Comparative genomics and metabolic reconstruction of bacterial pathogens

1. Comparative genomics and metabolic reconstruction of bacterial pathogens Mikhail Gelfand Institute for Information Transmission Problems, RAS GPBM-2004

2. Metabolic reconstruction • Identification of missing genes in complete genomes • Search for candidates • Analysis of individual genes to assign general function: • homology • functional patterns • structural features • Comparative genomics to predict specificity: • analysis of regulation • positional clustering • gene fusions • phylogenetic patterns

3. Enzymes • Identification of a gap in a pathway (universal, taxon-specific, or in individual genomes) • Search for candidates assigned to the pathway by co-localization and co-regulation (in many genomes) • Prediction of generalbiochemical function from (distant) similarty and functional patterns • Tentative filling of the gap • Verification by analysis of phylogenetic patterns: • Absence in genomes without this pathway • Complementary distribution with known enzymes for the same function

4. Transporters • Identification of candidates assigned to the pathway by co-localization and co-regulation (in many genomes) • Prediction of generalfunction by analysis of transmembrane segments and similarty • Prediction of specificity by analysis of phylogenetic patterns: • End product if present in genomes lacking this pathway (substituting the biosynthetic pathway for an essential compound) • Input metabolite if absent in genomes without the pathway (catabolic, also precursors in biosynthetic pathways) • Entry point in the middle if substituting an upper or side part of the pathway in some genomes

5. accA accD accB Missing link in fatty acid biosynthesis in Streptococci fabI(Enoyl-ACP reductase, EC 1.3.1.9) target of triclosan. Enzymatic activity, but no gene in Streptococci GenefabI of Enoyl-ACP reductase (EC 1.3.1.9) is missing in the genome 12B, and a number of Streptococci accC fabD fabF fabH fabG fabZ fabI acpP

6. fabG fabF fabZ accC accD accA fabI 6.3.4.15 hyp acpP fabD accB hyp TR? 3.5.1.? fabH Genome X acpP fabH ? fabG fabF fabZ accC accD accA fabD accB TR? 2.1.1.79 FRNS Genome Y acpP fabG fabF fabZ accC accD accA fabH fabD accB ? TR? 5.99.1.2 Clostridium acetobutylicum acpP fabH ? TR? fabG fabF fabZ accC accD accA fabD accB hyp Streptococcus pyogenes Identification of a candidate by positional clustering

7. Binding sites of FabR (“Tr?”, HTH) Fad (42.1.17) HTH acpP fabH fabK fabG fabF fabZ accC accD accA fabD accB 1 2 3 4

8. Metabolic reconstruction of the thiamin biosynthesis(new genes/functions shown in red) Purine pathway thiN(confirmed) Transport of HET Transport of HMP (Gram-positive bacteria) (Gram-negative bacteria)

9. Carbohydrate metabolism in Streptococcus and Lactococcus spp. Only biochemical data, genes unknown Experimentally verified genes Biochemical data and genomic predictions Only genomic predictions

10. An uncharacterized locus in invasive species S. pneumoniae S. pyogenes S. equi S. agalactiae S. suis

11. Structure of the genome loci S. pyogenes, S. agalactiae S. equi S. pneumoniae TIGR4 S. pneumoniae R6 S. suis

12. hyaluronidase(hyaluronate lyase) RegR Gene functions 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine PTS transporter hydrolase isomerase oxidoreductase dehydrogenase kinase aldolase pyruvate + D-glyceraldehyde 3-phosphate

13. Candidate regulatory signal

14. Structure of the genome loci - 2 S. pyogenes, S. agalactiae S. equi S. pneumoniae TIGR4 S. pneumoniae R6 S. suis

15. Possible function • Pathway exists in invasive species • Sometimes co-localized with hyaluronidase • Always co-regulated with hyaluronidase Thus: • Utilization of hyaluronate • May be involved in pathogenesis

16. Comparative genomics of zinc regulons Two major roles of zinc in bacteria: • Structural role in DNA polymerases, primases, ribosomal proteins, etc. • Catalytic role in metal proteases and other enzymes

17. Genomes and regulators nZURFUR family ??? pZURFUR family AdcR ?MarR family

18. nZUR- nZUR- Regulators and signals GAAATGTTATANTATAACATTTC GATATGTTATAACATATC GTAATGTAATAACATTAC TTAACYRGTTAA pZUR AdcR TAAATCGTAATNATTACGATTTA

19. Transporters • Orthologs of the AdcABC and YciC transport systems • Paralogs of the components of the AdcABC and YciC transport systems • Candidate transporters with previously unknown specificity

20. zinT: regulation zinT is regulated by zinc repressors (nZUR-, nZUR-, pZUR) zinTis isolated E. coli, S. typhi, K. pneumoniae Gamma-proteobacteria A. tumefaciens, R. sphaeroides Alpha-proteobacteria B. subtilis, S. aureus S. pneumoniae, S. mutans, S. pyogenes, L. lactis, E. faecalis Bacillus group Streptococcus group adcA-zinT is regulated by zinc repressors (pZUR, AdcR) (ex. L.l.) fusion:adcA-zinT

21. ZinT: protein sequence analysis TM Zn AdcA Y. pestis, V. cholerae, B. halodurans ZinT S. aureus, E. faecalis, S. pneumoniae, S. mutans, S. pyogenes E. coli, S. typhi, K. pneumoniae, A. tumefaciens, R. sphaeroides, B. subtilis L. lactis

22. ZinT: summary • zinT is sometimes fused to the gene of a zinc transporter component adcA • zinT is expressed only in zinc-deplete conditions • ZinT is attached to cell surface (has a TM-segment) • ZinT has a zinc-binding domain ZinT: conclusions: • ZinT is a new type of zinc-binding component of zinc ABC transporter

23. lmb phtD zinc regulation shown in experiment Zinc regulation of PHT (pneumococcal histidine triad) proteins of Streptococci S. pneumoniae S. pyogenes S. equi S. agalactiae phtE lmb phtD lmb phtD phtA phtB phtY

24. Structural features of PHP proteins • PHT proteins contain multiple HxxHxH motifs • PHT proteins of S. pneumoniae are paralogs (65-95% id) • Sec-dependent hydrophobic leader sequences are present at the N-termini of PHT proteins • Localization of PHT proteins from S. pneumoniaeon bacterial cell surface has been confirmed by flow cytometry

25. PHH proteins: summary • PHT proteins are induced in zinc-deplete conditions • PHT proteins are localized at the cell surface • PHT proteins have zinc-binding motifs A hypothesis: • PHT proteins represent a new family of zinc transporters

26. Zinc-binding domains in zinc transporters: EEEHEEHDHGEHEHSH HSHEEHGHEEDDHDHSH EEHGHEEDDHHHHHDED DEHGEGHEEEHGHEH (histidine-aspartate-glutamate-rich) Histidine triads in streptococci: HGDHYHY 7 out of 21 HGDHYHF 2 out of 21 HGNHYHF 2 out of 21 HYDHYHN 2 out of 21 HMTHSHW 2 out of 21 (specific pattern of histidines and aromatic amino acids) … incorrect 

27. HDYNHNHTYEDEEGH AHEHRDKDDHDHEHED LRR IR PHT internalin H-rich Analyis of PHP proteins (cont’d) • The phtD gene forms a candidate operon with the lmb gene in all Streptococcus species • Lmb: an adhesin involved in laminin binding, adherence and internalization of streptococci into epithelial cells • PhtY of S. pyogenes: • phtY regulated by AdcR • PhtY consists of 3 domains: 4 HIS TRIADS

28. PHH proteins: summary-2 • PHT proteins are induced in zinc-deplete conditions • PHT proteins are localized at the cell surface • PHT proteins have structural zinc-binding motifs • phtD forms a candidate operon with an adhesin gene • PhtY contains an internalindomain responsible for the streptococcal invasion Hypothesis PHT proteins are adhesins involved in the attachment of streptococci to epithelium cells, leading to invasion

29. Zinc and (paralogs of) ribosomal proteins nZUR pZUR AdcR

30. Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001) nZUR pZUR AdcR

31. Summary of observations: • Makarova-Ponomarev-Koonin, 2001: • L36, L33, L31, S14 are the only ribosomal proteins duplicated in more than one species • L36, L33, L31, S14 are four out of seven ribosomal proteins that contain the zinc-ribbon motif (four cysteines) • Out of two (or more) copies of the L36, L33, L31, S14 proteins, one usually contains zinc-ribbon, while the other has eliminated it • Among genes encoding paralogs of ribosomal proteins, there is (almost) always one gene regulated by a zinc repressor, and the corresponding protein never has a zinc ribbon motif

32. Zn-deplete conditions: all Zn utilized by the ribosomes, no Zn for Zn-dependent enzymes Bad scenario Zn-rich conditions

33. Regulatory mechanism Sufficient Zn ribosomes R repressor Zn-dependentenzymes Zn starvation R

34. Zn-deplete conditions: some ribosomes without Zn, some Zn left for the enzymes Good scenario Zn-rich conditions

35. Prediction …(Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9912-7.) …and confirmation (Mol Microbiol. 2004 Apr;52(1):273-83.)

36. Andrei A. Mironov Anna Gerasimova Olga Kalinina Alexei Kazakov (hyaluronate) Ekaterina Kotelnikova Galina Kovaleva Pavel Novichkov Olga Laikova (hyaluronate) Ekaterina Panina(zinc)(now at UCLA, USA) Elizabeth Permina Dmitry Ravcheev Alexandra B. Rakhmaninova Dmitry Rodionov (thiamin) Alexey Vitreschak(thiamin)(on leave at LORIA, France) Howard Hughes Medical Institute Ludwig Institute of Cancer Research Russian Fund of Basic Research Programs “Origin and Evolution of the Biosphere” and “Molecular and Cellular Biology”, Russian Academy of Sciences • Andrei Osterman (Burnham Institute, San-Diego, USA)(fatty acids)