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MICROBIOLOGY LETTERS

FEMS Microbiology Letters 145 (1996) 113-l 16

Construction of pRES 18 and pRES19, Streptomyces-Escherichiu coli shuttle vectors carrying multiple cloning sites

Jun Ishikawa *, Yasuhiro Niino, Kunimoto Hotta

Received 9 July 1996: revised 13 September 1996; accepted 18 September 1996

Abstract

We developed two Strepton?yces-E;sc,heuichiu co/i shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene (tsr) fragment of pIJ702, was combined with the E. coli plasmid vector pUCl8 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively. have relatively compact size (6.25 kb), low copy number. multiple cloning sites reciprocally arranged in opposite directions. and selection markers for both Streptomyces (tsr) and E. coli (p-lactamase (hlu) and P-galactosidase ([acz)). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. laven&lur and S. lividam. and are compatible with pIJ702.

Kqwords.- Recombinant DNA: Plasmid wctor; pRESl ; pRES102; pRES107: Stwptorrgws ~riseus; StwptonzJ~crs lividum

1. Introduction

Long-term cultivation and complicated manipula- tions are required for genetic study of streptomy- cetes. Consequently, researchers prefer Escherichia coli host-vector systems to those of Streptomyces in order to analyze cloned Streptomyces genes rapidly. A number of shuttle vectors have been reported in streptomycetes [l]. However, they have only a few cloning sites, and their sizes larger than unifunctional vector limit their use for genetic studies, except for pHJL401 [2]. We therefore attempted to develop compact Streptomyces-E. roli shuttle vectors carry- ing multiple cloning sites (MCS). The plasmid pRES1 (4.8 kb) is a cryptic low copy number plas-

* Corresponding author. Tel.: +8 I (3) 5185 1 II I ; Fax: +81 (3) 5285 1150; E-mail: jun@nih.go.jp

mid isolated from Streptomyces griseus SS-1429 (de- tails will be described elsewhere). Since the essential region of pRES1 lacks many recognition sites for commonly used restriction enzymes, we found the plasmid an advantageous source of construction of a useful shuttle vector with MCS.

2. Materials and methods

2.1. Strains, plasmids and culture conditions

S. lividans TK21 and Escherichia cnli JM109 were used as host strains for DNA manipulation. Plas- mids pUCl8 and pUC19 [3] were used for vector construction. Plasmid pRES1 (Fig. 1) was discovered in strain SS-1429, which is a soil isolate classified as S. griseus, in the course of plasmid screening in

0378-1097 /96/ $12.00 Copyright 80 1996 Published by Elsevier Science B.V. All rights reserved PI1s0378-1097(96)00397-7

J Mikaw et 01. IFEMS Microbiology Letters I45 (1996) 113-116

‘Hindlll ‘Sphl *PStl Sal1

‘Xbal ‘Bantil ‘Smal Kpnl

‘Sac1 ‘EcoRI

pRES1 2.5 kb Bell + plJ702 1.05 kb Bell (tsr)

Sacl Clal

pRES102 MM+ pUC18/19 Narl

/ pRES102 M/d + Sacl & Smal elimination pANT3-2 1.5 kb Mlul (kan)

\

(Mlul/Narl)

BarnHI, Mlul\ I”“”

M’-ll~sacl Bell

J. Ishikawu et al. IFEMS Microbiology Letters I45 (1996) 113-116 115

Fig. 1. Construction of pRES series plasmids. In the map of pRESlW19, suitable cloning sites are indicated with asterisks and eliminated sites are in parentheses. Arrows indicate marker genes and their directions of transwption. c

Streptomyces strains [4]. Plasmid pRES102 (Fig. 1) was constructed by ligating the 2.5 kb BclI fragment containing the replication region of pRES1 and the 1.05 kb BclI fragment containing the tsr gene of pIJ702 [5]. Details of pRES1 and pRESl02 will be described elsewhere. Plasmid pRESl07 (Fig. 1) was constructed from pRES102 and the 1.5 kb M/u1 fragment containing the kanamycin resistance gene (kan) of pANT3-2 [6,7]. Streptomyces strains were grown at 27°C in Tryptic soy broth (Difco) or on yeast-extract malt-extract agar (Difco). Transforma- tion of Streptomyces strains was carried out by the method previously reported [7].

2.2. DNA manipulution

Preparation of total DNA was previously reported [7]. Plasmid DNAs were extracted by alkaline lysis and purified by density-gradient centrifugation as re- ported by Andersson and Hjorth [8].

3. Results

3.1. Construction of’pRES18 and pRESl9

The plasmid pRES102 (Fig. 1) was used for vector construction by taking advantage of the unique A4luI site, whose half-site could be ligated with the unique NarI site of pUC plasmids [3]. In order to generate NurI compatible ends, pRES102 was digested with MluI followed by partial filling-in of 5’ overhangs, and ligated with NarI-digested pUC18 and pUC19 resulting in version 1 .O pRESl8 and pRES19, respec- tively. To increase the value of the MCS, we elimi- nated the Sac1 and the Smal sites located outside the polylinker region as follows. Polylinkers of the ver- sion 1.0 plasmids were replaced with the lambda-de- rived 1.4 kb EcoRI-Hind111 fragment in which no recognition sites for both Sac1 and SnzaI existed. The resulting plasmids (version 1 .l) were digested with Sac1 followed by Sl nuclease, self-ligated, and used to transform E. coli JM109. Ampicillin-resistant clones were selected and a plasmid without Sac1 site

was chosen (version 1.2). The same process was re- peated with XmaI, an isoschizomer of SmaI, instead of Sac1 (version 1.3). Finally, the lambda fragments were replaced with the original fragment containing a polylinker of pUC plasmid. The resulting plasmids (version 1.4) have unique BumHI, EcoRI, Hz’ndIII, PstI, SrrcI, SmaI, S&I and X&I sites as MCS, and selection markers for both Streptomyces (tsr) and E. coli (blu and lacz).

3.2. Characteristics of pRESl8 and pRES19

We attempted to transform several Streptomyces and E. coli strains with version 1.4 pRES18/19 plas- mids. The plasmids were well maintained in E. coli, S. griseus, S. lividans, and S. lavendulue [9]. These facts possibly imply a broad host range for pRES plasmids.

To determine the length of the DNA fragments pRES18/19 can carry, total DNA extracted from a S. kasugaensis strain was digested with BumHI, frac- tionated (5-20 kb), ligated with BarnHI-digested pRES19, and used to transform E. coli JM109. Twenty-four out of 100 ampicillin-resistant and white colonies were chosen and analyzed for their plasmids. They contained inserts ranging from 5 to 15 kb. S. lividans TK21 was also transformed with these recombinant plasmids suggesting that pRES19 was stably maintained in both S. lividuns TK2I and E. coli. There was a decrease in transformation effi- ciency of S. lividuns with plasmids propagated in E. coli JM109 cells. This is presumably due to the re- striction by S. lividans of the plasmids propagated in E. coli. This explanation was supported by the in- creased transformation efficiency of S. lividans with pRES plasmids after extraction from S. lividuns (data not shown).

To test compatibility with pIJ702, protoplasts of S. lividuns TK21 harboring pIJ702 were transformed with pRES107 (Fig. 1). Thiostrepton-resistant, mel- anin-producing and kanamycin-resistant clones were chosen, purified by single-spore isolation, and anal- yzed for their plasmids. As shown in Fig. 2, they harbored both pIJ702 and pRES107. This indicated

116 J. Ishikawa et al. I FEMS Microbiology Letters 145 (19961 113-116

M 1 2 3 4

Fig. 2. Agarose gel electrophoresis of plasmid DNA isolated from S. lividans TK21 carrying both pIJ702 and pRES107 (lanes I-3) or pIJ702 alone (lane 4). Plasmid DNA was isolated by the alkaline method, digested with MluI, and electrophoresed on 0.8% agarose gel. MIuI digestion of pIJ702 and pRES107 gave a 5.8 kb fragment and 3.55 kb and 1.5 kb fragments. respectively. HindIll-digested h DNA and HueHI-digested $X174 DNA were used as size standards (lane M).

that pRES plasmids were compatible with pIJ702. Fig. 2 also indicates that the copy number of plasmid pRES was low in Streptomyces. The copy number was estimated to be 5-10 per genome by comparison of its band intensity with those of pIJ702. This fea- ture may be useful because high copy number plas- mids sometimes change the physiological properties of the host strain in Streptomyces.

4. Discussion [cl

As described above, pRESl8119 have useful fea- tures for the genetic study of Streptomyces genes. In particular, it will be advantageous that pRESlS/ 19 are maintained in S. lividuns and are compatible with pIJlO1 derivatives, because they are the most widely used host and vector for the study of Strep- tomyces genes. Asymmetric MCS of pRES18/19 per- mits unidirectional deletion of inserts with exonu- clease III. Indeed, we carried out a structural study of an aminoglycoside-resistance gene cloned in pRES18 (unpublished data). The use of pRES18/19 saves time, since deletion derivatives could be di- rectly used for transformation without subcloning in order to test their expression in Streptomyces.

pRES18/19 are very similar in size and structure to pHJL401 [2]. However, our plasmids are distinct from pHJL401 in terms of reciprocally arranged MCS, a unique SphI site, and compatibility with pIJlO1 derivatives.

Acknowledgments

We thank N. Saito and S. Mizuno for isolation of pRES1 and encouragement, respectively.

References

[I] Hopwood, D.A., Bibb, M.J., Chater. K.F. and Kieser, T. (1987) Plasmid and phage vectors for gene cloning and anal- ysis in Streptomyces. Methods Enzymol. 153. 116-166.

[2] Larson, J.L. and Hershberger, C.L. (1986) The minimal repli-

[31

[41

[51

[71

con of a streptomycete plasmid produces an ultrahigh level of plasmid DNA. Plasmid 15, 199-209. Yanisch-Perron. C., Vieira, J. and Messing, J. (1985) Im- proved Ml3 phage cloning vectors and host strains: nucleo- tide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-l 19. Hotta, K., Saito, N. and Okami, Y. (1980) Studies on new aminoglycoside antibiotics, lstamycins, from an actinomycete isolated from a marine environment. I. The use of plasmid profiles in screening antibiotic-producing streptomycetes. J. Antibiot. 33, 1502-1509. Katz. E., Thompson, C.J. and Hopwood, D.A. (1983) Cloning and expression of the tyrosinase gene from Streptomyces anti-

bioticus in Streptomyces lividans. J. Gen. Microbial. 129, 2703-2714. Ishikawa, J. and Hotta, K. (1991) Nucleotide sequence and transcriptional start point of the kan gene encoding an ami- noglycoside 3-N-acetyhransferase from Streptomyces griseus

SS-1198PR. Gene 108. 127-132. Hotta, K., Ishikawa, J.. Ichihara, M.. Naganawa, H. and Mizuno, S. (1988) Mechanism of increased kanamycin-resist- ante generated by protoplast regeneration of Streptomyces griseus. I. Cloning of a gene segment directing a high level of an aminoglycoside 3-N-acetyltransferase. J. Antibiot. 41, 9&103.

[8] Andersson, K. and Hjorth, R. (1985) Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluor- oacetate (CsTFA) without the use of ethidium bromide. Plas- mid 13. 78-80.

[9] Kawamoto, S., Nakamura, M. and Yashima, S. (1993) Clon- ing. sequence and expression of the tyrosinase gene from Strepromyces lawndulae MA406 A-l. J. Ferment. Bioeng. 76, 345-355.