An 11 450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high-copy-number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DDpeptidase, this precursor possesses a cleavable 3 1-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. Note. This paper is part of a dissertation of C.P.F. presented as partial fulfilment for a Ph. D. thesis, at the University of Liege.Abbreviations. Ac, acetyl; bp, base pair; kb, 1000 base pairs; PBP, penicillin-binding protein.Enzymes. Bacterial alkaline phosphatase (EC 3.1.3.1); filactarnases (EC 3.5.2.6); DD-peptidases (EC 3.4.16.-); lysozyme (EC 3.2.1.17); restriction endonucleases(EC 3.1.21.4); T4 DNA ligase (EC 6.5.1.1). MATERIALS AND METHODS Bacterial strains and plasmidsStreptomyces R61 was from the Microbiology Department of the University of Liige. S. lividans TK24 (str-6; a strain cured of its natural phsmids) [6] and the non-conjugative high-copy-number plasmids pIJ702 [7] and pIJ385 [8] were from the John Innes Institute, Norwich, UK. Escherichia coli HBlOl [9] and plasmid pBR322 [lo] were also used. Growth conditions and mediaGrowth of Streptomyces cultures was carried out at 28 "C with vigorous orbital shaking. The following media were used: YEME medium [ll]; Merck peptone medium and E9 broth [12]; Difco brain heart infusion; 2 xTY broth (Amersham handbook); and glycerol-casein medium [13]. R2YE agar [ll] was also used. E. coli HBlOl was grown at 37°C in LuriaBertani or M9CA medium [14] with vigorous shaking. Enzymes, antiserum, proteins and antibioticsThe DD-peptidase of Streptomyces R61 was prepared as described in [15]. Rabbit anti-(R61 DD-peptidase) antiserum was prepared by Gamma S.A. (Tavier, Belgium). The enzymes used in the recombinant DNA techniques were from Bethesda

By using the promoter-probe plasmid pIJ424, genomic DNA fragments of Actinomadura R39 were shown to have promoter activity in Streptomyces lividans. The same 100-200-copy-number plasmid was used to clone in S. lividans TK24, the gene that encodes the Actinomadura R39 beta-lactamase. Gene cloning resulted in an amplified expression of the beta-lactamase when compared with the amounts of enzyme produced by the original strain (1 mg versus 0.008 mg.litre of culture-1).

Summary. The promoter region of the gene encoding the extracellular DD-peptidase/penicillin-binding protein of Streptomyces R61 has been identified by in vivo promoter probing and SI mapping. A secretion vector, pDML116, was constructed by inserting into the multicopy Streptomyces plasmid pIJ702, a 247 bp DNA sequence that contained the transcriptional, translational and secretory signals and the 12 amino acid N-terminal region-encoding sequence of the mature Streptomyces DD-peptidase/penicillin-binding protein. Insertion, downstream of this 247 bp segment, of the Streptomyces R61 DD-peptidase-encoding gene or the Escherichia coli R-TEM /%lactamase-encoding gene yielded plasmids pDML120 and pDML128, respectively, which allowed expression and secretion of the relevant enzymes by Streptomyces lividans. The maximal secretion levels obtained were 42 mg protein/ml for the autologous Streptomyces DD-peptidase and 0.9 mg protein/ml for the heterologous E. coli /%lactamase.

No abstract