J. Biochem. 110, 111-119 (1991) The Nucleotide Sequence for a Proline-Activating Domain of Gramicidin S Synthetase 2 Gene from Bacillus brevis1 Kazuko Hori,* Yoshihiro Yamamoto,* * Kazuhiko Tokita,* Fumihiko Saito,* Toshitsugu Kurotsu, * Masayuki Kanda, * Kaori Okamura, * Junichi Furuyama, and Yoshitaka Saito* *Department of Biochemistry and *'Department of Genetics , Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663 Received for publication, January 24, 1991 A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunore active proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PP, exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end. A comparison of the putative amino acid sequence of grs 2-pro gene product showed that the amino-terminal region was highly homologous to the available sequence of tyrocidine synthetase 2 (71% similarity) and the carboxy-terminal region of the gene product had significant homology to the amino-terminal half of GS 1(66% similarity), as well as to other antibiotic peptide synthetases, tyrocidine synthetase 1 and ƒÂ-(L-ƒ¿-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), an enzyme involved in penicillin biosynthesis. A cyclic decapeptide antibiotic, gramicidin S, (D-Phe- and included the full length of GS 1 gene. The sequence L-Pro-L-Val-L-Orn-L-Leu)2, is produced by various strains analysis of GS 1 gene was carried out using pGS309 (7). of Bacillus brevis at the beginning of the stationary phase of Three open reading frames of sufficient length to encode the growth. The synthesis of gramicidin S is catalyzed by protein were present in the 4.6-kb insert. GS 1 gene was the gramicidin S synthetase 1 (GS 1) [EC 5.1.1.11 ] and 2 (GS second open reading frame and the others were incomplete 2), which have molecular weights of 120,000 and 280,000, frames. The other clones, pGS708 and pGS719, contained respectively (1). GS 1 activates, thioesterifies, and race longer inserts (8.1kb) which were the same but inserted in mizes phenylalanine (2) and GS 2, a multifunctional opposite orientations (7). These inserts involved two other polypeptide, activates and thioesterifies other constituent open reading frames of about 1.5 and 3kb long, upstream amino acids (3, 4). Peptide elongation is initiated by the and downstream from the grs 1 gene, respectively. transfer of D-phenylalanine from GS 1 to GS 2 (5), subse Recently Kratzschmar et al. reported the DNA sequence quent peptidyltransfers to 4•L-phosphopantetheine and from of about 5.9 kb of the gramicidin S synthetase gene, the cofactor to the following specific amino acid give rise to including the full length of the GS 1 gene and a new gene a pentapeptide (6), and then two identical peptides cyclize encoding a 29.1-kDa protein of unknown function before GS to form gramicidin S by an unknown mechanism. 1 gene, as well as 732 base pairs of an incomplete open Recently we reported the cloning and sequencing of the reading frame considered to be a part of the GS 2 gene (8). entire GS 1 gene from B. brevis Nagano (7). We have They suggested that these genes are organized in an operon. isolated four clones which expressed GS 1 activity (7). In this paper, we report the nucleotide and deduced Three of these clones, pGS309, pGS708, and pGS719 amino acid sequences of the gene behind the grs 1 gene contained about 4.6-, 8.1-, and 8.1-kb inserts, respectively, using pGS708, pGS719 and show that this gene product is immunoreactive to GS 2 antibodies and has a specific ' This work was supported in part by a research grant from the Hyogo proline-activation activity and a molecular weight of about College of Medicine. 110,000. Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPTG, isopropyl ƒÀ-D-thiogalactoside; p-amidino PMSF, (p-amidinophenyl)methanesulfonyl fluoride. Vol. 110, No. 1, 1991 111 112 K. Hori et al. Sequence analysis was carried out by using the sequence MATERIALS AND METHODS analysis software package DNASIS (Hitachi Software Engineering). Bacterial Strains and Plasmids•\Gramicidin S producer Partial Purification of the Translated Products of B. brevis Nagano and its mutant strains were used as a GS708 and GS719 in Large Scale Preparation-E. coli source of chromosomal DNA. E. coli, JM103(ƒ¢P1) JM103 cells carrying pGS708 or pGS719 were grown at [4(lac-pro); thi, strA, supE, endAsbcB, hsdR, F'traD36, 37•Ž overnight in 5 liters of L-broth supplemented with proAB, lacIq, Zƒ¢M15] or E. coli DH-1 [recA1, endA1, ampicillin (100mg/ml) and IPTG (50ƒÊM). About 15g of gyrA96, thi-1, hsdR17 (rk-, mk-), supE44] were used as cells was harvested and stored at -20•Ž. Translated host cells for recombinant plasmids. products were purified from a lysate of the frozen cell paste The recombinant plasmids pGS309, pGS708, and by the procedure described previously (7). The lysate was pGS719 contained the GS 1 gene on 4.6-, 8.1-, and 8.1-kb fractionated with 50% saturated ammonium sulfate and the inserts in pUC18, respectively (7). dialyzed fraction was subjected to DEAE-Sepharose CL-6B Western Blot Analysis of the Translated Products of the column chromatography (1.5 X 45 cm) with a linear gradi Recombinant Plasmids-E. coli cells transformed by the ent of 0 to 0.28M KCl in buffer A [50mM potassium recombinant plasmids were grown at 37•Ž overnight in phosphate buffer (pH7.5), 1mM dithiothreitol, 1mM L-broth medium supplemented with ampicillin and with or MgCl2, 0.5mM EDTA, 10ƒÊM p-amidino PMSF, 10% without IPTG as described previously (7). Total cellular glycerol]. proteins from lysed cells or the fraction from DEAE- Enzyme Assay-Activities of the amino acid-dependent Sepharose CL-6B column chromatography were subjected ATP -32PP1 exchange reaction were measured as described to SDS-PAGE on 7.5% polyacrylamide gels as described by previously (3). Laemmli (9). Proteins were transferred to a Durapore filter Biochemicals•\[ƒ¿-32P]dCTP and 32PP1 were purchased membrane (Millipore) and the blots were treated with a from the Radiochemical Centre (Amersham, U.K.). Re 1 : 1,000 dilution of anti-GS 1 antibodies or 1:5,000 striction endonucleases were purchased from Takara Shuzo dilution anti-GS 2 antibodies and visualized with horse- (Kyoto) and Nippon Gene (Toyama). DNase and RNase radish peroxidase conjugated anti-rabbit IgG using 3,3•L-di were obtained from Sigma Chemical (St. Louis, U.S.A.). - aminobenzidine (7). The DNA ligase and E. coli alkaline phosphatase were Preparation of the Antibodies against Gramicidin S purchased from Takara Shuzo. All other chemicals were Synthetase 1 and Synthetase 2•\Antiserum against the standard commercial products. purified GS 1 was prepared as described previously (7). Polyclonal antibodies against the purified GS 2 were RESULTS prepared in a similar way to anti GS 1. GS 2 antibodies were further purified by using DEAE-cellulose column Detection of Immunoreactive Proteins to GS 2 Antibodies chromatography. GS 2 used for raising antibodies was in the Translated Products of the Recombinant Plasmids -Figure 1 shows the Western blot analysis of the transla purified from B. brevis as described previously (3) with some modifications. A DEAE-Sepharose CL-6B column ed products from the recombinant plasmids, pGS309, was used instead of a DEAE-cellulose column and this step pGS708, and pGS719. All these plasmids had the full was carried out before the Ornithine-Sepharose 4B column length of GS 1 gene and the sizes of the inserts were 4.6, chromatography. 8.1, and 8.1kb, respectively. DNA Sequence Analysis-DNA sequences were deter- The translated products from pGS309 included GS 1 mined for both strands by the dideoxy-chain termination protein as described previously (7). GS 1 protein was also method of Sanger et al. (10) as described previously (7). detected in the translated products from pGS708 or Fig. 1. Western blot analysis of total cellu lar proteins from E. coli cells transformed by recombinant plasmids. Total cellular proteins present in the supernatant of lysed cells were subjected to SDS-PAGE on 7.5% poly acrylamide gels and proteins were transferred electrophoretically to Durapore membrane and reacted with GS 1 antibodies (A) or GS 2 antibodies (B), and then visualized as described in "MATERIALS AND METHODS." (A) Lane 1, lysate from E. coli JM103ƒ¢P1 containing pUC18; lane 2, purified GS 1 from B. brevis; lanes 3-5, lysate from E. coli JM103ƒ¢P1 containing pGS309, pGS708, and pGS719, respectively. (B) Lane 1, lysate from E. coli JM103ƒ¢P1 containing pUC18; lane 2, purified GS 2 from B.