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 acp P fab D accA accDaccB accC fabHfab F fab G fabZ fabI Gene fabI of Enoyl-ACP reductase (EC ) is missing in the genome 12B, and a number of Streptococci fabI (Enoyl-ACP reductase, EC ) target of triclosan. Enzymatic activity, but no gene in Streptococci

Identification of a candidate by positional clustering Genome X TR? fabIhyp ? hyp TR? FRNS Genome Y Clostridium acetobutylicum TR? Streptococcus pyogenes ? hyp TR? ? fabH acpP ? fabGfabFaccAaccDaccC accB fabZ fabD fabGfabF accBfabD accAaccDaccCfabZ fabGfabFaccAaccDaccC accB fabZ fabDfabH acpP fabH acpP fabH acpP fabGfabFaccAaccDaccC accB fabZ fabD

Binding sites of FabR (“Tr?”, HTH) HTH fabK fabH acpP fabGfabFaccAaccDaccC accB fabZ fabD Fad ( ) 1234

Metabolic reconstruction of the thiamin biosynthesis (new genes/functions shown in red) thiN (confirmed) (Gram-positive bacteria) (Gram-negative bacteria) Transport of HMP Transport of HET Purine pathway

Carbohydrate metabolism in Streptococcus and Lactococcus spp. Only biochemical data, genes unknown Experimentally verified genes Biochemical data and genomic predictions Only genomic predictions

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 S. suis S. pneumoniae R6

Gene functions 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N- acetyl-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 S. suis S. pneumoniae R6

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 ??? AdcR ? MarR family pZUR FUR family

Regulators and signals nZUR-  nZUR-  AdcRpZUR TTAACYRGTTAA GATATGTTATAACATATC GAAATGTTATANTATAACATTTC GTAATGTAATAACATTAC TAAATCGTAATNATTACGATTTA

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 fusion: adcA-zinT E. coli, S. typhi, K. pneumoniaeGamma-proteobacteria Alpha-proteobacteria B. subtilis, S. aureus S. pneumoniae, S. mutans, S. pyogenes, L. lactis, E. faecalis Bacillus group Streptococcus group zinT is regulated by zinc repressors (nZUR- , nZUR- , pZUR) adcA-zinT is regulated by zinc repressors (pZUR, AdcR) (ex. L.l.) A. tumefaciens, R. sphaeroides

ZinT: protein sequence analysis E. coli, S. typhi, K. pneumoniae, A. tumefaciens, R. sphaeroides, B. subtilis L. lactis Y. pestis, V. cholerae, B. halodurans TMZnAdcA S. aureus, E. faecalis, S. pneumoniae, S. mutans, S. pyogenes 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 TM- segment) 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. equi S. agalactiae lmbphtDphtE phtBphtA lmbphtD S. pyogenes phtY lmbphtD zinc regulation shown in experiment

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: 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)

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: PHTinternalinH-rich 4 HIS TRIADS LRR IR HDYNHNHTYEDEEGH AHEHRDKDDHDHEHED

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

Zinc and (paralogs of) ribosomal proteins L36L33L31S14 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

Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001) L36L33L31S14 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

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 ribosomes Zn-dependent enzymes R Sufficient Zn Zn starvation R repressor

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 Aug 19;100(17): ) … and confirmation (Mol Microbiol Apr;52(1): )

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) 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 Andrei Osterman (Burnham Institute, San-Diego, USA) (fatty acids)