee Member

Narayan C. Rath

Third Committee Member

Mack Ivey

Abstract

ABSTRACT

As more whole genome sequences become available, there is an increasing need for high-throughput methods that link genes to phenotypes and facilitate discovery of new gene functions. The objective of this study was to develop a high-throughput method to study gene functions in bacteria and use this method to study gene functions of S. enterica serotype Typhimurium (S. Typhimurium) under various environmental conditions encountered during its life cycle. Chapter I of this dissertation reviews the history and evolution of functional genomics in bacteria with focus on Salmonella, along with the recent techniques available. Chapter II, deals with the development of new version of Tn-seq (Transposon sequencing) method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. The new version of Tn-seq method was applied to a genome-saturating S. Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted LB medium, LB medium + bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential in at least one of the above selective conditions. Phenotypic study of deletion mutants (pyrD, glnL, recD and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. The functional relevance of the genes identified was also studied. In chapter 3, Tn-seq method was applied from food safety perspective to a genome-saturating S. Typhimurium mutant library recovered from selection under 2 different in vitro growth conditions (4°C and -20°C), mimicking storage temperature of chicken meat. We identified an overlapping 42 genes conditionally essential in the selective conditions. The overall study demonstrated the utility and efficiency of the Tn-seq method in comprehensive identification of conditionally essential genes in Salmonella. The genes identified here could be an important resource for better understanding or control of Salmonella, including development of novel antimicrobials and vaccines. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established.

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