1. Discover the Microbes Within: Impacts of DNA-based technologies and PCR basics Bill Reznikoff Marine Biological Laboratory Woods Hole, MA

2. The Old Way Classical microbiology – phenotypic approach Culture Gram stain Shapes

3. The old, old way: Darwin and the first tree…

4. Genes to Cells (Cells to Organisms) DNA is the genetic material DNA encodes RNA RNA (mRNA) encodes proteins Proteins (and some RNAs) compose cells Cells compose organisms

6. The New way: The Evolutionary Tree of Life Evolution The process of genetic change in a line of descent over time that results in new varieties and species of organisms The process of genetic change in a line of descent over time that results in new varieties and species of organismsPhylogeny Evolutionary relationships between organisms Evolutionary relationships between organisms Relationships can be deduced by comparing genetic information (nucleic acid or amino acid sequences) in the different specimens Relationships can be deduced by comparing genetic information (nucleic acid or amino acid sequences) in the different specimens Ribosomal RNA (rRNA) are excellent molecules for determining phylogeny; Carl Woese – 1 st person to use it Ribosomal RNA (rRNA) are excellent molecules for determining phylogeny; Carl Woese – 1 st person to use it Can visualize relationships on a phylogenetic tree Can visualize relationships on a phylogenetic tree

7. 16S rRNA (ribosomal RNA)  Small ribosomal subunit involved in mRNA translation process  Ancient molecule, conserved function, universally distributed  Helps identify unknown bacterium to genus or species levels  Present in bacteria; eukaryote has very divergent copy that is named 18S rRNA; present in all cells  Plays a catalytic and structural role in the ribosome

8. 16S rRNA conservation (red)

9. Ribosomal RNA (rRNA) Gene Sequencing and Phylogeny Figure 2.16 Universal PCR primers for 16S rRNA

10. Microbial Systematics – genetic approach Gene sequences are identifiers of organisms More accurate, objective, and reproducible results Identifies unculturable bacteria (99%) Eliminates special growth requirements DNA sequence data are more easily shared and databased Rapid diagnosis Resolves evolutionary relationships through trees – metaphors for evolutionary diversification

11. The Evolutionary Tree of Life Comparative rRNA sequencing has defined three distinct lineages of cells called domains. Bacteria (prokaryotic) Bacteria (prokaryotic) Archaea (prokaryotic) Archaea (prokaryotic) Eukarya (eukaryotic) Eukarya (eukaryotic) Archaea and Bacteria are NOT closely related. Archaea are more closely related to Eukarya than Bacteria.

12. Evolutionary Tree of Life Based on 16S and 18S rRNA ARCHAEAARCHAEA BACTERIABACTERIA EUKARYAEUKARYA Everything we can see Carl Woese, 1977

13. Most microbes do not cause disease! 1 gram of soil = 1 billion bacteria More bacteria in your mouth than there are people in the world # beneficial bacteria in human intestine = # of cells in human body Your colon has the highest density of microbes recorded for any microbial habitat Oldest forms of life on earth are bacteria (3.8 billion years ago) Bacteria make up most of the biomass on earth, but only 1% have been cultured

14. Genes and Genomes: fun facts Bacteria have small genomes (1-7 million base pairs vs. 3 billion base pairs in human) 1995: First bacterial genome sequence, of Haemophilus influenzae Two years later: 12 genomes sequenced April, 2010: ~4900 prokaryotic genomes sequenced or in progress. Wow!

15. Prokaryotic Genomes: Sizes and Gene Contents Rule of thumb: A prokaryotic gene = 1,000 bp long As genome size increases gene content proportionally increases Prokaryotic genomes range in size from those of large viruses to those of eukaryotic microbes Unlike eukaryotes, prokaryotic genomes do not contain a large fraction of non-coding DNA

16. Correlation Between Genome Size and ORF Content Figure 13.1

17. Bacterial Lifestyle Correlates with Genome Size Variation

18. Mutational process in bacterial genes is biased towards deletions

19. Intracellular lifestyle alters genome size: Diagram modified from Mira, A., Ochman, H. & Moran, N.A. 2001. Trends Genet 17, 589-96. DNA Inflow DNA Outflow Eukaryotic cell

20. Genome Reduction The process by which a genome decreases in size relative to its ancestor Usually coupled with symbiotic, intracellular lifestyle Organelles and endosymbionts

21. Endosymbionts have the smallest bacterial genomes known Bordenstein, unpublished * Wolbachia (1.27 Mb)

22. Arthropods Nematodes Insects (20-75%!) How do we go from here…..? Crustaceans (35%) Chelicerates 2-6 million insect species are infected with Wolbachia!! Filarial nematodes (90%)

23. To studying the Wolbachia within? Credit: Mark Taylor

25. PCR (Polymerase Chain Reaction) 1- Amplifies defined DNA sequences 2- Uses basic properties of the DNA replicating enzyme (DNA Polymerase) 3- DNA polymerization using defined DNA template, two defined primers, 4 dNTPs

26. DNA Polymerase Action

27. Basics of PCR Template DNA - the starting DNA of interest. High temperature denatures template DNA into single strands and synthetic sequences of ssDNA (20-30 nucleotides) serve as primers Two different primers are used to bracket the target gene to be amplified DNA polymerase copies the complimentary strand starting at the primer. In one cycle, two identical strands are made.

28. PCR - Ready Beads Small quantity of DNA Primers Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl 2 All in test tube Buffered solution containing DNA polymerase Four base pairs of DNA Cofactor MgCl 2 Small quantity of DNA Primers

29. Temperature drives the reaction

30. Target gene: 16S rDNA of Wolbachia

31. PCR Animation http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

32. Two Key Innovations for Success of PCR Heat-stable DNA polymerase isolated from bacterium Thermus aquaticus which inhabits hot springs Polymerase remains active despite being heated many times DNA thermal cyclers – a computer that controls repetitive temperature changes required for PCR 70C hot springs in Yellowstone National Park Example of a thermal cycler from MJ Research

33. PCR and Pop Culture “Jurassic Park” and “CSI” “Jurassic Park” and “CSI” Some fun PCR facts to share with your students: …PCR has been used to amplify DNA from… a preserved quagga (a zebra relative that became extinct 100 years ago) crime scenes (e.g., O.J.) Abducted children to find parents Prisoners leading to exonerations Mummies to determine gender, bacterial infections

34. Our goal: Determine which of your insects harbor Wolbachia? DNA extraction: PCR: Gel electrophoresis: