Download Bacterial Genomics

Bioinformatic Insights into the
Evolution of Bacterial Genomes
H o w a rd O c h m a n
Department of Integrative Biology
University of Texas
Bacterial Genomics
(before 1995)
• Bacterial chromosomes are (typically) circular, with a single replication origin
• Bacterial genomes are tightly packed with coding genes & functional elements,
and have little repetitive or non-coding DNA
• Bacterial genomes are typically small, ranging in size from 0.5-10 Mb
• Bacterial coding genes have no introns, are arranged in operons, and are
assorted onto both strands
• Gene order is conserved among closely related bacteria
• Base composition varies among species (25-75% GC) and is similar among
closely related taxa
• Base composition is relatively homogeneous over the entire chromosome
• Rates & patterns of mutations vary with gene location & transcriptional status
The field of Bacterial Genomics is often
considered to have begun in
Haemophilus influenzae
Mycoplasma genitalium
What was learned from the first full genome sequences?
1. Possible to assemble full genomes by WGS with paired-end reads
2. Enabled resolution complete gene inventories (& encoded functions)
Mycoplasma pneumoniae
(816 kb; 736 ORFs)
Mycoplasma genitalium (580 kb; 564 ORFs)
1. Start to observe a consistent trend in genome size & gene number
2. Members of the same genus can differ in genome size & contents
What is the source of such differences in genome contents?
Loss of ancestral genes? Generation of new genes ?
Difficult to resolve without knowledge of ancestral (or outgroup) genome
genes acquired by lateral gene transfer (LGT)
--Geneswithatypicalfeatures(e.g.,G+Ccontent)areconsideredtoarisebyLGT-Strain- & species-specific genes often have
atypical base compositions
Sequenced genes present in Salmonell a , but not in E. coli (circa 1990)
Differences in base composition among species
are caused by mutational biases
Gene
Potential Function
Ma p
%G+C
cbi
Cobinamide synthesis
41
59.3
.233
fljA
Flagell ar synthesis
56
40.9
.210
fljB
Flagell ar synthesis
56
52.3
.216
inv/spa
Host recognition/invasion
59
45.5
.261
nanH
Sialidase
20.5
40.9
.263
ORF
Unknown
98
38.2
.296
pagC
Envelope protein
25
43.4
.274
phoN
Phosphatase
96
46.5
.248
rfc
LPS Synthesis
31
33.5
.175
sinR
Tra nscriptional control
7
39.8
.218
tctABCD
Tricarboxylate transport
57
55.0
.278
pgtE
Phosphoglycerate transport
–
45.3
.277
Salmonell a genome is 52% G+C
CAI
Inferring the Incidence of Lateral Gene Transfer (LGT) from
Sequence Heterogeneity along Bacterial Chromosomes
Neisseria meningitidis, 52% G+C
% G+C
(from Tettelin et al. 2000. Science)
⌇
(Mostly) Free-living bacteria
⌇
(Mostly) Pathogens
A relationship between between bacterial lifestyle and genome size
but with some exceptions (M. tuberculosis, 4.4 mb ; Aquifex, 1.6 Mb)
⌇ Intracellular pathogens & obligate endosymbionts
Remarkable relationship between genome size & gene number
across >10-fold range in genome size in organisms representing eight phyla
Mycobacteriumleprae
Then why haven’t pseudogenes accumulated in all
of the other sequenced bacterial genomes?
Since mutations occur as an on-going process & pseudogenes are
continually being generated, what about all those other
(big free-living & small symbiont) genomes that fall right on the diagonal?
Yersinia pestis
Shigella flexneri
Mycobacterium leprae
The genomes of other recent pathogens of humans possess high numbers
of inactivated genes (e.g., Shigella has 400; Yersinia pestis has 150)
In bacteria, non-functional regions are removed by a
pervasive mutational bias towards deletions
This deletional bias was detected computationally
and confirmed experimentally
Unlike most eukaryotes (in which
noncoding DNA accumulates),
bacteria show a strong mutational bias
towards deletions of all sizes
When comparing pseudogenes to
functional their counterparts,
deletions outnumber insertions
Lineages of bacteria with small genomes
derive from ancestors with larger genomes
< 2 Mb
2–4 Mb
> 4 Mb
The progression towards a compact genome
1.(Ancestral)free-living(large-genomed)bacteriamovesintoa(nutrient-rich)host
2.Hostassociationrendersmanygenessuperfluous/useless&deadgenesaccumulate
3.Deletionalbiasreducesgenomesizeandremovesinactivatedgenes
Sampling multiple strains within species
reveals wide variation in genome size
(concept of a “core” vs. “pan” genome)
1. Very large sampling of several phyla at various taxonomic levels
2. Minimal size of bacterial genome is ~500 kb (pathogens & symbionts)
A new lower limit to the size of a cellular genome
The symbiont of a psyllid collected on a hackberry tree in Tucson, Arizona
Carsonella ruddii: the smallest bacterial genome
(Nakabachi et al., 2005)
159,662 bp 182 ORFs
16.5% GC 98% coding
Other smallest cellular genome is 420 kb,
in the aphid symbiont Buchnera
…with the current record of 112 kb held by Nasuia deltocephalinicola
A thousand bacterial genomes sequenced
What has the sequencing of 1000s of genomes told us about factors
controlling the size & composition of bacterial genomes?
The (New) Observations:
• Bacterial genomes are typically small, ranging in size from 0.1–13 Mb
• Bacterial genomes are USUALLY tightly packed with coding genes, and USUALLY
have little repetitive & non-coding sequences (e.g., pseudogenes).
Therefore, in bacteria, genome size is tightly linked to gene number
• There is a pervasive mutational bias that removes non-functional regions
• Small bacterial genomes derive from lineages with large genomes
• Base composition varies among species (13-80% GC) and is relatively
homogeneous over the entire chromosome
• Many bacterial genomes harbor substantial amounts of laterally acquired DNA
• Strains within a named bacterial species can vary greatly in genome size
But what are the major determinants of
bacterial genome size & complexity?
First test if Bacterial Genome Size is
Adaptive (i.e., shaped by natural selection)
or Non-Adaptive (i.e., shaped by genetic drift)
The effects of genetic drift (changes in gene frequencies that take place strictly by chance)
are more dramatic in species with small effective population sizes (Ne) & will shape
the contents of genomes by affecting the fixation of deleterious mutations
Due to the link between genome size and gene density in bacteria,
evolutionary forces that act on individual genes will affect genome size
-- Fate of Bacterial Genes -No Benefit
genes whose presence is affected by drift
(decay)
Essential
(preserved)
Butassessingthelevelofdriftaffectingbacteriaisproblematic:
1.InBacteria,itisvery(very)difficulttoestimateNe
2.Methodsbasedonlevelsofintraspecificpolymorphismsare
confoundedbyproblemsindefiningbacterialspecies
Using Ka/Ks values as a proxy for level of drift
1. Because point mutations that cause amino acid replacements are often
deleterious, the rate of nonsynonymous changes (Ka) is expected to be less
than the rate of synonymous substitutions (Ks) in functional genes.
2. An increased level of drift, produced from reduced Ne (+ genome-wide
relaxation of selection) will result in an increased incidence in slightly
deleterious mutations and increase Ka/Ks genome-wide.
Examined genome-wide Ka/Ks in species pairs from eight phyla
Genome size exhibits a strong negative correlation
with the level of genetic drift
more efficient selection
less efficient selection
1. Same significant relationship is observed when considering all genes shared
by a genome-pair or only those genes common to the majority of genomes
2. Similarly significant relationship is observed when considering only pairs with
low, or only those with intermediate, levels of sequence divergence
3. Those with high genome-wide Ka/Ks have a lifestyle that reduces Ne
(obligate endosymbionts, vector-borne pathogens, extremophiles)
Gene density is related to the level of genetic drift
1. Genome-pairs with low levels of drift (i.e., more efficient selection)
display a relatively narrow range of coding densities (usually 85-90%)
2. Most genome pairs displaying high levels of drift have coding densities
that lie outside of the 85-90% range
This occurs due to pseudogene formation in recent pathogens (lowering coding density)
and to tight gene-packing in the highly reduced genomes (increasing coding density)
Genetic Drift as the Major Determinant
of Bacterial Genome Size & Complexity
But don’t bacteria have small genomes so that they can replicate quickly?
1a. Bacterial genome size is not caused by selection for replication efficiency
Comparisons across species
Genetic Drift as the Major Determinant
of Bacterial Genome Size & Complexity
2. The effect of drift on bacterial genomes is opposite to the pattern
proposed by Lynch
(Lynch & Conery 2003)
Eukaryotes
Bacteria
Smaller Ne –>
More drift
More drift
The difference arises from the fact that bacterial genomes comprise
sequences that are maintained by selection, or otherwise deleted
1. What is the basis of the variation in bacterial genome size?
✓
2. What is the basis of the variation in genomic base composition?
Proposed that differences in base composition among species
are caused by mutational biases (again, a non-adaptive process)
In bacteria, variation in genomic base composition has long been
thought to be due to mutational biases
but this has not slowed the search for an adaptive basis
for the observed differences
What might be a reason why some bacteria have G+C-rich
genomes whereas others have A+T-rich genomes?
PE!
Thermal tolerance?? GC basepairs (with 3 H-bonds) are stronger than are
AT basepairs, so high GC genomes would seem to be less prone to
denaturing at higher temperatures
O
N
--
What might be a reason why some bacteria have G+C-rich
genomes whereas others have A+T-rich genomes?
Wait, there are more.....
(Feil&Rocha.2010.PLoSGenetics)
Why are there GC-rich bacterial genomes?
Experimental analysis of mutations in E. coli and Salmonella
(both of which are >50% G+C)
Given these mutational patterns,
why are E. coli & Salmonella GC-rich?
of its mRNA. The folding energy of the entire between folding energy and expression did not
mRNA was not significantly correlated with flu- overlap with the Shine-Dalgarno (SD) sequence,
Due to the link between genome size and gene density in bacteria,
orescence (r = 0.16, P = 0.051), but the folding which suggested that SD occlusion by secondary
forces
that
act on
basestructure
composition
of individual
genes
energy of the first
third of
the mRNA
wasthe
strongly
(22, 23) did
not play a major
role will
in
affect
overall
composition
correlated: mRNAs with stronger
structure
pro-genomic
inhibiting base
expression,
probably because our conduced lower fluorescence (r = 0.60, P < 10–15). structs contained no noncoding mutations. By
A moving window analysis identified a region, contrast, the region of strongest effect overlapped
Gene-level selection on base composition: A simple experiment
GFP constructs with synonymous mutations in 3rd positions
(Kudla G, et al. 2009. Science. 324: 255)
Selected clones of low (40-42%), medium (46-48%) and high (51-54%) G+C content
All selected genes had similar codon usage biases: CAIE.coli (0.58-0.68)
Strains expressing GC-rich genes have higher growth rates!?!
(timepoints are hours after induction with IPTG)
The effect is observed with either of two anonymous genes
Conclusions: Observations
•
Bacterial genomes (and those of archaea & probably many eukarya)
display a mutational bias towards deletions among small indels
•
There is a strong negative association between bacterial genome
size and the level of drift, such that species with small Ne have
small genomes
•
Bacterial genomes are usually packed with functional genes,
but almost every genome contains some pseudogenes
•
Bacteria with small genomes (pathogens, symbionts) derive
from large-genomed ancestors; and during the transition to a
host-associated lifestyle, functional redundancy and lower
efficacy of selection causes accumulation of pseudogenes
Conclusions: Findings
• Variation in bacterial genome size, which has usually
been attributed to selection for replication efficiency,
is actually caused by non-adaptive processes
• Variation in the base composition of bacterial genomes,
long thought be determined by a strictly neutral mutational process,
is now known to be caused by selection.