From computer to the wet lab and back Comparative genomics in the discovery of a family of bacterial transporters with a new mode of action Mikhail Gelfand RECOMB-BE. UCSD, La Jolla, 15-III-2009

Transporters Two main classes –ATP-dependent TM-protein (permease) ATPase Substrate-binding protein –Secondary (symporters, antiporters) Difficult to study in experiment (compared to enzymes) Relatively easy to identify –Similarity to known transporters –Prediction of transmembrane segments Difficult to predict specificity H+H+

It is difficult to predict specificity by sequence analysis (nickel-oligopeptide family, substrate-binding NikA)

PnuC family of cofactor transporters

Riboflavin biosynthesis pathway

5’ UTR regions of riboflavin genes

RFN-element Capitals: invariant (absolutely conserved) positions. Lower case letters: strongly conserved positions. Dashes and stars: obligatory and facultative base pairs Degenerate positions: R = A or G; Y = C or U; K = G or U; B= not A; V = not U. N: any nucleotide. X: any nucleotide or deletion

RFN: the mechanism of regulation Transcription attenuation Translation attenuation

YpaA: riboflavin transporter 5 predicted TM segments => a transporter Upstream RFN element => co-regulation with riboflavin genes => transport of riboflavin / precursor S. pyogenes, E. faecalis, Listeria spp.: ypaA, no riboflavin pathway => transport of riboflavin Prediction: YpaA is riboflavin transporter (Gelfand et al., 1999) Verification: by genetic analysis (Kreneva et al., 2000) directly (Burgess et al., 2006) => RibU ypaA is regulated by riboflavin (Lee et al., 2001) … via attenuation of transcription (Winkler et al., 2003)

Biotin transporter BioY Identification: –co-localization –co-regulation –phylogenetic profiling Additional components –ATPase(?) bioM –Permease(?) bioN

Thiamin biosynthesis = thiN (confirmed) (Gram-positive bacteria) (Gram-negative bacteria) Transport of HMP Transport of HET

yuaJ(=thiT): thiamine transporter 6 predicted TM-segments Regulated by THI riboswitches Streptococci: ThiT, no thiamine pathway

Regulated by THI riboswitches Newer occurs in genomes lacking thiamine pathway Always co-occurs with thiD and thiE Sometimes occurs without thiC ykoFEDC: ATP-dependent HMP transporter

Cobalt and Nickel Co-localization –Ni transporters with genes for Ni- dependent enzymes –Co transporters with cobalamine biosynthesis genes Co-regulation –Ni transporters by transcription factor NikR –Co transporters by В12 riboswitich

Structure of the loci B12 riboswitchNikR binding sitegenes

Five families of transporters

New ATP-dependent transporters NikM CbiM Ni 2+ Co 2+ + CbiN + NikL, NikK + NikN + NikL

Dmitry Rodionov  Thomas Eitinger

Test 1: predicted specificity is correct Co Ni

Structure: too many components

Biotin transporter BioY ATPase BioM ~ CbiO = NikO Permease BioN ~ CbioQ = NikQ

Test 2: MN components are suffucient (ATPase and permease are dispensable) cbiMNQO cbiMNQ cbiMN cbiM control

Test 3: BioY is sufficient even if the genome had BioMNY; BioMNY has better kynetics

Tip of the iceberg?

Validations RibU: riboflavinThiT: thiamin FolT: folate (like BioY)

Universal energizing component + specific components

Transporters Identification of candidates by analysis of transmembrane segments and similarity Assignment to pathway by co-localization and co- regulation (in many genomes) 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

Dmitry Rodionov Alexei Vitreschak (RNA regulation) Andrei Mironov (software, discussion) Thomas Eitinger’s lab Anrei Osterman’s lab European Science Foundation HHMI, RFBR, RAS, INTAS