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Evolution of bacterial regulatory sytsems

Evolution of bacterial regulatory sytsems. Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia. Institute of Protein Research 40 th Anniversary Conference June 2007. Comparative genomics of zinc regulons.

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Evolution of bacterial regulatory sytsems

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  1. Evolution of bacterial regulatory sytsems Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia Institute of Protein Research 40th Anniversary Conference June 2007

  2. 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 Poisonous in large concentrations => the concentration of zinc is tightly controlled

  3. nZUR- nZUR- Regulators (zinc uptake) and motives GAAATGTTATANTATAACATTTC GATATGTTATAACATATC GTAATGTAATAACATTAC TTAACYRGTTAA pZUR AdcR TAAATCGTAATNATTACGATTTA

  4. adcA TM Zn lmb phtD zinT zinc regulation shown in experiment Predictions • Known 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 • ZinT is a new type of zinc-binding component of zinc ABC transporter • PHT (pneumococcal histidine triad) proteins of Streptococcus spp. • PHT proteins are adhesins involved in the attachment of streptococci to epithelium cells, leading to invasion. This process is regulated by zinc concentration. S. pneumoniae S. pyogenes S. equi S. agalactiae phtE lmb phtD lmb phtD phtA phtB phtY

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

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

  7. 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

  8. Zinc starvation and its consequences Bad scenario:all Zn utilized by the ribosomes, no Zn for Zn-dependent enzymes Good scenario:some ribosomes without Zn, some Zn left for the enzymes Zn-rich conditions: sufficient Zn for the ribosomes and the enzymes

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

  10. 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.)

  11. T-boxes: the mechanism (Grundy & Henkin; Putzer & Grunberg-Manago)

  12. Partial alignment of predicted T-boxes TGG: T-box Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

  13. … continued (in the 5’ direction) anti-anti (specifier) codon AT - GTA G CTTT GGA - > PN THRS THR ---- AG AG A CA AGT C 18 AGAGA GTGCGT GGTT --- G C TG G A A ACGCAT - 14 GA T -- AC T A CTCT TGA - > MN ileS Ile ---- CA AAA AC ACAA 17 AGCGA ATAGGT GAT ---- G G TG T A A G ACCTAT T 18 ----- ATC A TTTTG TT - > DF leuS Leu ---- CT AG A GC AGTA 19 AGAG G A AGT GGAA ----- G G TG A G A ACTAAT ATT 10 GA A -- CTT A CTAG ATT - > HD ARGS ARG ----- T GGG AG AGTA 20 AGCGA GTCGG GAT ---- - G G TG G G A G CCGAT - 14 GA AA - CGC A CCCA TGA - > DF proS Pro --- AAA GA A AT AGTA 18 AGAGA GAAAAC GGT ---- G G TG A G A GTTTTC -- 14 GA A -- CCT G TCTTT TA - > ZC lysS Lys --- AAG AGA AG AGTA 19 AGAGA GCTCT GGTA ---- G C TG A G A A AGAGC -- 15 GA AAA AAG A CTT GGAG - > BQ metS Met --- AAA GG A AA AGTA 19 AGAGA GCTTC GGTA ---- G C TG A G A A GAAGC -- 14 GA ACA ATG G CCTTT GA - > MN pheS Phe ---- TG AG A T T AGTA 18 AG G GA AT GCGGG GCGTG - A C TG G A A A CCCGC - 16 GA A -- TTC A CTCA GAA - > MN glyQ Gly --- AGA AAG AG AGT T 15 AG C GA ACCTGA GAG ---- A G TG T A A G TCAGGT 14 GA CT - GGC A CTTTCT C - > ST alaS Ala - AGTTA AG A AT T GT T 17 AGA A A AGTGAC GGTT --- G C TG C G A GTCATT - 17 ----- GCT A CTTAACT - > SA trpE Trp T CTAAA G AA AT AGTA 22 AGA A A GCTAAT GGGT --- G A TG G G A ATTAGC -- 14 GA AT - TGG A CTTTGGA - > BS ilvB Leu --- TGA GG A TA AGTA 20 AGAGA A CCGG GTTA ---- G C TG A G A A CCGG --- 16 GA A -- CTC G CCTCA GA - > CA ilvC Val ----- A GG A AG AGTA 17 AGAGA GTGAG ATACT --- G G TG G G A A CTCAT -- 13 GA AG - GTA G CCT TTGA - > BQ asnA Asn -- AGGA C GA GT AGTA 15 AG C GA G TCAGG GGT ---- G G TG T G A G CCTGA -- 15 GA AG - AAC C TCCT GGA - > BS proB Pro ----- A GG A TT AGTA 18 AGAGA GCAAAATG AACC - G C TG A A A CATTTTGC 15 GA A -- CCT G CCT TGGA - > SA cysE Cys -- CGAA GG A TT AGTA 18 AGAGA G TGTAC GGTT --- G C TG T G A GTACA --- 14 GA A -- TGC A CCTTCG T - > MN hisC His ----- A G AG A A AAAA 16 AGAGA GTATG GGAA ---- G C TG A A A A CATAC -- 15 ----- CAC A TTCT TGA - > DH A pheA Phe ----- A A AG A G AG C A 19 AG G GA ACTAAAG TCGGAG A C TG A A A G CTTTAGT 14 GA GA - TTC A CTCT GGA - > HD serA Ser ---- GA AG A TG AGGA 17 AGAG A GCTGGT G GTT --- G C TG T G A ACCAGCT - 18 ----- AGC C CTTC TGA - > BQ phhA Tyr AGAAT C G C A GT AGTA 17 AGAGA GCTAAT GGTC --- G G TG G A A ATTGGC - - 14 GA AT - TAC A ATTCT GG - > EF yxjH Met ----- T AGG AA AGTA 17 AGAGA GACTTT GGTT --- G G TG A A A AAAGTT -- 13 GA AAA ATG G CCTA GGA - > CA yckK Cys ---- AA GA A CC AGTA 17 AGAGA AAAATCTC CAAG - G C TG A A A GGGATTTT 15 GA A -- TGC A TCTT TGA - > DF yqiX Arg ----- A GAG AA AGTA 16 AG C GA GTTAGG G GTT --- G G TG T A A G CCTAGC - 14 GA AG - AGA G CTCT GGA - > HD BH0807 Lys ---- AG AG A AG AGTA 19 AGA A A GCCTGT AGTT --- G C TG A G A ACGGGT -- 14 GA AGC AAG A CTCT GAG - > EF yheL Tyr - TTATT AG C CC AGTA 19 AGA A A GTCGAT GGTT --- G C TG C G A ATCGAT -- 13 GA AT - TAC A CTAATAA - > BQ ykbA Thr -- GAGG A C A CG ATCA 16 AGAGA GGGAAGC CTTTG - G C TG T G A GCTTCCT - 14 GA TT - ACC A CCTC TGA - > BQ sdt2 Trp --- GCA AG A AG AGTA 18 AGAGA GCTGGG GGAA --- G G TG T G A G CCCGGT - 15 GA A -- TGG G CTTGC GA - > EF yusC Met ---- AA AGA AG AGTA 18 AGAGA G CCC TGTTT ---- G C TG A G A AT GGG --- 16 GA AG - ATG G TCTTT GA - > CA yhaG Trp ---- AA GG A AG AGTA 18 AGAGA GCTGAG GGT ---- G G TG T G A T CTCAGT - 15 GA A -- TGG A CCTT TTA - > BQ brnQ Ile ---- GA GA A CG AGTA 19 AGAGA GTTGGC GATTT -- G C TG A A A GCCAAC -- 15 GA AA - ATC A TCTC CGA - > REF01723 His -- TTAG G A C AT AGTA 18 AGAGA CTTTT TC ATTG -- G C TG A A specifier hairpin SC ===> ==> ===> <=== <== <=== SA SERS SER --- GTA GG A CA AGTA 19 AGAGA G CTT GT GGTT --- A G TG T G A ACAAG --- 15 GA A -- TCT A CCTAC TT - > DHA tyrZ Tyr ---- AA GA A CA AGTA 18 AGA A A GT T GCC G GCT --- G A TG A G A GGCGCTT 18 GA A -- TAC C TCTT TGA - > ST trpS Trp --- ATT AG A AG AGTA 16 AGAGA GTTAG TGGTT --- G G T G C A A G CTAAC - 12 GA AA - TGG A CTAAT GA - > CA ASPS ASP - ---- G AGA AA AGTA 18 AG C GA A TTGGG AAAT --- G G TG T G A G CCCAA - 15 GA AA - GAC A TCTC GGA - > DF VALS VAL - GAAG A AGA GG AGTA 16 AGAGA G GAAAAT TCACTG G C TG T A A G ATTTTC 17 GA Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters A GAAAAAG - 17 ----- CAC A CCTAA AA - > BS yvbW Leu ----- G GG A GC AGTA 18 AGAGA GCTGCG GGGT --- G G TG C G A CGCAGC -- 13 GA A -- CTC G CCC GGGA - >

  14. Why T-boxes? • May be easily identified • In most cases functional specificity may be reliably predicted by the analysis of specifier codons (anti-anti-codons) • Sufficiently long to retain phylogenetic signal • Thus T-boxes are a good model of regulatory evolution

  15. ~800 T-boxes in ~90 bacteria • Firmicutes • aa-tRNA synthetases • enzymes • transporters • all amino acids excluding glutamate (lysine and glutamine – rare) • Actinobacteria (regulation of translation) • branched chain (ileS) • aromatic (Atopobium minutum) • Delta-proteobacteria • branched chain (leu – enzymes) • Thermus/Deinococcus group (aa-tRNA synthases) • branched chain (ileS, valS) • glycine • Chloroflexi, Dictyoglomi • aromatic (trp – enzymes) • branched chain (ileS) • threonine

  16. Double and one-and-a-half T-boxes • TRP: trp operon (Bacillales, C. beijerincki, D. hafniense) • TYR: pah (B. cereus) • THR: thrZ (Bacillales); hom (C. difficile) • ILE: ilv operon (B. cereus) • LEU: leuA (C. thermocellum) • ILE-LEU: ilvDBNCB-leuACDBA (Desulfotomaculum reducens) • TRP: trp operon (T. tengcongensis) • PHE: arpLA-pheA (D. reducens, S. wolfei) • PHE: trpXY2 (D. reducens) • PHE: yngI (D. reducens) • TYR: yheL (B. cereus) • SER: serCA (D. hafniense) • THR: thrZ(S. uberis) • THR: brnQ-braB1 (C. thermocellum) • HIS: hisXYZ (Lactobacillales) • ARG: yqiXYZ (C. difficile)

  17. Predicted regulation of translation:ileS in many Actinobacteria • Instead of the terminator, the sequester hairpin (hides the translation initiation site) • Same mechanism regulates different processes – cf. riboswitches

  18. Same enzymes – different regulators (common part of the aromatic amino acids biosynthesis pathway) cf. E.coli: aroF,G,H: feedback inhibition by TRP, TYR, PHE; transcriptional regulation by TrpR, TyrR

  19. Recent duplications and bursts: ARG-T-box in Clostridium difficile

  20. More duplications: THR-T-box in C. difficile

  21. Duplications and changes in specificity:ASN/ASP/HIS T-boxes

  22. Blow-up

  23. Duplications and changes in specificity : branched-chain amino acids ATC CTC ATC

  24. Blow-up transporter: ATC GTC dual regulation of common enzymes: ATC CTC

  25. Summary / History

  26. [+Fe] [+Fe] [- Fe] [ Fe] - Irr Irr RirA RirA FeS heme degraded 2+ 3+ S i d e r o p h o r e F e / F e I r o n - r e q u i r i n g I r o n s t o r a g e F e S H e m e T r a n s c r i p t i o n u p t a k e u p t a k e e n z y m e s f e r r i t i n s s y n t h e s i s s y n t h e s i s f a c t o r s I r o n u p t a k [ i r o n c o f a c t o r ] e s y s t e m s FeS status IscR Fur Fur of cell Fe FeS [- Fe] [+Fe] Regulation of iron homeostasis in α-proteobacteria Experimental studies: • FUR/MUR: Bradyrhizobium, Rhizobium and Sinorhizobium • RirA (Rrf2 family): Rhizobium and Sinorhizobium • Irr (FUR family): Bradyrhizobium, Rhizobium and Brucella

  27. Distribution of transcription factors in genomes

  28. Regulation of genes in functional subsystems Rhizobiales Bradyrhizobiaceae Rhodobacteriales The Zoo (likely ancestral state)

  29. Reconstruction of history Frequent co-regulation with Irr Strict division of function with Irr Appearance of theiron-Rhodo motif

  30. Andrey Mironov (software): • genome analysis • conserved RNA patterns • Ekaterina Panina (now at UCLA, USA) • zinc and ribosomes • Alexey Vitreschak • T-boxes • Dmitry Rodionov • iron homeostasis • Support: • Howard Hughes Medical Institute • INTAS • Russian Fund of Basic Research • Russian Academy of Sciences (“Molecular and Cellular Biology”)

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