1. Fluidity of the 16S rRNA Gene Sequence within Aeromonas Strains Alessia Morandi Institute for Infectious Diseases University of Berne

2. Introduction Sequence analysis of  Classification ribosomal RNA (rRNA) of organisms

3. Three domains EubacteriaArchaeaEukarya

4. Introduction Sequence analysis of  Classification ribosomal RNA (rRNA) of organisms  Species identification of pathogenic bacteria for diagnostic purposes

5. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regions

6. Vertical Ancestor : AUCUGACCGUGACGGUCAUUC Descendent 1: AUCUCACCGUGACGGUCAUUC Descendent 2: AUCUCACCGUGACGUUCAUUC Descendent 3: AUCUCAACGUGACGUUCAUUC Descendent 4: AUCUCAACGUGACGGUCAUUC

7. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regionsF undamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny

8. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regionsF undamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny Multiple copies per genome

9. Concerted Evolution Copy 1: AUCUGACCGUGACGGUCA Copy 2: AUCUCACCGUGACGGUCA Copy 3: AUCUGACCGUGACGGUCA Copy 4: AUCUGACCGUGACGGUCA Copy 5: AUCUGACCGUGACGGUCA # 1: AUCUGACCGUGACGGUCAUUC # 2: AUCUGACCGUGACGGUCAUUC # 3: AUCUGACCGUGACGGUCAUUC # 4: AUCUGACCGUGACGGUCAUUC # 5: AUCUGACCGUGACGGUCAUUC # 1: AUCUCACCGUGACGGUCAUUC # 2: AUCUCACCGUGACGGUCAUUC # 3: AUCUCACCGUGACGGUCAUUC # 4: AUCUCACCGUGACGGUCAUUC # 5: AUCUCACCGUGACGGUCAUUC

10. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regionsF undamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny Multiple copies per genome 2 divergent 16S rRNA gene sequences present on the same chromosome found in T. chromogena and T. bispora

11. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regionsF undamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny Multiple copies per genome 2 divergent 16S rRNA gene sequences present on the same chromosome found in T. chromogena and T. bispora House-keeping gene

12. Horizontal Ancestor 1: AUCUGACCGUGACGGUCAUUC Ancestor 2: AUCUCAACGUGACGUUCAUUC X Descendent 1: AUCUCAACGUGACGGUCAUUC

13. 16S rRNA Universally distributed Conserved regions PCR -amplification Variable regionsF undamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny Multiple copies per genome 2 divergent 16S rRNA gene sequences present on the same chromosome found in T. chromogena and T. bispora House-keeping genes Horizontal gene transfer (HGT) of 16S rRNA is very rare and does not cause a significant problem for the species identification

14. Identification of Aeromonas species Aeromonas veronii biovar sobriahuman pathogen Aeromonas media environmental species Biochemical species identification: difficult  molecular methods using 16S rRNA gene sequence

15. RFLP-PCR Analysis Aeromonas strain to be identified Grow up a single colony Isolate genomic DNA PCR amplify the 16S rRNA gene Digest amplified 16S rRNA gene with restriction enzymes Agarose gel electrophoresis

16. RFLP-PCR analysis of 16S rRNA gene amplified from genomic DNA of A. media strain 622- 404- 307- 240- 190- 160- 110- 5u

17. Faint bands ?? Incomplete digestion of DNA

18. RFLP-PCR analysis of 16S rRNA gene amplified from genomic DNA of A. media strain 622- 404- 307- 240- 190- 160- 110- 5u 10u20u = excess amount of enzyme

19. Faint bands ?? Incomplete digestion of DNA Contamination with other DNA

20. Faint bands ?? Incomplete digestion of DNA Contamination with other DNA We suspected that these strains harbored multiple different copies of the 16S rRNA gene on their chromosome.

21. Faint bands ?? Incomplete digestion of DNA Contamination with other DNA We suspected that these strains harbored multiple different copies of the 16S rRNA gene on their chromosome. We tested this hypothesis by cloning and sequencing multiple copies of the 16S rRNA gene for each strain.

22. Our study Aeromonas strain Grow up a single colony Isolate genomic DNA PCR amplify the 16S rRNA gene Clone 16S rRNA gene

23. Cloning Single bacterium with multiple copies of 16S rRNA gene

24. Cloning Single bacterium with multiple copies of 16S rRNA gene Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies

25. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies

26. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies

27. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies Transformation of ultracompetent cells Amp R beta-gal = blue Amp R no beta-gal = white Amp S Amp R no beta-gal = white Amp R no beta-gal = white Amp R no beta-gal = white

28. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies Transformation of ultracompetent cells Amp S Amp R no beta-gal = blue Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Plasmid isolation from individual clones

29. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies Transformation of ultracompetent cells Amp S Amp R no beta-gal = blue Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Plasmid isolation from individual clones 4.5 kb 6 kb Restriction digest and gel electrophoresis  single insert

30. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies Transformation of ultracompetent cells Amp S Amp R no beta-gal = blue Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Plasmid isolation from individual clones PCR-amplification of individual copies of 16S rRNA gene 4.5 kb 6 kb Restriction digest and gel electrophoresis  single insert

31. Cloning Single bacterium with multiple copies of 16S rRNA gene + Ligation with Plasmid Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies Transformation of ultracompetent cells Amp S Amp R no beta-gal = blue Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Amp R beta-gal = white Plasmid isolation from individual clones PCR-amplification of individual copies of 16S rRNA gene Sequence analysis of a single copy 4.5 kb 6 kb Restriction digest and gel electrophoresis  single insert

32. Our study Aeromonas strain Grow up a single colony Isolate genomic DNA PCR amplify the 16S rRNA gene Clone 16S rRNA gene Sequence analysis of individual clones

33. Sequence Comparison within Strains

34.  As much variation occurs among the alleles present on the same chromosome as between the 16S rRNA gene from distantly related species These dramatic differences were surprising,and we wanted to verify that the cloned alleles corresponded to the sequence present on the genome.

35. RFLP-PCR analysis: comparison of the restriction patterns 16S rRNA alleles 16S rRNA genes amplified from vsamplified from plasmid DNAgenomic DNA

36. RFLP-PCR analysis: comparison of the restriction patterns of the A. veronii biovar sobria strain 5u 10u20u12345 Genomic DNACloned Allele

37. RFLP-PCR analysis: comparison of the restriction patterns of the A. media strain 5u 10u20u 622- 404- 307- 240- 190- 160- 110- 123456 Genomic DNACloned Allele

38. RFLP-PCR analysis: comparison All of the bands detected by digestion of the PCR-16S rRNA alleles amplified from plasmid DNA are also present on the same chromosome and are not due to cloning artifacts. !!!!! RFLP-PCR analysis: PCR-dependent approach.

39. Southern analysis: PCR-independent approach

40. Primer 27F 5‘-AGA GTT TGA TCM TGG CTC AG-3‘ All alleles

41. Southern analysis: PCR-independent approach Primer 27F 5‘-AGA GTT TGA TCM TGG CTC AG-3‘ 16S 230-232 A 5‘-GGG TCC ATC CAA TCG CG-3‘ All alleles Allele 1 A. media strain

42. Southern analysis: PCR-independent approach Primer 27F 5‘-AGA GTT TGA TCM TGG CTC AG-3‘ 16S 230-232 A 5‘-GGG TCC ATC CAA TCG CG-3‘ 16S 230-232 B 5‘-GGG CAT ATC CAA TCG CG-3‘ All alleles Allele 1 Allele 2-6 A. media strain

43. Southern analysis of the A. media strain 3000 1600 1000 2000 4000 12000 5000 27F all alleles 1 2 3 Restriction enzymes:

44. Southern analysis of the A. media strain 3000 1600 1000 2000 4000 12000 5000 230-232 A allele 1 27F all alleles 1 2 3 Restrition enzymes: 1 2 3

45. Southern analysis of the A. media strain 3000 1600 1000 2000 4000 12000 5000 230-232 A allele 1 27F all alleles 1 2 3 Restrition enzymes: 1 2 3 230-232 B allele 2-6 1 2 3

46. Southern analysis The results are consistent with our predictions based on the DNA sequence analysis. This suggests that the differences in the sequence of the cloned alleles are not due to cloning artifacts, PCR- errors or contamination but are present on the same chromosome.

47. Phylogenetic analysis Fundamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny.  We wanted to assess the significance of the presence of multiple divergent 16S rRNA gene sequences on the same chromosome by constructing phylogenetic trees.

48. Construction of phylogenetic trees Sequence 1 : AUCUGACCGUGACGGUCAUUC Sequence 2 : AUCUCACCGUGACGGUCAUUC Sequence 3 : AUCUCACCGUAACGUUCAUUC Sequence alignement and pairwise camparison

49. Construction of phylogenetic trees Sequence 1 : AUCUGACCGUGACGGUCAUUC Sequence 2 : AUCUCACCGUGACGGUCAUUC Sequence 3 : AUCUCACCGUAACGUUCAUUC Sequence alignement and pairwise camparison Distance matrix

50. Construction of phylogenetic trees Sequence 1 : AUCUGACCGUGACGGUCAUUC Sequence 2 : AUCUCACCGUGACGGUCAUUC Sequence 3 : AUCUCACCGUAACGUUCAUUC Sequence alignement and pairwise camparison Distance matrix Phylogenetic tree

51. Construction of phylogenetic trees Sequence 1 : AUCUGACCGUGACGGUCAUUC Sequence 2 : AUCUCACCGUGACGGUCAUUC Sequence 3 : AUCUCACCGUAACGUUCAUUC Sequence alignement and pairwise camparison Distance matrix % % = Bootstrap value Phylogenetic tree %

53. Phylogenetic analysis  Each strain harbors multiple divergent 16S rRNA gene sequences on its chromosome, that are related to different Aeromonas species. This result supports our cautious approach in using 16S rRNA gene sequences for the identification of Aeromonas species, because of the possible misidentification of pathogenic species for environmental species.

54. Sneath, 1992

55. The similarity of 16S rRNA gene sequences does not correlate with the similarity of the entire genome

56. Sneath, 1992 The similarity of 16S rRNA gene sequences does not correlate with the similarity of the entire genome Sneath‘s hypothesis: horizontal transfer of 16S rRNA genes between Aeromonas species and subsequent homologous recombination between the different gene copies.

57. Sneath, 1992 The similarity of 16S rRNA gene sequences does not correlate with the similarity of the entire genome Sneath‘s hypothesis: horizontal transfer of 16S rRNA genes between Aeromonas species and subsequent homologous recombination between the different gene copies. !!!Sneath missed the strains that harbored different alleles that supported his hypothesis.

58. Sequence Comparison within Strains

59. Phylogenetic analysis We therefore provide the missing strains that support Sneath‘s hypothesis of horizontal gene transfer between Aeromonas species. Maintenance of multiple alleles on the same chromosome = intermediate stage during homologous recombination

60. C onclusi ons

61. We described the presence of multiple divergent 16S rRNA alleles in 2 Aeromonas strains.

62. C onclusi ons We described the presence of multiple divergent 16S rRNA alleles in 2 Aeromonas strains. The variation that occurred in the 16S rRNA genes within one organism was as great as the variation between distantly related species.

63. C onclusi ons We described the presence of multiple divergent 16S rRNA alleles in 2 Aeromonas strains. The variation that occurred in the 16S rRNA genes within one organism was as great as the variation between distantly related species. We provide the missing strains that support Sneath‘s hypothesis of horizontal transfer of 16S rRNA genes between Aeromonas species.

64. C onclusi ons We described the presence of multiple divergent 16S rRNA alleles in 2 Aeromonas strains. The variation that occurred in the 16S rRNA genes within one organism was as great as the variation between distantly related species. We provide the missing strains that support Sneath‘s hypothesis of horizontal transfer of 16S rRNA genes between Aeromonas species. Our data suggest that horizontal gene transfer of the 16S rRNA gene occurred and that the degree of similarity of 16S rRNA gene sequences does not reflect phylogeny, which is based on the traditional view of evolution as a vertical process of inheritance.

65. C onclusi ons We described the presence of multiple divergent 16S rRNA alleles in 2 Aeromonas strains. The variation that occurred in the 16S rRNA genes within one organism was as great as the variation between distantly related species. We provide the missing strains that support Sneath‘s hypothesis of horizontal transfer of 16S rRNA genes between Aeromonas species. Our data suggest that horizontal gene transfer of the 16S rRNA gene occurred and that the degree of similarity of 16S rRNA gene sequences does not reflect phylogeny, which is based on the traditional view of evolution as a vertical process of inheritance. If this is the case, our results violate the fundamental assumption for using 16S rRNA gene to identify bacteria.

67. Our study 6  We tested Sneath‘s hypothesis by constructing distance matrices and phylogenetic trees only from the left or right region of the 16S rRNA gene sequence

68. Distance matrix: left and right region

69. Phylogenetic trees: left and right region

70. Phylogenetic analysis: left and right region The differences between the left and right region suggest that for both strains the left region of allele 1 was transfered from a distantly related Aeromonas species by horizontal gene transfer.

71. Phylogenetic analysis: left and right region We therefore provide the missing strains that support Sneath‘s hypothesis of horizontal gene transfer between Aeromonas species. Maintenance of multiple alleles on the same chromosome = intermediate stage during homologous recombination

72. Prevalence of multiple 16S rRNA alleles within Aeromonas RFLP-PCR analysis of 16S rRNA gene amplified from genomic DNA additional faint bands in the restriction = multiple 16S rRNA alleles patterns  Of 82 strains analyzed 21% contained multiple alleles This result indicates that the presence of multiple 16S rRNA alleles in Aeromonas is not an exception but rather common.

73. Cloned Alleles

74. Sequence Comparison within Strains

75. Concerted Evolution Copy 1: AUCUGACCGUGACGGUCA Copy 2: AUCUCACCGUGACGGUCA Copy 3: AUCUGACCGUGACGGUCA Copy 4: AUCUGACCGUGACGGUCA Copy 5: AUCUGACCGUGACGGUCA # 1: AUCUGACCGUGACGGUCAUUC # 2: AUCUGACCGUGACGGUCAUUC # 3: AUCUGACCGUGACGGUCAUUC # 4: AUCUGACCGUGACGGUCAUUC # 5: AUCUGACCGUGACGGUCAUUC # 1: AUCUCACCGUGACGGUCAUUC # 2: AUCUCACCGUGACGGUCAUUC # 3: AUCUCACCGUGACGGUCAUUC # 4: AUCUCACCGUGACGGUCAUUC # 5: AUCUCACCGUGACGGUCAUUC

76. If NO Concerted Evolution Copy 1: AUCUGACCGUGACGGUCA Copy 2: AUCUCACCGUGACGGUCA Copy 3: AUCUGACCGUGACGGUCA Copy 4: AUCUGACCGUGACGGUCA Copy 5: AUCUGACCGUGACGGUCA # 1: AUCUGACGGUGACGGUCA # 2: AUCUCACCGUGAAGGUCA # 3: AUCUGACCGUGACGGUCA # 4: AUCUGACCGUGACGGUCA # 5: AUCUGACCGUGACGGUCA # 1: AUCUGACGGUGACGGUCA # 2: AUCUCACCGUGAAGGUCA # 3: AUCUGACCGUGACGGUCA # 4: AUCUGAAGUGACGGUCA # 5: UUCUGACCGUGACGGUCA

77. Sequencing Primers, Both strands sequenced, only sequences shown that occurred at least twice.