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Comparative genomics and metabolic reconstruction of bacterial genomes

Comparative genomics and metabolic reconstruction of bacterial genomes. Mikhail S. Gelfand Meeting of HHMI International Research Scholars Tallinn, 2004. Metabolic reconstruction. Identification of missing genes in complete genomes Search for candidates

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Comparative genomics and metabolic reconstruction of bacterial genomes

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  1. Comparative genomics and metabolic reconstruction of bacterial genomes Mikhail S. Gelfand Meeting of HHMI International Research Scholars Tallinn, 2004

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

  3. Metabolic reconstruction of the lysine pathway • Predictions: • Genes for the acetylated pathway in Gram-positive bacteria • Positive regulation of the lysine catabolism genes in Thermoanaerobacter and Fusobacterium by LYS-elements: 1st example of activating riboswitches • New transporters

  4. Metabolic reconstruction of the methionine pathway • Predictions: • Genes for theSAM-recycling pathway • Transporters for methionine and methylthiribose • Other enzymes • Transcriptional regulation in Streptococci • Complicated S-box and Cys-T-box regulation of the ubiG-yrhBA operon in C. acetobutylicum: activation via repression of the antisense transcript

  5. Aromatic amino acid regulonsin Gram-positive bacteria

  6. Prediction of transporter specificity via analysis of regulation

  7. Some confirmed predictions

  8. 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

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

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

  11. 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

  12. 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

  13. 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

  14. ZinT: summary • zinT is sometimes fused to the gene of a zinc transporter 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

  15. 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

  16. 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

  17. 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

  18. 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 

  19. 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

  20. 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

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

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

  23. 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

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

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

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

  27. 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.)

  28. Andrei Mironov Anna Gerasimova Olga Kalinina Alexei Kazakov Ekaterina Kotelnikova Galina Kovaleva Pavel Novichkov Olga Laikova Ekaterina Panina(now at UCLA, USA) Elizabeth Permina Dmitry Ravcheev Dmitry Rodionov Alexey Vitreschak(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 Academu of Sciences

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