The impact of the neisserial DNA uptake sequence on genome evolution and stability

Transcription

1 The impact of the neisserial DNA uptake sequence on genome evolution and stability Ole Herman Ambur The Tønjum Group

2 Transformation

3 Neisserial transformation requires: DNA uptake sequence (DUS) Transformation apparatus, pili etc. Recombination components

4 DNA uptake sequence -DUS ATGCCGTCTGAA approx copies N. meningitidis Z2491

5 Bacterial sex

6 Transformation, like sex, is everywhere! Naturally competent prokaryotes (Johnsborg et al., 2007): Methanobacterium thermoautotrophicum Methanococcus voltae Deinococcus radioduransa Thermus aquaticus Thermus caldophilus Thermus flavus Thermus thermophilus Nostoc muscorum Synechococcus elongatusb Synechocystis spp.c Thermosynechococcus elongatus Chlorobium limicola Chlorobium tepidum Agrobacterium tumefaciens Methylobacterium organophilum Bradyrhizobium japonicumd Achromobacter spp. Eikenella corrodens Kingella denitrificans Kingella kingae Neisseria gonorrhoeae Neisseria meningitidis Ralstonia solanacearum Thiobacillus thioparus Thiobacillus sp. strain Y Acinetobacter baylyi Acinetobacter calcoaceticus Actinobacillus actinomycetemcomitans Actinobacillus pleuropneumoniae Aggregatibacter aphrophiluse Azotobacter vinelandii Cardiobacterium hominis Haemophilus influenzae Haemophilus parainfluenzae Haemophilus parasuis Legionella pneumophila Moraxella spp. Pseudomonas fluorescens Pseudomonas stutzeri and related species Pseudomonas spp.f Vibrio cholerae Vibrio parahaemolyticus Vibrio spp. Campylobacter coli Campylobacter jejuni Helicobacter pylori Bacillus amyloliquefaciens Bacillus licheniformis Bacillus subtilis Lactobacillus lactis Leuconostoc carnosum Streptococcus pneumoniae Streptococcus mitis Streptococcus oralis Streptococcus crista Streptococcus infantis Streptococcus gordonii Streptococcus sanguinisg Streptococcus anginosus Streptococcus intermedius Streptococcus constellatus Streptococcus thermophilush Streptococcus bovis Streptococcus mutans Thermoactinomyces vulgaris Mycobacterium smegmatis Streptomyces spp.

7 DNA uptake sequence DUS

8 DUS was identified in 1988 as a 10-mer 10-mer 12-mer degenerate Goodman and Scocca, 1988

9 DUS revised Transformation frequencies (10-7) M c M 400 Gc N400 ** * * * # # 10 p 0-DUS p 8-DUS p 9-DUS p 10-DUS p 11-DUS p 12-DUS Donor DNA 12 ** * # Ambur et al. J. Bacteriol. 2007

10 DUS inverted repeat forms transcriptional terminator/attenuator DUS 5 - A U A C G A U G C C G U C U G A A A U C G A U U C A G A C G G C A U U U A U U -OH 3 Complement DUS A U G C A 5 - A A G U C U G C C G U A A U A C G U U C A G A C G G C A U U U A U U -OH 3

11 A single DUS is sufficient for transformation 100 Mc MC58 Mc M400 Transformation frequencies (10-7) log 10 1 p0 psingle pdr pir Donor DNA Ambur et al. J. Bacteriol. 2007

12 DUS and neisserial sex Outline/hypothesis: Analysis of DUS conservation and distribution may reveal its evolutionary history and perhaps that of transformation/sex itself

13 M-GCAT global alignment of 6 neisserial genomes Treangen, Ambur et al., Genome Biology 9:2, 2008

14 6 neisserial genomes compared Genome Genome No of DUS distribution size (kb) genes % in CDS % alignment cdus Total DUS -1 N. men. Z , N. men. MC , N. men. FAM , N. men , N. gonorrhoeae , N. lactamica , Core genome More DUS in N. lactamica

15 Phylogeneny with strong consensus (+/ nt) surrounding DUS ubiquitous gene alignment entire concatenated multiple alignment Strong consensus phylogenetic tree that may guide us in detection of recombination

16 Two hypotheses for the manifestation of DUS DUS emerge by mutation DUS emerge by recombination (and sex)

17 DUS distribution Is there an association between DUS distribution and the recombinogenic past of neisserial genomes?

18 Comparing sequence columns DUS

19 DUS more conserved but in permissive regions DUS

20 DUS conservation 71% of the DUS in the multiple alignment were exactly conserved in all genomes. DUS, much more conserved (97%) than the average conserved sequence (~85%)

21 Purifying selection or mutation selection balanceof DUS non-degenerate, in all the other 5 genomes

22 Program: GENECONV (Sawyer, 1989) Detection of gene conversion elements finds the most likely candidates for aligned gene conversion events (signs of recombination) between pairs of sequences

23 DUS distribution is tuned N. meningitidis Z2491 Average DUS distance Ca nucleotides Average conversion fragment length Ca nucleotides

24 Signs of recombination DUS facing sequences with no similarity to DUS

25 2 lines of evidence for recombinational acquisition of DUS in populations The spacing of DUS matches the conversion fragment lengths Unique DUS in sequence columns are complete and show origin by recombination

26 DUS outside the core genome? Are DUS present in new laterally transferred sequences? New= present only in one genome (34 genes) Are DUS present in old lost sequences? Lost=absent in one and only one genome (29 genes)

27 DUS are absent in newly acquired and old lost sequences No. of DUS No. of genes p-value Ubiquitous genes New genes 0 34 <10-7 Lost genes 0 29 <10-6

28 Genetic variation is generated without DUS Phase variable genes are DUS-less Genes encoding outer membrane proteins are DUS-less

29 DUS and genome stability DUS are overrepresented in the core genome..underrepresented in regions under selective pressure driving diversification..totally absent in both recently acquired genes and recently lost genes

30 Summary of DUS evolution DUS is a 12-mer IR-DUS terminate/attenuate transcription Inter DUS distance is tuned Unique DUS suggest origin by recombination DUS are absent outside core genome

31 Sources of variation Spontaneous mutation Mutation rate modifiers (hypermutators) Mobile genetic elements (HGT) Evolved mechanisms for genetic variation Intrachromosomal recombination pili, phase variable genes encoding surface prot Antigenic variation Nucleotide tracts

32 Measuring genome instability Monitoring of phase variation Polynucleotide tract expansion and retraction Mutator phenotypes Effects of stress

33 DNA repair Davidsen and Tønjum, 2006

34 Acknowledgements Tone Tønjum Kristian Alfsnes Seetha Balasingham Tonje Davidsen Stephan Frye Håvard Homberset Sheba Lothe Emma Lång International: Eduardo Rocha (Pasteur Institute) Todd Treangen (ABI, Paris) CMBN Rognes group Koomey group Bjørås group Ottersen group Forskerlinje Marie Bergheim Ohr Jarle Breivik