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Comparative genomics and metabolic reconstruction of bacterial pathogens Mikhail Gelfand Institute for Information Transmission Problems, RAS GPBM-2004 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 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 general biochemical 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 Transporters • Identification of candidates assigned to the pathway by co-localization and co-regulation (in many genomes) • Prediction of general function 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 Missing link in fatty acid biosynthesis in Streptococci accA accD accB Gene fabI of Enoyl-ACP fabI (Enoyl-ACP reductase, (EC 1.3.1.9) is ECreductase 1.3.1.9) target of triclosan. missing in the genomebut 12B, Enzymatic activity, no and agene number of Streptococci in Streptococci accC fab D fab F fab G fabZ fabI acp P fabH Identification of a candidate by positional clustering hyp TR? 3.5.1.? fabH fabD acpP fabG fabF fabZ accB accC accD accA fabI hyp 6.3.4.15 Genome X TR? fabH acpP ? fabD fabG fabF accB fabZ accC accD accA fabZ accC accD accA 2.1.1.79 FRNS Genome Y TR? fabH acpP ? fabD fabG fabF accB 5.99.1.2 Clostridium acetobutylicum TR? fabH Streptococcus pyogenes acpP ? fabD fabG fabF accB fabZ accC accD accA hyp Binding sites of FabR (“Tr?”, HTH) 1 Fad (42.1.17) 2 HTH fabH acpP 3 4 fabK fabD fabG fabF accB fabZ accC E. faecalis E. faecalis E. faecalis CONSENSUS HTH-1 HTH-2 HTH-3 acTTTGAtwaTCAAAgt AgTTTGggTATCAAAGT AgTTTGAacATCAAAtg GtTTTGATAATCAAAGT E. faecium E. faecium E. faecium HTH-1 HTH-2 HTH-3 ACTTTGATAATCAAAaT AgTTTGAacATCAAAag gaTTTGATAATCAAAcT S. pyogenes S. pyogenes S. pyogenes S. pyogenes 4.2.1.17 HTH-1 fabK-1 fabK-2 GaTTTGATTATCAAAtg AaTTTGATTgTCAAAGT CtTTTGATAtTCAAAtT AgTTTGATTATCAAAtT S. pneumoniae S. pneumoniae S. pneumoniae 4.2.1.17 HTH-1 fabK-1 ACTTTGAcAgTgAAAta gtTTTGATTgTaAAAGT AgTTTGAcTgTCAAAtT S. mutans S. mutans S. mutans S. mutans S. mutans 4.2.1.17-1 4.2.1.17-2 HTH-1 fabK-1 fabK-2 ACTTTGATTtTCAAAcT AaTTTGATTATCttAaT ACTTTGATAgTCAAAGT AgTTTGAcAtTCAAAtc AgTTTGAcTgTCAAAtT 1 2 3 4 accD accA Metabolic reconstruction of the thiamin biosynthesis (new genes/functions shown in red) Purine pathway Transport of HMP thiN (confirmed) Transport of HET (Gram-positive bacteria) (Gram-negative bacteria) unknown arabinose arbutin cellobiose dextran esculin fructose fucose galactose glucose inulin lactose maltose mannitol mannose melibiose N-AcGlu raffinose ribose salicin sorbitol sorbose sucrose tagatose trehalose xylose L. mesenteroides Oenococcus oeni L. brevis P. pentosaceus L. delbrueckii L. gasseri L. casei L. lactis S. suis S. thermophilus S. mutans S. agalactiae S. uberis S. equi S. pyogenes S. pneumoniae Carbohydrate metabolism in Streptococcus and Lactococcus spp. Only biochemical data, genes unknown Experimentally verified genes Biochemical data and genomic predictions Only genomic predictions unknown arabinose arbutin cellobiose dextran esculin fructose fucose galactose glucose inulin lactose maltose mannitol mannose melibiose N-AcGlu raffinose ribose salicin sorbitol sorbose sucrose tagatose trehalose xylose L. mesenteroides Oenococcus oeni L. brevis P. pentosaceus L. delbrueckii L. gasseri L. casei L. lactis S. suis S. thermophilus S. mutans S. agalactiae S. uberis S. equi S. pyogenes S. pneumoniae An uncharacterized locus in invasive species S. pneumoniae S. pyogenes S. equi S. agalactiae S. suis Structure of the genome loci S. pyogenes, S. agalactiae S. equi S. pneumoniae TIGR4 IS S. pneumoniae R6 IS S. suis IS Gene functions 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-Nacetyl-D-glucosamine PTS transporter hydrolase isomerase oxidoreductase dehydrogenase kinase aldolase pyruvate + D-glyceraldehyde 3-phosphate hyaluronidase (hyaluronate lyase) RegR Candidate regulatory signal Structure of the genome loci - 2 S. pyogenes, S. agalactiae S. equi S. pneumoniae TIGR4 IS S. pneumoniae R6 IS S. suis IS 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 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 component 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 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) • Andrei Osterman (Burnham Institute, San-Diego, USA) (fatty acids) • 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