1. An Introduction to “Bioinformatics to Predict Bacterial Phenotypes” Jerry H. Kavouras, Ph.D. Lewis University Romeoville, IL

2. Why study bioinformatics? It’s a rapidly growing field in biology and computer science that analyzes the genetic information stored in DNA.

3. What are phenotypes? Phenotypes are the observed manifestations of genes, which are linear sequences of nucleotides on chromosomes and plasmids. Examples: Cell shapes (bacillus, coccus) Metabolic capacity (glucokinase) Antibiotic resistance (  -lactamase)

4. How are phenotypes determined? Traditionally, microbiologists determine phenotypes by culture methods and microscopic techniques. Examples: TestPhenotype Gram stainCell wall Catalase testDegrades H 2 O 2

5. What phenotypes do you observe in this Gram stain preparation?

6. Phenotypic observations Bacillus Length is 3  m Gram negative 1mm1mm Gram positive Coccus

7. Homologous genes Homologous genes share similar sequences -- Related by evolution -- Good chance have similar function Similar sequences are likely to be versions of the same gene, or at least similar genes, meaning there is a very good chance the PHENOTYPE is present.

8. Use bioinformatics to predict cellular phenotypes! Possible to predict cellular phenotypes from a linear sequence of chromosomal or plasmid DNA if the gene is already known. Why is this important? This means microbiologists can determine the phenotypes for a microbe without culture methods and microscopy.

9. Why use bioinformatics instead of standard methods? Microbiologists estimate the overwhelming majority of bacteria in nature have not been cultured (based on analyses of 16S rDNA sequences) Microbiologists can predict the phenotypes of UNCULTURED bacteria! -- Very powerful tool

10. Genomes & Bioinformatics The number of complete genome sequences for organisms is increasing steadily. Someday microbiologists may rely on DNA sequences for determining cell phenotypes. This does not mean culture methods and microscopy will not be important!

11. Real world applications How can the same species of bacteria be pathogenic and nonpathogenic? Answer: Different strains! Example: Escherichia coli K-12 (nonpathogenic) Escherichia coli O157:H7 (pathogen)

12. Real world applications (cont’d) Because the chromosomal and plasmid sequences are known for these strains, bioinformatics can determine differences in the set of genes (genome) for each organism. Now use the Signature Genes tool at NMPDR to actually answer this question.

13. Compare whole genomes Go to www.nmpdr.orgwww.nmpdr.org Select the Signature Genes tool from the left navigation bar Choose E. coli O157:H7 as the reference Choose another strain of the O157:H7 serotype, strain EDL933, in set 1 Choose two strains of avirulent E. coli K12 in the exclusion set 2 Click Go

14. Find genes responsible for virulence phenotype 847 genes common to two strains of O157:H7 and absent from two lab strains of E. coli Most of these are phage-associated Included are 6 toxin genes What might the remaining 841 genes be doing?

15. How do you explore physical & biological context of virulence genes in E. coli ? 1.Study phylogenetic profiles -- evolutionary relationships among genes 2.Study protein families -- functional (analogous) relationships among genes

16. Acknowledgements Thanks to Leslie McNeil for her additions, assistance, and comments in preparing this exercise introduction.