BMC Genomics 2002, 3 http://www.biomedcentral.com/1471-2164/3/8 Helix-1 Helix-2 Helix-3 Strand-1 Strand-2 Strand-3 PHD Sec. Structure -HHHHHHHHHHH---------------------------HHHHH-----HHHHHHH-----EEEE------EEEEEE----EEEEEEEEEEE----------------- ERF_P22_9635505 5 FYARLAEIQEHL--------NAPK--NQYN-SFGKYKYRSCEDIL----EGVKPLLK--GLFLSIS-----DEIVLI--GDRYYVKATATITD-----GENS------- ERF_HK97_9634188 5 FYARLAAIQENL--------NAPK--NQYN-SFGKYKYRSCEDIL----EGVKPLLN--GLFLSIS-----DEVVLI--GDRYYVKATATITD-----GENS------- orf17_phiPV83_9635694 10 INKAMVAFRKEV--------KQPLK-DKNN-PFFKSKYVPLENVV----EAIDEAATPHGLSYT------QWALNDVD-GRVG-VATMLMHES-----GEYI------- lmo2318_Lm_16804357 10 IAKAMAAIQKEV---------KPLEKSAAN-PFTDSKYTPLDKIV----EAIFKVAPGHGVSFT------QWPISGDN-GTIG-IGTMLMHES-----GEWI------- orf216_phiSLT_12719409 7 LYQKIADVKANI----------AGFTKDTK-GY-NFSYVSGSQIL----HRIREKMIEHNLLLVPNTLNNEWTTHTF--KNK--KGQEVTEFIVEMDLNYTWINAD--- ORF4_Bbcp8.3_458219 18 FISDMKTLRMNL----------AGIDKNLK-GY-GYKYQNFNEIV----REIKNVINKHNLYLDFK----QFPTFTVVEGQQV-LHVVRTTFY-STNTGYKDSFDTPIL BBL29_Bbcp32-8_11497308 41 FRKDMKTLKMNL----------PGIDKSLK-GY-GYKYQNFNEIV----REIKNVIKKHNLELDIE----QYPISIE--GQYGIVDYIRTTFY-STSTGYEFSFDTRIP BBO29_Bbcp32-7_11497280 36 FRKDMKTLKMNL----------PGIDKSLK-GY-GYKYQNFNEIV----REIKNVIKKHNLELDIE----QYPISIE--GQYGIVDYIRTTFY-STSTGYEFSFDTRIP BBR29_Bbcp32-4_11497168 47 FRKDMSTLIRNL----------PRIDKSLK-GY-GYKYQDFNDIV----EVIYSVIDKHNLDLFFT----QAPISVE--GQYGIVDYIRTTFY-STSTVYKYSFDTRIH BBC05_Bbcp9_11527300 19 FLRDMETLRMNL----------PGIDKNLK-GY-GYKYQNFNEIA----REIKKVIBKHNLCLDFK----QFPTFTVV-GEQQVLHVVRTTFY-STNTGYKDSFDTPIL ORF7_ST7201_9634634 2 EEMTFTELQQKI--------QLEK---KKE-GTAKYASRHVEDIY----NVFKNLKS--NWNVVV-----NYELVEF--SGKTFIKAIATASNKDEK------------ Orf52_D3_9635643 85 FNASMAAMQSEL-------ERGPSIAAITVNGQKRSNYATFEDIN----DIVKPIMQRFGFAVSFR-----VETVQT--GV--SVTGILMHCA-----GHRE------- Orf223_Lj771_13491642 13 WAMHYAQVKANIKQPEKTHKVTVSGKTKQGTPY-SYDYNYADLNDIDAAVMDGIKKVTDKDGNVVF----SYFFDIRTENNTVEVQTILVDSS-----GFTL------- ERF_Unk_6015511 7 LYEALAETQNNI--------EQPKK-DASN-PMFKSSYVTLDAVI----NAIVKARKASGAKFFF------TNIVQD--GV--MFTRIIGYGE-----TLDL------- ORF244_BPmv4_11138335 10 IFGALSKFRAQV--------KQPAK-TAKN-PYFNSNYVTLEGVM----QSIDAALPGTGLAYC------QLVENGDN-GV--SVSTLITHSS---------------- ERf_bIL67_9627938 1 MESKILKLINEI--------EVPK--SQYN-SYGKYNFRNNEDIQ----TALKPLLLKYGLMEVA-----GTEMLEM--NNELMLHVHVEIFDPENPNDV--------- ERf_c2_9628668 1 MESKVLKLINEI--------KVPK--SQYN-SFGKYNFRNNEDIQ----TALKPLLLQFGLMEKA-----TTEMLEM--NNELMLHVHIDIFDPDNPNDI--------- ERF_MM1_15088753 2 ADLTFAELQRKM--------QIEK---QTK-QGVKYPFRTAEDIN----NKFKSLDS--GWSVSFP----EDDIIQK--GDKLYYKAVAVVKRESDGTI---------- consensus/85% h...h..hb.pl..........s......p.sb...pa.s.p.l.....p.h..h....sh..................s....h........................ PHD Sec. Structure ERF_P22_9635505 Gp40_HK97_9634188 orf17_phiPV83_9635694 lmo2318_Lm_16804357 orf216_phiSLT_12719409 ORF4_Bbcp8.3_458219 BBL29_Bbcp32-8_11497308 BBO29_Bbcp32-7_11497280 BBR29_Bbcp32-4_11497168 BBC05_Bbcp9_11527300 ORF7_ST7201_9634634 Orf52_D3_9635643 Orf223_Lj771_13491642 ERF_Unk_6015511 ORF244_BPmv4_11138335 ERf_bIL67_9627938 ERf_c2_9628668 ERF_MM1_15088753 consensus/85% Strand-4 Helix-4 Helix-5 -----EEE------------------------HHHHHHHHHHHHHHH--------------------------------------HHHHHHHHHH--- HSASAIAREE-----ENKK----GMDAAQVTGATSSYARKYCLNGLFGIDD-AKDADTEEHKQQQN--------AAPAKQTKSSPSSPAPEQVLKAFS 157 HTATALAREE-----ESKK----GMDSAQVTGATSSYARKYCLNGLFGIDD-AKDADTDEHKHQQ------------NAAAKQSKPSPTPEQVLKAFT 153 EYDPVFMNAE--------K------NTPQGAGSLISYLKRYSLSAIFGITS-DQDDDGNEASGKNNNPK---QQTRTQWASSETIGILRKEVISFTKL 164 EYDPLYMTVI-----TNKK-----MSSAQEAGGTITYAKRYVIAAVFGIVS-DEDKDGNIQSSPSKYPKNN-SYKSN-NYNQNSNSQQQTKQSQQNDN 169 KPEEQYEVSYHA---YGQQ-----NDISQAHGTALTYAERYFLMKFFNIPTDEDDADAKQKQDKY-----------------STVSQEFKDILTKEVN 165 TENLKWYNEN------GAKN---VNTVPQLVGSSITYFKRYALVAYLNIES-EVDTDAAPINNNYKNEN-SNMPSK--QVIVNQEQKKDINQNQNQIK 189 TENLQWNNEN------GSKV---TNTVYQMFGSGITYVKRYALVAALGIES-EIDTDAAPIYNNHENEN--SMSSK--QVSVNQKQEQKREQKQEINQ 210 TENLQWNNEN------GSKV---TNTVYQMFGSGITYVKRYALVAALGIES-EIDTDAAPIYNNHENEN--SMPSK--QSSVNQKQEQKREQKQEINQ 205 TDKLQWNSEN------GSKN---MNTMPQFVGSAITYFKRYALVGHLCIRS-EMDTDAAPIYNNYENRN--SMPSK--QSSVNQKQEQKREQKQEINQ 216 TENLKWNNEN------GSKNV--VNTVPQLVGSSITYFKRYALVAYLNIES-EVDTDAAPIYNNHENEN--SMPSK--QAGVNQNQIKNFDKKLKTGK 190 VQAQAFAELSPVPILKTRNGELKQMNEPQWVGAVQSYAGKYALQALFAIG--EEDVDHFEVAEQ-------SMRPN--QNHNQMQSHQQANYINQQQH 160 ---QTTMLVPLDT--SGSK------NAVQSLGSSVSYGKRYVLSALLNITTRGEDDDGNAAVPP--------------KKLITKAQAQQLKALLSQCL 234 --KTNKVVFQ------NNK-----AWDAQATASLISYAKRYSLSGAFGI---AADNDDDAQDQKTIYE------------PKILTKQELEDYKVYYNG 174 --AGSKVADDL-----GNR----GTNSAQAEGSALTYARRYSLSMAFGI---ASDVDDDGNSAGG------SKRKPEAPKTISQEKLVLLEKLITDTS 157 ---GEWMIVGPLTL-APTK------RDPQGQGSAITYAKRYQLASAFGI---SSDIDDDGNACSFG----EDRQSGYQRQSAQNKSYRGQNANQGNRQ 160 TSGDGWAVID------VNKK---GMDKAQATGASQSYASKYAYGQALKLDD-IKDADSTNKGQNN--------AQRPKAVPKASYQYKLSDLKKMVAN 158 ASGDGWAVID------INKK---GMDKAQATGASQSYASKYAYGQALKLDD-TKDADSTNKGPNN--------ATQMKSRPKSNYQYNLSDLKKKVAN 158 EKAIGWAREEDVPIFHTQKGDVKQMQDPQWTGAVGSYARKYALQGLFAIG--GEDVDEYPVEESQEQGQNNQQQKPNNQQAQGQNQVRYIDNTQYQEI 172 .....b..........sp+......p.sQ..Gss.oYhp+Ysl..hhsI.....DsDs.........................p.p....p....... Figure 3 Multiple sequence alignment of the ERF protein superfamily. The coloring reflects the amino acid conservation at 85% consensus. The coloring scheme and secondary structure assignment abbreviations are as in Fig. 1. Species abbreviations are as follows: Bbcp32-4: Borrelia burgdorferi cicular plasmid (cp) 32-4, Bbcp32-7: B. burgdorferi cp 32-7, Bbcp32-8: B. burgdorferi cp 32-8, Bbcp8.3: B. burgdorferi cp 8.3, Bbcp9: B burgdorferi cp 9, bIL67: bacteriophage IL67, c2: Lactococcus phage c2, D3: Pseu- domonas phage D3, HK97: bacteriophage HK97, Lj771: Lactobacillus johnsonii prophage Lj771, Lm: Listeria monocytogenes, MM1: Streptococcus pneumoniae bacteriophage MM1, BPmv4: Bacteriophage mv4, P22: Bacteriophage P22, phiPV83: Bacteriophage φPV83, phiSLT: Staphylococcus aureus temperate phage φSLT, ST7201: Streptococcus thermophilus bacteriophage 7201, Unk: Unknown. ERF defines a superfamily of SSAPs that are evolutionarily distinct from the RecT/Redβ super family The ERF protein of phage P22 is involved in the circulari- zation of the linear dsDNA phage genome upon entry into the host cell [21–23]. Experimental studies have shown that, mutations in ERF are complemented by Redβ and that in vitro ERF adopts quaternary structures analogous to those of Redβ and RecT [14,17,24,25]. However, in the comprehensive analysis of the RecT/Redβ superfamily no statistically significant similarity could be detected be- tween these proteins and the ERF proteins. To explore the evolutionary affinities of the ERF domains, we carried out a sequence profile analysis as described above for the RecT case using transitive PSI-BLAST analysis. As a result of these searches, homologs of ERF encoded in several bacte- rial and phage genomes from diverse taxa were identified. The alignments generated in these searches consistently pointed to a region of approximately 150 amino acids that is conserved in all these proteins, with a characteristic motif of the form: GuXXoYhp + YXhXXhh (where G is gly- cine, Y-tyrosine, u is a tiny residue, h-hydrophobic, p is a polar residue, o is an alcohol residue, + is a basic residue, and X is any residue; Fig. 3). This suggested that ERF was the prototype of a family of conserved bacterial domains. Secondary structure prediction based on the multiple alignment of the ERF domain suggests a globular α + β fold with five helices and three or four strands (Fig. 3). The above-mentioned motif that is typical of this family is as- sociated with helix 4 of this domain; given the presence of conserved basic residues, it may be critical for DNA-bind- ing and strand-transfer activity of the ERF-like proteins. Page 5 of 11 (page number not for citation purposes)