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

Fluidity of the 16S rRNA Gene Sequencewithin Aeromonas Strains

Alessia Morandi

Institute for Infectious DiseasesUniversity of Berne

Introduction

Sequence analysis of Classification ribosomal RNA (rRNA) of organisms

Three domains

Eubacteria Archaea Eukarya

Introduction

Sequence analysis of Classification ribosomal RNA (rRNA) of organisms

Species identification of pathogenic bacteria for diagnostic purposes

16S rRNA

Universally distributed

Conserved regions PCR-amplification

Variable regions

Vertical

Ancestor : AUCUGACCGUGACGGUCAUUCDescendent 1: AUCUCACCGUGACGGUCAUUCDescendent 2: AUCUCACCGUGACGUUCAUUCDescendent 3: AUCUCAACGUGACGUUCAUUCDescendent 4: AUCUCAACGUGACGGUCAUUC

16S rRNA



Variable regions Fundamental assumption for using 16S rRNA to identify bacteria: the degree of sequence similarity correlates with phylogeny

16S rRNA




Multiple copies per genome

Concerted Evolution

Copy 1: AUCUGACCGUGACGGUCACopy 2: AUCUCACCGUGACGGUCACopy 3: AUCUGACCGUGACGGUCACopy 4: AUCUGACCGUGACGGUCACopy 5: AUCUGACCGUGACGGUCA

# 1: AUCUGACCGUGACGGUCAUUC# 2: AUCUGACCGUGACGGUCAUUC# 3: AUCUGACCGUGACGGUCAUUC# 4: AUCUGACCGUGACGGUCAUUC# 5: AUCUGACCGUGACGGUCAUUC

# 1: AUCUCACCGUGACGGUCAUUC# 2: AUCUCACCGUGACGGUCAUUC# 3: AUCUCACCGUGACGGUCAUUC# 4: AUCUCACCGUGACGGUCAUUC# 5: AUCUCACCGUGACGGUCAUUC

16S rRNA




Multiple copies per genome 2 divergent 16S rRNA gene sequences present on the same chromosome found in T. chromogena and T. bispora

16S rRNA





House-keeping gene

Horizontal

Ancestor 1: AUCUGACCGUGACGGUCAUUC

Ancestor 2: AUCUCAACGUGACGUUCAUUC X

Descendent 1: AUCUCAACGUGACGGUCAUUC

16S rRNA





House-keeping genes Horizontal gene transfer (HGT) of 16S rRNA is very rare and does not cause a significant problem for the species identification

Identification of Aeromonas species

Aeromonas veronii biovar sobria human pathogenAeromonas media environmental species

Biochemical species identification: difficult

molecular methods using 16S rRNA gene sequence

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

RFLP-PCR analysis of 16S rRNA gene amplified from genomic DNA of A. media strain

622-

404-

307-

240-

190-

160-

110-

5u 5u

Faint bands ??

Incomplete digestion of DNA

RFLP-PCR analysis of 16S rRNA gene amplified from genomic DNA of A. media strain

622-

404-

307-

240-

190-

160-

110-

5u 5u 10u 20u = excess amount of enzyme

Faint bands ??


Contamination with other DNA

Faint bands ??



We suspected that these strains harbored multiple different copies of the 16S rRNA gene on their chromosome.

Faint bands ??



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.

Our study

Aeromonas strain


Isolate genomic DNA


Clone 16S rRNA gene

CloningSingle bacterium with multiple copies of 16S rRNA gene


Isolation of genomic DNA and PCR-amplification of 16S rRNA gene copies


+Ligation with Plasmid








Transformation ofultracompetent cells

Amp Rbeta-gal= blue

Amp R no beta-gal= white

Amp S








Amp S Amp Rno beta-gal= blue

Amp Rbeta-gal= white




Plasmid isolation from individual clones











4.5 kb 4.5 kb 4.5 kb 6 kb

Restriction digest andgel electrophoresis single insert











PCR-amplification of individual copies of

16S rRNA gene

4.5 kb 4.5 kb 4.5 kb 6 kb












PCR-amplification of individual copies of

16S rRNA gene

Sequence analysisof a single copy

4.5 kb 4.5 kb 4.5 kb 6 kb


Our study

Aeromonas strain


Isolate genomic DNA


Clone 16S rRNA gene

Sequence analysis of individual clones

Sequence Comparison within Strains


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.

RFLP-PCR analysis: comparison of the restriction patterns

16S rRNA alleles 16S rRNA genesamplified from vs amplified fromplasmid DNA genomic DNA

RFLP-PCR analysis: comparison of the restriction patternsof the A. veronii biovar sobria strain

5u 5u 10u 20u 1 2 3 4 5

Genomic DNA Cloned Allele

RFLP-PCR analysis: comparison of the restriction patternsof the A. media strain

5u 5u 10u 20u

622-

404-

307-

240-

190-

160-

110-

1 2 3 4 5 6Genomic DNA Cloned Allele

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.

Southern analysis: PCR-independent approach


Primer 27F 5‘-AGA GTT TGA TCM TGG CTC AG-3‘

All alleles



16S 230-232 A 5‘-GGG TCC ATC CAA TCG CG-3‘

All alleles

Allele 1

A. media strain



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

Southern analysis of the A. media strain

3000

1600

1000

2000

4000

12000

5000

27F all alleles

1 2 3Restriction enzymes:


3000

1600

1000

2000

4000

12000

5000

230-232 Aallele 1

27F all alleles

1 2 3Restrition enzymes: 1 2 3


3000

1600

1000

2000

4000

12000

5000

230-232 Aallele 1

27F all alleles

1 2 3Restrition enzymes: 1 2 3

230-232 Ballele 2-6

1 2 3

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.

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.

Construction of phylogenetic trees

Sequence 1 : AUCUGACCGUGACGGUCAUUCSequence 2 : AUCUCACCGUGACGGUCAUUCSequence 3 : AUCUCACCGUAACGUUCAUUC

Sequence alignementand pairwise camparison




Sequence 1 2 31 0 1 32 1 0 23 3 2 0

Distance matrix




Sequence 1 2 31 0 1 32 1 0 23 3 2 0

Distance matrix

Phylogenetic tree




Sequence 1 2 31 0 1 32 1 0 23 3 2 0

Distance matrix

% % = Bootstrap value

Phylogenetic tree%

Phylogenetic tree


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.

Sneath, 1992

Sneath, 1992

The similarity of 16S rRNA gene sequencesdoes not correlate with

the similarity of the entire genome

Sneath, 1992



Sneath‘s hypothesis: horizontal transfer of 16S rRNA genes between Aeromonas species and subsequent homologous recombination between the different gene copies.

Sneath, 1992



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.



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

Conclusions

Conclusions

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

Conclusions


The variation that occurred in the 16S rRNA genes within one organism was as great as the variation between distantly related species.

Conclusions



We provide the missing strains that support Sneath‘s hypothesis of horizontal transfer of 16S rRNA genes between Aeromonas species.

Conclusions




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.

Conclusions




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.

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

Distance matrix: left and right region

Left region Right region

A. media alleles 1 2 3 4 5 6 A. media alleles 1 2 3 4 5 61 0 10 8 10 10 10 1 0 9 11 2 7 52 10 0 2 0 0 0 2 9 0 2 7 8 63 8 2 0 2 2 2 3 11 2 0 9 6 84 10 0 2 0 0 0 4 2 7 9 0 7 55 10 0 2 0 0 0 5 7 8 6 7 0 26 10 0 2 0 0 0 6 5 6 8 5 2 0

A. veronii alleles 1 2 3 4 5 A. veronii alleles 1 2 3 4 51 0 14 13 7 13 1 0 6 8 5 32 14 0 1 7 2 2 6 0 2 9 73 13 1 0 6 1 3 8 2 0 7 54 7 7 6 0 6 4 5 9 7 0 25 13 2 1 6 0 5 3 7 5 2 0

Phylogenetic trees: left and right region

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.

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

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 allelespatterns

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.

Cloned Alleles

Strain Allel Plasmid Strain Allel Plasmid Strain Allel Plasmid Strain Plasmid Strain Allel Plasmid Strain Allel Plasmid Strain Plasmid Strain Plasmid409 6 410 417 1 501 628 1 341 646 603 662 1 802 664 1 307 666 208 705 1008

1 426 1 503 1 702 601 1 804 1 302 207 10011 401 1 506 2 338 604 1 805 2 303 209 10021 408 1 502 2 346 605 1 806 2 308 210 10044 409 1 505 3 101 606 2 309 211 10034 406 1 511 3 337 2 306 213 10074 427 1 509 3 331 2122 420 1 510 3 350 2062 402 1 507 3 3393 415 2 512 3 3423 423 2 508 3 3435 414 3 1105 413 3 1065 405 3 3355 412 3 7045 411 3 3345 418 7035 4245 4035 4075 4225 4195 404


Species/Acc# Allele # 76 129

131

132

154

155

156

165

166

167

199

218

230

231

232

250

258

264

457

458

459

460

461

462

463

464

469

470

471

472

473

474

475

476

647

649

650

A. veronii 1 2 LM13695 2 4

3 64 35 2

A. media 1 5AmCDC0862 2 2

3 24 45 96 5

A. media/X74679-1A. hydrophila/X74677A. trota/X60415A. caviae/X60408A. caviae/X60409A. salmonicida/X74681A. bestiarum/AB034759A. encheleia/AJ224309A. popoffii/AJ223180A. popoffii/AJ223181A. eucrenophila/X74675A. sobria/X74683A. allosacchrophila/S39232A. veronii/X74684A. jandaei/X74678A. schubertii/X60416

A A A U A G U A C U C G A U G U A U U G G C G C C U C G U G U C A A C A GG G U C U A C G U A G U G A A A G C C A A U A G U G U C C A C U G G U G A

G U A G

Concerted Evolution


# 1: AUCUGACCGUGACGGUCAUUC# 2: AUCUGACCGUGACGGUCAUUC# 3: AUCUGACCGUGACGGUCAUUC# 4: AUCUGACCGUGACGGUCAUUC# 5: AUCUGACCGUGACGGUCAUUC

# 1: AUCUCACCGUGACGGUCAUUC# 2: AUCUCACCGUGACGGUCAUUC# 3: AUCUCACCGUGACGGUCAUUC# 4: AUCUCACCGUGACGGUCAUUC# 5: AUCUCACCGUGACGGUCAUUC

If NO Concerted Evolution


# 1: AUCUGACGGUGACGGUCA# 2: AUCUCACCGUGAAGGUCA# 3: AUCUGACCGUGACGGUCA# 4: AUCUGACCGUGACGGUCA# 5: AUCUGACCGUGACGGUCA

# 1: AUCUGACGGUGACGGUCA# 2: AUCUCACCGUGAAGGUCA# 3: AUCUGACCGUGACGGUCA# 4: AUCUGAAGUGACGGUCA# 5: UUCUGACCGUGACGGUCA

Sequencing

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

Documents

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