135 Gene, 126 (1993) 135-139 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00

GENE 07008

The pub gene of Streptovnyces griseus, encoding p-aminobenzoic acid synthase, is located between genes possibly involved in candicidin biosynthesis

(Recombinant DNA; PABA synthase; thioesterases; p-coumarate CoA ligase; DNA deletions in vivof

Luis M. Criado, Juan F. Martin and Jo& A. Gil

Departamento de Ecologia, Gendtica y Microbiologia, Area de Microbiologia. Uniuersidad de L&n, Lecin, Spain

Received by K.F. Chater: 27 July 1992; Revised/Accepted: 30 October/Z November 1992; Received at publishers: 29 December 1992

SUMMARY

The nucleotide (nt) sequence of the gene (pub) encoding p-aminobenzoic acid (PABA) synthase, a key enzyme in the biosynthesis of candicidin by St~e~to~yces griseus IMRU3570, was determined and an open reading frame (ORF) of 2171 nt was found. The predicted amino acid sequence demonstrated extensive sequence identity with PABA synthases (Pab) from Gram-negative Enterobacteria. The protein encoded by ORF pub shows a clear relationship at the N terminus with PabA and at the C terminus with PabB from Escherichia coli, Serrutia and Klebsiella. We also determined the extent of a spontaneous deletion that removed the ORF located upstream from pub near the 5’ end of the cloned fragment. The deletion occurred when the gene was cloned in the BumHI site of pBR322 and allowed pab expression in E. coli.

INTRODUCTION

Candicidin (Cd) is an aromatic polyene (heptaene) mac- rolide antibiotic produced by S. griseus IMRU3570. Cd consists of a macrolide ring with seven double bonds, the amino sugar mycosamine and a p-aminoacetophenone moiety (Waksman et al., 1965). The aromatic moiety is formed from chorismic acid via PABA, and it was pro- posed that PABA-CoA serves as a starter group for head- to-tail condensation of malonyl-CoA and methylmalo-

Correspondence to: Dr. J.F. Martin, Departamento de Ecologia, Genet- ica y Mi~robiolog~a, Area de ~icrobiolog~a, Universidad de Leon, 24071 Leon, Spain. Tel. (34~87)-291505, Fax: (34-87)-291506; e-mail: JAGIL (a ELEULEl 1

Abbreviations: aa, amino acid(s); bp, base pair(s); Cd, candicidin; CoA, coenzyme A, E., Escherichia; Exo III, E. cofi exonuclease III; kb, kilo- base(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PABA, p-aminobenzoic acid; pub, gene(s) coding for Pab; Pab, PABA syn- thase(s); PolIk, Klenow (large) fragment of E. coli DNA polymerase I; R.. Rattus: RBS, ribosome-binding site(s); S., Srre~rom~~ees: Ths, thiost- erase(s); [I, denotes plasmid-carrier state.

nyl-CoA in the biosynthesis of the macrolide ring of Cd (Martin, 1977; Gil et al., 1980).

The S. gviseus gene (pab) for PABA synthase(s) (Pab) was cloned in a 4.5-kb fragment and was expressed in several ~~~e~~o~yces strains, but not in E. coli (Gil and Hopwood, 1983). Expression occurred in E. coli after deletion in vivo of 1 kb of DNA upstream from the pub

gene. The resulting 3.5-kb fragment could complement mutations in both of the unlinked pubA and pabB loci of E. coli. Recently Green and Nichols (1991) showed that the conversion of chorismic acid to PABA in E. coli

occurs in two separate protein-catalyzed steps, not one, as previously thought. PabA and PabB act together to convert chorismic acid and glutamine to a diffusible inter- mediate (aminodeoxychorismic acid), which is acted upon by a third protein (encoded by pubC) to form PABA. This protein has no counterpart in the anthranilate synthase of E. coli. The pabC gene of S. griseus, if it is present, is apparently separate from pabA and pabB.

Because DNA homologous with pub is present only in

136

. . . . . . . . . . . . . . . . . . . . . GGATCCTGTTCGGGCTGCACGRGCTGCTGCCGGTGCGCTGGTAGCCGGGCGCCGCCGACGGAC

CCCCCATCCAC~CAACGATCATGCGTGTCACCGTCGACAGCGAGCAGTGCGTA

RBS? OK=-I>> M R " T " D S E Q C"

GGAGCGGGCCAGTGCGTCCTGAACGCGCGCCGGAGGTCTTCGACCAGGACGACGACGGCGTC

GAGQCVLNAPEVFDQDDDG"

GTGGTCCTGCTCCGCGCGGACCCGACGAGCGGGRCCACCACGAGGCGGTCCGCACGGCGGGCG

""L L R A D P TSGTTRRSARRA

ACCTGTGCCCGTCGGCCTCGGTCGTCCTCCAGGAGGACTGAACCAGCCGGGTGCGCGGAC

T C AR R P RSSSRRTEPAGCAD

CGGCACCGGTGCCCCGTCCGGCCCGGCCCTCGCACCCGCGGACCCGCGGACCCGCCGCCG

R H RCPVRPGPRTRGPADPPP

GCGAACACCGACCGGCGCCAGAACCATCGRAGGGACTCCCCCGTGACCACCGCTGACGAC

ANTDRR QNHRRDSPVTTADD

ACCGCGGCCCGCTGGCTGCGGCGGTACCACCCGGCCGAGGCCGACGCGGTACGGCTGGTG

TAARWL RRYHPAEADAVRL"

TGCTTCCCGCACGCCGGCGGCTCCGCCAGCTTCTACCACCCGGTCTCGGCGCGGTTCGCG

CFPHAGGSASFYHPVSARFA

CCGGGCGCCGAGGTCGTCTCGCTCCRGTACCCCGGCGGAGCCC

P GAE""SLQYPGRQDRRKEP

TGTGTCCCGGACCTCGGCACGCTGGCCGACCTGATCACCGAGCAGCTGCTCCCGCTGGAC

C" P D L G T LADLIT EQLLPLD

GAGCGGCCCACCGTCTTCTTCGGGCACAGCATGGGGGCCGCGCTCGCCTTCGAGACGGCG

E R P T ” F FGHSMGAALAF E T A

TGGCGGCTGGAGCAGAAGGGCGCCGGTCCCCGCACCGTCATCGCCTCCGGACGGCGCGGC

WRLEQKGAGPRTVIASGRRG

CCCTCGACCACCCGCGCCGACGGGTCCACACGAGGGACGACGACGGGATCGTCGCGGAG

P STTRAEHVHTRDDDGIVAE

ATGRAGCGGCTGAATGGCACGGCGGCCGGTGTCCTCGGCGACGAGGAGATCCTCCGCATG

M KRLNGTAAGVLGDEEI L R M

GCGCTGCCCGCGCTGCGCGGCGACTACCGCGCCATCGAGACCTACACCTGCCCTCCGGAC

A L P ALRGDY RAIETY T C P P D

CGCCGGCTGGCCTGCGGGCTGACCGTGCTGACGGGCGAGGACGACCCGCTGACCACCGTC

R RLACGLTVLTGEDDPLTT"

GAGGAGGCCGAGCGGTGGCGCGACCACACCACCGGGCCGTTCCGGCTGCGGGTCTTCACG

E E A E RWRDHT T G P FRLRVFT

GGCGGGCACTTCTTCCTCACCCAGCACCTCGACGCGGTCACACGGAGATCGCCCAGGCC

G G H F FLTQHLDAVNTEIAQA

. . . . . . . . . . . . . . . . . . . . . CTCCACCCCGACCGGGCCGCCCCGGCCGCCTGAGCGTCCGGCCCGGCCGGCGGCGGGACC

L H P DRAAPAA-

GCCGGCCGGGCAGGCGCTGGAGCCGTGACCGACGCACCGCCGACGGCGTACGCGCGTACG

CGGCGGACGTCGTCCGCACCACCGGAGAGCCCGCACAGGCCCGGACGGCGATCCCGTCCG

ACCGCCTCCCCCGTCACTTTCCGCTCACGCGCC~~GGACACATGCGCACCCT

RBS? ORF-2>> M R T L

TCTCGTCGACAACTACGACTCGTTCACCTACAACCTCTCTTCCACTACCTCTCCCGGGCC~

LVDNYDSFT YNLFHYL SRAN

CGGCCGGGRACCCGAGGTCATCCGCAACGACGACGACCCGGCCTGGCGGCCGGGTCTGCTCGA

GREPEVIRNDDPAWRPGLLD

CGCGTTCGACAACGTGGTGCTCTCCCCGGGGCCGGGCACCCCGCACCGCCCGGCCGACTT

A F D NV" L SPGPGTPHRPADF

CGGCCTGTGCGCCCGGATCGCCGAGGAGGGCCGGCTGCCGGTGCTCGGCGTCTGCCTGGG

GLCARI AEEGRLPVLGVCLG

CCACCAGGGCATGGCCCTCGCCCACGGCGCCCGGGTGGGCCGGGCCCCCGAGCCCCGTCA

HQGMALAHGARVGRAPEPRH

CGGCCGCACCTCGGCGGTACGGCACGACGGCACCGGGCTCTTCGAGGGGCTGCCGCAGCC

G R T SAVRHD GTGLFEGLPQP

GCTGGAGGTGGTGCGGTACCACTCCCTCGCGGTGACGGRACTGCCGCCGGAGCTGGAGGC

L E""RYH SLAVTELPPELEA

CACCGCCTGGTCGGAGGACGGGGTCCTGATGGCGCTGCGCCACCGCACGCTGCCGCTGTG

T A W S E DGVLMALRHRTLPLW

GGGAGTGCAGTTCCACCCCGAGTCGATCGGAC

GVQFHP ESIGTQDGHRLLAN

CTTCCGCGACCTCACCGAGCGCCACGGCCGRACGCGCGCCACGGCGGCCGGGCGGGACACGG

F R D L T E R H G RTRHGGRAGHG

CACGCTCCCGCCCCCCGCGCCCGCCCGGGAGACGAAGGCCACCACCGGCACCCCACGGCG

T L P P PAPARE T K A T TGTPRR

GCTCCGGGTCATCGCAAAGTCGCTGCCCACGCGCTGGGACGCCGAGGTCGCCTTCGACTC

LRVIAKSLPT RWDAEVAFDS

GCTGTTCCGCACCGGCGACCACCCCTTCTGGCTCGACAGCAGCCGTCCC~~~~~~~A~CT

L F R T G D HPFWLD SSRPGGEL

GGGCCAGCTCTCCATGATGGGCGACGCCTCAGGTCCCCTCGCCCGGACCGCC~GGCCGA

GQLSMMGDASGPLARTAKAD

CGTGCACGCCGGAACCGTCACGGTGAGAGCCGACGGCGCCAGCAGCACGGTCGAGAGCGC

"HAGTVTVRADGASSTVESA

CTTCCTGACCTGGCTGGAGAACGACCTGGCGGGGCTGCGCACCGAGGTGCCCG~CTTCC

F L T W L E NDLAGLRT E" P E L P

Fig. 1. Nucleotide sequence and predicted ORFs of the 4.5kb S. griseus fragment carrying the pabAB gene (The GenBank accession No. is M93058).

Putative RBSs are in boxes. Transcription terminators are represented by facing arrows, The dotted nt in the 5’ end and around nt 1164 are regions

of partial homology where recombination appears to have occurred. The asterisk and double asterisks denote the PuuII sites. The aa motif GXSXG

present in Ths is underlined. Methods: The fragment was sequenced using the dideoxy nucleotide chain termination method of Sanger et al. (1977;

1980). Restriction fragments from the 4.5 and 3.5.kb fragments were subcloned in the sequencing vectors directly or after treatment with Exo III, Sl

and PolIk to create deletions (Erase-a-Base, Promega Corporation, Madison, WI, USA). pBluescript KS(+) and KS(-) (Stratagene, La Jolla, CA,

USA) and Ml3mplO and 11 (Messing and Vieira, 1982) were used as sequencing vectors. Ml3K07 (Vieira and Messing, 1987) was used as helper

phage. [35S]dATP (Amersham, Bucks., UK) and Sequenase (US Biochemical, Cleveland, OH) were used instead of [a-32P]dATP and PolIk. To avoid

the compressions found when sequencing Streptomyces DNA, dITP was used instead of dGTP (Mills and Kramer, 1979) and Tuq polymerase instead

of T7 DNA polymerase. Data analysis and homology studies were done with the DNASTAR program (DNASTAR, London, UK).

1320

1380

4

1440

24

1500

44

1560

64

1620

84

1680

104

1740

124

1800

144

1860

164

1920

184

1980

204

2040

224

2100

244

2160

264

2220

284

2280

304

2340

324

CATCTCTAGGG-CACGGGCCCCGTGGCAAGAGCCTGG~CCGTTCCCCCCTCTGACG

GCGACGCTCGACATCC4;GGTGATGGTGCCCGTCCACGCACACGACTACGTGACCGAT

ORF3>> RBS? M"P"HAHDY"TD

CCGCCCTCCACCACCGGCAGGACCCTGGACGGGCTGACGCTGCCACGGGTGTTCGCCGAC

P P STTGRT LDGLTLP R" F A D

GCCGTGCACCGGGGCGGCGACGCCGTGGCCCTGGTGGACGGGGAGTACGCCCTGACCTGG

AVHRGGDAVALVDGEYALTW

AGCGCCTGGCGGACGGCGGTGGACGCGCTGGCGCGCGGCCTCCAGGAGTCCGGCGTCGTC

SAWRTAVDALARGLQESGVV

: TCCGGCGACGTGGTGGCCCTGCACCTGCCCAACAGCTGGGGAGTACCTGACGCTCCATCTG

SGDVVALHLPNSWEYLTLHL

GCCGCCGCCTCGGTCGGGGCGGTCACGATGCCGGTGCACCAGGGC~CGCTCCCTCGGAC

AAASVGAVTMPVHQGNAPSD

GTCCGGGCCCTGCTGGAACGGGTCCGGCCCGCCGCCGTCGTCCTGACGGCGCGGACCCAG

" RALLERVRPAAVVLTARTQ

GAGGGTGGGGGCCCGCTCACCGGCCCGGCGCTGCGCGAGGTCCTGCCCGAGCTGCGCGCC

E G G G P LTGPALREVLPELRA

GTGCTCGTCACGGGCGACGCGGCGGGCGAGGGCACCGAGACGGTGACCGAGATGCTGGAG

"LVTGDAAGEGT E TV T EM L E

CGGTGGTCCGGAGAGGACCCGCTGCCCGTCGAGGTGCGCCCGGACTCGCCGTTCCTGCTG

R W S G E D P I. P" E" R P D S P F L L

CTGCCGTCCTCGGGCACCACCTCGGCGCGGCCCAAGATCTGCCTCCACTCGCACGAGGGG

L P SSGTTSARPKICLHSHEG

CTGCTCACCAACTCCCGGGCCGCCACCGAGGACACCGCGGACGCCTACGCGGGCACCCTG

L L T N S R A A T E DTADAYAGTL

ATCACCGCGTGCCCCCTGACCCACTGCTTCGGCCTCCAGTCGGCGTACTCGGCGCTCTTC

I T A C P L T H C FGLQSAY S A L F

CGCGCCGGCCGCCAGGTGCTGCTGTCCGGGTGGGACGTGGGCCGGTTCCTGGAGCTGGCC

RAGRQVLLSGWDVGRFLELA

CGCCGGGAGCGGCCCAGCGTGGTGGTGGCGGTCCCCGCCAGCTGCACGACCTGGTCACCC

R R E R P S"""A"PASCTTWSP

GGGTGCGCGAGGACGCGGACGGCCCCGGCTTCGGCCCGGCCGGATCC

GCARTRTAPASARPD

3660

3720

12

3780

32

3840

52

3900

72

3960

92

4020

112

4080

132

4140

152

4200

172

4260

192

4320

212

4380

232

4440

252

4500

272

4560

292

4607

307

60

120

11

180

31

240

51

300

71

360

91

420

111

480

131

540

151

600

171

660

191

720

211

780

231

840

251

900

271

960

291

1020

311

1080

331

,140

351

1200

361

1260

CTTCGCGTTCGCGCTCGGCTGGGTCGGCTGCCTGGGCTACGAGTTG~GGCCGAGTGCGA

FAFALGWVGCLGYELKAECD

CGGCGACGCCGCGCACCGCTCGCCCGATCCCGACGCCGTGCTGGTCTTCGCCGACCGGGC

GDAAHRSPDP DAVLVFADRA

CCTGGTG~TGGACCAC~G~AC~~G~ACCA~~TA~~TG~TGGCG~TGGTGGAGGA~GACGC

L" L D HRTRTTYLLALVEDDA

CGAGGCCGAGGCGCGCGCCTGGCTCGCGGCGGCCTCCGCCACCCTGGACGCCGTCGCCGG

EAEARAWLAAASATLDAVAG

G~GGGAGC~~GAGCCGTG~CCCGAGG~GCC~GTGTG~A~GA~GGGT~~GGTGGAG~TG~G

R E P E P CPEAPVCTT GPVELR

CCACGACCGGGACGGCTACCTGRAGCTGATCGATCGACGTCTGCCAGCAGGAGATAGCCGCCGG

H 0 RDGYLKLIDVCQQEIRAG

GGAGACCTACGAGGTCTGCCTGACCAACATGGCCGAGGCCGAGGCGGACACCGACCTCACCCCGTG

E T Y E" c LTNMAEADT D L T P W

GGCGGCCTACCGCGCGCTGCGCCGGGTGAGCCCCGCCCCGTTCGCCGCGTTCCTGGACTT

A A Y RALRRVSPAPFAAF L D F

CGGTCCCATGGCCGTGCTCAGCAGCTCTCCGGAGCGGTTCCTGCGCATCGACCGGCACGG

GPMAVLSSSP ERFLRI D R H G

GCGGATGGAGTCCAAGCCGATCAAGGGGCGGCCACGCGGCCACGCGGCGCCACCCCGCAGGAGGA

RMESKPI KGTRPRGAT P * E D

CGCCGCGCTCGTACGTGCCCTGGCCACCTGCGAGAAGGACCCTGATGAT

A A L" RALATC EKDRAENLMI

CGTCGACCTGGTCCGCCACGACCTGGGGCGGTGCGCCGAGGTCGGCTCGGTCGTCGCCGA

"D LVRHDLG RCAE"GS""AD

* CCCGGTGTTCCAGGTCGAGACGTACGCGACCGTGCACCAGCTGGTCAGTACCGTCACGGC

P "FQVETYATVHQLVSTVTA

GCGGCTGCGCGAGGACAGCAGCCCGGTGGCGGCGGTCCGGGCGGCCTTCCCCGGCGGGTC

R L R E D s S PVAAVRAAFP G G S

GATGACCGGGGCGCCGAAGATCCGCACCATGCAGATCATCGACCGGCTGG~GGCGGGCC

MTGAPKI RTMQII DRLEGGP

GCGCGGTGTCTACTCGGGCGCCATCGGCTACTTCTCCCTCACCGGCGCGGTAGACCTGTC

RGVYSGAIGYFSLTGAVDLS

CATCGTGATCCGCACGGTGGTGCTCAGCGGCGGCAGGCTGCGCTACGGCGTCGGCGGCGC

I" I RTVVLSGGRLRYGVGGA

CGTCATCGCGCTCTCCGACCCGGCCGACGAGTTCGAGGAGACGGCGGTC~GGCCGCCCC

" I ALSDPADEFE ETAVKAAP

GCTGCTGCGTCTCCTCGACACCGCCTTCCCGGGCCGTGAGCGTCCCGGC~GGACCTCGA

L L R L L D T A F PGRERPGKDLD

CGGTGAACCCGACGACGGCACGGACGCGGGTGCTCCGAAGGACCTCGTCCTGCCCGGGTG

GEPDDGTDAGRPKD LVLPG-

ACCTCACGGGTGGGGCCGGCGGTCCGGCGGGCCGGGGCCCCGCGCGGCCGGCACCGGCCG

2400

344

2460

364

2520

384

2580

404

2640

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2700

444

2760

464

2820

484

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524

3000

544

3060

564

3120

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604

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624

3300

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684

3480

704

3540

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3600

:

13

*

S-end of 4.5 kb fragment

1 GGATCCTGTTCGEGCTGCACGAGCTGCCGGTGC... III II III I III I

1163ntregion . . . GGCCGCCCCGGCCGCCTGAGCGTCCGGCCC... 4.4

+

see~~~~3e;l:kb ~GGATCCTGTTCG~GCCGCCTGAGCGTCCGGCCC... *

Fig. 2. Nucleotide sequences of relevant regions at the 5’ end of the

4%kb fragment, in the 1164-bp region and at the 5’ end of the deleted

3.5-kb fragment.

strains that produce aromatic polyene antibiotics (Gil et al, 1991), it has also been used to find new producers of such compounds.

The aim of this study was to sequence the pub gene, to analyse genes located around pub and to determine a plausible molecular mechanism for the occurrence of the 1 -kb deletion.

EXPERIMENTAL AND DISCUSSION

(a) Nucleotide sequence of the pub gene from

Streptomyces griseus The sequence of the entire cloned 4.5-kb insert of

pIJ814 was determined (Fig. 1). The sequence across the deletion giving rise to pIJ819, the in vivo derivative of pIJ814 that complements E. coli pub,4 and pabl? mu- tations, was also determined and revealed that the dele- tion occurred at or around nt 13 and 1164 of the 4.5-kb

PabA

MRTLLVDNYDSFTYNLFHYLSRANGREPEVIRNDDPAWRPGLLDAFDNW

M-ILLIDNYDSFTwNLYQYFc-El,GAD”Lviw.NDALTLADIDALKPQKI”

M-LLLIDNYDSFTYNLYQYFc-ELG*~“““KRNDEL*LTDIERLAP*“L” * **.*******.**.,*., * * *+*. .._*

LSPGPGTP”RPADFGLCARIAEEGRLP”LG”cLGfiQGMALAHGAR”GRAP

ISPGPCTPDE-AGISLDVIRHYAGRLPILGVCLGHQAMAOAFGGKVVRAA

ISPGPCTPND-AGISLAVIRHYAGRIPMLGVCLGH&MA~AFGASVVRAA

I.SPGPCTPDE-SGIsLAAIRHFSGQTPILGVCLGHQAIAQVFGAAI"RAA

ISPGPCTPNE-AGISVAAIRHFAGKLPILGVCLGHQALGQAFGAEWRAR

.**** **. .*. *,**.*****,_, *, **

EPRHGRTsAVRHDGTGLFEGLPQPLEVVRYHSLAVTE--LPPsL~ATAWs

KVMHGKTSPITHNGEGVFRGLANPLTVTRYHSLVVEPDSLPACFDVTAWS

KVMHGKTsPVTHNGQGVFRGLpSpLTVTRY~SLI"DPATLp~CFEITANs

KVMHGKTSPVSHTGQGVFLGLNNPLTVTRYHSLLIDPRTLPECFEVTARS

A"MHGKTSAIRHLGVG"FRGLsDpLTVTRY"SLVLKADTLpDCFE"TAWS ,**,**,, * * *,* ** **,*_*I*** ,_ ** l *.*

EDG"L----MALRHRTLPLWGVQFHPESIGTQDGHRLLANFRDLTERHGR ETR----EIMGIRHRQWDLEGVQFHpEsILSEQGHQLLANFLH------R

ETQ----EIMGIRHREWoLEGVQFHPESlLSEQGHALLKNFLR------R

EEG----EIMGIRHRVFDLEGVQF~P~SILSEQG~QLL~FLN------R

ERDGVRDEIMGIRHRALALEGVQFHPEsVLS~QGHQLLDNFLN------R * *..*** * ********. __,** ** ** *

50

48

48

48

48

100

97

97

97

97

148

147

147

147

147

194

187

187

187

191

Fig. 3. Sequence relationships between PabAB from S. griseus and the

PabA proteins from listed microorganisms. The aa sequences were com-

pared using the CLUSTAL (PCGENE) program. K-tuple, 1; gap

penalty, 5; window size, 10; filtering level, 2.5; open gap cost, 10 and

unit gap cost, 10. (*) identical aa; (.) similar aa. Sg, S. griseus; EC, E. coli; St, S. typhimurium; Ka, Klehsielfa aerogenes; Sm, Serratia marcescens.

PabB

sL7 Ec St Ka

.% EC St Ka

sg EC St Ka

sg EC St Ka

%I EC St .Ka

sg EC St Ka

sg EC St

Ka

Sg EC

St

Ka

Sg EC

St

Ka

Sg EC

St

Ka

Sg EC

St

Ifa

---DAEVAFDSLFRTGDHPFWLDSSRPGGELGQLSMMGDASGPLAR----

M-KTLSPAVITLLWRQDAAEFYFSRLSHLPWAMLLHSGYADHPYSRFDIV

MMKTLSPTVITLPWRPDAAEHYFAPVNHLPWAMLLHSGDAIHPYNRFDIL

M---LSPAMISLPWRPDAAEYYFSPLSSQPWA"LLHSGFAEHAHNRFDII

_*_*. _* ** ._*

----TAKADV"AGTVT"RADGASSTVESAFLTWLENDLAGLRTEV-PELP

VAEPICTLTTFGKETWSESEKRTTTTDDPLQVLQQVLDRAD*RpTHNE~

"ADP"TTLTTRAQETT"CTARTTTVTLDDPL""LQTQLEALPPHPQPDpD

~AQPRATLVTHGQLTTLREGETVSTSAADPLTLVHQQLAHCNLQPQPHPH

..* *.

FAFALGWVGCLGYELKAECDGDAAHRSPD---PDAVLVFADRAL"LDHRT

LPFQGGALGLFGYDLGRRFESLPEIAEQDIVLPDMRVGIYDWALIVDHQR

LPFQGGALGLFGYDLGRRFE~LPDTAARDIALPDMRIGLYDWALI"~~QK

LPFLGGALGLFGYDLGRRFEHLPARADADIELPDMAVGIYDWALIVDHQR

..* * .* .**.* * ** __ *_**..**_

RTTYLLALVEDDAEAEARAWLAAASATLDAVAGREPEPCPEAPVCTTGPV

HTVSLLSHNDVNAR---RAWtESQQFS-------------PQEDFTLTSDW

OVVSLISYHDADAR---YRWLTSORAP------------TRTPFRLTSAW

REVSLFSYDDPQAR---LAWLEAQTAP------------~AATFTLT~AW

..*.. ..* **... *_

ELRHDRDGYLKLIDVCQQEIAAGETYEVCLTNMAEADTDLTPWAAYRALR

OSNMTREOYGEKFROVOEYLHSGDCYQVNLAQRFHATYSGDEWOAFLQLN

QSNMTRCEYGEKFRQVQAWLHSGDCYQVNLSQRFQASYEGDEWQAFERLN

RANMSREEYGEKFRQIQAYLHSGDCYoVNLRQRFTATYRGLN

..* *__ *..:_*.**... *. .*.t. '.

RVSPAPFAAFLDFGPMAVLSSSPERFLRIDRHGRMESKPIKGTRPRGATP

QANRAPFSAFLRLEQGAILSLSPERFILCD-NSEIQTRPIKGTLPRLPDP

RANRApFSAFLRLHDGAILSLSPERFIQLE-NGHIQTRPIKGTLPRLNDP

RANRAPFSAFIRLDEGAVLSLSp~RFIQLR-QGD~QTRPIKGTLPRLADp

. ...*'*.**. l .** *****. . ...***** ** .*

QEDAALVRALATCEKDRAENLMIVDLVRHDLGRCAEVGSVVADPVFQVET

QEDSKQAVKLANSAKDRAENLMIVDLMRNDIGRVAVAGSVKVPELFVVEP

QADRQQAQKLANSMKDRRENLMIVDLMRNDIGRVAVPGSVKVPELFVVEP

EQDALQQQRLANSPKDRAENLMIVDLMRNDIGRVAEPGSVR"P~LF"V~P

..* l *_, ,*******+,**~i,*~** \ *** .* **.

YATVHQLVSTVTARLREDSSPVAAVRAAFPGGS~TGAPKIRT~QI~DRLE

FPAVHHLVSTITAQLPEQLHASDLLRAAFPGGSITGAPKVRAMEIIDELE

FPAVHHLVSTITRRLPDSLHATDLLRARFPGGSITGAPKV~~ID~L~

FPAVHHLVSTVTARLPAHLHAADLLRAAFPGGSITGAPKVRAMEIIDELE ,..**,****.*t,*.. .********,t****,*,*,*** **

GGPRGVYSGAlGYFSLTGAVDLSIVIRTWLSGGRLRYGVGGAVIALSDP

PQRRNAWCGSIGYLSFCGNMDTSITIRTLTAINGQlFCSAGGGIVADSQE

PQRRNAWCGSIGYLSFCGKMDTS~TIRTVTATQGQLYCSAGGGIVADSNE

pQRRNAWCGSIGYLSFCGNMDSSITIRTLTAWQGHLYCSRGGGI"A~S~~

.*,.._*_***.*_ * .* **_***.. +_. .._**.. * *.

ADEFEETAVKAAPLLRLLDTAFPGRERPGKDLDGEPDDGTDAGAPK~LV~ EAEyQ~TF~~vNR~LKQL~~_-_____--______--____-________

~~~yQ~~~~~“*~~~~p~~~---____----__________--_______

AAEyQETFDKVNRILHQLE~_-_____________-______________

,*. .** *__._*_ *.,

PG 723

-- 453

-- 454

-- 451

137

279

49

50

47

324

99

100

97

371

149

150

147

421

184

185

182

471

234

235

232

521

283

284

281

571

333

334

331

621

383

384

381

671

433

434

431

721

453

454

451

Fig. 4. Sequence relationships between PabAB from S. griseus and the

PabB proteins from the listed microorganisms. See Fig. 3 legend for

parameters and microorganisms used in this comparison.

fragment of S. griseus DNA. In each of these regions there is a series of identical nt that form part of a longer sequence of 21 nt with partial identity (Fig. 2). In vivo deletions resulting in the novel expression of Streptomyces

genes in E. coli by recombination between short DNA repeats has already been described (Albertini et al., 1982; Murray et al., 1989). The presence of a hairpin-loop immediately downstream from the recombination site (nt 118 1 to 12 11; AG = - 44.2 kcal) may also be involved in the deletion event. This hairpin-loop might be a transcrip- tion terminator of the first ORF of the sequenced DNA because it is located 7 nt downstream from the stop codon.

138

Three ORFs were found in the 4.5-kb fragment (Fig. 1);

they showed the marked asymmetry in mol% G + C at

each codon position expected for a typical Streptomyces gene (Bibb et al., 1984). The pub ORF should be ORF-2

since it is the only complete ORF present in the 3.5-kb

fragment that complemented pabA and pabB mutations

in E. coli. The 2-kb BamHI-PuuII* region subcloned from

pIJ819 complemented only E. coli AB3292 (pabA) whereas the 2.7-kb BamHI-PvuII$ region complemented

both E. coli AB3292 (pubA) and AB3295 (pabB) (for posi-

tions of PuuII sites marked * and g see Fig. 1). Moreover,

a putative terminator (AG= - 17.3 kcal) was found at

the end of ORF-2.

(b) Sequence identity between thepab gene product Pab,

and enterobacterial Pab

Alignment of the Pab aa sequence with those for Pab

of enterobacteria indicates strong sequence identity

between the N end of the S. griseus enzyme and PabA

from E. coli, Salmonella typhimurium, Klebsiella uerogenes and Serratia marcescens (Fig. 3). In 200 aa 129 residues

(64.5%) are similar in all four proteins. The C end of the

deduced S. griseus aa sequence showed marked identity

with PabB from E. coli, S. typhimurium and K. aerogenes (Fig. 4). In 502 aa there are 283 similar (56.5%) aa posi-

tions. Thus, pab from S. griseus encodes the PabA and

PabB functions in a single gene, and is therefore named

pabAB. The Pab sequence from S. griseus has 723 aa and

an deduced M, of 77 900. The purified Pab from S. griseus or from E. coli[pIJ819] has an M, of 60 000. The differ-

ence in M, may be due to an abnormal behavior of the

enzyme in the calibration column. Pab purified from S.

liuiduns had a M, of 47 000 (Rebollo, 1987) and presuma-

bly consisted of only the PabB component (see the

accompanying paper by Arhin and Vining, 1993).

(c) Evidence that pabAB may be located between genes

involved in Cd biosynthesis

Alignment of the predicted aa sequence of the product

of ORFl (protein X) with sequences in the PIR data bank

of DNASTAR showed similarities with gramicidin-S

synthetase from Bacillus breuis (74% similarity in an over-

lap of 225 aa) and with the thioesterase-II-S-acyl fatty

acid synthase from Ruttus norvegicus (72.6% similarity in

a overlap of 223 aa). ORF-1 encodes a polypeptide of

361 aa with a deduced M, of 39 559, slightly higher than

the Ths of B. brevis (29 191) or R. norvegicus (29 471) (Kratzschmar et al., 1989). The active-site Ser residue that

is a part of the motif GXSXG in Ths was found in protein

X (Fig. 5). Thus, protein X may be a Ths involved in the

biosynthesis of Cd. An enzyme with this activity should

be needed at the end of polyketide synthesis to release

Bb

Fvi

sq

CF

RF

R2

PA

Ca

PI

An

SBG S Y

S R GMT

S R G S T

L G L

L

L

Fig. 5. Sequence similarity between the deduced product of ORF-I

(protein-X) and Ths from listed organisms. Data were taken from

Gutierrez et al. (199!), Kratzschmar et al. (1989), Naggert et al. (1987).

Bh, Bacillus hrevis gramicidin-S synthetase; Rn, R. noruegicus S-acyl

FAS; Sg, S. griseus protein-X; GF, goose FAS Ths; CF, chick FAS Ths;

RF, R. norvegicus FAS Ths; R2, R. norvegicus FAS ThsII; PA; Penicil-

lium chrysogenum ACV synthetase; Ca, Cephalosporium acremonium

ACV synthetase; PI, P. chrysogenum IPN acyhransferase; An, Aspergil-

lus nidulans IPN acyltransferase.

the macrolide ring from the polyketide synthase

(Hopwood and Sherman, 1990).

Alignment of the truncated protein Y (encoded by

ORF-3) with the protein data bank showed similarity to

p-coumarate:CoA ligase from Petroselinum hortense (parsley), with 61.7% similarity in an overlap of 269 aa

(Fig. 6). p-Coumarate:CoA ligase is a key enzyme in the

biosynthesis of plant polyketide secondary metabolites,

such as flavonoids (Lozoya et al., 1988). Thus, ORF-3

may code for a PABA-CoA ligase that activates PABA

to start the biosynthesis of Cd. Such enzymatic activity

was predicted by Martin (1977).

(d) Conclusions

(1) The pubAB gene of the Cd-producer Streptomyces griseus IMRU3570 codes for a protein with two domains:

a PabA domain at the N end and a PabB domain at the

C end.

(2) The in vivo deletion of a I-kb DNA segment, when

propagated in E. coli, could have been due to recombina-

tion between short direct repeats.

(3) A gene upstream from pabAB seems to code for a

Ths. Such an activity may be involved in the release of

the completed macrolide ring of Cd from the polyketide

synthase.

139

(4) A gene downstream from pabAB (not complete in the cloned fragment) may code for a PABA-CoA ligase, the function of which would be activation of PABA for use as a starter unit in the head-to-tail condensation of malonyl-CoA and methylmalonyl-CoA.

ACKNOWLEDGEMENTS

This work was supported by grants from Junta de Cas- tilla y Lecin (Ref. 1203/90) and CICYT, Madrid (Bio 90- 0556). L.M.C. acknowledges the facilities provided by Antibibticos-Farma (Madrid) to carry out part of this work. We thank Dr. Francisco Malpartida for help in analysis of the pub nt sequence.

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