Do bacteria have sex? Rosie Redfield University of British Columbia
Do bacteria have any processes that evolved to make new combinations of chromosomal genes? Why is this an important questi...
Cycles of meiosis and gamete fusion that randomly recombine alleles from two parents into offspring. Sexual reproduction (...
Why does sexual reproduction exist? Genes that cause sexual reproduction must end up in more fit genomes than they would h...
How should we decide?  (What kind of evidence is best?) Do bacteria have any processes that evolved to make new combinatio...
How should we decide?  (What kind of evidence will best show how natural selection has acted?) Have ‘parasexual’ processes...
How should we decide?  Have ‘parasexual’ processes evolved for genetic exchange? What has real-world selection produced? W...
How should we decide?  Have ‘parasexual’ processes evolved for genetic exchange? Make mathematical  models and computer si...
Case study #1:  RecA Discovery:  Needed for homologous recombination in bacteria. Phenotype of mutant:   Recombination dow...
Roles of RecA in DNA repair:  RecA binds to damaged DNA strands This induces other DNA repair proteins RecA pairs damaged ...
Recombination: Do ‘parasexual’ processes exist for genetic exchange? All of these genes have important 'housekeeping' func...
Transduction and conjugation are caused by genetic parasites.  Do ‘parasexual’ processes exist for genetic exchange? Conju...
Do ‘parasexual’ processes exist for genetic exchange? Why do bacteria take up DNA? Templates for DNA repair? Nucleotides a...
DNA is a good source of nutrients: Nucleotide synthesis is very expensive. DNA is widely available in the environment:  <u...
Animation by Matt Baumann Competence in  Haemophilus influenzae
Why do bacteria take up DNA? How does H. influenzae decide  when  to take up DNA? How does H. influenzae decide  which  DN...
Why do bacteria take up DNA? How does H. influenzae decide  when  to take up DNA? Competence genes are induced when both s...
CRP-S sequence
1.  The sugar signal: First, CRP activates energy-supply genes (and  sxy ): Then, CRP and Sxy activate DNA uptake genes: C...
Purine nucleotides and nucleosides prevent competence by inhibiting Sxy translation purines pyrim-idines purine bases
10 -5 10 -6 10 -7 purR + purR - Constitutive production of purines prevents competence induction The  purR  gene encodes t...
10 -4 10 -5 10 -6 10 -7 sxy + Hypercompetent  sxy  mutants Cells with hypercompetence mutations in  sxy  don’t care about ...
Combining  sxy-1  and  sxy 3  restores base pairing and eliminates hypercompetence A A U U U U C U C A G U C A U C U U U A...
Mutations in  sxy  stem 1 do alter mRNA secondary structure 7 1
Why do bacteria take up DNA? How does H. influenzae decide  when  to take up DNA? How does H. influenzae decide which DNAs...
H. influenzae  cells (and their relatives) preferentially take up DNA containing a short sequence called the uptake signal...
Molecular drive explanation:  Sequences preferred by the uptake machinery accumulate in the genome by recombination, repla...
Molecular drive explanation:  Sequences preferred by the uptake machinery accumulate in the genome by recombination, repla...
USS Randomized 200bp 300bp core s-2 s-3 Uptake sequences are bent. Core Seg 2 Seg 3 Core Seg 2 Seg 3 Neighbors are correla...
New research direction:  What role do uptake sequences play in the mechanism of DNA uptake? Want to see it?  Our lab web p...
If uptake sequences didn’t evolve for sex, bacteria don’t have ANY processes that evolved to generate recombination. Bacte...
With funding from CIHR & NIH
 
Uptake sequences are not species-specific. Redfield et al. 2006 80 96 100 80 A. actinomycetemcomitans P. multocida H. somn...
136 260 459 581 648 640 384 180 436 583 628 445 174 43 Percent identities to homologs Mean uptake sequences per gene Uptak...
Laterally transferred genes have few uptake sequences: Mean number of uptake sequences per gene N. meningitidis H. influen...
Uptake sequences are less variable than other sequences in the genome. BLAST comparison of homologous USS in  H. influenza...
Molecular drive explanation:  Sequences preferred by the uptake machinery accumulate in the genome by recombination, repla...
Core Seg 2 Seg 3 Genomic USS motif DNA uptake of fragments with mutant USS Why doesn’t the uptake bias more closely match ...
Tripeptide frequency  (per 1000 amino acids) Peptides encodable by  Hin  uptake sequences are enriched only in the  Hin  p...
Competence genes are regulated similarly in many bacteria Gene present
Artificially inducing  E. coli  Sxy turns on  many E. coli  genes:
Does  E. coli  have natural competence? Same competence genes:    Same induction by Sxy and CRP acting at CRP-S sites: S...
 
CAGTTAGTA -10 -35 A 0 50 100 150 200 250 300 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 Transformation frequency Time...
So do  Neisseria  species. Bacteria in the Pasteurellaceae preferentially take up ‘self’ DNA.
734 copies in + orientation 731 copies in - orientation Smith  et al . 1995 Science 269:538-540 The H. influenzae genome c...
Uptake sequences are motifs, not mobile elements. AAGTGCGGT SAV SAL CAV SAI H. influenzae protein Homologs in species with...
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  1. 1. Do bacteria have sex? Rosie Redfield University of British Columbia
  2. 2. Do bacteria have any processes that evolved to make new combinations of chromosomal genes? Why is this an important question? Why is natural competence the only process we need to investigate? Why do bacteria take up DNA? How does Haemophilus influenzae decide when to take up DNA? How does H. influenzae decide which DNAs to take up? What kinds of evidence are most useful?
  3. 3. Cycles of meiosis and gamete fusion that randomly recombine alleles from two parents into offspring. Sexual reproduction (in eukaryotes): Do bacteria have any processes that evolved to make new combinations of chromosomal genes? Why is this an important question?
  4. 4. Why does sexual reproduction exist? Genes that cause sexual reproduction must end up in more fit genomes than they would have without sex.
  5. 5. How should we decide? (What kind of evidence is best?) Do bacteria have any processes that evolved to make new combinations of chromosomal genes? What are the candidates? Parasexual processes of bacteria:
  6. 6. How should we decide? (What kind of evidence will best show how natural selection has acted?) Have ‘parasexual’ processes evolved for genetic exchange? Make mathematical models or computer simulations? Run long-term evolution experiments (like Lenski)? Use the ‘comparative method’?
  7. 7. How should we decide? Have ‘parasexual’ processes evolved for genetic exchange? What has real-world selection produced? What genes are responsible for the process? What genes does the process benefit? Do these genes affect other processes? What goes wrong when a gene is defective? Is the process regulated? If so, how? First, characterize these processes in more detail, paying attention to evolutionary issues.
  8. 8. How should we decide? Have ‘parasexual’ processes evolved for genetic exchange? Make mathematical models and computer simulations. How can selection act (under very simplified conditions.)? How does selection act (under very unnatural conditions)? Run long-term evolution experiments. Use the ‘comparative method’. If selection is still unclear: What has selection produced (under other conditions)?
  9. 9. Case study #1: RecA Discovery: Needed for homologous recombination in bacteria. Phenotype of mutant: Recombination down >1000x. Mechanism: More mutant phenotypes: Viability down ~10%. Death from DNA damage up >100x. RecA senses DNA damage and induces the ‘SOS response’. RecA carries out ‘recombination repair’ of otherwise-lethal DNA damage.
  10. 10. Roles of RecA in DNA repair: RecA binds to damaged DNA strands This induces other DNA repair proteins RecA pairs damaged strands with complementary intact strands. The intact strand provides a template for repair of the damage. The same steps cause recombination with incoming DNA:
  11. 11. Recombination: Do ‘parasexual’ processes exist for genetic exchange? All of these genes have important 'housekeeping' functions in DNA replication and repair.
  12. 12. Transduction and conjugation are caused by genetic parasites. Do ‘parasexual’ processes exist for genetic exchange? Conjugation: Usually plasmid transfer Rarely host DNA transfer Transduction: Recipient Phage-infected source
  13. 13. Do ‘parasexual’ processes exist for genetic exchange? Why do bacteria take up DNA? Templates for DNA repair? Nucleotides and other nutrients (food)? Sources of environmental DNA Recipient cell x Could be for New combinations of genes (sex)?
  14. 14. DNA is a good source of nutrients: Nucleotide synthesis is very expensive. DNA is widely available in the environment: <ul><li>biofilms </li></ul><ul><li>host mucosa </li></ul><ul><li>dead eukaryotes </li></ul>10-100 µg/ml in Serving size = 1 genome (3 pg) Fragments per serving = 12 Calories Carbon (as ribose) Phosphate Nitrogen Fats Amino acids Amounts Per Serving 0.000005 1 fg 1 fg 1 fg 0 fg 0 fg Percent daily values are based on a 3 mb genome. Not a significant source of calories from fat, saturated fat, cholesterol, dietary fiber, vitamin A, vitamin C, calcium, sulfur or iron.
  15. 15. Animation by Matt Baumann Competence in Haemophilus influenzae
  16. 16. Why do bacteria take up DNA? How does H. influenzae decide when to take up DNA? How does H. influenzae decide which DNAs to take up? If DNA serves mainly as food, we predict regulation by nutritional signals
  17. 17. Why do bacteria take up DNA? How does H. influenzae decide when to take up DNA? Competence genes are induced when both sugars and nucleotides are scarce.
  18. 18. CRP-S sequence
  19. 19. 1. The sugar signal: First, CRP activates energy-supply genes (and sxy ): Then, CRP and Sxy activate DNA uptake genes: CRP is activated (by cAMP) when preferred sugars are depleted. If…
  20. 20. Purine nucleotides and nucleosides prevent competence by inhibiting Sxy translation purines pyrim-idines purine bases
  21. 21. 10 -5 10 -6 10 -7 purR + purR - Constitutive production of purines prevents competence induction The purR gene encodes the purine repressor. In purR + cells the enzymes that synthesize purines are only made when the cell runs out of purines. In purR - cells the proteins are always made, so the cell never runs out of purines. Transformation frequency
  22. 22. 10 -4 10 -5 10 -6 10 -7 sxy + Hypercompetent sxy mutants Cells with hypercompetence mutations in sxy don’t care about purine levels. purR + purR -
  23. 23. Combining sxy-1 and sxy 3 restores base pairing and eliminates hypercompetence A A U U U U C U C A G U C A U C U U U A G A A G U U A G U A G G U C A A A U G U U U C . . . . . . . . 5’-G A A G U A C U 1 4 5 3 2 RBS A U G The 5 hypercompetence mutations all disrupt sxy mRNA base pairing sxy+ C : G sxy-3 T x G C x A sxy-1 sxy-1+3 T : A
  24. 24. Mutations in sxy stem 1 do alter mRNA secondary structure 7 1
  25. 25. Why do bacteria take up DNA? How does H. influenzae decide when to take up DNA? How does H. influenzae decide which DNAs to take up? Regulation is by nutritional signals.
  26. 26. H. influenzae cells (and their relatives) preferentially take up DNA containing a short sequence called the uptake signal sequence (USS). Their genomes have these sequences at thousands of sites. USS locations in the genome
  27. 27. Molecular drive explanation: Sequences preferred by the uptake machinery accumulate in the genome by recombination, replacing allelic versions that are not as easily taken up. Computer simulation modeling confirms that this drive can explain the observed properties of uptake sequences. Sex explanation: Species with uptake sequences in their genomes and a corresponding uptake bias benefit more from recombination. Why are uptake sequences so abundant in the genome?
  28. 28. Molecular drive explanation: Sequences preferred by the uptake machinery accumulate in the genome by recombination, replacing allelic versions that are not as easily taken up. But why are the uptake sequences preferred by the uptake machinery? Hypothesis: Uptake sequences are easier to kink Sex explanation: Species with uptake sequences in their genomes and a corresponding uptake bias benefit more from recombination. DNA must kink to enter the pore. outer membrane inner membrane
  29. 29. USS Randomized 200bp 300bp core s-2 s-3 Uptake sequences are bent. Core Seg 2 Seg 3 Core Seg 2 Seg 3 Neighbors are correlated at bend positions Ethylation of some positions enhances uptake
  30. 30. New research direction: What role do uptake sequences play in the mechanism of DNA uptake? Want to see it? Our lab web pages include our grant proposals.
  31. 31. If uptake sequences didn’t evolve for sex, bacteria don’t have ANY processes that evolved to generate recombination. Bacteria apparently get whatever recombination they might ‘need’ by accident. So why do eukaryotes need so much more?
  32. 32. With funding from CIHR & NIH
  33. 34. Uptake sequences are not species-specific. Redfield et al. 2006 80 96 100 80 A. actinomycetemcomitans P. multocida H. somnus M. succiniciproducens H. influenzae H. ducreyi M. haemolytica A. pleuropneumoniae 100 100 1465 1760 927 1216 1485 742 199 973 Consensus ‘cores’ Hin-type USS Apl -type USS AAGTGCGGT ACAAGCGGT
  34. 35. 136 260 459 581 648 640 384 180 436 583 628 445 174 43 Percent identities to homologs Mean uptake sequences per gene Uptake sequences are rare in strongly conserved genes. Findlay and Redfield 2009 H. influenzae N. meningitidis
  35. 36. Laterally transferred genes have few uptake sequences: Mean number of uptake sequences per gene N. meningitidis H. influenzae Number of standard genomes with homologs Number of standard genomes with homologs Mean uptake sequences per gene Uptake sequences don’t promote LGT; they accumulate after LGT. Findlay and Redfield 2009
  36. 37. Uptake sequences are less variable than other sequences in the genome. BLAST comparison of homologous USS in H. influenzae strains
  37. 38. Molecular drive explanation: Sequences preferred by the uptake machinery accumulate in the genome by recombination, replacing allelic versions that are not as easily taken up. Both predict that the USS motif found in the genome should reflect the uptake bias, but… Sex explanation: Species with uptake sequences in their genomes and a corresponding uptake bias benefit more from recombination.
  38. 39. Core Seg 2 Seg 3 Genomic USS motif DNA uptake of fragments with mutant USS Why doesn’t the uptake bias more closely match the sequences in the genome? 3 0 1 2 n n n n A A A G T G C G G T n A A A T T T n n n n n n A T T T T T n n n n n % variation Genetic variation at USS positions 0 10 20 30 A A G T G C G G T n AA n TT % uptake C C C G C A C C G . . . . . . . . . TT . G . . . T GG
  39. 40. Tripeptide frequency (per 1000 amino acids) Peptides encodable by Hin uptake sequences are enriched only in the Hin proteome Biased DNA uptake modifies tripeptide frequencies: Findlay and Redfield 2009 H. influenzae proteome ( Hin ) A. pleuropneumoniaeae proteome ( Apl ) N. meningitidis proteome ( Nme) E. coli proteome (control) Reversed peptides show no enrichment Peptides encodable by Nme uptake sequences are enriched only in the Nme proteome Peptides encodable by Apl uptake sequences are enriched only in the Apl proteome
  40. 41. Competence genes are regulated similarly in many bacteria Gene present
  41. 42. Artificially inducing E. coli Sxy turns on many E. coli genes:
  42. 43. Does E. coli have natural competence? Same competence genes:   Same induction by Sxy and CRP acting at CRP-S sites: So why won’t E. coli become competent? Perhaps because we don’t know how to turn Sxy on...
  43. 45. CAGTTAGTA -10 -35 A 0 50 100 150 200 250 300 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 Transformation frequency Time in rich medium (min) No colonies Wildtype cells sxy-1 mutant WT sxy-1 sxy-2 sxy-3 sxy-4 sxy-5 Transformation frequency 10 -3 10 -4 10 -5 10 -6 10 -7 WT sxy-1 sxy-2 sxy-3 sxy-4 sxy-5 log-phase late-log 5 hypercompetent sxy mutants CTACTGACT TCA 5 4 3 Gln A 2 1 Val Ile
  44. 46. So do Neisseria species. Bacteria in the Pasteurellaceae preferentially take up ‘self’ DNA.
  45. 47. 734 copies in + orientation 731 copies in - orientation Smith et al . 1995 Science 269:538-540 The H. influenzae genome contains 1471 copies of the uptake sequence AAGTGCGGT.
  46. 48. Uptake sequences are motifs, not mobile elements. AAGTGCGGT SAV SAL CAV SAI H. influenzae protein Homologs in species without uptake sequences Pattern found with the Gibbs motif sampler program

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