JOURAUL OF BACTERIOLOGY, OCt. 1992, p. 6699-6702
Vol. 174, No. 20
0021-9193/92/206699-04$02.00/0 Copyright © 1992, American Society for Microbiology
Determination of the Sequence of spaE and Identification of a Promoter in the Subtilin (spa) Operon in Bacillus subtilis YONG JOON CHUNG AND J. NORMAN HANSEN* Department of Chemistpy and Biochemistry, University of Maryland, College Parkc Maryland 20742 Received 18 February 1992/Accepted 10 August 1992
An 851-residue open reading frame (ORF) called SpaE has been discovered in the subtilin (spa) operon. Interruption of this ORF with a chloramphenicol acetyltransferase gene destroys the ability of Bacilus subtilis LH45Ac (a derivative ofB. subtilis 168) to produce subtilin, which is an antimicrobial peptide belonging to the class of ribosomally synthesized peptide antibiotics called lantibiotics. SpaE shows strong homology to NisB, which is in the nisin (nis) operon in Lactococcus lactis ATCC 11454. Despite the strong sequence homology between SpaE and NisB, the spaE and nisB genes occupy very different locations in their respective operons, indicating that they have been evolving separately for a long time. Primer extension analysis was employed to identify a promoter upstream from the spaE gene, which appears to define the 5' end of the spa operon, which contains four other ORFs (Y. J. Chung, M. T. Steen, and J. N. Hansen, J. Bacteriol. 174:1417-1422, 1992).
Nisin and subtilin are structurally homologous members of a class of antimicrobial peptides called lantibiotics. Nisin is a 34-residue peptide produced by Lactococcus lactis ATCC 11454 (6), and subtilin is a 32-residue peptide produced by Bacillus subtilis ATCC 6633 (5). Nisin, subtilin, and the other lantibiotics contain unusual lanthionine and dehydro residues and are formed from ribosomally synthesized peptide precursors that undergo extensive posttranslational modification and export outside the cell. The genes for the prepeptides of subtilin and of nisin have been cloned, and their kinetics of expression have been characterized (1, 2). Because lantibiotics have novel structures, it is probable that the maturation pathway involves genes that encode novel proteins. We have been searching for genes involved with lantibiotic maturation within the operons that contain the genes for the precursor peptides. The nisin structural gene (nisA) appears to be cotranscribed with a downstream gene (nisB) (11), and the subtilin structural gene (spaS) is in the same transcriptional unit as several genes (spaD, spaB, and spaC) that lie upstream from spaS (3). The deduced SpaB protein sequence shows extensive homology to a variety of transport proteins, including the hemolysin B and multidrug resistance proteins (3), and may be involved in subtilin export. The nucleotide sequence upstream from the previously sequenced (3) spa genes was determined by cloning restriction fragments into M13mpl8 or M13mpl9 and sequencing by the dideoxy chain termination method with the Sequenase kit supplied by U.S. Biochemical Corp. (Cleveland, Ohio). DNA was isolated by standard methods (9). Long restriction fragments were cloned into pTZ18R and incrementally shortened with the Erase-a-base kit supplied by Promega Corp. (Madison, Wis.). The shortened plasmids were purified with GeneClean provided by Bio 101 (La Jolla, Calif.). To obtain the DNA containing this upstream region, a synthetic oligonucleotide based on the sequence of the 5' end of the spaD gene was used as a hybridization probe to clone a 10-kb PstI restriction fragment that contained the 5' end of spaD and its upstream flanking region. The bacterial strain used was LH45, which is a chloramphenicol-resistant *
Corresponding author.
subtilin-producing strain of B. subtilis (8) derived from B. subtilis 168 by transformation with DNA from B. subtilis ATCC 6633, which is the natural producer of subtilin. Strain LH45Ac is a chloramphenicol-sensitive derivative of LH45 derived by spontaneous loss of the cat gene (8). PstI fragments of genomic DNA prepared from B. subtilis LH45 were size selected on agarose and cloned into Eschenichia coli, and clones containing the 10-kb PstI fragment were identified by hybridization with the probe. This fragment was mapped with restriction enzymes, subcloned, and partially sequenced. Some primers were synthesized with a Biosearch model 8500 synthesizer. Approximately 3.5 kb of the sequence is shown in Fig. 1. The sequence reveals an 851-residue open reading frame (ORF). The gene for this ORF is designated spaE, and the deduced protein sequence is designated SpaE. The lack of any obvious terminator sequence between the spaE gene and its immediately downstream spaD gene argues that spaE and spaD are in the same transcriptional unit. Further sequence analysis revealed an intergenic space upstream from the spaE gene. This space contained a promoter-like sequence, and upstream from this was an inverted repeat with characteristics of a p-independent terminator. This putative terminator is located immediately downstream from an ORF called ORF X since its sequence is incomplete and it is not yet known whether it is involved with subtilin biosynthesis. The presence of this terminator suggests that expression of the spa operon is dependent on the promoter sequence that lies between this terminator and the spaE gene. Experiments to identify this promoter are described below. Interruption of the spaE gene prevents production of active subtilin. The role of the spaE gene in subtilin production was tested by integration of a cat gene into the SpaE coding region, thus interrupting the continuity of the reading frame. The strategy was similar to that previously used to show that the spaB gene is required for subtilin production (3). The spaE-containing SstI-PstI restriction fragment (see Fig. 2) was cloned into pTZ18R, and then the cat gene was cloned into the XbaI site of the insert. This plasmid was linearized by restriction with SstI and PstI, introduced into LH45Ac cells by transformation, and selected on chloramphenicol plates. The transformants should be the result of integration 6699
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J. BACrERIOL.
NOTES
TTCGGCCGCCACTTTCTCATGTCGGTTTGCGGCGTATTGTTG7CCGCTGTTGTCGGGGTTCCGGTCGGAATCCTCATTGCGCATTATCGCCGCTTATCA ORF X-->H
F
M
L
S
V
Y
G
V
L
F
A
A
V
V
G
V
P
V
G
L
I
I
A
H
R
Y
L
R
TTGTAA79CT7GCAT GGGGCTCGGCGCAAACACCGTG 198
A W V F A V T N V I Q T I P A L A M L A V A TTAT TCATTA7TF CATTCCC707GCCGA700A0CGAAACAC9TACACAGrCA I I S L F L Y S L L P I I R N T Y T G I V
L
ATGGGCA7GACGAAA7TCCAGGTTCGCCGGAG
GTTGAGCTCCCG
M
L
V
M
G
L
A
G
N
FIG. 1. Nucleotide sequence of the spaE gene. The organization of the spaE gene within the spa operon is shown in Fig. 2. The transcription start site of the spaE gene was determined by primer extension (Fig. 4) to give an mRNA that begins with the sequence 5'-GAGTATTGTAAATA--*. The -10 and -35 regions of the promoter that correspond to this start site are underlined. Upstream from spaE is part of an ORF of unidentified function (ORF X). Immediately downstream from ORF X is an inverted repeat (underlined) with a stretch of T residues that is typical of a p-independent terminator. The translation start codon for the SpaE ORF is shown as the first methionine downstream from the transcription start site, although the second methionine has a better ribosome binding site. The deduced protein sequence from the SpaE ORF has strong homology to the NisB ORF from the nis operon (Fig. 3). The EcoRI restriction site in ORF X shown in Fig. 2 is located approximately 10 nucleotides upstream from residue 1 of the sequence. a, the sequence of the rest of the operon, including spaD, spaB, spaC (3), and spaS (2), has been published previously.
99
S
GCCTGGGTGTTCGCCGTAACAAATGTTATCCAGACAATCCCGTCGCTCGCGA7GTGGCCG
T V
AGCA7TG9CA9CTACTTAGAATACGGGAAAAGCA 297 S
I
H
E
A
Y
L
E
G K
S
A
ITTATGGCAGGCTTGCGCACGGCTCGTCATTGCCATC
396
Q V L R M V E L P L A L S V I M A G L R T A L V I A I CTCAA ACGCG ACA AACGGAAC7CCGAGATT7CTCGCA 495 GGAATTACAGCAA7CGGCAC0T6G7 TCGGCGCCG GTGG9 ATATGA7TGCCGCGG
M
G
T
M
K
F
G I T A I G T F V G A GGCGCGACCCCGACTGCGGTAAT G A T P T A V M A I G
G L G D M I V R G S N A T N G T A I I L A GGCTAGGAG_ wTTCTTGAATICCGGTGAAACAAAAAAGCAGAAGAAAG 5 94 D L I M A W I E R F L N P V K Q K S R R K 9TAA7AAGTTGA7AGA97rAAGA AIY AGA'rlTG 5FGTG -.AT TATITACTGGT7GGA70=ATT7TT1TT 693 V I S V -35 inverted repoat rAATAGAATAATATGG GGAoCATAAGCAGAGGGATGTAGAATTGTTrnATT 792 ATTTTAGAATvAG&,AAATATGA(TATTATAAATArrl -10 I------------ > mRNA G
A
AAGAAGCGGATAAATARGlGGCTATAGACTAATrA7GAcTTATATACACCTACAGATTATTATATIGATTCGGGTTCCTATCATuCAAGCr 891 spaE--> M K S L Y T P T D Y Y M I R V P L V H Q D L TAAAGGATTAGTT CGATCGTTTCTACTrGACGTClATTG;AACAAATACTIGGTATCTAGCAGGACACTAT 990 K N E N S-Q D I D Q L L H D L C N D S L F R E Q I L V S S R T L Y AT,GAAACAATACATACntV7GAGCGCCGGATAAATTAAAAGGGAAAAAAAAGCGCAACTrTCAACAGGCTATTTTGA GTATGCAACAAGAAGAG 1089 E T I H T F L Q A P D K L K G K K K R N F Q Q A I L K Y A T R R A CAACAAGAACCACAC TTCATTTTACAAAA 1188 T R T T P F G L F S S V G I G S F S D K N H L S F N Q H S F Y K K AGGCTCGTGTTGA?TG A rGTrCATATrGAATGAAGAATACACCGACCGACTTTrCTTTTACATTAAA7TTCGCTTGTTATA 1287 A R V D F E W L Y Q L I R K L E N E Y T D R L S F T L N S A C Y I kh TrrAAGGGTIGACCGGGcTTACTTrGTTGTACAGCICA sAGTTAGTGTTCG'GTrGCGACATCMTGTTTCTATMT rGA ;ATAAATGAACTGT K G D R A Y L L Y S T D G K S E E V S V R A T S V F Y L I N E L C ,TCCAhATACTMCC CAL'kTAAATAAAATTAATICAOTA( 'GTAGCAGACCTTA GTGGTG;AATCTGCT'GCATATCAAGATATAATX 'CGTTTTATASTACAkT G E S A A Y Q D I I R C L I D N Y P N T P I N K I N Q Y V A D L I TACAA CTTAG( ;CCGCCGATGACTGCTrlrCAGAI,TCAATTTCAGTA, MTTAATTGA7CAAGCOGAAAGC .CGCCATATTCCAA TTrACAGAGTcA D K E F L I S N L R P P M T V S D Q F Q Y L I D Q A E S R H I P N ATIGAACTCATTCAGGCTTGTAAGAACATITCA. kTATCAGATAGATGCQTATAUz MTCGGATICACTATR :GGAGAGGGAGAGCAT'CAGTAI rIAAA7C'TGATTG E L I Q A c K N I Q Y Q I D A Y N R I T I G E G E H Q Y L N L I E AMAT.AAAAC _TAG(CATAC MCCTCTGCAAGTAGACGCTG= X;GCTG GAGACTC( ItCCATTCAArrAGATAA7r.AJ LkCATC7C'TCGCCA T M N K L I K A S S P L Q V D A G A G D S S I Q L D N E T S L A I rAAGTGAATT'GGCAAGCTTTAC TATT ;GCTtUTC7t=CTGCCATACJATTAGACCACTT GAAAAATACAAA AATGTATT n'PGGAACGCTATG S E L A S M F T Y M A S P S A N T L D H L E K Y K N V F L E R Y G G G.TTAAGAAAAIICTTTTAAA ;CTA7GTTCCAGCACTGOCAIIrr rAGTCCWATCTCrAkw ,mGGTGClCTW CT) MGAATTCT7TGAAG Y E R E V P L L E M L C S S T G I G A P A T Y T N P A N EE F F E E hMAAcAAT7TGGGGAGCT7MACCGGA, UTGAAACAATTTT TCAT'GAG3A AAAGTATTTTGAAAI U7CG CTGAA ACTC' rATTCAATTAGACG T S F G E Q F S P E M K Q F F M R X Y F E S V R K K A P I Q L D D UATPGCTrGATGAGG AATUTCCi:ATTATCATTT,GA kCGAAAT'GGGAGAG E T F H R I C N S E I A D E E I P L S F E L N F F V K L R N G R V nTTAAGCT7ATGCCCAACGTCGGAMC rACCCGCGCAGGGAAAACA=T 'TGGGAGATTTTC PCATATG7=TATTICAATCAGI rGAAATCATAAAGA K L Y L G P N V G S T R A G K T F G R F S H M S D S I S E I I K T CCTTACATAAAGAAAGAGTrAACAGAC ;TGTAhTACGAAAGTTTGTGAJsACTGAOTATTGT( 3CCTAATCAAACTrAGGTCTGG, LAATGTrAACAAGAA L H N R E K E L T E C N T K V C E L S I V P N Q T R S G N V T R N Ah7TGTAAGCTACCGAGAAAAkAGAGATGTCT=r rrPTTTAC GAACAGT'GCTCtrCA'kT ,TCTCAATGATMCCCC 'GTCAAAGCTGAAGATATTCT.rAA LATTGGAATCAATA V S Y R E K E M S L F T N S A L H L N D S V K A E D I L I G I N K A AC14GC,GAAATTrCMTGrrlr n'GAGTCAAATrCA' ATrATGTTTAA7CCT7TATTAAIX ;ACCAACGCTGTAC D H I F Y A R H K T T G E I L S F E S N H M F N P L L M T N A v R GGCTTTATTAGAGATTIrAAGAGATGGTAA) L%GAAAATOGAATGATtTTCCCCGTGGTTTAGTATN .TATAGTGA7TMTAAGTATA7 rsCTGAAA7CAAAT L L L E I S R D G K R K W N D F P W F S I Y S D F K Y I P E I K Y ATAAAGAGATAACCTTATCTTGTGAACAATG ;MAAACAATGATI% TT)P'AAGCATGCACTCC .AATIGCA7ClCTGAAGAAT) LkAATCTGCTTTrT K E I T L S C E Q W L I Y K N D L S M H S N A S L E E I K S A F F TTGAATT7CATCGTACTTA7r,"CTGCCGCAi UCATITTTATATCG7rAACGCi 'AGACkAATCG ATTJ kTTGATTGATATAGAGAATGA' MTTACTTTGAT'G E F H R T Y E L P Q T F Y I V N A D N R L L I D I E N D C T L D V TT7TTrCGGAAMAAAAACGACCAIkTA 'GGCTGTTGAGCA'ATC ZAACCAGCCA7TACAACTTUGTIrG rGATIGCAGATIGCGTTAATGGAkTA MAGAAACCAAAATG P F W E L K K T N H N Q P L Q L V A V E H D A D A L M D R N Q N D
13866
14855 1 584
1683 1782
1881 19800
of the cat gene into spaE by a double crossover between the flanking regions, resulting in interruption of the spaE gene at the XbaI site, which is located about three-fourths of the way into the gene. A halo assay (9) showed that these integrants were unable to produce active subtilin (data not shown). By using the same approach, ORF X was interrupted by integration of the cat gene into the BalI site shown in Fig. 2. That the correct insertion occurred was established by Southern analysis of chromosomal DNA (data not shown). Interruption of ORF X at the BalI site had no effect on subtilin production. Homology between SpaE and NisB. The sequence data bases were searched for homologies to the deduced SpaE protein sequence. One very strong similarity, to NisB, was revealed (Fig. 3). We conclude that SpaE and NisB are likely to perform analogous roles in the biosynthesis of subtilin and nisin, respectively. It is notable that, despite this sequence homology, their locations in their respective operons are quite different, as illustrated in Fig. 2. This provides additional support for our earlier conclusion (2) that the spa and nis operons have been evolving separately for a long time. The roles of SpaE and NisB in lantibiotic synthesis are
2 07 99 2178 2 277 2 37 6 2 47 55
2 57 44 2 67 3 277 2 2 871 2 97 00
TGTAAACCATTGTCT CAT ATACCGGTTCTTAACGCAATAGAGGGATAGT9GG9TT7GACA L L R K Q P V K P L Y L P V L N A I - - - - GGC XTGTTTATrAAGCTTTATTGDMACACAGGAAGAAGAGCTAATTGCTTT TTGAAATAGCGGATTTT I K M P F E D W LI I RK L Y C K F Q T R E E E L I A E I A D F A(,CCAGATTTCTGAICAATATCCAG74TV CA _AAACCTCAT :AGA ATAAGACTTAGAT7 T'rAATOGAAAAGCCGAA Q I S D Q Y P V RF H F F M R Y R D P K P H I R L R F N G K A E 'GTACAGCTTCCCAATTATTnTG) JAATTGGCTGAAAAGcFTA99A9999190799717CA99GAG974Cr7ATCAC CTCAATACGAGCG(;GAG Y S L F P Q L L N W L K S L R E K G L V S E S V I T ACTATTCOGGAGAAATC Y
s
G
E
I
V
V
P
Gi
R Y G G spaD--> M E A A E Q L F C E D S K V V E M M I R H R M R D TATTA CGATAAGC AAGGAALATTGCAGGCAT,GGTTTCGGT TATACAGT7TTTTAGAACAGTT,CGAGCTAACGTTTGAAGAACAGTTAACTT7T TAGAGAG I T I S K E I A G M V S V I Q F L E Q F E L T F E E Q L T F L E R a AAATTCCTTACAGAAT -N S L Q N --------->
spa operon Eo RI
termntor
p_
NarI BaI
/
RI|(
kb
0
Hind1II
Hind EOI
2
1
8_3
5
4
nis operon
kb
0
nisA
I
I
1
2
Hind
III
CB *spa1. .7o I
[
nisB
5 3
6
termnr
B
EF X
B at
PVUII
BamHI
FSPD||
spaE
X
III
Xba
EcsR jEco RV
EOORI%%.om
ORF
EwoRV HL dI I
l
terminator
Hind III Bam Hi
4
6
56
7
s
S
VOL. 174, 1992
NOTES 10
20
30
40
50
60
SpaE
MKSLYTPTDYYMIRVPLVHQDLKNENSQDIDQLLHDLCNDSLFREQILVSSRTLYETIHT
NisB
MI KSSFKAQP- FLVRNTI LS PNDKRSFT- EYTQVI ETVSKNKVFLEQLLLANPKLYDVMQK 10 20 30 40 50
70 SpaE NisB
80
NisB
100
110
70
80
90
100
110
GIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNV 130
180
140
190
150
160
210
200
170
220
230
SpaE
FYLINELCGES-AAYQDIIRCLIDNYPNTPINKINQYVADLIDKEFLISNLRPPMTVSDQ
NisB
YQIISEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISSNLQKDLLSDFS
:1:1:1:::
180
1:11
:
:11:::I1
::
::
190
240 NisB
200
250
210
270
260
:::11111:
220
230
280
290
FQYLIDQAESRHIPNELIQACKNIQYQIDAYNRITIGEGEHQYLNLIETMNKLIKASSPL 240
250
260
310
270
320
280
330
QVDAGAGDSSIQLDNETSLAISELASMFTYMASPSANTLDHLEKYKNVFLERYGYEREVP :1 1:1 :11:1::1::::::11:::: ::: :1::11: 1:1:11:1: QIDL - ISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRT- -YLDDYKDKFI EKYGVDQEVQ 300 310 320 330 340 350
360
370
380
390
400
410
LLEMLCSSTGIGAPATYTNPANEFFEETSFGEQFSPEMKQFFMRKYFESVRKKAPIQLDD : 1:: 1: 11111 :1::1 1:1:1::: : :: ::::I 1:1:::: 1:111 :i ITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDD 360
370
420
380
430
400
390
440
450
410
460
470
NisB
ETFHRICNSEIADEEIPLSFELNFFVKLRN--GRVKLYLGPNVGSTRAGKTFGRFSHMSD ::: ::: ::: :: :1::1::1 : 11 : 1111 :1 II::: --LESHYQKMDLEKKSELQ-GLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSP
SpaE
SISEIIKT-LHNKEKELTECNTKVCELSIVPNQTRSGNVTRNVSYREKEMSLFTNSALHL
NisB
ELTSYHRTIVDSVERENENKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTS---
420
430
480 :1
1:1
:::
480
NisB
SpaE
450
500 ::
:::
11: ::1:::1
490
460
510
520 :11
500
470 530
:::11:::
::
510
520
540 550 570 580 560 590 NDSVKAEDILIGINKDHIFYARHKTTGEILSFESNHMFNPLLMTNAVRLLLEISRDGKRK :::I ::I III:::: 1111: :1 1:1:1 1:1 1::I::1:1 III 1:
HNEVLLTNIYIGIDEKEKFYARDIOSTQEVLKFYITSMYNKTLFSNELRFLYEISLDD--K 530
NisB
440
490
::::
SpaE
540
550
560
570
580
600 610 620 630 640 650 WNDFPWFSIYSDFKYIPEIKYKEITLSCEQWLIYKNDLSMHSNASLEEIKSAFFEFHRTY ::::II 11:11:111 : ::11::1 ::I 1: :1: : :: 1: :: FGNLPWELIYRDFDYIPRLVFDEIVISPAKWKIWGRDV ---------NSKMTIRELIQSK 590 600 610 620 630
SpaE
660 670 680 690 700 710 ELPQTFYIVNADNRLLIDIENDCTLDVFFWELKKTNHNQPLQLVAVEHDADALMDRNQND
NisB
EIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGEKG 640 650 670 680 660 690
SpaE
720 730 740 760 770 750 YSGEIVVPLLRKQPVKPLYLPVLNAIEGSGSDRIKMPFEDWLFIKLYCKQTREEELIAFE
NisB
RVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQNEFLLSY
:::11::1:::
700
780
710
790
:
:::
720
800
1:11::11::111: :1::1::
i :
730 810
740
820
750 830
SpaE
IADFYNQISDQYPVRHFFMRYRDPKPHIRLRFNGKAEVL-YSLFPQLLNWLKSLREKGLV
NisB
LPDI-QKIVANLGGNLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILKRSRKNRIMSTF 760
SpaE
NisB
A-T G-C A-T
-
G-C T-A A-T T-A T-A G-C-d
350
NisB
SpaE
A-T, T-A
290
340
SpaE
NisB
° G r
WNTFLTKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYI 300
SpaE
Nb 2
120
130 140 150 160 170 KARVDFEWLYQLI RKLENEYTDRLSFTLNSACYI KGDRAYLLYSTDGKS -EEVSVRATSV 1:1 :11 :1::1:1 :::::1111 1:1 III:: :1:::::: III::: 1:1 120
SpaE
sequencing reactions i _
FLQAPDKLKGKKKRNFQQAILKYATRRATRTTPFGLFSSVGIGSFSDKNHLSFNQHSFYK Y--NAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKT-TK
60 SpaE
90
6701
770
780
790
800
830
840
FIG. 4. Identification of promoter by primer extension. A 19-mer primer (5'-GATGTACrAAAGGAACCCG-3') that is complementary to a sequence within the N-terminal region of the spaE mRNA was used in a primer extension reaction as described in Materials and Methods. The same primer was used in dideoxy sequencing reactions of the double-stranded EcoRI fragment that contained this region (Fig. 2). The sequencing reaction products and the primer extension reaction products were electrophoresed simultaneously on a denaturing sequencing gel and autoradiographed. The mRNA used as a template was isolated from strains ATCC 6633, the natural subtilin producer, and LH45, which is a subtilin-producing strain of B. subtilis 168 as described by Liu and Hansen (8). The mRNA was isolated from cells that had been incubated for 22 h, which is a time that these cells are known to produce subtilin mRNA (1). The autoradiogram shows the results for sequencing reaction products, the primer extension products from LH45 and ATCC 6633, and products of a control reaction which contained all reaction components except RNA. In the pair of sequences to the left of the autoradiogram, the right-hand sequence was read from the bracketed region of the gel and the left-hand sequence is the reverse complement that corresponds to the mRNA sequence shown in Fig. 1. The single sharp band that corresponds to a G residue in the reverse complement indicates that this residue is a transcription initiation site. The corresponding promoter sequence, including its -10 and -35 regions, is shown in Fig. 1.
810
840 850 SESVITQYEREIERYGGX : 1: :1 1:11111 DISIYDQ --- EVERYGGFDTLELSEAIFVPILKLFQICLHX 820
UAT C L1
850
FIG. 3. Sequence homologies between SpaE and NisB. The deduced protein sequence encoded by spaE is compared with the corresponding sequence from the previously described ORF that is downstream from the nisin structural gene (nisA). The gene for this downstream ORF and the deduced protein sequence are referred to as nisB and NisB, respectively. The homology comparison was carried out with programs FASTA and GAP (4). Residues connected by lines are identical, and those connected by dots are similar. Dashes in the sequences represent gaps introduced to improve sequence homology. There are 29% identity and 53% similarity between the two proteins.
unclear. The absence of sequence similarity to other (nonlantibiotic) proteins requires that functional tests be performed to elucidate the roles of SpaE and NisB. Neither protein has a recognizable export signal region or transmembrane helices, so they appear to be restricted to the cytoplasmic side of the membrane. Results presented above showed that interruption of the spaE gene destroys the ability of the cell to produce subtilin, although the experiment could not distinguish between the effect being the result of destroying the function of the SpaE protein and being the result of interference with transcription of other downstream spa genes. Identification of a promoter upstream from the spaE gene. The presence of a p-independent terminator sequence in the
FIG. 2. (A) Organization of genes within the spa operon. Major restriction sites are shown. The promoter and its orientation are shown (P--). ORF X is an incomplete reading frame of unknown function. The sequence of spaE is given in Fig. 1. The sequences for spaD, spaB, spaC, and spaS have been published previously (2, 3). Horizontal arrows show the direction of transcription. (B) Organization of genes within the nis operon. The nisA gene encodes the primary structure of the nisin precursor peptide (1), and nisB is an ORF that is transcribed by read-through from nisA (11). ORF X is a partially sequenced ORF that has homology to a transposase (1). The terminator structure has characteristics of a p-independent terminator that probably defines the 3' end of the nis operon (11). The double-headed shaded arrow indicates homologous spaE and nisB genes, shown in Fig. 3. The promoter of the nis operon (not shown) lies several kilobases upstream (11).
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J. BACTERIOL.
NOTES
intergenic space upstream from spaE suggests that expression of the spa genes requires an active promoter between this putative terminator and the spa operon. A promoter-like sequence shown in Fig. 1 is a candidate. Primer extension analysis of the mRNA isolated from subtilin-producing cells was used to identify spaE transcripts. The cells were isolated after 22 h of growth under the conditions used previously (1), and primer extension experiments were performed with the Primer Extension System kit from Promega by using the protocol provided with the kit. Total cellular RNA was isolated as previously described (12). Figure 4 shows that the only detectable transcripts using a primer sequence from just inside the spaE gene have 5' termini that are consistent with having been transcribed from this promoter. This 5' terminus is identified in Fig. 1. The same result was obtained for both the natural subtilin producer, B. subtilis ATCC 6633, and a subtilin-producing strain that had been constructed (8) from B. subtilis 168 by transformation with a restriction fragment of the ATCC 6633 chromosome that contained the spa genes. The sequence of this promoter is similar to the vegetative 0A promoter of B. subtilis (10), except that the -10 and -35 regions are separated by 20 nucleotides instead of 17. This promoter is temporally regulated and is preferentially expressed during late growth stages (unpublished observations). The results presented here complete the sequence of a 7-kb operon that contains five ORFs that are required for subtilin biosynthesis, although the number of proteins that are actually produced has not been determined. For example, it is possible that frameshifting may fuse SpaB and SpaC (3). Also, another laboratory has recently reported the sequence of the 3' end of spaE (which is spaB by their nomenclature) and concluded, because of a discrepancy of a single nucleotide, that the SpaE and SpaD ORFs are fused (7). An argument against this fusion is that it would put a long C-terminal extension onto SpaE. The homologous NisB protein has no counterpart to this C-terminal extension. This discrepancy cannot be resolved with certainty until the expressed proteins are actually isolated and characterized. In any event, it is possible that the five ORFs in the spa operon could represent as few as three expressed proteins. One can legitimately wonder whether three proteins, or even five, are enough to carry out all the identifiable steps of the biosynthetic pathway of subtilin as well as provide immunity. If these are not sufficient, the rest of the genes must be in one or more additional operons. Experiments to determine whether the spa operon contains all of the genes required for subtilin biosynthesis are in progress.
Nucleotide sequence accession number. Nucleotide sequence accession no. M99263 has been assigned by the GenBank/EMBL Data Bank to the sequence in this article. This work was supported by NIH grant AI24454, the National Dairy Promotion and Research Board, and Applied Microbiology, Inc., New York, N.Y. We thank Tomas Kempe of the University of Maryland ProteinNucleic Acid Laboratory for synthesis of oligonucleotides.
1. 2.
3.
4.
5. 6. 7. 8.
9. 10.
11.
12.
REFERENCES Baneijee, S., and J. N. Hansen. 1988. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J. Biol. Chem. 263:9508-9514. Buchman, G. W., S. Banerjee, and J. N. Hansen. 1988. Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J. Biol. Chem. 263:16260-16266. Chung, Y. J., M. T. Steen, and J. N. Hansen. 1992. The subtilin gene of Bacillus subtilis ATCC 6633 is encoded in an operon that contains a homolog of the hemolysin B transport protein. J. Bacteriol. 174:1417-1422. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. Gross, E., H. H. Kiltz, and E. Nebelin. 1973. Subtilin. Part 6: the structure of subtilin. Hoppe-Seyler's. Z. Physiol. Chem. 354: 810-812. Gross, E., and J. L. Morell. 1971. The structure of nisin. J. Am. Chem. Soc. 93:4634 4635. Klein, C., C. Kaletta, N. Schnell, and K.-D. Entian. 1992. Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl. Environ. Microbiol. 58:132-142. Liu, W., and J. N. Hansen. 1991. Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation. J. Bacteriol. 173:7387-7390. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Moran, C. P., Jr. 1989. Sigma factors and the regulation of transcription, p. 167-184. In I. Smith, R. A. Slepecky, and P. Setlow (ed.), Regulation of procaryotic development. American Society for Microbiology, Washington, D.C. Steen, M. T., Y. J. Chung, and J. N. Hansen. 1991. Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis 11454. Appl. Environ. Microbiol. 57:1181-1188. Ulmanen, I., K. Lundstrom, P. Lehtovaara, M. Sarvas, M. Ruohonen, and I. Palva. 1985. Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. J. Bacteriol. 162:176-182.
Vol. 174, No. 20
0021-9193/92/206699-04$02.00/0 Copyright © 1992, American Society for Microbiology
Determination of the Sequence of spaE and Identification of a Promoter in the Subtilin (spa) Operon in Bacillus subtilis YONG JOON CHUNG AND J. NORMAN HANSEN* Department of Chemistpy and Biochemistry, University of Maryland, College Parkc Maryland 20742 Received 18 February 1992/Accepted 10 August 1992
An 851-residue open reading frame (ORF) called SpaE has been discovered in the subtilin (spa) operon. Interruption of this ORF with a chloramphenicol acetyltransferase gene destroys the ability of Bacilus subtilis LH45Ac (a derivative ofB. subtilis 168) to produce subtilin, which is an antimicrobial peptide belonging to the class of ribosomally synthesized peptide antibiotics called lantibiotics. SpaE shows strong homology to NisB, which is in the nisin (nis) operon in Lactococcus lactis ATCC 11454. Despite the strong sequence homology between SpaE and NisB, the spaE and nisB genes occupy very different locations in their respective operons, indicating that they have been evolving separately for a long time. Primer extension analysis was employed to identify a promoter upstream from the spaE gene, which appears to define the 5' end of the spa operon, which contains four other ORFs (Y. J. Chung, M. T. Steen, and J. N. Hansen, J. Bacteriol. 174:1417-1422, 1992).
Nisin and subtilin are structurally homologous members of a class of antimicrobial peptides called lantibiotics. Nisin is a 34-residue peptide produced by Lactococcus lactis ATCC 11454 (6), and subtilin is a 32-residue peptide produced by Bacillus subtilis ATCC 6633 (5). Nisin, subtilin, and the other lantibiotics contain unusual lanthionine and dehydro residues and are formed from ribosomally synthesized peptide precursors that undergo extensive posttranslational modification and export outside the cell. The genes for the prepeptides of subtilin and of nisin have been cloned, and their kinetics of expression have been characterized (1, 2). Because lantibiotics have novel structures, it is probable that the maturation pathway involves genes that encode novel proteins. We have been searching for genes involved with lantibiotic maturation within the operons that contain the genes for the precursor peptides. The nisin structural gene (nisA) appears to be cotranscribed with a downstream gene (nisB) (11), and the subtilin structural gene (spaS) is in the same transcriptional unit as several genes (spaD, spaB, and spaC) that lie upstream from spaS (3). The deduced SpaB protein sequence shows extensive homology to a variety of transport proteins, including the hemolysin B and multidrug resistance proteins (3), and may be involved in subtilin export. The nucleotide sequence upstream from the previously sequenced (3) spa genes was determined by cloning restriction fragments into M13mpl8 or M13mpl9 and sequencing by the dideoxy chain termination method with the Sequenase kit supplied by U.S. Biochemical Corp. (Cleveland, Ohio). DNA was isolated by standard methods (9). Long restriction fragments were cloned into pTZ18R and incrementally shortened with the Erase-a-base kit supplied by Promega Corp. (Madison, Wis.). The shortened plasmids were purified with GeneClean provided by Bio 101 (La Jolla, Calif.). To obtain the DNA containing this upstream region, a synthetic oligonucleotide based on the sequence of the 5' end of the spaD gene was used as a hybridization probe to clone a 10-kb PstI restriction fragment that contained the 5' end of spaD and its upstream flanking region. The bacterial strain used was LH45, which is a chloramphenicol-resistant *
Corresponding author.
subtilin-producing strain of B. subtilis (8) derived from B. subtilis 168 by transformation with DNA from B. subtilis ATCC 6633, which is the natural producer of subtilin. Strain LH45Ac is a chloramphenicol-sensitive derivative of LH45 derived by spontaneous loss of the cat gene (8). PstI fragments of genomic DNA prepared from B. subtilis LH45 were size selected on agarose and cloned into Eschenichia coli, and clones containing the 10-kb PstI fragment were identified by hybridization with the probe. This fragment was mapped with restriction enzymes, subcloned, and partially sequenced. Some primers were synthesized with a Biosearch model 8500 synthesizer. Approximately 3.5 kb of the sequence is shown in Fig. 1. The sequence reveals an 851-residue open reading frame (ORF). The gene for this ORF is designated spaE, and the deduced protein sequence is designated SpaE. The lack of any obvious terminator sequence between the spaE gene and its immediately downstream spaD gene argues that spaE and spaD are in the same transcriptional unit. Further sequence analysis revealed an intergenic space upstream from the spaE gene. This space contained a promoter-like sequence, and upstream from this was an inverted repeat with characteristics of a p-independent terminator. This putative terminator is located immediately downstream from an ORF called ORF X since its sequence is incomplete and it is not yet known whether it is involved with subtilin biosynthesis. The presence of this terminator suggests that expression of the spa operon is dependent on the promoter sequence that lies between this terminator and the spaE gene. Experiments to identify this promoter are described below. Interruption of the spaE gene prevents production of active subtilin. The role of the spaE gene in subtilin production was tested by integration of a cat gene into the SpaE coding region, thus interrupting the continuity of the reading frame. The strategy was similar to that previously used to show that the spaB gene is required for subtilin production (3). The spaE-containing SstI-PstI restriction fragment (see Fig. 2) was cloned into pTZ18R, and then the cat gene was cloned into the XbaI site of the insert. This plasmid was linearized by restriction with SstI and PstI, introduced into LH45Ac cells by transformation, and selected on chloramphenicol plates. The transformants should be the result of integration 6699
6700
J. BACrERIOL.
NOTES
TTCGGCCGCCACTTTCTCATGTCGGTTTGCGGCGTATTGTTG7CCGCTGTTGTCGGGGTTCCGGTCGGAATCCTCATTGCGCATTATCGCCGCTTATCA ORF X-->H
F
M
L
S
V
Y
G
V
L
F
A
A
V
V
G
V
P
V
G
L
I
I
A
H
R
Y
L
R
TTGTAA79CT7GCAT GGGGCTCGGCGCAAACACCGTG 198
A W V F A V T N V I Q T I P A L A M L A V A TTAT TCATTA7TF CATTCCC707GCCGA700A0CGAAACAC9TACACAGrCA I I S L F L Y S L L P I I R N T Y T G I V
L
ATGGGCA7GACGAAA7TCCAGGTTCGCCGGAG
GTTGAGCTCCCG
M
L
V
M
G
L
A
G
N
FIG. 1. Nucleotide sequence of the spaE gene. The organization of the spaE gene within the spa operon is shown in Fig. 2. The transcription start site of the spaE gene was determined by primer extension (Fig. 4) to give an mRNA that begins with the sequence 5'-GAGTATTGTAAATA--*. The -10 and -35 regions of the promoter that correspond to this start site are underlined. Upstream from spaE is part of an ORF of unidentified function (ORF X). Immediately downstream from ORF X is an inverted repeat (underlined) with a stretch of T residues that is typical of a p-independent terminator. The translation start codon for the SpaE ORF is shown as the first methionine downstream from the transcription start site, although the second methionine has a better ribosome binding site. The deduced protein sequence from the SpaE ORF has strong homology to the NisB ORF from the nis operon (Fig. 3). The EcoRI restriction site in ORF X shown in Fig. 2 is located approximately 10 nucleotides upstream from residue 1 of the sequence. a, the sequence of the rest of the operon, including spaD, spaB, spaC (3), and spaS (2), has been published previously.
99
S
GCCTGGGTGTTCGCCGTAACAAATGTTATCCAGACAATCCCGTCGCTCGCGA7GTGGCCG
T V
AGCA7TG9CA9CTACTTAGAATACGGGAAAAGCA 297 S
I
H
E
A
Y
L
E
G K
S
A
ITTATGGCAGGCTTGCGCACGGCTCGTCATTGCCATC
396
Q V L R M V E L P L A L S V I M A G L R T A L V I A I CTCAA ACGCG ACA AACGGAAC7CCGAGATT7CTCGCA 495 GGAATTACAGCAA7CGGCAC0T6G7 TCGGCGCCG GTGG9 ATATGA7TGCCGCGG
M
G
T
M
K
F
G I T A I G T F V G A GGCGCGACCCCGACTGCGGTAAT G A T P T A V M A I G
G L G D M I V R G S N A T N G T A I I L A GGCTAGGAG_ wTTCTTGAATICCGGTGAAACAAAAAAGCAGAAGAAAG 5 94 D L I M A W I E R F L N P V K Q K S R R K 9TAA7AAGTTGA7AGA97rAAGA AIY AGA'rlTG 5FGTG -.AT TATITACTGGT7GGA70=ATT7TT1TT 693 V I S V -35 inverted repoat rAATAGAATAATATGG GGAoCATAAGCAGAGGGATGTAGAATTGTTrnATT 792 ATTTTAGAATvAG&,AAATATGA(TATTATAAATArrl -10 I------------ > mRNA G
A
AAGAAGCGGATAAATARGlGGCTATAGACTAATrA7GAcTTATATACACCTACAGATTATTATATIGATTCGGGTTCCTATCATuCAAGCr 891 spaE--> M K S L Y T P T D Y Y M I R V P L V H Q D L TAAAGGATTAGTT CGATCGTTTCTACTrGACGTClATTG;AACAAATACTIGGTATCTAGCAGGACACTAT 990 K N E N S-Q D I D Q L L H D L C N D S L F R E Q I L V S S R T L Y AT,GAAACAATACATACntV7GAGCGCCGGATAAATTAAAAGGGAAAAAAAAGCGCAACTrTCAACAGGCTATTTTGA GTATGCAACAAGAAGAG 1089 E T I H T F L Q A P D K L K G K K K R N F Q Q A I L K Y A T R R A CAACAAGAACCACAC TTCATTTTACAAAA 1188 T R T T P F G L F S S V G I G S F S D K N H L S F N Q H S F Y K K AGGCTCGTGTTGA?TG A rGTrCATATrGAATGAAGAATACACCGACCGACTTTrCTTTTACATTAAA7TTCGCTTGTTATA 1287 A R V D F E W L Y Q L I R K L E N E Y T D R L S F T L N S A C Y I kh TrrAAGGGTIGACCGGGcTTACTTrGTTGTACAGCICA sAGTTAGTGTTCG'GTrGCGACATCMTGTTTCTATMT rGA ;ATAAATGAACTGT K G D R A Y L L Y S T D G K S E E V S V R A T S V F Y L I N E L C ,TCCAhATACTMCC CAL'kTAAATAAAATTAATICAOTA( 'GTAGCAGACCTTA GTGGTG;AATCTGCT'GCATATCAAGATATAATX 'CGTTTTATASTACAkT G E S A A Y Q D I I R C L I D N Y P N T P I N K I N Q Y V A D L I TACAA CTTAG( ;CCGCCGATGACTGCTrlrCAGAI,TCAATTTCAGTA, MTTAATTGA7CAAGCOGAAAGC .CGCCATATTCCAA TTrACAGAGTcA D K E F L I S N L R P P M T V S D Q F Q Y L I D Q A E S R H I P N ATIGAACTCATTCAGGCTTGTAAGAACATITCA. kTATCAGATAGATGCQTATAUz MTCGGATICACTATR :GGAGAGGGAGAGCAT'CAGTAI rIAAA7C'TGATTG E L I Q A c K N I Q Y Q I D A Y N R I T I G E G E H Q Y L N L I E AMAT.AAAAC _TAG(CATAC MCCTCTGCAAGTAGACGCTG= X;GCTG GAGACTC( ItCCATTCAArrAGATAA7r.AJ LkCATC7C'TCGCCA T M N K L I K A S S P L Q V D A G A G D S S I Q L D N E T S L A I rAAGTGAATT'GGCAAGCTTTAC TATT ;GCTtUTC7t=CTGCCATACJATTAGACCACTT GAAAAATACAAA AATGTATT n'PGGAACGCTATG S E L A S M F T Y M A S P S A N T L D H L E K Y K N V F L E R Y G G G.TTAAGAAAAIICTTTTAAA ;CTA7GTTCCAGCACTGOCAIIrr rAGTCCWATCTCrAkw ,mGGTGClCTW CT) MGAATTCT7TGAAG Y E R E V P L L E M L C S S T G I G A P A T Y T N P A N EE F F E E hMAAcAAT7TGGGGAGCT7MACCGGA, UTGAAACAATTTT TCAT'GAG3A AAAGTATTTTGAAAI U7CG CTGAA ACTC' rATTCAATTAGACG T S F G E Q F S P E M K Q F F M R X Y F E S V R K K A P I Q L D D UATPGCTrGATGAGG AATUTCCi:ATTATCATTT,GA kCGAAAT'GGGAGAG E T F H R I C N S E I A D E E I P L S F E L N F F V K L R N G R V nTTAAGCT7ATGCCCAACGTCGGAMC rACCCGCGCAGGGAAAACA=T 'TGGGAGATTTTC PCATATG7=TATTICAATCAGI rGAAATCATAAAGA K L Y L G P N V G S T R A G K T F G R F S H M S D S I S E I I K T CCTTACATAAAGAAAGAGTrAACAGAC ;TGTAhTACGAAAGTTTGTGAJsACTGAOTATTGT( 3CCTAATCAAACTrAGGTCTGG, LAATGTrAACAAGAA L H N R E K E L T E C N T K V C E L S I V P N Q T R S G N V T R N Ah7TGTAAGCTACCGAGAAAAkAGAGATGTCT=r rrPTTTAC GAACAGT'GCTCtrCA'kT ,TCTCAATGATMCCCC 'GTCAAAGCTGAAGATATTCT.rAA LATTGGAATCAATA V S Y R E K E M S L F T N S A L H L N D S V K A E D I L I G I N K A AC14GC,GAAATTrCMTGrrlr n'GAGTCAAATrCA' ATrATGTTTAA7CCT7TATTAAIX ;ACCAACGCTGTAC D H I F Y A R H K T T G E I L S F E S N H M F N P L L M T N A v R GGCTTTATTAGAGATTIrAAGAGATGGTAA) L%GAAAATOGAATGATtTTCCCCGTGGTTTAGTATN .TATAGTGA7TMTAAGTATA7 rsCTGAAA7CAAAT L L L E I S R D G K R K W N D F P W F S I Y S D F K Y I P E I K Y ATAAAGAGATAACCTTATCTTGTGAACAATG ;MAAACAATGATI% TT)P'AAGCATGCACTCC .AATIGCA7ClCTGAAGAAT) LkAATCTGCTTTrT K E I T L S C E Q W L I Y K N D L S M H S N A S L E E I K S A F F TTGAATT7CATCGTACTTA7r,"CTGCCGCAi UCATITTTATATCG7rAACGCi 'AGACkAATCG ATTJ kTTGATTGATATAGAGAATGA' MTTACTTTGAT'G E F H R T Y E L P Q T F Y I V N A D N R L L I D I E N D C T L D V TT7TTrCGGAAMAAAAACGACCAIkTA 'GGCTGTTGAGCA'ATC ZAACCAGCCA7TACAACTTUGTIrG rGATIGCAGATIGCGTTAATGGAkTA MAGAAACCAAAATG P F W E L K K T N H N Q P L Q L V A V E H D A D A L M D R N Q N D
13866
14855 1 584
1683 1782
1881 19800
of the cat gene into spaE by a double crossover between the flanking regions, resulting in interruption of the spaE gene at the XbaI site, which is located about three-fourths of the way into the gene. A halo assay (9) showed that these integrants were unable to produce active subtilin (data not shown). By using the same approach, ORF X was interrupted by integration of the cat gene into the BalI site shown in Fig. 2. That the correct insertion occurred was established by Southern analysis of chromosomal DNA (data not shown). Interruption of ORF X at the BalI site had no effect on subtilin production. Homology between SpaE and NisB. The sequence data bases were searched for homologies to the deduced SpaE protein sequence. One very strong similarity, to NisB, was revealed (Fig. 3). We conclude that SpaE and NisB are likely to perform analogous roles in the biosynthesis of subtilin and nisin, respectively. It is notable that, despite this sequence homology, their locations in their respective operons are quite different, as illustrated in Fig. 2. This provides additional support for our earlier conclusion (2) that the spa and nis operons have been evolving separately for a long time. The roles of SpaE and NisB in lantibiotic synthesis are
2 07 99 2178 2 277 2 37 6 2 47 55
2 57 44 2 67 3 277 2 2 871 2 97 00
TGTAAACCATTGTCT CAT ATACCGGTTCTTAACGCAATAGAGGGATAGT9GG9TT7GACA L L R K Q P V K P L Y L P V L N A I - - - - GGC XTGTTTATrAAGCTTTATTGDMACACAGGAAGAAGAGCTAATTGCTTT TTGAAATAGCGGATTTT I K M P F E D W LI I RK L Y C K F Q T R E E E L I A E I A D F A(,CCAGATTTCTGAICAATATCCAG74TV CA _AAACCTCAT :AGA ATAAGACTTAGAT7 T'rAATOGAAAAGCCGAA Q I S D Q Y P V RF H F F M R Y R D P K P H I R L R F N G K A E 'GTACAGCTTCCCAATTATTnTG) JAATTGGCTGAAAAGcFTA99A9999190799717CA99GAG974Cr7ATCAC CTCAATACGAGCG(;GAG Y S L F P Q L L N W L K S L R E K G L V S E S V I T ACTATTCOGGAGAAATC Y
s
G
E
I
V
V
P
Gi
R Y G G spaD--> M E A A E Q L F C E D S K V V E M M I R H R M R D TATTA CGATAAGC AAGGAALATTGCAGGCAT,GGTTTCGGT TATACAGT7TTTTAGAACAGTT,CGAGCTAACGTTTGAAGAACAGTTAACTT7T TAGAGAG I T I S K E I A G M V S V I Q F L E Q F E L T F E E Q L T F L E R a AAATTCCTTACAGAAT -N S L Q N --------->
spa operon Eo RI
termntor
p_
NarI BaI
/
RI|(
kb
0
Hind1II
Hind EOI
2
1
8_3
5
4
nis operon
kb
0
nisA
I
I
1
2
Hind
III
CB *spa1. .7o I
[
nisB
5 3
6
termnr
B
EF X
B at
PVUII
BamHI
FSPD||
spaE
X
III
Xba
EcsR jEco RV
EOORI%%.om
ORF
EwoRV HL dI I
l
terminator
Hind III Bam Hi
4
6
56
7
s
S
VOL. 174, 1992
NOTES 10
20
30
40
50
60
SpaE
MKSLYTPTDYYMIRVPLVHQDLKNENSQDIDQLLHDLCNDSLFREQILVSSRTLYETIHT
NisB
MI KSSFKAQP- FLVRNTI LS PNDKRSFT- EYTQVI ETVSKNKVFLEQLLLANPKLYDVMQK 10 20 30 40 50
70 SpaE NisB
80
NisB
100
110
70
80
90
100
110
GIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNV 130
180
140
190
150
160
210
200
170
220
230
SpaE
FYLINELCGES-AAYQDIIRCLIDNYPNTPINKINQYVADLIDKEFLISNLRPPMTVSDQ
NisB
YQIISEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISSNLQKDLLSDFS
:1:1:1:::
180
1:11
:
:11:::I1
::
::
190
240 NisB
200
250
210
270
260
:::11111:
220
230
280
290
FQYLIDQAESRHIPNELIQACKNIQYQIDAYNRITIGEGEHQYLNLIETMNKLIKASSPL 240
250
260
310
270
320
280
330
QVDAGAGDSSIQLDNETSLAISELASMFTYMASPSANTLDHLEKYKNVFLERYGYEREVP :1 1:1 :11:1::1::::::11:::: ::: :1::11: 1:1:11:1: QIDL - ISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRT- -YLDDYKDKFI EKYGVDQEVQ 300 310 320 330 340 350
360
370
380
390
400
410
LLEMLCSSTGIGAPATYTNPANEFFEETSFGEQFSPEMKQFFMRKYFESVRKKAPIQLDD : 1:: 1: 11111 :1::1 1:1:1::: : :: ::::I 1:1:::: 1:111 :i ITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDD 360
370
420
380
430
400
390
440
450
410
460
470
NisB
ETFHRICNSEIADEEIPLSFELNFFVKLRN--GRVKLYLGPNVGSTRAGKTFGRFSHMSD ::: ::: ::: :: :1::1::1 : 11 : 1111 :1 II::: --LESHYQKMDLEKKSELQ-GLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSP
SpaE
SISEIIKT-LHNKEKELTECNTKVCELSIVPNQTRSGNVTRNVSYREKEMSLFTNSALHL
NisB
ELTSYHRTIVDSVERENENKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTS---
420
430
480 :1
1:1
:::
480
NisB
SpaE
450
500 ::
:::
11: ::1:::1
490
460
510
520 :11
500
470 530
:::11:::
::
510
520
540 550 570 580 560 590 NDSVKAEDILIGINKDHIFYARHKTTGEILSFESNHMFNPLLMTNAVRLLLEISRDGKRK :::I ::I III:::: 1111: :1 1:1:1 1:1 1::I::1:1 III 1:
HNEVLLTNIYIGIDEKEKFYARDIOSTQEVLKFYITSMYNKTLFSNELRFLYEISLDD--K 530
NisB
440
490
::::
SpaE
540
550
560
570
580
600 610 620 630 640 650 WNDFPWFSIYSDFKYIPEIKYKEITLSCEQWLIYKNDLSMHSNASLEEIKSAFFEFHRTY ::::II 11:11:111 : ::11::1 ::I 1: :1: : :: 1: :: FGNLPWELIYRDFDYIPRLVFDEIVISPAKWKIWGRDV ---------NSKMTIRELIQSK 590 600 610 620 630
SpaE
660 670 680 690 700 710 ELPQTFYIVNADNRLLIDIENDCTLDVFFWELKKTNHNQPLQLVAVEHDADALMDRNQND
NisB
EIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGEKG 640 650 670 680 660 690
SpaE
720 730 740 760 770 750 YSGEIVVPLLRKQPVKPLYLPVLNAIEGSGSDRIKMPFEDWLFIKLYCKQTREEELIAFE
NisB
RVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQNEFLLSY
:::11::1:::
700
780
710
790
:
:::
720
800
1:11::11::111: :1::1::
i :
730 810
740
820
750 830
SpaE
IADFYNQISDQYPVRHFFMRYRDPKPHIRLRFNGKAEVL-YSLFPQLLNWLKSLREKGLV
NisB
LPDI-QKIVANLGGNLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILKRSRKNRIMSTF 760
SpaE
NisB
A-T G-C A-T
-
G-C T-A A-T T-A T-A G-C-d
350
NisB
SpaE
A-T, T-A
290
340
SpaE
NisB
° G r
WNTFLTKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYI 300
SpaE
Nb 2
120
130 140 150 160 170 KARVDFEWLYQLI RKLENEYTDRLSFTLNSACYI KGDRAYLLYSTDGKS -EEVSVRATSV 1:1 :11 :1::1:1 :::::1111 1:1 III:: :1:::::: III::: 1:1 120
SpaE
sequencing reactions i _
FLQAPDKLKGKKKRNFQQAILKYATRRATRTTPFGLFSSVGIGSFSDKNHLSFNQHSFYK Y--NAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKT-TK
60 SpaE
90
6701
770
780
790
800
830
840
FIG. 4. Identification of promoter by primer extension. A 19-mer primer (5'-GATGTACrAAAGGAACCCG-3') that is complementary to a sequence within the N-terminal region of the spaE mRNA was used in a primer extension reaction as described in Materials and Methods. The same primer was used in dideoxy sequencing reactions of the double-stranded EcoRI fragment that contained this region (Fig. 2). The sequencing reaction products and the primer extension reaction products were electrophoresed simultaneously on a denaturing sequencing gel and autoradiographed. The mRNA used as a template was isolated from strains ATCC 6633, the natural subtilin producer, and LH45, which is a subtilin-producing strain of B. subtilis 168 as described by Liu and Hansen (8). The mRNA was isolated from cells that had been incubated for 22 h, which is a time that these cells are known to produce subtilin mRNA (1). The autoradiogram shows the results for sequencing reaction products, the primer extension products from LH45 and ATCC 6633, and products of a control reaction which contained all reaction components except RNA. In the pair of sequences to the left of the autoradiogram, the right-hand sequence was read from the bracketed region of the gel and the left-hand sequence is the reverse complement that corresponds to the mRNA sequence shown in Fig. 1. The single sharp band that corresponds to a G residue in the reverse complement indicates that this residue is a transcription initiation site. The corresponding promoter sequence, including its -10 and -35 regions, is shown in Fig. 1.
810
840 850 SESVITQYEREIERYGGX : 1: :1 1:11111 DISIYDQ --- EVERYGGFDTLELSEAIFVPILKLFQICLHX 820
UAT C L1
850
FIG. 3. Sequence homologies between SpaE and NisB. The deduced protein sequence encoded by spaE is compared with the corresponding sequence from the previously described ORF that is downstream from the nisin structural gene (nisA). The gene for this downstream ORF and the deduced protein sequence are referred to as nisB and NisB, respectively. The homology comparison was carried out with programs FASTA and GAP (4). Residues connected by lines are identical, and those connected by dots are similar. Dashes in the sequences represent gaps introduced to improve sequence homology. There are 29% identity and 53% similarity between the two proteins.
unclear. The absence of sequence similarity to other (nonlantibiotic) proteins requires that functional tests be performed to elucidate the roles of SpaE and NisB. Neither protein has a recognizable export signal region or transmembrane helices, so they appear to be restricted to the cytoplasmic side of the membrane. Results presented above showed that interruption of the spaE gene destroys the ability of the cell to produce subtilin, although the experiment could not distinguish between the effect being the result of destroying the function of the SpaE protein and being the result of interference with transcription of other downstream spa genes. Identification of a promoter upstream from the spaE gene. The presence of a p-independent terminator sequence in the
FIG. 2. (A) Organization of genes within the spa operon. Major restriction sites are shown. The promoter and its orientation are shown (P--). ORF X is an incomplete reading frame of unknown function. The sequence of spaE is given in Fig. 1. The sequences for spaD, spaB, spaC, and spaS have been published previously (2, 3). Horizontal arrows show the direction of transcription. (B) Organization of genes within the nis operon. The nisA gene encodes the primary structure of the nisin precursor peptide (1), and nisB is an ORF that is transcribed by read-through from nisA (11). ORF X is a partially sequenced ORF that has homology to a transposase (1). The terminator structure has characteristics of a p-independent terminator that probably defines the 3' end of the nis operon (11). The double-headed shaded arrow indicates homologous spaE and nisB genes, shown in Fig. 3. The promoter of the nis operon (not shown) lies several kilobases upstream (11).
6702
J. BACTERIOL.
NOTES
intergenic space upstream from spaE suggests that expression of the spa genes requires an active promoter between this putative terminator and the spa operon. A promoter-like sequence shown in Fig. 1 is a candidate. Primer extension analysis of the mRNA isolated from subtilin-producing cells was used to identify spaE transcripts. The cells were isolated after 22 h of growth under the conditions used previously (1), and primer extension experiments were performed with the Primer Extension System kit from Promega by using the protocol provided with the kit. Total cellular RNA was isolated as previously described (12). Figure 4 shows that the only detectable transcripts using a primer sequence from just inside the spaE gene have 5' termini that are consistent with having been transcribed from this promoter. This 5' terminus is identified in Fig. 1. The same result was obtained for both the natural subtilin producer, B. subtilis ATCC 6633, and a subtilin-producing strain that had been constructed (8) from B. subtilis 168 by transformation with a restriction fragment of the ATCC 6633 chromosome that contained the spa genes. The sequence of this promoter is similar to the vegetative 0A promoter of B. subtilis (10), except that the -10 and -35 regions are separated by 20 nucleotides instead of 17. This promoter is temporally regulated and is preferentially expressed during late growth stages (unpublished observations). The results presented here complete the sequence of a 7-kb operon that contains five ORFs that are required for subtilin biosynthesis, although the number of proteins that are actually produced has not been determined. For example, it is possible that frameshifting may fuse SpaB and SpaC (3). Also, another laboratory has recently reported the sequence of the 3' end of spaE (which is spaB by their nomenclature) and concluded, because of a discrepancy of a single nucleotide, that the SpaE and SpaD ORFs are fused (7). An argument against this fusion is that it would put a long C-terminal extension onto SpaE. The homologous NisB protein has no counterpart to this C-terminal extension. This discrepancy cannot be resolved with certainty until the expressed proteins are actually isolated and characterized. In any event, it is possible that the five ORFs in the spa operon could represent as few as three expressed proteins. One can legitimately wonder whether three proteins, or even five, are enough to carry out all the identifiable steps of the biosynthetic pathway of subtilin as well as provide immunity. If these are not sufficient, the rest of the genes must be in one or more additional operons. Experiments to determine whether the spa operon contains all of the genes required for subtilin biosynthesis are in progress.
Nucleotide sequence accession number. Nucleotide sequence accession no. M99263 has been assigned by the GenBank/EMBL Data Bank to the sequence in this article. This work was supported by NIH grant AI24454, the National Dairy Promotion and Research Board, and Applied Microbiology, Inc., New York, N.Y. We thank Tomas Kempe of the University of Maryland ProteinNucleic Acid Laboratory for synthesis of oligonucleotides.
1. 2.
3.
4.
5. 6. 7. 8.
9. 10.
11.
12.
REFERENCES Baneijee, S., and J. N. Hansen. 1988. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J. Biol. Chem. 263:9508-9514. Buchman, G. W., S. Banerjee, and J. N. Hansen. 1988. Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J. Biol. Chem. 263:16260-16266. Chung, Y. J., M. T. Steen, and J. N. Hansen. 1992. The subtilin gene of Bacillus subtilis ATCC 6633 is encoded in an operon that contains a homolog of the hemolysin B transport protein. J. Bacteriol. 174:1417-1422. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. Gross, E., H. H. Kiltz, and E. Nebelin. 1973. Subtilin. Part 6: the structure of subtilin. Hoppe-Seyler's. Z. Physiol. Chem. 354: 810-812. Gross, E., and J. L. Morell. 1971. The structure of nisin. J. Am. Chem. Soc. 93:4634 4635. Klein, C., C. Kaletta, N. Schnell, and K.-D. Entian. 1992. Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl. Environ. Microbiol. 58:132-142. Liu, W., and J. N. Hansen. 1991. Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation. J. Bacteriol. 173:7387-7390. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Moran, C. P., Jr. 1989. Sigma factors and the regulation of transcription, p. 167-184. In I. Smith, R. A. Slepecky, and P. Setlow (ed.), Regulation of procaryotic development. American Society for Microbiology, Washington, D.C. Steen, M. T., Y. J. Chung, and J. N. Hansen. 1991. Characterization of the nisin gene as part of a polycistronic operon in the chromosome of Lactococcus lactis 11454. Appl. Environ. Microbiol. 57:1181-1188. Ulmanen, I., K. Lundstrom, P. Lehtovaara, M. Sarvas, M. Ruohonen, and I. Palva. 1985. Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. J. Bacteriol. 162:176-182.
