April 15 2011 talk by Rosie Redfield at the University of Louisville. Title "Do bacteria have sex?"

1. Do bacteria have sex? Rosie Redfield University of British Columbia

2. Do bacteria have any processes that evolved to make new combinations of chromosomal genes? Why is natural competence the only process that still needs investigating? Why does Haemophilusinfluenzaetake up DNA? When does H. influenzaetake up DNA? Which DNAs does H. influenzaetake up, and why? What happens to the DNA? But first, why should we care?

3. Sexual reproduction (in eukaryotes): Why do we bother? Why is sexual reproduction so common in eukaryotes?

4. Genes that cause sex must somehow improve the fitness of the genomes they’re in. But how? Why does sexual reproduction exist? The object of this process is to create those individual differences which form the material out of which natural selection produces new species. Weismann 1886

5. “It is very much sexual reproduction!” Everyone assumes that bacteria have sex, but do they really? Sex is as old as bacteria are. Now, a team at the University of California-Davis has developed a mathematical model of the speed of bacterial sex. The model... “The major bacterial sex mechanism is conjugation.” “Bacteria, too, have sex” “Conjugation is bacterial sex. This name refers simply to the fact that it is a mechanism SPECIFICALLY DESIGNED for transfer of DNA ...”

6. Do bacteria have any processes that evolved to make new combinations of chromosomal genes? Parasexual processes of bacteria: Why is natural competence (transformation)the only process that needs investigation?

7. Do ‘parasexual processes’ exist for genetic exchange? Recombination: All of these genes have important functions in DNA replication and repair. They’re not ‘genes for recombination’.

8. Conjugation: Usually plasmid transfer Rarely host DNA transfer Do ‘parasexual processes’ exist for genetic exchange? Transduction and conjugation are caused by genetic parasites. Transduction: Recipient Phage-infected source

9. New combinations of genes (sex)? Could be for Do ‘parasexual processes’ exist for genetic exchange? Naturally competent recipient cell Why do bacteria take up DNA? Templates for DNA repair? Nucleotides and other nutrients (food)?

11. host mucosa

14. Regulation of competence

15. CRP-S sequence

16. 1. The sugar signal: CRP is activated (by cAMP) when preferred sugars are depleted. First, CRP activates energy-supply genes (and sxy): Then, CRP and Sxy activate DNA uptake genes: If…

17. pyrim-idines purine bases purines 2. The nucleotide-pool signal: Purine nucleotides and nucleosides prevent competence by inhibiting Sxy translation

18. Competence genes are regulated similarly in many related bacteria

19. Why do bacteria take up DNA? How does H. influenzae decide when to take up DNA? Regulation is by nutritional signals. How does H. influenzaedecide which DNAs to take up? Why does it prefer fragments with a particular sequence motif? And why is this motif so common in its own DNA? What happens to the DNA that H. influenzae takes up?

20. Bacteria in the Pasteurellaceae preferentially take up ‘self’ DNA. So do Neisseria species.

21. Uptake sequences: The H. influenzaegenome contains about 2000 strong matches to the USS consensus. Uptake sequences are motifs, not mobile elements. Uptake sequences are not species-specific. Why are the preferred motifs so common in the genomes of bacteria that take them up?

22. Molecular drive explanation: Sequences preferred by the uptake machinerypassively accumulate in the genome by recombination, replacing allelic versions that are not as easily taken up. But why are the USS sequences preferred by the uptake machinery? Maughan, H. et al.2010 Genetics;186:613-627

23. Hypothesis:The motif is preferred because it is easily kinked, allowing the DNA to pass through the secretin pore.

24. Uptake sequences are bent. Neighbors are correlated at bend positions Seg 3 Seg 2 Seg 3 Seg 2 Core Core core s-2 s-3 USS Randomized Ethylation of some positions enhances uptake 300bp 200bp

25. To test this hypothesis we first need to know exactly what the uptake specificity is. DNA uptake Periplasm DNA pool Input DNA pool ~109 cells in 1 ml competent culture ~1010 taken-up fragments purified from the periplasm ~1011 synthetic fragments with a degenerate USS Results of sequencing 200 bp Degenerate USS: 24% mismatch from consensus at each position (31 bp) Flow cell priming site A and sequencing priming site Flow cell priming site B A complex pool of DNA fragments is mixed with cells Competent cells with a rec2 mutation take up DNA without translocation

26. Uptake sequences in the genome reflect the true uptake specificity roughly but not exactly. Genome sequence motif Measured uptake preference

27. Why do bacteria take up DNA? How does H. influenzae decide when to take up DNA? Regulation is by nutritional signals. How does H. influenzae decide which DNAs to take up? Uptake sequences may favour the local kinking needed for DNA uptake. What happens to the DNA that H. influenzae takes up?

28. What happens to the DNA that H. influenzae takes up?

29. What happens to the DNA that H. influenzae takes up? How long are recombination tracts? Are they contiguous or interupted? How many does a cell get? The experiment: Competent cells* took up DNA from a diverged strain*. Four transformants were completely sequenced. * NovR-1 NalR-1 * * NovR-2 NalR-2 * * * *Donor and recipient strains had both already been sequenced.

30. All of the recombination tracts in the genomes of four H. influenzae recombinants I II III IV V Region:

31. Nov1 A B I. Nal1 G donor recipient * * C D Nov1 E F II. I IV II III V donor Nov2 L K recipient

32. III. Nal2 Nov2 M N O Nal1 IV. H I … I IV II III V … Nal1 donor J … recipient V. Nal2 P … … donor … recipient donor recipient

33. Recombination of structural variation Indels and more complex structural variants: sometimes recombined perfectly. often were found at the endpoints of recombination tracts. sometimes recombined in complex ways. Transformation at structural variation in recombinant Nov1 B A 1241 bp 128 bp inferred intermediate NP NP Rd Rd 0 bp 52 bp observed recombinant 286 bp 1241 bp

34. Lots and lots of control sequencing and analysis: Coming soon: 100 recombinant genomes, Using sequencing to map phenotypes (every bp variant is a genetic marker).

35. Do bacteria have any processes that evolved to make new combinations of chromosomal genes? The evidence from gene regulation says that natural competence is a way of obtaining nucleotides from DNA, not a mechanism for recombination. Uptake specificity is uncommon and may be best explained by molecular drive acting on a mechanistic bias in uptake. Whatever recombination bacteria have needed must have occurred by accident (like mutations). Eukaryote sexual reproduction must have evolved to solve a problem specific to eukaryotes. And yet this problem must be shared by almost all eukaryotes.

36. Other contributors: NRC Ottawa: Wendy Findlay John Nash Imperial College: Simon Kroll Janine Bosse With funding from CIHR, Genome BC and NIH