1. Microbial Genome/Proteome Architectures – Signatures of Environmental Adaptation CHITRA DUTTA Structural Biology & Bioinformatics Division Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road Kolkata 700 032

2. Molecular Evolution – Alternate views Evolution of genes/proteins – Mutation versus Selection

3. Types of Mutations Neutral Mutation : Synonymous base changes Synonymous base changes Base changes in introns, pseudogenes, other non-coding and non-regulatory regions Base changes in introns, pseudogenes, other non-coding and non-regulatory regions Even some non-synonymous base changes can be neutral if they don’t affect protein function Even some non-synonymous base changes can be neutral if they don’t affect protein function Advantageous Mutation – Positive Selection Deleterious Mutation – Purifying Selection

4. Tests for selection on sequences d S = # synonymous substitutions per nucleotide site d S = # synonymous substitutions per nucleotide site in the sequence in the sequence d N = # non-synonymous (replacement) substitutions d N = # non-synonymous (replacement) substitutions per nucleotide site in the sequence per nucleotide site in the sequence

5. Molecular Evolution – Alternate views Evolution of genes/proteins – Mutation versus Selection Current view Mutation and Selection

6. (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 (B) INTRAGENOMIC VARIATIONS : Intergenic variation - Translational Selection & other forces Intergenic variation - Translational Selection & other forces Interstrand variation – (i) Replicational-transcriptional Selection Interstrand variation – (i) Replicational-transcriptional Selection (ii) Thermophilic Adaptation (ii) Thermophilic Adaptation Horizontally Transferred genes Horizontally Transferred genes VARIATIONS IN GENOME COMPOSITION : UNICELLULAR ORGANISMS

7. Codon Bias : Mutation versus Selection 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 gene. 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 gene. The measure ranges from 61 for a gene using all codons with equal frequency to 20 for a gene that is effectively using only one codon to translate its corresponding amino acid. The measure ranges from 61 for a gene using all codons with equal frequency to 20 for a gene that is effectively using only one codon to translate its corresponding amino acid. Higher is selection pressure, higher is codon bias and lower is Nc value. Higher is selection pressure, higher is codon bias and lower is Nc value.

8. Translational Selection in Synonymous Codon Usage

9. Translational Selection In unicellular organisms, a significant correlation exists between the extent of codon bias and expression levels of genes. In unicellular organisms, a significant correlation exists between the extent of codon bias and expression levels of genes. Highly expressed genes, in general, exhibit a strong preference for a subset of synonymous codons recognized by the abundant tRNAs in such species, while the lowly or moderately expressed genes have a more uniform pattern of codon usage. Highly expressed genes, in general, exhibit a strong preference for a subset of synonymous codons recognized by the abundant tRNAs in such species, while the lowly or moderately expressed genes have a more uniform pattern of codon usage. Microbial genes : Microbial genes :

10. 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.

11. 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

12. Amino Acid Selection in Bacterial Proteins Multivariate analyses of various bacterial proteome 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

13. Asymmetric Mutational Bias AND Replicational -Translational Selection

14. 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.

15. Asymmetric Mutational Bias & Replicational - Translational Selection Overall GC-skew = (G-C) / (G+C) (GC) 3 - skew = (G 3 -C 3 ) / (G 3 +C 3 ) Overall AT-skew = (A-T) / (A+T) (AT) 3 - skew = (A 3 -T 3 ) / (A 3 +T 3 )

16. Influence of Replicational –Transcriptional Selection on Codon Usage

17. Influence of Replicational –Transcriptional Selection on Amino Acid Usage

18. 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.

19. Influence of Environment / Life-style (i) Thermophilic Adaptation (ii) Halophilic Adaptation Influence of Environment / Life-style (i) Thermophilic Adaptation (ii) Halophilic Adaptation

20. Thermophilic 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 ?

21. Thermophilic 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

22. Mutivariate Analysis of Amino Acid Usage in N. equitans

23. Thermophilic Adaptation of N. equitans  Comparison of 109 common orthologs between N. equitans, S. tokodaii and M. maripaludis reveals that thermophile proteins are usually characterized by  relatively high aliphatic index,  marked overrepresentation of positively charged residues,  underrepresentation of Ser, Thr and Cys,  fewer sulfur atoms and  higher propensities of alpha-helix formation in secondary structure.  Homology modeling reveals that surface charge distribution is significantly different in the orthologous proteins of N. equitans and M. maripaludis.  Comparison of isoelectric points indicates that hyperthermophiles have relatively more basic proteomes than mesophiles.

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

25. Halophilic Adaptation

26. Halophilic Adaptation - Amino Acid Usage Halophilic Adaptation - Amino Acid Usage

27. Halophilic Adaptation – Codon & Amino Acid Usage

28. Halophilic Adaptation - Amino Acid Usage

29. 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.

30. Microbial 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.