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Zpa-14, a Gene, Involved in the Production of Lipopeptide Antibiotics, Regulates the Production of a Siderophore, 2,3-Dihydroxybenzoylglycine, in Bacillus sub tilis RB 14

Research Laboratory of Resources Utilization, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan
Received 31 July 1998/Accepted 21 September 1998 A gene designated lpa-14, which was cloned from Bacillus subtih RB14 and was associated with the production of lipopeptide antibiotics, was found to be involved in the production of an iron-chelating siderophore, 2,3-dihydrozybenzoylglycine (2,3-DHBG). Although strain RAl, an Zpa-14 defective mutant of RB14, showed no growth in iron-deficient medium in contrast to the marked growth of RB14 in the same medium, the addition of chemically synthesized 2,3-DHBG at approximately lOtIppm caused the growth of RAl to be resumed. [Key words: Bacillussubtilis, lipopeptide antibiotic, siderophore, 2,3-dihydroxybenzoylglycine] We isolated Bacillus subtilis RB14, a coproducer of the cyclic lipopeptide antibiotics iturin A and surfactin (l-3), which is a potential biocontrol agent (4). Using the B. subtilis M1113, a nonproducer of these antibiotics, as the cloning host, we cloned a fragment from the RB14 genome which permitted the MI113 strain to be a surfactin producer. When the fragment was destructed in the chromosome of RB14, the strain was altered to produce neither surfactin nor iturin A. The gene was designated lpa-14 (lipopeptide antibiotic production of RB14), and the existence of an open reading frame consisting of 224 amino acid residues was identified by determination of the nucleotide sequence (5, 6). lpa-14 showed high sequence homology with sfpof B. subtilis OKB105 (7), gsp of Bacillus brevis ATCC9999 (8, 9), psf-I of Bacillus pumilus A-l (10) and entD of Escherichia coli (6, 11). The intensive analysis by Lambalot et al. (12) demonstrated that (i) Sfp has phosphopantetheinyl transferase activity which will modify the consensus serine residue in all seven amino-acid-activating sites in SrfABC and (ii) EntD has the same enzymic activity, which is specific for the activation of enterobactin synthetase in E. coli. They also stated that the lpa-14 product most probably has an equivalent role, phosphopantetheinylation of each amino-acid-activating domain, in B. subtifis iturin A synthetase. An iron-chelating metabolite classified as a siderophore is synthesized and secreted in response to an irondeficient medium by microorganisms. So far, 2,3-dihydroxybenzoylglycine (2,3-DHBG) (13), shown in Fig. 1, is the only known siderophore produced by the grampositive B. subtilis. The gram-negative E. coli produces a siderophore, enterobactin, and this system has been well studied (11). The E. coli entD mutant was complemented for enterobactin production by the sfp gene of the B. subtilis 168 strain (7) which differs from sfp by five base substitutions and one base insertion. These mutations change a 224-amino-acid peptide in SFP to a * Corresponding author. 605 165amino acid peptide of SFP (7). This indicates that there is functional interchangeability between sfp and entD. Furthermore, we found that lpa-14 showed 72% sequence homology with sfp (6). From these data we speculated the involvement of lpa-14 in siderophore production in B. subtilis. In this paper, 2,3-DHBG was both purified from the culture broth of RB14 and chemically synthesized, and the relationship between the gene, lpa-14, and 2,3-DHBG was clarified. Growth in iron-deficient medium B. subtilis RAl is a lpa-14 defective mutant that was derived from RB14 (5). Plasmid pC115 has a fragment encoding lpa-14, and RAl/pCllS indicates a transformant of Rhl with pC115. The characteristics of these strains were described in detail in previous papers (4, 6). RB14, RAl and RAl/pC115 were precultured overnight in Luria-Bertani (LB) medium containing log of Polypepton (Nihon Pharmaceutical Co., Tokyo), 5 g of yeast extract, and 5 g of sodium chloride per liter (pH 7.2), and washed with 1 mM Hepes buffer. Then, the suspension of each strain was used to inoculate synthetic iron-deficient medium (1 g of potassium sulfate, 3 g of dipotassium phosphate, 3 g of ammonium acetate, 20 g of sucrose, 1 g of citric acid, 2mg of thiamin, 0.0196mg of copper (II) sulfate, 0.154mg of manganese sulfate, 8.79mg of zinc sulfate, and 0.81 g of magnesium sulfate per liter of water, pH7.0) (13) and the growth curves of RB14, RAl, and RAl/pC115 were monitored by measuring the OD at 660nm (UV-1200, Shimadzu, Kyoto) as shown in Fig. 2. The iron-deficient medium contained an extremely low concentration of iron which was estimated to be around 0.1 PM (14). RB14 and RAl/pCIlS grew well under iron-stressed conditions by producing a siderophore, but

OH \ n /



FIG. 1. Structure of 2,3-dihydroxylbenzoylglycine.





0 0


20 Tie

30 01)



Time (h)
FIG. 2. The growth of RB14 (0), RAl (0) and RAl/pCllS in iron-deficient medium.


FIG. 3. The growth of RAl in iron-deficient medium (0) and in iron-deficient medium fortified with 10 pM of FeC& ( W).

RAl showed no growth. When 10pM FeC& was added to the iron-deficient medium, growth of RAl was observed as shown in Fig. 3, suggesting that lpa-14 in RB14 is related to iron uptake.

Purification and identification of 2,3-DHBG produced Purification of 2,3-DHBG was conducted by RB14
using a 5-day-old culture broth of RB14 in an irondeficient medium using almost the same method as described in a previous paper (13), except that recrystallization of the siderophore in hot water was repeated several times in this study. The purified material was used for the following analyses. Spectrometric analysis of the purified product was performed using fast atom bombardment-mass spectrometers (FAB-MS, models JMX-DX303 and JMA-DASlOO) and showed a peak at the molecular weight of 2,3-DHBG (212[M+H]+). H-NMR (JEOL JNM-EX-90) analysis of the product demonstrated a peak at 12.26 ppm for COOH, at 9.1 ppm for ArCONHR-(Ar:phenyl), at 7.35-6.60 ppm for CsH3, and at 4.06ppm for CH&OOH. The Fourier transform infrared spectrum chart showed absorption at 1739cm- resulting from the C-O stretching mode for COOH, at 3352 cm-l from the N-H stretching mode for CONH, at 1648cm-r from the stretching mode for CONH, and at 738 cm- from C6H3. The melting point was determined to be between 200 and 206C. The Rf value as determined by TLC (PSC-Fertigplatten Kieselgel 60 F254, Merck & Co. Inc., NJ, USA, solvent system (volume ratio): t-butyl alcohol, 10; methyl ethyl ketone, 10; water, 5; diethyl amine, 1) was 0.36, while previously, a value of 0.39 was reported (13). By comparing the data obtained here with those in a previous paper (13), the purified substance was judged to be 2,3-DHBG. Quantitative analyses of 2,3-DHBG by HPLC To determine the concentration of 2,3-DHBG produced by RB14, a HPLC system was established by modification of a previously reported system (15). After the purification of 2,3-DHBG from the culture broth as mentioned above, the sample extracted into ethyl acetate was dissolved in 100% methanol and then filtered through a 0.2pm PTFE membrane (JPO20, Advantec Ltd., Tokyo). The filtrate was subjected to HPLC with the elution solvent consisting of a mixture of methanol-0.1% phosphoric acid (1 : 1 [v/v]) (16). The elution time of 2,3-DHBG was 3.5 min, and the concentration produced

by RB14 grown in an iron-deficient medium for 5 d was determined to be approximately 70 ppm. As RAl showed almost no growth in the iron-deficient medium after 5 d, no peak corresponding to 2,3-DHBG was observed in the HPLC trace of the filtrate derived from this culture. The fact that RAl/pCl15 also produced about 70 ppm 2,3-DHBG indicates that lpa-I4 is associated with the production of the siderophore, 2,3-DHBG.

Growth of RAl in iron-deficient medium containing chemically synthesized 2,3-DHBG To clarify that the
lack of growth of RAI in the iron-deficient medium is related to the defective production of 2,3-DHBG, 2,3DHBG was chemically synthesized and added to the iron-deficient medium. Although the synthesis of 2,3DHBG was reported in previous papers (13, 17), a simpler procedure to achieve higher purity and yields of 2,3DHBG than previously report (13) was developed because 2,3-DHBG of high purity is critical for observing the effects on the growth of RAI. 2,3-Dihydroxybenzoic acid (2,3-DHB) (5 mmol), glycine ethyl ester hydrochloride (6 mmol) and water soluble l-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (6 mmol), a dehydrant, were dissolved in 25 ml of chloroform. Then, 0.83 ml of triethylamine (6 mmol) was slowly added, and the mixture was stirred overnight at room temperature under a Nz atmosphere. The reaction mixture was dried by evaporation, and the residue was acidified with 2 N HCI (pH 3) and then extracted 3 times with ethyl acetate. The organic phase was washed with Hz0 and 0.5 N NaHC03. The crude 2,3-DHBG ethyl ester thus obtained was eluted with ethyl acetate: n-hexane (1 : 1 [v/v]) as an elution solvent through an EDTA-treated silica gel column. Recrystallization of the eluted solution produced a crystal of 2,3-DHBG ethyl ester in 25% yield. The crystal was dissolved in 2N NaOH, and stirred at room temperature for 1 h. The reaction mixture was acidified with dilute HCl (pH 4), extracted 3 times with ethyl acetate and washed with 10% citrate and H20. After removing the solvent, the residue was recrystallized from diethyl ether-hexane. Several repeats of the recrystallization process gave pure white 2,3-DHBG in 10% yield. The structure and purity of the synthesized sample were confirmed using the previously mentioned methods. When chemically synthesized 2,3-DHBG was added to the iron-deficient medium at 10 ppm, 50ppm, 100 ppm,

VOL. 86, 1998 antibiotic from (1978). 4. Asaka, 0. and damping-off of ron. Microbial.,
5. Bacillus subtilis. Biochemistry,



17, 3992-3996


7. 0








Time (h)

FIG. 4. Growth of RB14 (4) in iron-deficient medium and Ral in iron-deficient medium containing 2,3-DHBG. 2,3-DHBG: 0 ppm (0), 10 ppm (V), 50 ppm (0 ), loo ppm (a), 200 ppm ( q ).


and 2OOppm, the growth rate of RAl increased with each increase in the concentration of 2,3-DHBG as shown in Fig. 4. The growth of RB14, which produced about 70 ppm 2,3-DHBG, was between that of RAl in medium with 50 ppm and 100 ppm of added 2,3-DHBG, indicating that the growth of RAl in the presence of 2,3-DHBG was almost equivalent to that of RB14 in the iron-deficient medium. This confirms that the lpa-14 gene is involved in the synthesis of 2,3-DHBG. We showed in this study that lpa-14 functions not only in the synthesis of the lipopeptide antibiotics, iturin A and surfactin, but also in siderophore synthesis in the RB14 strain. We previously found (6) that transformation with psf-I of B. pumilus A-l into the lpa-14 defective mutant RAl gave the transformant the ability to produce both iturin A and surfactin, even though B. pumilus A-l is a non-producer of iturin A. This indicates that Psf-1 has a function similar to Lpa-14. As entD could complement sfp mutants (18) and B. subtilis genes involved in siderophore synthesis were isolated by complementing enterobactin mutants (19), these proteins have similar and interchangeable functions in different bacteria. Bacterial siderophores are known to help plant growth by supplying iron via iron-chelation termed the PGPR (Plant Growth Promoting Rhizobacteria) effect (20-22). The fact that B. subtilis RB14 produced not only antibiotics which suppress plant pathogens but also a siderophore, and that these products are commonly regulated by the gene lpa-14, indicate the possibility of enhancing biocontrol using such agents if Ipa- is appropriately manipulated.
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