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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
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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
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Regulators (zinc uptake) and motives
nZUR- nZUR- GAAATGTTATANTATAACATTTC GATATGTTATAACATATC GTAATGTAATAACATTAC TTAACYRGTTAA pZUR AdcR TAAATCGTAATNATTACGATTTA
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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. TM Zn adcA zinT S. pneumoniae S. pyogenes S. equi S. agalactiae zinc regulation shown in experiment lmb phtD phtE lmb phtD lmb phtD phtA phtB phtY
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Zinc and (paralogs of) ribosomal proteins
E. coli, S.typhi – – + K. pneumoniae – – Y. pestis,V. cholerae – B subtilis – + – S. aureus – – – Listeria spp. E. faecalis – – – S. pne., S. mutans S. pyo., L. lactis nZUR pZUR AdcR
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Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001)
L36 L33 L31 S14 E. coli, S.typhi (–) – (–) + K. pneumoniae (–) – Y. pestis,V. cholerae (–) B subtilis (–) + – S. aureus (–) – – Listeria spp. E. faecalis (–) – – S. pne., S. mutans S. pyo., L. lactis nZUR pZUR AdcR
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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
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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
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Regulatory mechanism Sufficient Zn Zn starvation ribosomes R repressor
Zn-dependent enzymes Zn starvation R
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Prediction … (Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9912-7.)
… and confirmation (Mol Microbiol Apr;52(1): )
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T-boxes: the mechanism (Grundy & Henkin; Putzer & Grunberg-Manago)
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Partial alignment of predicted T-boxes
TGG: T-box Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters
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… continued (in the 5’ direction)
anti-anti (specifier) codon 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 - > AT - GTA G CTTT GGA > PN THRS THR ---- AG A CA AGT C 18 AGAGA GTGCGT GGTT --- TG ACGCAT 14 GA T -- AC CTCT TGA MN ileS Ile AAA ACAA 17 AGCGA ATAGGT GAT ACCTAT T 18 ----- ATC TTTTG TT DF leuS Leu CT GC AGTA 19 AGAG GGAA ACTAAT ATT 10 CTT CTAG HD ARGS ARG GGG 20 GTCGG CCGAT AA CGC CCCA DF proS Pro GAAAAC GGT GTTTTC CCT TCTTT TA 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 ZC lysS Lys --- AAG AGA AG AGTA 19 AGAGA GCTCT GGTA ---- G C TG A AGAGC -- 15 GA AAA CTT GGAG - > BQ metS Met GG AA GCTTC GAAGC 14 ACA ATG CCTTT MN pheS Phe T 18 AT GCGGG GCGTG CCCGC 16 TTC CTCA GAA MN glyQ Gly AGT ACCTGA GAG TCAGGT CT GGC CTTTCT ST alaS Ala AGTTA GT 17 AGTGAC GGTT GTCATT ----- GCT CTTAACT SA trpE Trp CTAAA AA AT AGTA 22 AGA A GCTAAT GGGT --- G TG ATTAGC -- 14 GA - TGG CTTTGGA > BS ilvB Leu TGA GG TA 20 AGAGA CCGG GTTA ---- C 16 CTC CCTCA CA ilvC Val ----- AG 17 GTGAG ATACT CTCAT 13 GTA CCT TTGA BQ asnA Asn AGGA GT 15 TCAGG GGT T CCTGA AAC TCCT GGA BS proB Pro TT 18 GCAAAATG AACC CATTTTGC TGGA SA cysE Cys CGAA Amino acid biosynthetic genes TGTAC GGTT --- G C TG T A GTACA 14 GA -- TGC CCTTCG - > MN hisC His ----- AG AAAA 16 AGAGA GTATG GGAA ---- CATAC 15 CAC TTCT TGA DH A pheA Phe 19 ACTAAAG TCGGAG CTTTAGT TTC CTCT GGA HD serA Ser AGGA 17 AGAG GCTGGT GTT ACCAGCT 18 AGC CTTC BQ phhA Tyr AGAAT GT AGTA GCTAAT GGTC ATTGGC AT TAC ATTCT GG EF yxjH Met AGG AA GACTTT AAAGTT -- 13 GA AAA ATG G CCTA GGA - > CA yckK Cys ---- AA A CC AGTA 17 AGAGA AAAATCTC CAAG C TG GGGATTTT 15 TGC TCTT TGA DF yqiX Arg ----- GAG 16 AG GTTAGG GTT --- T CCTAGC 14 AGA CTCT HD BH0807 Lys 19 GCCTGT AGTT ACGGGT AGC AAG EF yheL Tyr TTATT GTCGAT GGTT ATCGAT AT TAC CTAATAA BQ ykbA Thr GAGG CG ATCA GGGAAGC CTTTG Amino acid transporters G A GCTTCCT - 14 GA TT ACC CCTC TGA > BQ sdt2 Trp --- GCA AG AGTA 18 AGAGA GCTGGG GGAA TG T CCCGGT 15 -- TGG CTTGC EF yusC Met ---- AA AGA CCC TGTTT C AT GGG 16 ATG TCTTT CA yhaG GG GCTGAG GGT CTCAGT CCTT TTA BQ brnQ Ile CG 19 GTTGGC GATTT GCCAAC ATC TCTC CGA REF01723 His TTAG CTTTT TC ATTG 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 - >
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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
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~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) threonine
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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)
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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
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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
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Recent duplications and bursts: ARG-T-box in Clostridium difficile
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More duplications: THR-T-box in C. difficile
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Duplications and changes in specificity: ASN/ASP/HIS T-boxes
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Blow-up
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Duplications and changes in specificity : branched-chain amino acids
ATC ATC CTC
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Blow-up transporter: dual regulation of common enzymes: ATC GTC ATC
CTC
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Summary / History
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Regulation of iron homeostasis in α-proteobacteria
RirA Irr FeS heme degraded Fur Fe I r o n u p t a k e s y m S i d h F / T c f 2+ 3+ g H - q z [ ] IscR [- Fe] [+Fe] [ Fe] FeS status of cell Experimental studies: FUR/MUR: Bradyrhizobium, Rhizobium and Sinorhizobium RirA (Rrf2 family): Rhizobium and Sinorhizobium Irr (FUR family): Bradyrhizobium, Rhizobium and Brucella
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Distribution of transcription factors in genomes
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Regulation of genes in functional subsystems
Rhizobiales Bradyrhizobiaceae Rhodobacteriales The Zoo (likely ancestral state)
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Reconstruction of history
Frequent co-regulation with Irr Strict division of function with Irr Appearance of the iron-Rhodo motif
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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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