Evidence of Selection on Genomic GC Content in Bacteria Falk Hildebrand Adam Eyre-Walker

Genomic G+C content

Genomic GC content

Codons ATA CCC CTA CCT Non-synonymous Synonymous 2-fold : TTT TTC 4-fold : CCT CCC CCA CCG

Genomic GC content

Variation

Correlations

Explanations Mutation bias Suoeka (1961) & Freese (1962) Intrinsic and/or extrinsic Selection Many authors Biased gene conversion Anonymous referees

Correlates Genome size positive correlation Lifestyle higher GC in free living Aerobiosis higher GC in aerobic Nitrogen utilization higher amongst N fixers Temperature higher amongst thermophiles?

Evidence of selection I Escherichia coli Mutation pattern 273 GC  AT versus 131 AT  GC Predicted GC content = 0.32 Observed GC content = 0.50 Observed GC at neutral sites = 0.58 Lynch (2007) Origins of genome architecture

Evidence of selection II Phylogenetic analyses Mycobacterium leprae (Lynch 2007) Escherichia coli (Balbi et al. 2009) 5 pathogenic bacteria (Hershberg and Petrov 2010)

Phylogenetic analysis GAAGGG

Evidence of selection II Phylogenetic analyses Mycobacterium leprae (Lynch 2007) Escherichia coli (Balbi et al. 2009) 5 pathogenic bacteria (Hershberg and Petrov 2010) Excess of GC  AT

Test of mutation bias If GC content is Due to mutation bias alone Stationary And the infinite sites assumption holds Then # GC  AT mutations = # AT  GC mutations

Why? If GC stationary #GC  AT subs = #AT  GC subs All neutral mutations have same chance of fixation #GC  AT muts = #AT  GC muts

Identifying mutations Strain 1 ACT GCT TTG GCT TTA TGG Strain 2 ACT GCT TTG GCT TTA TGA Strain 3 ACT GCT TTG GCT TTA TGG Strain 4 ACT GCT TTC GCT TTA TGA Strain 5 ACC GCT TTC GCT TTA TGG Strain 6 ACT GCT TTG GCT TTA TGG TCTCCGCGGAGA

Orienting mutations Outgroup ACT GCT TTC GCT TTA TGG Strain 1 ACT GCT TTG GCT TTA TGG Strain 2 ACT GCT TTG GCT TTA TGA Strain 3 ACT GCT TTG GCT TTA TGG Strain 4 ACT GCT TTC GCT TTA TGA Strain 5 ACC GCT TTC GCT TTA TGG Strain 6 ACT GCT TTG GCT TTA TGG TCTCCGCGGAGA GC  AT = 1 AT  GC = 1

Orienting mutations Strain 1 ACT GCT TTG GCT TTA TGG Strain 2 ACT GCT TTG GCT TTA TGA Strain 3 ACT GCT TTG GCT TTA TGG Strain 4 ACT GCT TTC GCT TTA TGA Strain 5 ACC GCT TTC GCT TTA TGG Strain 6 ACT GCT TTG GCT TTA TGG TCTCGCGCGAGA GC  AT = 1 AT  GC = 1

Test of mutation bias If GC content is Due to mutation bias alone Stationary And the infinite sites assumption holds Then # GC  AT = # AT  GC

Four-fold synonymous sites

Codons ATA CCC CTA CCT Non-synonymous Synonymous 2-fold : TTT TTC 4-fold : CCT CCC CCA CCG

Data Popset Keyword “bacteria” 8 or more sequences from same species 149 bacterial species 8 phyla, 15 classes and 77 genera 1 or more genes 10 or more synonymous polymorphisms 4-fold diversity < 0.1

Overall result No. of SNPs GC  AT11045 AT  GC8309 P<

Bias versus GC4 Z = GC  AT GC  AT No. speciesZ > 0.5P-value GC-rich8269< GC-poor

Phylogenetic distribution PhylumClassNo. of speciesGC4 range Mean Z (GC4<0.34) Mean Z (GC4>0.34) Actinobacteria no species0.64 Bacteroidetes Chlamydiae+Chlamydiae no species CyanobacteriaChroococcales no species0.53 CyanobacteriaNostocales no species CyanobacteriaOscillatoriales 20.41no species0.38 CyanobacteriaStigonemales 10.40no species0.59 FirmicutesBacilli FirmicutesClostridia no species ProteobacteriaAlphaproteobacteria ProteobacteriaBetaproteobacteria no species0.67 Proteobacteriadelta/epsilon Proteobacteria Gammaproteobacte ria Spirochaetes TenericutesMollicutes no species

Potential problems Infinite sites assumption Sequencing error

Infinite sites assumption Each mutation occurs at a site which is not polymorphic

Infinite sites assumption If GC content stationary #GC  AT subs = #AT  GC subs All neutral mutations have same chance of fixation #GC  AT muts = #AT  GC muts

Finite sites assumption If GC content stationary #GC  AT subs = #AT  GC subs All neutral mutations have same chance of fixation #GC  AT muts = #AT  GC muts But some mutations not evident as poly

Finite sites GC rich sequence Implies rate of AT  GC > rate of GC  AT Mutation rate low #AT  GC poly = # GC  AT poly Mutation rate high #AT  GC poly < # GC  AT poly

Finite sites theory GCAT uμ vμ Assume : stationary popn stationary GC

Finite sites theory

Predicting Z Assume finite sites neutrality Use GC4 to get f Use observed diversity to estimate μ Predict Z

Z pred

Z-Z pred No. of speciesZ-Z pred > 0P-value GC-rich8261< GC-poor

Mutation rate variation

Z-Z pred (exponential rates) No. of speciesZ-Z pred > 0P-value GC-rich GC-poor

Sequencing error No. of speciesZ > 0.5P-value GC-rich8260<0.0001

Explanations Non-stationary base composition Selection for translational efficiency Biased gene conversion Selection upon base composition

Explanations Non-stationary base composition Selection for translational efficiency Biased gene conversion Selection upon base composition

Non-stationary GC content

Non-stationary base composition

Explanations Non-stationary base composition Selection for translational efficiency Biased gene conversion Selection upon base composition

Selection on codon usage Amino AcidCodonHigh usageLow usage PhenylalanineUUU UUC ValineGUU GUC GUA GUG

Translational efficiency No. of speciesZ > 0.5P-value GC-rich3129<0.0001

Explanations Non-stationary base composition Selection for translational efficiency Biased gene conversion Selection upon base composition

Biased gene conversion ATAT CGCG AGAG CTCT CGCG CGCG

Four gamete test G A G T C A G A G T C A C T No recombination Recombination

Biased gene conversion No. speciesZ > 0.5P-value GC-rich GC  ATAT  GCP-value No. of SNPs <0.0001

Biased gene conversion GCAT -ww if N e w >> 1 BGC effective if N e w << 1 BGC ineffective

Biased gene conversion r / mp-value GC Z Z-Z pred GC4 pred species with estimate of r / m Vos & Didelot (2009) ISME J.

Biased gene conversion θ r / mp-value GC Z Z-Z pred GC4 pred

Explanations Non-stationary base composition Selection for translational efficiency Biased gene conversion Selection upon base composition

Selection on GC content GCAT uμ vμ +s-s

Selection on GC content

Selection on GC4

f = α + β GC4 f = GC4

Selection on GC4 f = α + β GC4 f = GC4

Summary Large excess of GC  AT mutations at 4-fold sites Particularly in GC-rich species Not due to Infinite sites Sequencing error Translational selection Biased gene conversion Therefore Selection on GC4

Selection on genomic GC Genomic GC GC4

Environmental meta-genomics Foerstner et al. (2005) EMBO Reports

Environmental meta-genomics

Correlates Genome size positive correlation Lifestyle higher GC in free living Aerobiosis higher GC in aerobic Nitrogen utilization higher GC amongst N fixers Temperature higher amongst thermophiles?

Thanks Falk Hildebrand Axel Meyer

Further reading Hildebrand et al. (2010) PLoS Genetics Hershberg and Petrov (2010) PLoS Genetics Rocha and Feil (2010) PLoS Genetics

Protein coding sites