Agricultural Microbes Genome 2 Conference

Workshop: CSREES Grant Awardees
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Salmonella enterica serovars Enteritidis (LK5), Dublin, Pullorum, and Choleraesuis

¹ University of Illinois, Urbana-Champaign

² University of Tennessee

³ Keck Center for Functional Genomics, University of Illinois, Urbana-Champaign

ORGANISM STRAIN:
Salmonella enterica serovars Enteritidis (LK5), Dublin, Pullorum, and Choleraesuis

BRIEF SUMMARY OF SEQUENCING STRATEGIES:
We are determining genome sequences of four Salmonella serovars that are host-adapted to farm animals, S. Enteritidis, S. Dublin, S. Pullorum, and S. Choleraesuis. For comparative analysis, each of the genomes is being sequenced to approximately 3-4x coverage using a whole genome shotgun approach. The genome sequences will be compared with each other and with other closely related Salmonella serovars to gain insight into the evolution of host adaptation and virulence. Purified chromosomal DNA from each of the Salmonella serovars was sheared by passage through a nebulizer under nitrogen gas to obtain fragments with an average size of 1-2 Kb. The resulting random fragments were ligated into a plasmid vector. For the Enteritidis library we cloning the DNA fragments into the plasmid vector pZErO-2 (Invitrogen). This plasmid contains a multiple cloning site upstream of the ccdB gene. The ccdB gene product poisons the essential bacterial enzyme, DNA gyrase. When the pZErO plasmid is transformed into a sensitive host, CcdB is expressed, killing the cell. Insertion of a DNA fragment into the multiple cloning site prevents expression of the ccdB gene, allowing efficient transformation of a sensitive host. Although this approach was efficient, a low percentage of plasmids survived that lacked an insert, thereby decreasing the sequencing efficacy. Therefore, we switched to the plasmid vector pCR4Blunt-Topo (Invitrogen) for construction of the remaining libraries. pCR4Blunt-Topo relies on Vaccinia topoisomerase I to perform the ligation reaction, and has yielded 100% clones with inserts. Clones were patched into 96 well microtiter plates with selection for the plasmid. Plasmids were purified using a simultaneous robotic 96-colony microtiter-plate based purification protocol (Qiagen). Aliquots of the plasmid-carrying strains are also stored frozed at ÿ70°C in microtiter dishes. The DNA sequence is determined via ABI Big Dye Terminator sequencing on ABI 3700 instrumentation.

DATA RELEASE PLAN:

• Website Address(es): http://salmonella.org
• Frequency of Data Release & Updates: Data is released as it becomes available and sequence alignment/analysis is released as soon as it is completed, typically once a week.

TIMELINE FOR COMPLETION:

• Start date: October 1, 2000
• B. Anticipated completion date: Sequence determination within 2 years, data analysis within 3 years.
• C. Milestones: The S. Enteritidis sequence has been completed to ca. 4.5x coverage, the S. Dublin sequence has been completed to ca. 3x coverage, and the S. Pullorum sequence will begin within a month. The sequence database has been extensively used by researchers from around the world.

PLAN FOR MAINTENANCE & DISTRIBUTION OF REAGENTS:
The database will be maintained indefinately. The strains and clones are readily available to researchers upon request (with evidence of appropriate CDC and USDA permits for working with Salmonella).

PROJECT CONTACT:
Stanley Maloy, Phone: (217) 333-3122, Fax: (217) 244-6697, Email: s-maloy@life.uiuc.edu