JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 86, No. 6, 605-607. 1998

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

CHIEH-CHEN HUANG, ZHA MIN LIAO, MITSUYO HIRAI, TAKASHI ANO, AND MAKOTO SHODA*

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 sidero- phore, 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 lOtI ppm caused the growth of RAl to be resumed.

[Key words: Bacillus subtilis, 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 antibio- tics, 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 con- sisting of 224 amino acid residues was identified by deter- mination of the nucleotide sequence (5, 6). lpa-14 showed high sequence homology with sfp of B. subtilis OKB105 (7), gsp of Bacillus brevis ATCC9999 (8, 9), psf-I of Bacillus pumilus A-l (10) and entD of Escheri- chia coli (6, 11).

The intensive analysis by Lambalot et al. (12) demon- strated 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 sidero- phore is synthesized and secreted in response to an iron- deficient medium by microorganisms. So far, 2,3-di- hydroxybenzoylglycine (2,3-DHBG) (13), shown in Fig. 1, is the only known siderophore produced by the gram- positive 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 com- plemented 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.

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 chemi- cally 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 describ- ed 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 me- dium (1 g of potassium sulfate, 3 g of dipotassium phos- phate, 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 OH

-

n \ / CONH-CH2-COOH

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

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606 HUANG ET AL. J. FERMENT. BIOENG.,

Time (h)

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

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

Purification and identification of 2,3-DHBG produced by RB14 Purification of 2,3-DHBG was conducted using a 5-day-old culture broth of RB14 in an iron- deficient medium using almost the same method as described in a previous paper (13), except that recrystalli- zation 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 206°C. 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 previous- ly, 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 purifica- tion of 2,3-DHBG from the culture broth as mentioned above, the sample extracted into ethyl acetate was dis- solved 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

0 0 10 20 30 40 50

Tie 01)

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

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, 2,3- DHBG was chemically synthesized and added to the iron-deficient medium. Although the synthesis of 2,3- DHBG was reported in previous papers (13, 17), a sim- pler procedure to achieve higher purity and yields of 2,3- DHBG than previously report (13) was developed be- cause 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 hydrochlo- ride (6 mmol) and water soluble l-ethyl-3 (3-dimethyl- aminopropyl) carbodiimide hydrochloride (6 mmol), a de- hydrant, 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 pro- duced 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 recry- stallization 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 NOTES 607

0 0 10 20 30 40 50 60 70

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 transforma- tion with psf-I of B. pumilus A-l into the lpa-14 defec- tive 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 indi- cates 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 anti- biotics which suppress plant pathogens but also a sidero- phore, and that these products are commonly regulat- ed by the gene lpa-14, indicate the possibility of enhanc- ing biocontrol using such agents if Ipa- is appropriately manipulated.

REFERENCES

Arima, K., Kakinuma, A., and Tamura, G.: Surfactin, a crys- talline peptidelipid surfactant produced by Bacillus subtilis: iso- lation, characterization and its inhibition of fibrin clot forma- tion. Biochem. Biophys. Res. Commun., 31, 488-494 (1968). Hiraoka, H., Asaka, O., Ano, T., and Shoda, M.: Characteri- zation of Bacillus subtilis RB14, coproducer of peptide antibiot- ics iturin A and surfactin. J. Gen. Appl. Microbial., 38, 635- 640 (1992). Peypoux, F., Guinand, M., Michel, G., Delcambe, L., Das, B.C., and Lederer, E.: Structure of iturin A, a peptidolipid

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

antibiotic from Bacillus subtilis. Biochemistry, 17, 3992-3996 (1978). Asaka, 0. and Shoda, M.: Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Envi- ron. Microbial., 62, 4081-4085 (1996). Hiraoka, H., Ano, T., and Shoda, M.: Molecular cloning of a gene responsible for the biosynthesis of the lipopeptide antibio- tics iturin and surfactin. J. Ferment. Bioeng., 74, 323-326 (1992). Huang, C. C., Ano, T., and Shoda, M.: Nucleotide sequence and characteristics of the gene, lpa-14, responsible for biosyn- thesis of lipopeptide antibiotics iturin A and surfactin from Bacillus subtilis RB14. J. Ferment. Bioeng., 76, 445-450 (1993). Nakano, M. M., Corbell, N., Besson, J., and Zuber, P.: Isola- tion and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacil- lus subti1i.s. Mol. Gen. Genet., 232, 313-321 (1992). Borchert, S., Stachelhaus, T., and Marahiel, M. A.: Induction of surfactin production in Bacillus subtilis by gsp, a gene locat- ed upstream of gramicidin S operon in Bacillus brevis. J. Bac- teriol., 176, 2458-2462 (1994). Eratzsmar, J. M., Krause, M., and Marahiel, M. A.: Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J. Bacterial., 171, 5422-5429 (1989). Morikawa, M., Ito, M., and Imanaka, T.: Isolation of a new surfactin producer Bacillus pumilus A-l, and cloning and nucleotide sequence of regulator gene, psf-I. J. Ferment. Bioeng., 74, 255-261 (1992). Armstrong, S. K., Pettis, G. S., Forrester, L. J., and McIn- tosh, M. A.: The Escherichia coli enterobactin biosynthesis gene, e&D: nucleotide sequence and membrane localization of its protein product. Mol. Microbial., 3, 757-766 (1989). Lambalot, R. H., Gehring, A.M., Flugel, R. S., Zuber, P., LaCehe, M., Marahiel, M. A., Reid, R., Khosla, C., and Walsh, C. T.: A new enzyme superfamily- the phosphopan- tetheinyl transferases. Chem. Biol., 3, 923-936 (1996). Ito, T. and Neilands, J. B.: Products of “low iron fermenta- tion” with Bacillus subtilis: isolation, characterization and syn- thesis of 2,3-dihydroxybenzoylglycine. J. Am. Chem. Sot., 80, 46454647 (1958). Neilands, J. B.: Effects of iron deprivation on outer membrane protein expression. Methods Enzymol., 235, 344-352 (1994). Berner, I., Greiner, M., Metzger, J., Jung, G., and Winkel- mann, G.: Identification of enterobactin and linear dihydroxy- benzoylserine compounds by HPLC and ion spray mass spec- trometry. Biol. Metals, 4, 113-118 (1991). O’Brien, I. G. and Gibson, F.: The structure of entrochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli. Biochim. Biophys. Acta, 215, 393-402 (1970). Sheehan, J. C. and Hess, G. P.: A new method of forming peptide bonds. J. Am. Chem. Sot., 77, 1067-1068 (1955). Grossman, T. H., Tuckman, M., Ellestad, S., and Osbume, M. S.: Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: relationship between B. subtilis sfp” and Escherichia coli entD genes. J. Bacterial., 179, 6203-6211 (1993). Rowland, B. M., Grossman, T. H., Osbume, M. S., and Taber, H. W.: Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3_dihydroxybenzoate biosynthetic enzymes. Gene, 178, 119-123 (1996). Crowley, D. E., Wang, Y. C., Reid, C. P. P., and Szaniszlo, P. J.: Mechanisms of iron acquisition from siderophores by microorganisms and plants. Plant and Soil, 130, 179-198 (1991). Kloepper, J. W., Leong, J., Teintze, M., aud Schroth, M. H.: Enhanced plant growth by siderophores produced by plant growth promoting Rhizobacteria. Nature, 286, 885-886 (1980). Leong, J.: Siderophores: their biochemistry and possible role in the biocontrol of plant pathogen. Ann. Rev. Phytopathol., 24, 187-209 (1986).