Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

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MICROBIOLOGY LETTERS FEMS Microbiology Letters 132 ( 1995) 6 l-66

Bleomycin-induced P-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues Katsutoshi Yuasa, Masanori Sugiyama * Institute

of Pharmaceutical Sciences, Hiroshima University School of Medicine. Kasumi l-2-3, Minami-ku, Hiroshima 734, Japan Received 29 June 1995; revised 25 July 1995; accepted 31 July 1995

Abstract

A bleomycin-resistance gene, designated bltd, has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 15 1 (1994) 1 l-16). The present study shows that Escherichia coli harboring the blnul-carrying pUC plasmid overproduced plactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA. We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blm4 and a Streptomyces tyrosinase gene located under the control of plactamase promoter: E. coli harboring pMABS0 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues. Keywords:

Bleomycin; Bleomycin-binding protein; PLactamase; Induction; Streptomyces verticilfus; Escherichia co/i

1. Introduction Bieomycin (Bm) is a glycopeptide antibiotic which is used clinically as an anti-tumor agent; it inhibits

DNA synthesis in bacterial and mammalian cells [l]. Antibiotic-producing microorganisms must be protected from the lethal effects of their own products. We have cloned and sequenced two independent genes, designated blmA and blmB, encoding determinants which confer resistance to Bm, from the

* Corresponding author. Tel.: +81 (82) 257 5280; Fax: + 81 (82) 257 5284; E-mail: [email protected].

chromosomal DNA of Bm-producing Streptomyces verticillus ATCC 15003 [2]. The blmA gene product, designated BLMA (M, 13 1791, encodes a Bm-binding protein [2,3]. Another gene, blmB, encodes a Bm N-acetyltransferase [2,4]. We found by chance that Escherichiu coli TGl cells harboring b1m.A inserted into pUC plasmid overproduced a 29-kDa protein when cultured in the presence of Bm. Here we describe that the Bm-induced protein is P-lactamase encoded by the plasmid. In addition, we constructed an E. coli vector sensing Bm which is designated pMAB50. E. coli transformed with the vector produced the melanin

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pigment in the presence of Bm, suggesting that P-lactamase promoter functions as a sensor for Bm.

2. Materials and methods 2. I. Bacterial strain and plasmid E. coli strains TGl (supE hsdA5 thi A(lacproAB)/F’[ traD36 proAB + la@ lacZAMlS]), HBlOl (supE44 hsdS20(r;m,) recA13 ara-14 proA lacy1 galK2 rpsL20 xyl-5 mtl-1) and JM109 (recA1 supE44 endA hsdR17 gyrA96 relA1 thi laclY A(lac_proAB)/F’[traD36 proAB + lacZAM151) were used as hosts [5]. The plasmids pUC18 and pUC19 were used to generate pl81EBl and pl91EBl described below, respectively, which carry blmA from the Bm-producing Streptomyces oerticillus ATCC 15003 ( = Streptouerticillium sp. ATCC 15003). The pUC-based plasmid, pUC4KIXX, was purchased from Pharmacia LKB Biotechnology (Sweden). 2.2. Detection of a 29-kDa protein in the cell extract from E. coli cells E. coli TGl (pl81EBl) were grown overnight in Luria-Bertani (LB) [5] liquid medium containing 100 wg ml-’ of ampicillin (Ap) or 200 pg ml-’ of bleomycin A z sulfate (Bm). The harvested cells suspension (5 ~1) were added to 100 ~1 of sample buffer for SDS-PAGE [6] and boiled for 5 min. The cell extracts were centrifuged at 12000 rpm for 10 min and resulting supernatant fluids were subjected to SDS-PAGE [6] or two-dimensional PAGE as described [7] and then stained with Coomassie brilliant blue R250. The area of the gel containing an 29-kDa protein overexpressed in the presence of Bm was excised and transferred to a ProBlott membrane filter (Applied Biosystems) and used for N-terminal amino acid sequence analysis. 2.3. Culture media and condition to produce melanin pigment

the

E. coli harboring pMAB50 was pre-cultured overnight at 37°C in LB medium supplemented with 100 pg ml-’ of Ap. The culture (4 ~1) was trans-

Letters 132 (1995) 61-66

ferred into 4 ml of the same medium, incubated for 10 h at 37°C and subsequently diluted with 100 volume of LB medium. After a 4-ml portion of the diluted solution was overlaid onto Tyr-agar medium (0.2% glucose, 1% yeast extract (Difco), 0.5% NaCl, 0.05% L-tyrosine, 0.2 mM Cu(NO,),, 0.2 mM FeCl, and 1.5% agar, pH 7.2), which was used to visualize melanin synthesis, the excess culture fluid was decanted. Stainless steel cylinders (inner diameter 6 mm, hight 10 mm) for anti-bacterial assay containing Bm or its analogues (200 ~1) at the given concentrations were placed onto the layer on top of the Tyr-agar medium and plates were incubated in the dark at 37°C.

3. Results and discussion 3.1. Overproduction of a 29-kDa protein in E. coli A 658-bp EcoRI-BamHI fragment containing blmA from S. verticillus ATCC 15003 [2] was subcloned into the same sites in pUC18 and pUC19 to generate pl81EBl and pl91EB1, respectively. E. coli TGl (pl81EBl or pl91EBl) grew on LB agar medium supplemented with 1000 pg ml-’ of Bm, whereas the host cells did not, even in medium containing 5 pg Bm ml-‘. In the course of experiments designed to study the expression of the cloned blmA in E. coli, we observed that 200 pg ml-’ of Bm added to LB liquid medium (pH 7.2) for maintenance of pl81EBl and pl91EBl gave rise to overproduction of a 29-kDa protein in E. coli cells (Fig. 1). Addition of Ap instead of Bm into the culture did not lead to increased synthesis. To determine the minimal amount of Bm needed for induction, E. coli TGl(pl81EBl) was exposed to a series of dilutions of Bm and analysed by SDS-PAGE. At a concentration of 1 pg ml-‘, no induction of the 29-kDa protein was observed; however, induction was observed at concentrations of > 10 pg Bm ml-’ (data not shown). The Bm-induced protein was observed in E. coli TGl and HBlOl, but only scarcely in E. coli JM109. Additionally, the pl81EBl-harboring cells overproduced the protein when grown at 37°C but did not at 18 and 27°C. Overproduction of the protein was observed in cells cultured in LB liquid medium adjusted at pH 7.2-9.0 but not at pH 7.0 and below (data not shown).

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3.3. Identification of the Bm-induced 29-kDa protein

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Fig. 1. SDS-PAGE analysis of a 29-kDa protein induced by Bm. Lanes: 1, Ap-cultured E. coli(pUC18); 2, Aptultured E. coli(plllEB1); 3, Bm-cultured E. coli(plllEB1); 4, Ap-cultmed E. coli(pUC19); 5, Ap-cultured E. coli(pl91EBl); 6, Bm-cultured E. cofi(pl91 EB 1). The 29-kDa proteins are indicated by arrow heads. Numbers indicate sizes in kDa.

3.2. EfSect of the other Bm-resistance genes on Bminducible activity In addition to bbn.4, we have cloned bbnB which confers resistance to Bm [2] and codes for Bm N-acetyltransferase from S. verticillus ATCC 15003 [2,4]. We constructed the pUC18-based p181B2 plasmid carrying blmB and introduced it into E. cofi TGl. The pl81B2-containing cells did not produce the Bm-inducible protein (data not shown). A transposon Tn5mediated resistance to Bm is known to exist in Gram-negative bacteria [8,9]. The pUC-based plasmid pUCCKIXX contains a 1.6kb DNA cassette with both kanamycin (neoj- and Bm-resistance genes derived from transposon Tn5. E. coli (pUC4KIXX) did not overproduce the 29-kDa protein in the presence of Bm (data not shown). Therefore, we suggest that the overproduction of Bm-induced protein was specific to binA.

Cell extracts from E. cofi TGl(pl81EBl) were subjected to two-dimensional PAGE using the method described by O’Farrell [7]. As shown in Fig. 2a, when cultured in the presence of Bm, a 29-kDa protein, observed as a major spot, was produced. However, E. coli grown in the presence of Ap produced only low levels of the corresponding protein (Fig. 2b). The Bm-induced 29-kDa protein spot in the two-dimensional PAGE was transferred to a membrane filter by electro-blotting method and analysed for N-terminal amino acid sequence using an automated sequencer (Model 477A; Applied Biosysterns). The N-terminal amino acid sequence of the protein, determined by Edman degradation, was HisPro-Glu-Thr-Leu-Val-Lys-Val-Lys-Asp-Ala-GluAspGln-Leu-Gly-Ala-Arg-Val-Gly-Tyr-Ile-Glu, indicating that the N-terminal 23-amino acids sequence of the 29-kDa protein is identical to that of the mature &lactamase (EC 3.5.2.6) deduced from the nucleotide sequence of Ap-resistance gene on pBR322 [lo]. It was to be expected that the Bm-induced 29-kDa protein would not be produced if the pl81EBl plactamase gene is disrupted. Therefore, the DNA fragment carrying thr neo gene but not the Bm-resistance gene, isolated from pUC4-KIXX by digestion with SmuI, was inserted into ScaI-digested pl81EBl to generate plasmid pl81SSl which was introduced into E. cofi TGl. Cells harboring the plasmid disrupted with plactamase gene, produced an aminoglycoside 3’-phosphotransferase II protein that migrated on SDS-PAGE at about M, 27500 [l 11, but not plactamase, regardless of the presence of Bm or Ap (Fig. 3). 3.4. Effect of the Bm derivatives on the p-lactamase induction Bm derivatives, such as phleomycin, pepleomycin ( = peplomycin), liblomycin and cleomycin, consist of a bleomycinic acid moiety common to all Bm analogues and each has a different terminal amine structure. We examined P_lactamase induction in the presence of Bm derivatives when added to the culture of E. cofi(pl8 1EBl). Phleomycin MOP-copper complex, pepleomycin, liblomycin and cleomycin A, were effective for the overproduction of the

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Fig. 2. Two-dimensional PAGE of the cell extract from E. coli(pl8lEB 1) grown in the presence of Bm (a) or Ap (b).The induced protein is indicated by an arrow head. The protein was not visible in (b).Numbers indicate sizes in kDa.

29-kDa protein, P-lactamase, whereas bleomycinic acid was hardly effective (Fig. 4), suggesting that the terminal amine moiety in Bm molecule may be essential for the Bm-induced /?-lactamase gene expression associated with blm.4. 3.5. Construction of a vector that senses Bm and produces melanin pigment We attempted to construct a chimeric plasmid, designated pMAB50 (Fig. 5), carrying a Strepto-

myces tyrosinase gene necessary for the synthesis of melanin pigment, under the control of the P-lactamase promoter (Pump> which senses Bm, together with blmA. The strategy is as follows: a sense (5’-ATTGAAAAAGGAAGAGCATGCTCGAG-3’) and an anti-sense(S’-CTCGAGCATGCTCTTCCTTTITCAAT-3’) nucleotide were chemically synthesized. The complemental oligonucieotides were annealed to become a linker for the connection of Pamp and tyrosinase structural gene from S. antibi-

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DraI

Sph I ssp I

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31 Fig. 5. Structure of pMAB50. amp, p-lactamase.structural gene; blmA, a gene encoding a Bm-binding protein from S. verticillus; mel, tyrosinase sttuctural gene for the synthesis of melanin pigment; Pamp, p-lactamase promoter; ori, replication origin. Fig. 3. SDS-PAGE analysis of the Bm-induced protein by insertion of the neo gene into the /3&ctamase gene of pl81EBI. Lanes: I, Ap-cultured E. coli(pl81EBl); 2, Bm-cultumd E. coli(plllEB1); 3. kanamycin-cultured E. coli(pl81SSl); 4, Bmcultured E. coli(pl81SSl). Induced protein is indicated by an arrow head. Numbers indicate sizes in kDa.

oticus [12]. This linker, having S’hI and SspI sites, was phosphorylated with T4 polynucleotide kinase and inserted by blunt-end ligation into the 2.4-kb SspI-DraI DNA fragment of pl8lEBl to generate pMAB 10. The SphI site located downstream of Pump was made unique by elimination of the other SphI site from the multi-cloning sites of the pMABl0 plasmid. That is, pMAB 10 was digested with HindIB and XbaI, and subsequently the resulting 2.4-kb fragment was filled in with T4 DNA polymerase and self-ligated to generate pMAB20. Next, to express the tyrosinase gene under the control of the Pamp,

Fig. 4. Effect of Bm analogues on ~lactamase induction. E. coli TGl(pl81EBl) were grown overnight in LB liquid medium containing Ap &JO pg ml-‘) or Bm analogues (200 pg ml-‘) described below. The cell extracts from E. cob’ grown in the presence of each compound were subjected to 13% acrylamide SDS-PAGE. An arrowhead indicates the 29-kDa protein (P_ lactamase). Lanes: 1, Ap; 2, Bm A, (sulfate); 3. Bm A, (hydrochloride); 4, copper (II)-chelated Bm A, (hydmchloride); 5, copper (II&elated bleomycinic acid; 6, copper (I&chelated phleomycin-MOP; 7, pepleomycin; 8, liblomycin; 9, cleomycin A,.

the 1.3-kb &&I-SphI fragment containing both Pamp and bZmA from pMAB20 was inserted into EC&I-SphI sites in a promoter-probe vector pMX 180 (submitted elsewhere) to construct pMAB30. Likewise, the 2.4-kb DNA fragment from BumHI- and DruIdigested pMAB30 was ligated to the 1.7-kb BumHI-DraIdigest containing bfm.4 and

3 Fig. 6. Bm-induced melanin pigment production in E. coli TGl(pMAB50). After E. coli TGl(pMAB50) was poured on the Tyr-agar medium, Eve stainless steel cylinders containing Bm at the given concentrations or the supematant from the culture broth of S. verticillus were placed onto the medium. The plate was incubated overnight at 37°C and then for 2-3 days at 27°C. Wells: I, 2 pg; 2.20 pg; 3,200 pg; 4, the supematant from Bm-producing microorganism; 5, distilled water.

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the replication origin of pl81EBl. The 4.1-kb DraI digest of the resulting plasmid was ligated to a 0.7-kb DruI fragment from p18 lEB1 to generate pMAB50 (Fig. 5). E. coli cells transformed with pMAB50 produced the melanin pigment in the presence of significant amounts of Bm (20 and 200 pg) or the supematant fluid from a culture of 5. uerficillus ATCC 15003, a Bm-producing strain, but not in the presence of less than 2 pg of Bm or in the absence of the drug (Fig. 6). Pepleomycin and phleomycin were also effective (data not shown). We observed that further incubation of the test organism for 2-3 days at 27°C after growing overnight at 37°C and high concentrations of antibiotic were necessary to produce the melanin pigment efficiently. Thus, E. coli cells transformed with pMAB50 can be useful as a reporter organism for screening microorganisms which produce the Bm family of antibiotics.

Acknowledgements This work was supported in part by a grant to M.S. from the Ministry of Education, Science, and Culture of Japan, and a grant of the Tsuchiya foundation, Hiroshima, Japan. The Bm family of antibiotics were kindly donated by from Nippon Kayaku Co., Ltd., Japan.

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