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
424
2700
444
2760
464
2820
484
2880
504
2940
524
3000
544
3060
564
3120
584
3180
604
3240
624
3300
644
3360
664
3420
684
3480
704
3540
723
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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