Microbial Genome/Proteome Architectures – Microbial Genome/Proteome Architectures –

Signatures of Environmental AdaptationSignatures of Environmental Adaptation Microbial Genome/Proteome Architectures – Microbial Genome/Proteome Architectures –

Signatures of Environmental AdaptationSignatures of Environmental Adaptation

CHITRA DUTTACHITRA DUTTA

Structural Biology & Bioinformatics DivisionStructural Biology & Bioinformatics Division

Indian Institute of Chemical BiologyIndian Institute of Chemical Biology

4, Raja S. C. Mullick Road4, Raja S. C. Mullick Road

Kolkata 700 032Kolkata 700 032

Molecular Evolution – Molecular Evolution – Alternate viewsAlternate views

Evolution of genes/proteins – Evolution of genes/proteins –

Mutation versus SelectionMutation versus Selection

Types of MutationsTypes of Mutations

Neutral Mutation :Neutral Mutation :

Synonymous base changesSynonymous base changes Base changes in introns, pseudogenes, other Base changes in introns, pseudogenes, other

non-coding and non-regulatory regionsnon-coding and non-regulatory regions Even some non-synonymous base changes can Even some non-synonymous base changes can

be neutral if they don’t affect protein functionbe neutral if they don’t affect protein function

Advantageous Mutation –Advantageous Mutation – Positive SelectionPositive Selection

Deleterious Mutation –Deleterious Mutation – Purifying SelectionPurifying Selection

Tests for selection on sequencesTests for selection on sequences

ddSS = # synonymous substitutions per nucleotide site = # synonymous substitutions per nucleotide site

in the sequencein the sequence ddNN = # non-synonymous (replacement) substitutions = # non-synonymous (replacement) substitutions

per nucleotide site in the sequenceper nucleotide site in the sequence

Molecular Evolution – Molecular Evolution – Alternate viewsAlternate views

Evolution of genes/proteins – Evolution of genes/proteins –

Mutation versus SelectionMutation versus Selection

Current view

Mutation and Selection

(A) INTERGENOMIC VARIATIONS - (A) INTERGENOMIC VARIATIONS -

Mutational Bias – Mutational Bias – (i) Base-equifrequent genomes(i) Base-equifrequent genomes (ii) G+C-rich genomes(ii) G+C-rich genomes(iii) A+T-rich genomes(iii) A+T-rich genomes

(B) INTRAGENOMIC VARIATIONS :(B) INTRAGENOMIC VARIATIONS :

• Intergenic variationIntergenic variation - - Translational Selection & other forcesTranslational Selection & other forces

• Interstrand variationInterstrand variation – – (i)(i) Replicational-transcriptional Replicational-transcriptional Selection Selection

(ii) Thermophilic Adaptation(ii) Thermophilic Adaptation

• Horizontally Transferred genes Horizontally Transferred genes

VARIATIONS IN GENOME COMPOSITION : UNICELLULAR ORGANISMS

Codon Bias : Mutation versus SelectionCodon Bias : Mutation versus Selection

Nc (Effective Number of Codons used by a gene) – It is a Nc (Effective Number of Codons used by a gene) – It is a measure of how small a subset of codons are being used by a measure of how small a subset of codons are being used by a gene. gene.

The measure ranges from 61 for a gene using all codons with The measure ranges from 61 for a gene using all codons with equal frequency to 20 for a gene that is effectively using only equal frequency to 20 for a gene that is effectively using only one codon to translate its corresponding amino acid. one codon to translate its corresponding amino acid.

Higher is selection pressure, higher is codon bias and lower Higher is selection pressure, higher is codon bias and lower is Nc value. is Nc value.

Translational Selection Translational Selection

inin

Synonymous Codon UsageSynonymous Codon Usage

Translational SelectionTranslational Selection

In unicellular organisms, a significant correlation exists between In unicellular organisms, a significant correlation exists between the extent of codon bias and expression levels of genes.the extent of codon bias and expression levels of genes.

Highly expressed genes, in general, exhibit a strong preference Highly expressed genes, in general, exhibit a strong preference for a subset of synonymous codons recognized by the abundant for a subset of synonymous codons recognized by the abundant tRNAs in such species, while the lowly or moderately expressed tRNAs in such species, while the lowly or moderately expressed genes have a more uniform pattern of codon usagegenes have a more uniform pattern of codon usage..

Microbial genes :Microbial genes :

Relative Synonymous Codon Usage (RSCU) of different codons in a set of highly expressed genes of any organism :

where, Xij = No. of the jth codon for the ith amino acid, ni = Total no. of synonymous codons for the ith amino acid.

The Normalized RSCU or Relative Adaptiveness (W) for a set of genes:

The Codon Adaptation Index (CAI) of a particular gene ( 0 ≤ CAI ≤ 1) :

where L is the number of codons in the gene.

Greater is the value of CAI, higher is the potential of expression.

ij

ij

1

XRSCU = (1)

1 i

ij

i

n

j

Xn

max max

(2)ij ij

ij

i i

RSCU XW

RSCU X

18

1 1

1exp ln (3)

i

ij ij

n

i j

CAI X WL

Translational selection usually operates in accordance with mutational bias of the genome

Most of the unicellular organisms exhibit Translational Selection.

Exceptions include

• Some genomes with extremely high mutational bias

• Genomes of some obligatory intracellular organisms with strand-specific mutational bias

• Species adapted to extreme environments

Translational Selection – Some Observations

Amino Acid Selection in Bacterial Proteins

Multivariate analyses of various bacterialproteome reveals the following as primary sources of intra-proteome variations :

(i) Hydrophobicity

(ii) Aromaticity

(iii) Mean molecular Weight

(iv) Biosynthetic cost of Production

(v) Gene Expression Level

In most of the free-living bacteria, the

Principle of Cost minimization holds good

Asymmetric Mutational Bias

AND

Replicational -Translational Selection 

Asymmetric Mutational Bias & Replicational -Translational Selection

 Strong compositional asymmetries between the genes lying on the leading versus lagging strands were observed in many other prokaryotic organisms at the level of nucleotides, codons and even in amino acids.

Bacterial chromosome replication usually starts at a single origin, and two replication forks propagate in opposite directions up to termination signals. As the replication mechanism differs for the two strands of the duplex DNA, this process often gives rise to compositional asymmetries between the leading strand and the lagging strand.

During transcription, the non-template strand is in an open single stranded configuration that is more prone to specific mutations like C T (U) deamination. The template strand is less susceptive to this process and is protected by transcription dependent DNA repair. As a result, the leading strand contains an excess of G/T over C/A.

Replicational selection is responsible for the higher number of genes on the leading strands, and transcriptional selection appears to be responsible for the enrichment of highly expressed genes on these strands.

Example: the spirochaetes Borrelia burgdorferi and Treponema pallidum, the endosymbiotic bacteria Blochmannia floridanus, human pathogens Bertonella henselae and Bertonella quintana etc.

Asymmetric Mutational Bias & Replicational - Translational Selection

Overall GC-skew = (G-C) / (G+C)

(GC)3 - skew = (G3-C3) / (G3+C3)

Overall AT-skew = (A-T) / (A+T)

(AT)3 - skew = (A3-T3) / (A3+T3)

Influence of Replicational –Transcriptional Selection on Codon Usage

Influence of Replicational –Transcriptional Selection on Amino Acid Usage

Asymmetric Mutational Bias & Replicational -Translational Selection

 

Striking features :

Strong strand-specific skews in nucleotide composition - Leading strand in replication is richer in G and T than lagging strand.    Higher number of genes on the leading strands - Replicational selection

Enrichment of highly expressed genes on leading strands – Transcriptional selection

Distinct codon as well as amino acid usage patterns depending on whether the gene is transcribed on the leading or lagging strand of replication.

Replicational-transcriptional selection is very common in obligatory intra-cellular bacteria which are not much exposed to recombinational processes.

Influence of Environment / Life-style Influence of Environment / Life-style

(i) Thermophilic Adaptation (i) Thermophilic Adaptation

(ii) Halophilic Adaptation (ii) Halophilic Adaptation

Thermophilic Adaptation – A case studyThermophilic Adaptation – A case study

Nanoarchaeum equitans –

• Only known obligatory symbiotic archaeon. It must be in contact with the crenarchaeon host Ignicoccus for survival and growth

• Genome size is only 490 kb - the smallest microbial genome known to date

• Yet it has the highest coding capacity, with little non-coding regions • Genes for several vital metabolic pathways appear to be missing . It cannot synthesize most nucleotides, amino acids, lipids, and cofactors

• Possesses most of the DNA repair enzymes and the complete genetic mechinary necessary for transcription, translation and DNA replication

• Apparent lack of translational selection, like other strictly symbiotic /parasitic microorganisms

Ancient species, or Reduced Genome ?

Thermophilic AdaptationThermophilic Adaptation

• Coding regions are significantly overrepresented by purine bases

• A significant positive correlation exists (r=0.89,p<0.0001) between overall purine-pyrimidine ratio (R:Y) and the optimal growth temperature (OGT)

• Higher is the OGT, higher is the selection for purine nucleotides in coding sequences

• Prevalence of purine bases in mRNAs might prevent distracting RNA-RNA interactions and formation of local double-strands within the molecule

Mutivariate Analysis of Amino Acid Usage in N. equitans

Thermophilic Adaptation of Thermophilic Adaptation of N. equitansN. equitans

Comparison of 109 common orthologs Comparison of 109 common orthologs between between N. equitans,N. equitans, S. tokodaiiS. tokodaii and and M. M. maripaludismaripaludis reveals that thermophile reveals that thermophile proteins are usually characterized by proteins are usually characterized by

relatively high aliphatic index, relatively high aliphatic index,

marked overrepresentation of positively marked overrepresentation of positively charged residues, charged residues,

underrepresentation of Ser, Thr and Cys, underrepresentation of Ser, Thr and Cys,

fewer sulfur atoms and fewer sulfur atoms and

higher propensities of alpha-helix higher propensities of alpha-helix formation in secondary structure.formation in secondary structure.

Homology modeling reveals that Homology modeling reveals that surface surface

charge distribution is significantly charge distribution is significantly different in the orthologous proteins of different in the orthologous proteins of N. N. equitansequitans and and M. maripaludisM. maripaludis. .

Comparison of isoelectric points indicates Comparison of isoelectric points indicates that that hyperthermophiles have relatively hyperthermophiles have relatively more basic proteomes than mesophilesmore basic proteomes than mesophiles..

Halophilic AdaptationHalophilic Adaptation Halophilic organisms require Halophilic organisms require very high concentrations of salt (at very high concentrations of salt (at

least 2 M, approximately ten times the salt level of ocean water) least 2 M, approximately ten times the salt level of ocean water)

for optimal growth and can be found in environments such as for optimal growth and can be found in environments such as

Dead Sea, the Great Salt Lake, or man made salterns. Dead Sea, the Great Salt Lake, or man made salterns.

Salient features of extreme halophiles:Salient features of extreme halophiles:

cytoplasm is nearly saturated with KCL (Lanyi 1974). cytoplasm is nearly saturated with KCL (Lanyi 1974).

proteins of these organisms require high salt for activity and proteins of these organisms require high salt for activity and stability and at less than 1–2 M NaCl or KCl most haloarchaeal stability and at less than 1–2 M NaCl or KCl most haloarchaeal proteins unfold and lose their activity (Madern et al. 2000).proteins unfold and lose their activity (Madern et al. 2000).

Organism Abb Group GC (%)

Ch I HMAR1 62 Haloarcula marismortui Ch II HMAR2

Euryarchaeota 57

Halobacterium salinarum HSAL Euryarchaeota 68

Halobacterium sp. NRC-1 HALO Euryarchaeota 67

Haloquadratum walsbyi HWAL Euryarchaeota 48

Natronomonas pharaonis NPHA Euryarchaeota 63

Hal

ophi

lic

Salinibacter ruber SRUB Bacteroidetes/Chlorobi 66

Azoarcus sp. AZOA Betaproteobacteria 65

Bifidobacterium longum BLON Actinobacteria 60

Caulobacter crescentus CCRE Alphaproteobacteria 67

Escherichia coli ECOL Gammaproteobacteria 50

Pelobacter propionicus PPRO Deltaproteobacteria 58

Pelodictyon luteolum PLUT Bacteroidetes/Chlorobi 57

Polaromonas sp. POLA Betaproteobacteria 62

Pseudomonas putida PPUT Gammaproteobacteria 61

Shigella boydii SBOY Gammaproteobacteria 47

Non

hal

ophi

lic

Yersinia pestis YPES Gammaproteobacteria 48

Halophilic Adaptation

Halophilic Adaptation - Amino Acid UsageHalophilic Adaptation - Amino Acid Usage

Halophilic Adaptation – Halophilic Adaptation – Codon & Amino Acid UsageCodon & Amino Acid Usage

-0.18 0.18

Axis 1

0.15

-0.15

2 4 6 8 10 12 14

1816141210 8 6 4 2 0

Isoelectric Point

% o

f gen

es

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

16

12

8

4

0

Hydrophobicity

% o

f g

en

es

Halophilic Adaptation - Amino Acid Halophilic Adaptation - Amino Acid UsageUsage

Halophilic Adaptation - Summary

Extreme Halophilic organisms are clustered according to their unique amino acid composition and synonymous codon usage irrespective of their taxonomic position and GC content.

Higher ratio of negative to positive charged amino acid residues and lower hydrophobicity are the major factors contributing for halophilic adaptation of proteins.

Negatively charged amino acid residues increase at the cost of increase in positively charged and non-polar residues in Halophilic orthologs.

Large hydrophobic residues are replaced by small and borderline hydrophobic residues.

There is a lack of regular secondary structure i,e, decrease in alpha helical content and increase in coil region for halophilic proteins. These features may be important to prevent aggregation and, at the same time, retain structural flexibility and activity of proteins at high salt concentrations.

Microbial Genome/Proteome ArchitecturesMicrobial Genome/Proteome Architectures

Factors influencing amino acid usages :

Directional mutational pressure

Functional/structural constraints

Gene expressivity

Bioenergetic Requirements (Cost minimization)

Environmental Adaptation

Origins of codon/nucleotide biases :

Directional mutation Pressure

Translational Selection – Gene Expressivity

Coupled Replicational –Transcriptional Selection

Environmental Adaptation

Other minor factors like context-dependence etc.