Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Pathogenomics wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Designer baby wikipedia , lookup
History of genetic engineering wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Metabolic network modelling wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Gene expression profiling wikipedia , lookup
Minimal genome wikipedia , lookup
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 – 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 L-aspartate lysC,thrA,metL Metabolic reconstruction of the lysine pathway lysC,dapG,yclM -aspartyl-phosphate asd aspartate semialdehyde • Predictions: – Genes for the acetylated pathway in Gram-positive bacteria – Positive regulation of the lysine catabolism genes in Thermoanaerobacter and Fusobacterium by LYSelements: 1st example of activating riboswitches – New transporters dapA hom homoserine thrA, metL dihydrodipicolinate dapB tetrahydrodipicolinate dapD N-succinyl-2-amino-6-ketopimelate dapC(argD) N-succinyl-L,L-diaminopimelate dapE dapD N-acetyl-2-amino-6-ketopimelate patA N-acetyl-L,L-diaminopimelate ykuR L,L-diaminopimelate dapF, dal meso-diaminopimelate Lysine transport lysA ddh • Predictions: – A Metabolic reconstruction Genes for the SAM-recycling pathway of the methionine pathway – Transporters for methionine and methylthiribose Threonine – Other enzymes – Transcriptional regulation in Streptococci – Complicated S-box and Cys-T-box regulation of the ubiG-yrhBA operon in C. acetobutylicum: S-ribosylhomocysteine activation via repression (SRH) of the antisense mtn transcript Aspartate semialdehyde hom Homoserine cysH-... metX metB O-acetylhomoserine metI Sulfide metY ubiG yrhA yrhB S-box sense transcript Cystathionine metC yrhA Homocysteine methyl-THF betaine yxjH* metE , metH , dimethylglycine S-adenosylmethionine S-adenosylhomocysteine (SAH) (SAM) THF metK CH3 Cysteine Methionine mtnKSUVWXYZ antisense transcript S-adenosylmethionine methylene-THF metF A Cys-T-box yrhB MTA mtn Methylthioribose (MTR) Aromatic amino acid regulons in Gram-positive bacteria Prediction of transporter specificity via analysis of regulation Pasteurella ceae NMB S ON-2 S ON-1 VC-1 VC-2 BH SON-3 clostridia OB FN 0978 OB1118 HP C AC0744 LysW CB EF -nhaC 1 PPE Archaea LP-nha2 LGA LME LP -nha 1 LB EF-nhaC2 TyrT BC14 34 FN1414 BT1270 CB NMB0536 FN0352 BC4121 TTE-nhaC SA2117 CJ OB2874 269. 47 CT C CPE DF BL1111 MetT BS-yh eL FN0650 BC1709 CTC00901 FN062 4 CTC02520 BB 0637 CPE2317 FN1420 CTC0 2529 VCA0193 S O10 87 FN1422 BC0373 B B0 638 FN207 7 BH3946 BS-mleN VC2037 SA2292 HI1107 V V 21061 MleN Some confirmed predictions PREDICTION GENOME REF – Prediction REF – Verification Mechanism of regulation of riboflavin metabolism and transport genes Bacteria (Bacillus subtilis, Escherichia coli) Vitreschak et al., 2002 Winkler et al., 2002b; Mironov et al., 2000 Mechanism of regulation of thiamin metabolism and transport genes Bacteria and archaea (Bacillus subtilis, Escherichia coli) Rodionov et al., 2002b Winkler et al., 2002a Transcription regulatory signal for the nitrogen-fixation pathway Methanogenic archaea (Methanococcus maripaludis) Gelfand et al., 2000 Kessler and Leigh, 1999; Lie and Leigh, 2003 Acyl-CoA-dehydrogenase FadE is encoded by gene yafH Gamma-proteobacteria (Escherichia coli) Sadovskaya et al., 2001 Campbell and Cronan, 2002 ThiN, an enzyme (MTH861) or ThiD domain functionally equivalent to ThiE T. maritima, archaea (Methanobacterium thermoautotrophicum) Rodionov et al., 2002b Morett et al., 2003 Riboflavin transporter YpaA: specificity and regulation Gram-positive bacteria (Bacillus subtilis) Gelfand et al., 1999 Kreneva et al., 2000 Oligogalacturonide ABC-transporter Gamma-proteobacteria (Erwinia ogtABCD (togMNAB) chrysanthemi) Rodionov et al., 2000 Hugouvieux-CottePattat et al., 2001 Arginine ABC-transporter yqiXYZ: specificity and regulation Bacteria (Bacillus subtilis) Makarova et al., 2001 Sekowska et al., 2001 Methionine transporter MetD Bacillus subtilis, Escherichia coli Zhang et al., 2003 Zhang et al., 2003 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 Genomes and regulators ??? nZUR FUR family pZUR AdcR ? FUR family MarR family nZUR- Regulators and signals GATATGTTATAACATATC nZUR- GAAATGTTATANTATAACATTTC GTAATGTAATAACATTAC TTAACYRGTTAA pZUR TAAATCGTAATNATTACGATTTA AdcR 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: regulation zinT is isolated zinT is regulated by zinc repressors (nZUR-, nZUR-, pZUR) E. coli, S. typhi, K. pneumoniae Gamma-proteobacteria A. tumefaciens, R. sphaeroides Alpha-proteobacteria B. subtilis, S. aureus Bacillus group S. pneumoniae, S. mutans, S. pyogenes, L. lactis, E. faecalis Streptococcus group fusion: adcA-zinT adcA-zinT is regulated by zinc repressors (pZUR, AdcR) (ex. L.l.) ZinT: protein sequence analysis Y. pestis, V. cholerae, B. halodurans S. aureus, E. faecalis, S. pneumoniae, S. mutans, S. pyogenes E. coli, S. typhi, K. pneumoniae, A. tumefaciens, R. sphaeroides, B. subtilis L. lactis TM Zn AdcA ZinT 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 TMsegment) • ZinT has a zinc-binding domain ZinT: conclusions: • ZinT is a new type of zinc-binding component of zinc ABC transporter Zinc regulation of PHT (pneumococcal histidine triad) proteins of Streptococci S. pneumoniae S. pyogenes S. equi S. agalactiae zinc regulation shown in experiment lmb phtD phtA phtE phtB lmb phtD phtY lmb phtD 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. pneumoniae on bacterial cell surface has been confirmed by flow cytometry 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 … incorrect • Zinc-binding domains in zinc transporters: • Histidine triads in streptococci: EEEHEEHDHGEHEHSH DEHGEGHEEEHGHEH HGDHYHY HGDHYHF HGNHYHF HYDHYHN HMTHSHW (histidine-aspartateglutamate-rich) (specific pattern of histidines and aromatic amino acids) HSHEEHGHEEDDHDHSH EEHGHEEDDHHHHHDED 7 out of 21 2 out of 21 2 out of 21 2 out of 21 2 out of 21 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 PHT LRR IR HDYNHNHTYEDEEGH AHEHRDKDDHDHEHED internalin H-rich 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 internalin domain responsible for the streptococcal invasion Hypothesis PHT proteins are adhesins involved in the attachment of streptococci to epithelium cells, leading to invasion AdcR pZUR nZUR Zinc and (paralogs of) ribosomal proteins L36 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 – L33 – – – –+– ––– –– ––– ––– ––– L31 –+ –– –+ –+ – – – – – S14 – – – –+ –+ –+ –+– – –+ Zn-ribbon motif AdcR pZUR nZUR (Makarova-Ponomarev-Koonin, 2001) L36 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 (–) L33 – – – (–) + – (–) – – (–) – (–) – – (–) – – (–) – – L31 (–) + (–) – (–) + (–) + – – – – – S14 – – – (–) + (–) + (–) + (–) + – (–) (–) + 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 Bad scenario Zn-rich conditions Zn-deplete conditions: all Zn utilized by the ribosomes, no Zn for Zn-dependent enzymes Regulatory mechanism Sufficient Zn ribosomes repressor R Zn-dependent enzymes Zn starvation R Good scenario Zn-rich conditions Zn-deplete conditions: some ribosomes without Zn, some Zn left for the enzymes 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.) • 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