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. 2009 Apr;20(2):149-57.
doi: 10.1016/j.copbio.2009.02.002. Epub 2009 Mar 13.

Novel genetic tools for studying food-borne Salmonella

Helene L Andrews-Polymenis  1 , Carlos A Santiviago, Michael McClelland
Affiliations

Novel genetic tools for studying food-borne Salmonella

Helene L Andrews-Polymenis et al. Curr Opin Biotechnol. 2009 Apr.

Abstract

Nontyphoidal Salmonellae are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates. Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM), pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Complete genome sequences permit the construction of pools of targeted in-frame deletions that have improved STM by minimizing the number of clones and the polarity of each mutant. Together, genome sequences and the continuing development of new tools for functional genomics will drive a revolution in the understanding of Salmonellae in many different niches that are critical for food safety.

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Figures

Figure 1
Figure 1. The Influence of Complete Genome Sequencing on Salmonella Genetics
Complete genome sequencing has revolutionized comparative genomics of Salmonellae, and allowed the development of DNA microarrays and targeted deletion libraries. These tools are accelerating both the accuracy and the coverage obtained in gene expression studies and forward genetic analysis of mutants.
Figure 2
Figure 2. Forward Genetic Method to Identify Salmonella Genes Selected in an Environment of Interest
Forward Genetic Method to Identify Salmonella Genes Selected in an Environment of Interest. Libraries of targeted deletion mutants are constructed. Mutants are passed through a selective condition as a pool. Genomic DNA from both the input pool and the output pool is sheared, polyadenylated, and nested PCR is used to specifically amplify junction fragments containing the T7 promoter. The resulting amplified product is used for T7 in vitro transcription with direct incorporation of fluorescent nucleotides. Mutants selected for or against are determined by comparison of the labeled transcripts in the input pool to the labeled transcripts in the output pool using an oligonucleotide microarray of genomic sequences directly adjacent each mutant.
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