Nucleotide sequence and characteristics of the gene, lpa-14, responsible for biosynthesis of the lipopeptide antibiotics iturin A and surfactin from Bacillus subtilis RB14

JOURNAL OP FERMENTATIONAND BIOENGINEEI~q'G

Vol. 76, No. 6, 445-450. 1993

Nucleotide Sequence and Characteristics of the Gene, lpa-14, Responsible for Biosynthesis of the Lipopeptide Antibiotics Iturin A and Surfactin from Bacillus subtilis RB 14 C H I E H - C H E N HUANG, TAKASHI ANO,* ~ D MAKOTO SHODA Research Laboratory o f Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan Received 18 August 1993/Accepted 30 August 1993

Bacillus subtilis RB14 is a coproducer of the lipopeptide antibiotics iturin A and surfactin, lturin A and surfactin have a similar structure consisting of a seven-amino-acid cyclic peptide, linked by either hydroxy or ester peptide linkage, respectively, to the fatty acid part. A 10-kb fragment responsible for the production of the lipopeptide antibiotics iturin A and surfactin in B. subtilis RB14 was minimized and the nucleotide sequence of the region essential for the synthesis of the lipopeptides was determined. A large open reading frame consisting of 224 amino acid residues was found. The gene, designated as lpa-14 (lipopeptide antibiotic production of RB14), showed high homology with sfp and psf-I (regulators for surfactin production in B. subtilis and BaciUuspumilus, respectively) and an unknown open reading frame, offX, in the upstream region of the peptide antibiotic gramieidin S biosynthesis operon of Bacillus brevis. The biosynthesis of surfaetin and of iturin A was shown to be coregulated by the same gene, lpa-14. It is suggested that a common regulation system might exist among these genes for the production of peptide antibiotics in Bacillus species.

We have previously isolated several bacteria suppressive against phytopathogenic microorganisms (1-3), and have shown that they are good candidates for environmentally safe biological pesticides (1--4). Among them, Bacillus subtilis RB14 was shown to be a coproducer of the lipopeptides iturin A and surfactin (3). Iturin A has the structure of a cyclolipopeptide, containing seven residues of aamino acids (L-Asn-D-Tyr-D-Asn-L-Gln-l.-Pro-D-Asn-L-Ser-) and one residue o f a E-amino acid (Fig. 1A, 5, 6). The other cyclic lipopeptide, surfactin, contains a/~-hydroxy fatty acid linked by ester peptide linkage to a seven-aminoacid cyclic peptide (L-GIu-L-Leu-D-Leu-L-VaI-L-Asp-DLeu-L-Leu-) (Fig. 1B, 7). Since the antifungal activity of iturin A is enhanced by the presence of surfactin synergistically (3), the application of the coproducer of these lipopeptides as a biological pesticide is promising. Surfactin is a potent surface-active reagent (8) and antibiotic (9). Biochemical analyses of the surfactin synthetase multi-enzyme system (10, 11) as well as genetic analyses of the biosynthesis genes by Nakano et al. (12-15), have recently been carried out. Although a preliminary enzymatic analysis of the coproducer has been conducted (16), no genetic analysis has been done except for the cloning of a gene fragment involved in surfactin and iturin A coproduction (17). In this report, we describe the nucleotide sequence of the gene lpa-14, which is required for the production of surfactin and iturin A in B. subtilis RB14, and compare it to other similar genes.

pressive against phytopathogenic microorganisms. This strain produces the lipopeptide antibiotics surfactin and iturin A (3), and it has a cryptic plasmid designated p a l . Bacillus pumilus A-1 was isolated as a surfactin producer by Morikawa et al. (18). Media L medium contained (per liter) 10 g of Polypepton (Nihon Pharmaceutical Co., Tokyo) 5 g of yeast extract, and 5 g of NaCI, and was adjusted to pH 7.2. Number 3 medium, containing (per liter) 10 g of Polypepton, 10 g of glucose, 1 g of KH2PO4, and 0.5 g of MgSO4.7H20 (pH 7.0), was used for the production of the antibiotics. Each medium was solidified with 1.5% agar. To select or cultivate the transformants, the antibiotics chloramphenicol (Cm), or tetracycline (Tc) were added at concentrations of 5 and 25 pg/ml, respectively. Transformation o f B. subtilis M I l l 3 Competent cells of B. subtilis MI113 were prepared by the method of Spizizen (19) with the slight modification of adding required amino acids (50 and 5 pg/ml for the first and second growth media, respectively). Transformation of the iturin producer and its derivative Transformation of B. subtilis RB14 and its derivative RA1 with plasmid DNA was carried out by electroporation using a Cell Porator linked to a booster (BRL Life Technologies, Inc., Gaithersberg, MD, USA) with a A

B ]~~o--~L-Asn-~ D-Tyr-~D-Asn ~O--~L-Glu --~L-Leu--~D-Leu

MATERIALS AND METHODS H~-L-Ser ~-D-Asn~-L-Pro O.~---L-Leu~-D-Leu~-L-Asp FIG. 1. (A) Structure of iturin. (B) Structure of surfactin. R denotes one of the following aliphatic chains: CH3(CH2)I0-,

Bacterial strains and plasmids Bacterial strains and plasmids are listed in Table 1. B. subtilis RB14 was isolated from a compost and characterized as a bacterium sup-

CH3CH2(CH3)CH(CH2)s-, (CHs)2(CH)(CH2)9-, CH3(CH2)I2-, arid (CH3)2CH(CH2)zo-. R' denotes (CH3)2CH(CH2)9-.

* Corresponding author. 445

446

HUANG ET AL.

J. FERMENT.BIOENG., TABLE 1. Strains and plasmids

Strain or plasmid

Genotype, phenotype, or plasmid markera

B. subtilis MI113 MI113 (pC194) MIll3 (pTB522-2 kb) MIll3 (pCll2) RB14 RA1 RAI (pC194) RAI (pC115) RA1 (pCSF1) B. pumilus A-1

arg-15 trpC2 hsmM hsrM Cm r SF ÷ Tcr SF ÷ Cm r SF+ IT + SF- IT- Em r SF IT- Em f Cm r SF + IT +Em r Cm ~ SF + IT +Em ~ Cm r SF +

Plasmids pC194 pTB522-2 kb pC 112 pC113 pC115 pC112A pCSF1

Source or reference 17 This work This work, 18 17 3 17 This work This work This work 18

Cm r, 2.9 kb SF +, Tcr, 12.5 kb SF+, reduced plasmid from pC 111 SF +, reduced plasmid from pC 112 SF +, reduced plasmid from pCll3 SF-, PstI fragment was removed from pC112 SF +, psf-1 was cloned from pTB522-2 kb

30 18 17 This work This work 17 This work

a SF, Surfactin production; IT, iturin production; Tc, tetracycline; Cm, chloramphenicol; Em, erythromycin. 0.15 cm cuvette, as described previously (20). Cells o f a 150-ml late logarithmic growth phase culture o f B. subtilis were harvested a n d washed three times with cold 1 m M Hepes (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid) buffer (pH 7.0) and were resuspended in a final volume o f 400 Fl. Aliquots (40 pl) were mixed with 5 Ftl o f D N A and 40 pl o f 50% P E G 6000 in distilled water. F r o m this mixture, 20 ~tl was transferred to a chilled cuvette (0.15 cm electrorode gap) and a single pulse o f 16.0 k V / c m (2.4 kV, 4 kfl, 2 p F ) was applied. The cell suspension was p u t into 0.5 ml o f L m e d i u m and allowed to stand for 3 h at 37°C. Then, the cell culture was spread onto antibioticcontaining selective plates, which were incubated overnight at 37°C. Preparation of plasmid D N A The r a p i d alkaline lysis procedure (21) used for screening clones was p e r f o r m e d with modifications as follows: glucose in solution I was replaced by 25% (w/v) sucrose, the lysozyme concentration was increased to 5 m g / m l and cells were suspended in the solution and incubated at 37°C. F o r large-scale preparation, plasmids extracted b y the alkali lysis m e t h o d were further purified b y CsCl-ethidium b r o m i d e density gradient ultracentrifugation (22). Restriction endonuclease or T4 D N A ligase treatment and electrophoretic analysis o f D N A in agarose gel were d o n e by following the stand a r d m e t h o d s (22). D N A sequencing and analysis D N A sequencing o f d o u b l e - s t r a n d e d D N A cloned in pUC19 was p e r f o r m e d by an A B I 373A Auto-sequencer at T a k a r a Shuzo Co. and was reconfirmed by a P h a r m a c i a A . L . F . D N A sequencer at the Gene Experimental L a b o r a t o r y o f T o k y o Institute o f Technology b y the dideoxy-chaln termination m e t h o d o f Sanger et al. (23) using a T7 Sequencing kit. Detection of biosurfaetant activity of the lipopeptides surfactin and iturin A The presence o f lipopeptides was detected on tributyrin L agar plates (TB plates), using 20 pl o f tributyrin spread o n t o a L agar plate by a glass rod. W h e n a halo was m a d e a r o u n d a colony spotted on a TB plate, a biosurfactant was j u d g e d to be p r o d u c e d . Fungai growth inhibition test by bacteria A phytopathogenic fungus, Fusarium o x y s p o r u m f. sp. lycopersici race J1 S U F I 1 9 (2) was grown on a potato-dex-

trose agar m e d i u m (potato-dextrose agar 39g, distilled water 1 l, p H 5.6) at 30°C for 5 d a n d suspended in sterile distilled water. A p o r t i o n o f this suspension was mixed into L agar m e d i u m before it was solidified. A f t e r spotting the bacterium to be tested at the center o f this plate, its suppressiveness was investigated by observing the zones inhibitory to the growth o f the fungus. HPLC analysis of iturin A and surfactin Iturin A was assayed by reversed-phase H P L C as described previously (1). A f t e r 5 d cultivation o f the bacteria in 100 ml o f no. 3 m e d i u m , the culture was centrifuged at 11,000 × g for 10 min, and the supernatant was acidified to p H 2 with 12 N HCI. Then, the precipitate collected by centrifugation was extracted with 2 m l methanol (100%) for 2 h . A f t e r the extracted solution was centrifuged at 10,000 × g for 2 min, the supernatant was filtered t h r o u g h a 0.2 p m P T F E m e m b r a n e (JP020, Advantec, Ltd., T o k y o ) and injected into a H P L C column (column: ODS-2, 4.64 × 250 mm, G L Sciences, Tokyo). The system was operated at a flow rate o f 1.0 m l / m i n with acetonitrile-ammonium acetate (10 raM) 3 : 4 (v/v). The elution pattern was monitored at 205 nm. The c o m p o n e n t s o f iturin are denoted as peaks 1 to 6 according to the elution time order. Peaks 1

H pCll2

pCll2A

H

P h hE

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I

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Surfactln production

H Pvull

I

+

I I

+

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FIG. 2. Physical map of the Ipa-14 region and localization of the lpa-14 gene. The open boxes indicate the fragment cloned onto the vector plasmid pC194. The thin lines between the open boxes mean the part removed from pC112. The arrow indicates the orientation of the lpa-14 open reading frame. +, Surfactin positive production; - , surfactin negative production in B. subtilis MI113. Restriction endonuclease sites are as follows: E, EcoRI; H, HindIII; h, HpaI; P, PstI.

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NUCLEOTIDE SEQUENCE OF A GENE FOR ITURIN AND SURFACTIN

447

L t d . , Osaka.

to 6 c o r r e s p o n d to the c o m p o n e n t s with n-C~4-/3-amino acid, anteiso-C~s-~-amino acid, iso-Cls-/3-amino acid, nCl6-/3-amino acid, /so-Cl6-/~-amino acid, and n-C17-/3a m i n o acid, respectively (1). The a m o u n t o f peak 6 was usually t o o small to be detected. The concentration o f each c o m p o n e n t o f iturin was determined t h r o u g h a calib r a t i o n curve m a d e b y each purified-component. Surfactin was analyzed by a reversed-phase H P L C system on the same c o l u m n used for the identification o f iturin. The system was o p e r a t e d at a flow rate o f 1.5 m l / m i n and m o n i t o r e d at 205 n m with the solvent acetonitriletrifluoroacetic acid (3.8 m M ) (80 : 20, v/v). Authentic surfactin was purchased f r o m W a k o Pure Chemical Ind.,

RESULTS AND DISCUSSION Subcioning of pCl12 B. subtilis R B I 4 was previously shown to c o p r o d u c e the lipopeptides iturin A and surfactin (3). A gene which converts a derivative o f B. subtilis 168, a n o n - p r o d u c e r o f surfactin, to a surfactin-producer was previously isolated from B. subtilis RB14 as a 10-kb insert comprising four-HindlII fragments; two o f these HindlII fragments, which were cloned and designated as pC112, were f o u n d to be essential for the production o f surfactin (17). F u r t h e r reduction in the sizes o f the

Hpa I ATACGCGATCTCCGGGCGGOCAACOTCCOTTCATTAAAAACAAAOCCOAAACOGTTTTG

65

TTTTTAATGAACGGACAGCTTTCGTCTGATATGATAGGATGGTTTTGACAATATTTTCAG

125

rbs

Psi I

ACGGAGGATCTGGACATGAAAATTTACGGAGTATATATGGACCGCCCGCTTTCTGCAGGG M K I Y O V Y M D R P L S A G

185

OAAOAOOATCGGATGATOOCOOCCGTGTCCOCCOAAAAOCOGGAAAAATGCCOGCGCTTT E E D R M M A A V S A E K R E K C R R F

245

TACCATAAGGAGGATGCTCACCGCACCTTOATCOOCOACATOCTGATCCOCACCOCTGCO Y H K E D A H R T L I O D M L I R T A A

305

OCOAAOOCTTATGGACTTOATCCGGCCGOGATTTCATTCOGCOTCCAGOAATACGOAAAO A K A Y 0 L D P A O I S F G V Q E Y O K

365

CCGTACATCCCCGCOCTTCCOOACATGCACTTTAACATTTCCCACTCCGGGCOCTGGATC P Y I P A L P D M H F N I S H S O R W I

425

OTOTOCOCCGTTGATTCAAAACCGATCGOCATTOATATTOAAAAAATOAAGCCCOOCACO

485

V C A V D S K P I

0

1D

I

E K M K P G T

ATTGATATCGCCAAACGOTTTTTTTCGCCOACGOAATACAOTOATCTGCAAGCGAAACAC I D I A K R F F S P T E Y S D L O A K H

545

Hind ]If

CCCGATCAOCAOACCOATTATTTTTACCACCTGTGOTCGATOAAAOAAAGCTTTATCAAA 605 P

D

Q

Q

T

D

Y

F

Y

H

L

W

S

M

K

E

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F

l

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CAGOCCGOAAAAOOGCTTTCCCTOCCOCTTOATTCATTCAOCOTCCOCCTCAAAGACOAC Q A O K O L $ L P L D S F S V R L K D D

665

GOCCATGTGTCCATTGAOCTTCCOOACOOOCATOAACCTTOTTTCATCCOCACATATOAT O H V S I E L P D O H E P C F I R T Y D

7P.5

GCGGACOAGGAGTATAAGCTOGCCOTTTGTGCGOC(ICATCCCOATTTTTOTGACOGGATT A D E E Y K L A V C A A H P D F C D O I

785

GAGATGAAAACGTATGAAGAGCTGTTATAAGCAATCCGTCAGCATTTTGATGCCTCGCGT 845 E M K T Y E

E

L L *

GATATCCCCTGTCTTGACGTTGGACACGTTGAGTTTGATGATATTTTCTTTCGGAAAATC905 CGATAGGTAATGGCTGTCAATCGGCTCCACCAGCACGCCTTGCGCTTTCAGACGCTGCAC 965 AACTCTTGAAGCGGAAAGGCTTTTCTCCAGCACGAGATGGGTATGGATCGACGTTTGCTT I025

TACAGAAAAACCGTCGTACTCPstI 1052 FIG. 3. Nucleotide sequence of lpa-14. The HindlII, HpaI, and PstI sites are shown. The putative ribosome-binding site (rbs) is identified above the sequence and also underlined. The letters below the nucleotide sequence indicate the deduced amino acid sequence of LPA-14, using the single-letter notation. *, Stop codon. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number D21876.

448

HUANG ET AL.

J. FEgM~NT.BmeNo.,

fragments in pC112 was attempted, as shown in Fig. 2, to determine the region indispensable for surfactin production, by using B. subtilis M I l l 3 , a non-producer of surfactin, as a host strain. Plasmid pC112 was cut by HpaI and the 0.5-kb HpaI fragment was removed and ligated to make pCl13. As pC113 clearly transformed MI113 into a surfactin producer, the region required for surfactin production was estimated to be to the right or left of the HpaI site of p C l l 3 in Fig. 2. A 2.0-kb fragment was removed from pC113 by the double digestion of HpaI and PvuII followed by blunt end ligation. The resulting plusmid, pC115, also transformed MI 113 into a surfactin producer. Surfactin productivity in no. 3 medium by these surfactin-positive plasmids, pCl12, p C l l 3 , and pCllS, showed no significant variation (data not shown). Thus, we considered that the reduction in the size of the fragment was accomplished without the loss of any important part required for surfactin-production encoded on pC112.

fragment in pC112 (Fig. 2), were respectively introduced into RA1. While the clear recovery of the production of both lipopeptides was observed with pCl15, no production of these substances by pCII2A was observed (Table 2). From these results, the possibility described above was eliminated. We confirmed that the gene product from this ORF was indispensable for the production of the lipopeptides iturin A and surfactin in B. subtilis RB14, and the gene containing the ORF was named lpa-14 (lipopeptide antibiotic production of RBI4). Characteristics o f B. pumilus A-1 and psf.1 A gene named psf-l, which is necessary for surfactin production in B. pumilus A-1 as well as in M I l l 3 , was previously cloned and sequenced (18). As relatively high amino acid homology (41%) was observed between Psf-1 and Lpa-14 (Fig. 4), the productivity of iturin A or other antifungal substance(s) of B. pumilus A-1 was tested (Figs. 5 and 6). The production of iturin A of strain A-I was tested after cultivating it in no. 3 medium or spotting it on the center of a plate containing Fusarium oxysporum, as described in Materials and Methods. No antifungal zone in the bioassay and no peak of iturin A in H P L C analysis were detected from the cells or the culture broth of strain A-l, as shown in Figs. 5B and 6B. The gene psf-1 was cloned into pC194 from pTB522-2kb by HindIII and T4 ligase treatments and the resulting plasmid, pCSFI, was introduced into B. subtilis RA 1 by electroporation. The transformants exhibited the production of both iturin A and surfactin (Table 2) while the control strain RA1 with the vector plusmid pC194 showed neither production of the peptides (Table 2) nor inhibitory effect against F. oxysporum (Fig. 5E). The recovery of the lipopeptides in the strain is interesting in that psf-1 isolated from B. pumilus, a non-iturin producer, has the function of producing the two peptide antibiotics in B. subtilis, indicating the common features and uniqueness of peptide antibiotics of Bacillus species.

Analysis o f the region essential for surfaetin-production The nucleotide sequence of the 1. l-kb region from

HindIII to HpaI in pC115 (Fig. 2) was determined. Three overlapping fragments: HindIII to HindIII, PstI to PstI, and HindIII to EcoRI in p C l l 3 from left to right in Fig. 2 were subcloned into pUC19, respectively. The nucleotide sequence determined from these subclones is shown in Fig. 3. The sequence of this region revealed the existence of a large open reading frame (ORF) consisting of 224 amino acids; this ORF showed high homology with that of sfp (15), which is known to be a regulatory gene for surfactin production (Fig. 4). B. subtilis RAI is an iturin-surfactin non-producer which has been derived from B. subtilis RB14, a coproducer of the lipopeptides. A 10-kb insert comprising fourHindIII fragments originally isolated from B. subtilis RB14 could restore the productivity of both iturin A and surfactin in B. subtilis RA1 (17). The possibility that the region required for the production of iturin A and surfactin might be differently located on this fragment was first considered. The smallest plasmid, pC115, containing the ORF found above, and plasmid pCI12A without the ORF, which had been constructed by removing the PstI :

~

V

i " ~~

A'" "G' "

E " " ~E' " D

Productivity o f surfactin in M I l 1 3 and transformants o f RA1 Surfactin production of M I I I 3 ( p C l l 2 ) in no.

3 medium showed about two-fold higher productivity than that of the parental strain, RB14 (24). The vector plasmid of pC112 is pC194, which has a copy number of

L P A 1 4

1

SFP PSF-1 ORFX

1 ~ I~,~Q E~ NE ~ F ~ T F ] ' ~ P ~ ~ ' ~ , ~ 1 NK I'FA~(ILQPI.~Di(NARKQI EQL K P F ~ F ~ 1 : .... I ORHVFNF L S S N ~ K ~ Y ~ ' S "

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LPA14 SFP PSF-1 ORFX

48 : ~ I I . ~ T A A A K A ~ G ~ P A G ~ S [ G V ~ , , ~ ¥ ~ A ~ I ~ 48 ,~V~,~S V I SRQ~Q~KS D~RE.,$T ~ C ~ R O ~ , ~ , ~ ¥ I.G 51 E V ~ H I IHEH~A[PHEQ~I~ETE,GN,,~V RQIp.S~,,L,[[~,D~V~,G 37 E L ~ K Y L I QVI~Ni PNSN~ L~ RK N ~ . . ~ F Y-- ~ ~ D 6~V ~

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148 : 148 : 151 : 137:

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~ ........ ................... " . . . . . . . . . . . . . . . . . . ~. . . . . . . ~ A E ~ K ~ ~'~'(~~ ! ! ! , ~ ! ! R~li ! ~ ]~L[jLI~A~!~LT~I~YG.~'SUTA,~SE,I~Q6TLR~ I~ Q~..... ! T'I'~,~V~A I ~ N ~ l l I DkHQTQT~ I YK~k'kKEPYT I YEP ELF E

FIG. 4. Amino acid residue sequence comparison for Lpa-14 of B. subtilis RBI4, Sfp of B. subtilis OKB 105, OrfX ofB. brevis, and Psf-I of B. pumilus. Regions of identity are shown as shaded areas. - , Amino acid deletion.

VoL 76, 1993

NUCLEOTIDE SEQUENCE OF A GENE FOR ITURIN AND SURFACTIN 449

TABLE 2. Recoveryof iturin A and surfactin Strain B. B. B. B. B.

subtilis RBI4 subtilis RAI/pC194 subtilis RA1/pC112A subtilis RAI/pC115 subtilis RAI/pCSFI

Iturin A (ppm) 140 0 0 152 128

Surfactin (ppm) 335 0 0 41 71

about 15 per chromosomal equivalent (25), and lpa-14 was cloned onto pUB110, which has a much higher copy number of about 50 per chromosomal equivalent (26). However, the amounts of surfactin produced in the medium from both plasmids were almost the same (data not shown), and no gene dosage effect of this gene was observed. The productivity of iturin A and surfactin was clearly restored in B. subtilis RA1 by plasmid p C l l 5 or pCSF1, as shown in Table 2. Although the levels of iturin A production by these transformants were almost the same of that of the parental strain, RB14, the recovery levels of surfactin in the transformants were only 10 to 20% that of RB14. The true reason for the latter poor restoration levels in RA1 is not known. A regulatory gene for surfactin production in B. subtilis JH642, sfp, is reported, when cloned onto a multicopy plasmid, to repress the lacZ transcriptional fusion of the srfA operon, which encodes enzymes that catalyze surfactin synthesis (15). The poor recovery observed here may be partly explained by a similar effect of Lpa-14, because it has 72% homology with Sfp (Fig. 4). Besides this, some factors involved in iturin A production by RB14 may be associated with the phenomenon. The effect of ipa-14 on the production of lipopeptides will be clarified when the gene is introduced into a single defective strain of RB14, i.e. a non-producer of either iturin A or surfactin. Isolation of such a mutant is now in progress. H o m o l o g y a m o n g lpa-14, sfp, psf-1, and orfX A gene responsible for the production of iturin A and surfactin was analyzed in a derivative of the original wild strain as well as in a derivative of strain 168. The gene, lpa-14, permitted only surfactin production in the derivative of strain 168, and it had a high homology with sfp, a genetic locus responsible for surfactin production, which had been transferred from the original surfactin producer B. subtilis ATCC21332 to JH642, a derivative of strain 168. As sfp is located in the B. subtilis genome at a site closely linked to srfA, which encodes the surfactin synthetase enzymes (12), ituA [a putative gene encoding the iturin A synthetase enzyme(s)] might be coded just upstream of ipa-14 in B. subtilis RB14 by the same analogy (27). Conversely, the possibility that B. subtilis M I l l 3 , a derivative of strain 168, might be a defective strain of ituA is also suggested. Analysis of the flanking region of lpa-14

A

B

C

i I

A

I ,...r-m. I

[

0

10 Tim~min)

I

I

20

0

I

10 Time(rain)

i

20

FIG. 6. HPLC analysis of the production of iturin. (A) and (B) are the separation patterns of methanol extracts from B. subtilis RB14 and B. pumilus A-l, respectively. Peaks 1 to 5 correspond to the components of iturin as described in Materials and Methods. in B. subtilis RB14 and the shotgun cloning of ituA in MI113 in the presence of lpa-14 are now in progress. The deduced amino acid sequence of sfp had no significant homology to other known proteins, and the function of this protein is not yet known (14, 15). Only a relatively high homology (47%) was found between the sequence of sfp and an unknown open reading frame, o r f X (28), in the upstream region of the grs operon for gramicidin S biosynthesis. Although enzyme activities such as ATP-PPi exchange (amino acid activating) activity, and thioesterase II, esterase, and surfactin degrading activity of the Psf-1 protein have not yet been detected (18), the relatively high homology among the sfp, orfX, psf-1, and ipa-14 gene products is a clue to analyzing the role of those new gene products in the complex mechanism of peptide synthesis. If they share the same function(s) in their peptidic antibiotics, surfactin, gramisidine S, and iturin A, they are suggested to function in the upstream region in the synthetic metabolic pathways of the peptides because there is no apparent homology among the substances. An analogous structure consisting of a seven-aminoacid cyclic peptide linked to the fatty acid part is found in iturin A and surfactin, but the linkage modes and amino

D

E

ii!:°

FIG. 5. Iturin production assayed on agar plates containingF. oxysporum prepared as described in Materials and Methods. (A) B. subtilis RBI4; (B) B. pumilus A-l; (C) B. subtilis RAI (pCll5); (D) B. subtilis RA1 (pCSF1); (E) B. subtilis RAI (pC194).

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HUANG ET AL.

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acid components are quite different in the two compounds (5-7). However, two synergistic effects of surfactin and iturin A have been reported: very extensive haemolysis induced by the presence of surfactin and iturin A (29), and increased antifungal activity of iturin A in the presence of surfactin (3). The natural occurrence of these two lipopeptides in the same strain of B. subtilis, their synergistic effects, and the coregulation of their synthesis by the same gene, lpa-14, might provide useful information for developing more effective biological control agents. ACKNOWLEDGMENTS We are grateful to Dr. T. Imanaka and Mr. M. Morikawa of Osaka university for their kind gift of strain A-1 and the gene psf-], and thankful to Dr. K. Shishido and Mr. H. Amano of Tokyo Institute of Technology for their help in the nucleotide sequence analysis.

13. 14. 15.

16. 17.

18. REFERENCES

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