AU6077499A - Genes for the biosynthesis of anticapsin and bacilysine, their isolation and their use - Google Patents

Description

I Genes for the Biosynthesis of Anticapsin and Bacilysin, Their Isolation and Use Description The invention concerns genes for the biosynthesis of anticapsin and bacilysin, their isolation and use, in particular, in medicine and agriculture. Bacilysin is a known dipeptide that was described as an antibiotic agent (Zuber and Marahiel, 1992). It is produced, species-specifically, by Bacillus subtilis and by individual strains of other Bacillus species of the Bacillus subtilis group (Loeffler et al., 1986) and secreted out of the cell into the surrounding culture medium. The N-terminal end of bacilysin consists of L-alanine and the C-terminal end of the non proteinogenic epoxy amino acid L-anticapsin (Rogers et al., 1965; Walker and Abraham, 1970a; Neuss et al., 1970), and bacilysin was described chemically as L-alanyl-L-p-(2,3 epoxycyclohexanono-4)alanine (Walker and Abraham, 1970b; Chmara et al., 1981). After its uptake into sensitive cells, bacilysin is split proteolytically, the antibiotically effective anticapsin thus being released. In bacteria and fungi, anticapsin interferes with glucosamine synthetase and, thus, with the biosynthesis of the amino sugars glucosamine and N acetylglucosamine, which are required for the synthesis of cell walls. Synthesis of the cell wall is therefore inhibited and cell lysis triggered (Whitney and Funderburk, 1970; Kenig et al., 1976; Milewski, 1993). Bacilysin and anticapsin are inhibited competitively by other di- and tripeptides (Perry and Abraham, 1979) as well as by glucosamine and N-acetylglucosamine (Walton and Rickes, 1962; Chmara, 1985) and can thus also be distinguished from other antibiotic agents. An additional method for identifying anticapsin and bacilysin consists of the fact that their biosynthesis is prevented by mutations in aro genes for the biosynthesis of the aromatic amino acids phenylalanine and tyrosine (Hilton et al., 1988a). Through Bacillus subtilis A14 (Walker and Abraham, 1970a, b) and a strain of Streptomyces griseoplanus (Whitney et al., 1970), extracellular anticapsin is also formed directly, but such findings are restricted to these exceptional cases. In addition, it is known that bacilysin also has a relatively broad, unspecific activity against gram-positive and gram-negative bacteria as well as against budding fungi and filamentous fungi and that it is uptaken effectively by sensitive cells via dipeptide and oligopeptide permease systems (Diddens et al., 1979; Perry and Abraham, 1979; Chmara et al., 1981). Defect mutations in this permease system lead to resistance to bacilysin. Extracellular anticapsin is, in most cases, not incorporated by bacteria and therefore has a relatively low antibacterial activity, except against Streptococcus pyogenes and Salmonella gallinarum (Neuss et al., 1970). But anticapsin has a strong antifungal activity, including against Candida albicans and Botrytis cinerea and other filamentous fungi (Neuss et al., 1970; Kenig et al., 1976; Chmara et al., 1980).

2 As regards the biosynthesis of bacilysin by Bacillus subtilis 168 we know that this biosynthesis branches from prephenate of the aromatic amino acid synthesis pathway, suggesting that prephenate serves as the primary metabolic precursor for the synthesis of anticapsin (Hilton et al., 1988a). The enzymatic reactions in that connection and in connection to the following bonding of anticapsin with L-alanine to form bacilysin are still unknown. Excretion of the formed bacilysin out of the cell is presumably based on a peptide transport system (Diddens et al., 1979). The findings relating to the regulation of the bacilysin biosynthesis suggest its suppression by growth-promoting supplements (including ammonium, nitrate, protein hydrolysates, phenylalanine, L-alanine) and other growth-promoting conditions (e.g. complete media or an optimum temperature of 37'C). The end product inhibition through accumulated bacilysin is also of particular interest as regards an overproduction (Ozcengiz and Alaeddinoglu, 1991). As regards the genetics of the bacilysin biosynthesis, only a rough mapping of a bac-l-locus within a region of approximately 90 kb between the genes ctrA and sacA was achieved using conventional genetic methods (chemical mutagenesis, transduction) (Hilton et al., 1988b), whereas encoding genes or gene groups and the gene products are still unknown. For a bacilysin-negative (Bac~) mutant Bacillus subtilis NG79 bac-1 used for this purpose, a lesion in the bonding reaction of L-alanine with L-anticapsin to form bacilysin was identified, but a corresponding amino acid ligase has not yet been isolated. From a few Bacillus strains, halogenated bacilysin derivatives (chlorotetaine, bromotetaine) were also formed, whose biosynthesis and genetics are also not characterized. But these halogen derivatives are interesting insofar as they show, compared to bacilysin, a few differences in their mode of action and in the spectrum of their producers (Loeffler et al., 1990; Katzer, 1991). With respect to the status of results, several successful studies (Wild, 1994; Baldwin et al., 1995) relating to the chemical synthesis of anticapsin, bacilysin and chlorotetaine should be mentioned that have led to detailed knowledge about the molecular structure, but not to an economically usable production procedure, as little as has the bacilysin biosynthesis. The invention was based on the task to isolate, and characterize in structural and functional aspects, genes for the biosynthesis of anticapsin and bacilysin as well as to render them usable specifically for an overproduction of antibiotic agents and an efficient and versatile antibiosis against microbial pathogens and putrefactive agents. According to the invention, the task is solved by first genetically cloning and amplifying genes or a gene group for the biosynthesis of anticapsin and bacilysin, using a suitable bacterial host vector system, which then are available to a structural and functional characterization. Due to gene amplification and the resulting gene-dosage effect as well as by using, if applicable, other bacterial host-vector systems with increased gene amplification and enhanced and/or 3 induced gene expression, an economically usable biosynthesis of the agents can be achieved, which are then applied against microbial pathogens and putrefactive agents. An excretion of the agents is favourable so that they can be isolated in a more cost effective manner from the culture supernatants. Such organisms that naturally excrete anticapsin or bacilysin from the cell are ideal for this purpose as host strains. What is also favourable is the suppression of the end product inhibition, as in the bacilysin biosynthesis through accumulated bacilysin, so that the overproduction of the antibiotic agents is not inhibited by a high concentration of the antibiotic agents themselves. Due to the structural similarity of the halogenated bacilysin derivatives and their, at least partially, equal biosynthesis, the present invention can also be used for the biotechnological production of chlorotetaine and bromotetaine or of new, more strongly or specifically acting bacilysin and anticapsin derivatives. Alternatively, the recombinant agent formers can be used as cellular antagonists by placing them in the vicinity of microbial purtrefactive agents and pathogens, e. g. in the soil, in culture liquids or onto the surface of other organisms, the antibiotic agents excreted thus becoming effective. Another opportunity of applying cloned bacilysin genes is their introduction into the organisms to be protected antibiotically so that these themselves form the antibiotic agent and achieve self-protection against microbial pathogens and putrefactive agents. As an example, the genes that are directly required for the biosynthesis of anticapsin or bacilysin can be transferred to and expressed in useful plants so that, through the transgenic synthesis of anticapsin or bacilysin, an increased tolerance or resistance to a broad spectrum of various phytopathogenic agents can be achieved. The task of the invention is solved, in particular, by isolating corresponding genes, in compliance with the invention, from a bacterial strain of the Bacillus subtilis group, preferably from Bacillus subtilis 168, with methods usually applied in genetic engineering. The object of the invention is genes for the in-vitro and in-vivo biosynthesis of anticapsin and bacilysin as well as their fragments, variants and mutants that are characterized by base exchanges or other mutations, and RNA nucleic acid sequences, in which T is exchanged by U, and nucleic acid sequences that are completely or partially complementary or homologous. The genes and their products themselves are diagnostic and/or therapeutic substances or serve as targets for the search for therapeutically effective substances. In accordance with the invention, the genes are preferably bacilysin genes of Bacillus subtilis 168, here in the following termed "bac " genes, within a 7471 bps DNA sequence according to SEQ ID No. 1 (Exhibit 1), their mutants, variants and their completely or partially complementary DNA sequences.

4 For the primary cloning (embodiment 1) of the bac genes, positive-selection vectors with a high cloning capacity and stability are preferably used and gene libraries with large chromosomal DNA inserts isolated so that it is possible to isolate not only individual genes but also gene groups with sufficient probability. The palindrome, unipositive-selection vector pPS15 (Steinborn, 1996) has proven to be a favourable cloning vector. This palindrome plasmid vector and also the non-palindrome plasmid vector pSB595 used for subsequent clonings (see below) belong to the incompatibility group incl8, and their particularly high cloning capacity as well as their structural and segregative plasmid stability are based on their Theta replication and a partition gene parS, respectively. Bacterial bacilysin-negative strains, preferably of Bacillus subtilis 168, are used as a host strain for the primary cloning of the genes in accordance with the invention. Due to a chromosomal bac mutation, they do not show a chromosomally encoded bacilysin formation, and the cloning of the bac genes can be evidenced because of the complementation of the chromosomal mutation. A mutant NG79 bac-1 (Hilton et al., 1988b) or derivatives from NG79 of the strain family Bacillus subtilis 168 are preferably used as bacilysin-negative host strains. In particular, a stabilizing strain Bacillus subtilis GSB298 hip-I bac-l or a comparable strain is used as bacilysin-negative host strain, which shows an increased transformation capacity and stabilizing activity on recombinant plasmid derivatives with a large insert, whereby this GSB298 is derived as spontaneous hip (host increased plasmid stability) mutant from NG79 bac-1 and isolated due to its stabilizing activity on palindrome, dipositive selection vectors (Steinborn, 1996). Gene libraries with large chromosomal DNA inserts are obtained preferably utilizing partial restrictase cleavage of the donor DNA and insertion of preselected, large (> 10 kb) DNA fragments. As an example, a gene bank is isolated after partial BamHI cleavage, from which a recombinant strain Bacillus subtilis GSB298 (pSB657) with bac genes is isolated from Bacillus subtilis 168. This secretes plasmid-encoded bacilysin, which is evidenced through the inhibition of a bacilysin-sensitive indicator strain, preferably of Escherichia coli B or Proteus vulgaris, and through the inhibition of bacilysin through N-acetylglucosamine. An additional proof for the plasmid-encoded expression of bacilysin is provided by the fact that pSB657 does not express it in a host strain Bacillus subtilis GSB285 aroI906 (Steinborn, 1996), since the mutation aroI906 prevents the biosynthesis of prephenate as precursor for anticapsin. An optimum formation and antibiotic activity of bacilysin is achieved through the utilization of a PA minimal medium (Perry and Abraham, 1979) and cultivation at 30*C. Agar diffusion tests serve for the half-quantitative determination of the bacilysin activity using inhibition zones around growing colonies or punch holes that are charged with culture supernatants. In accordance with the invention, the bac genes are contained in the recombinant plasmid pSB657, generated through primary cloning, within a 21.5 kb-BamHI insert. Through subcloning, a further recombinant strain Bacillus subtilis GSB298 (pSB660) is derived from 5 the 21.5 kb-BamHI insert of pSB657, whereby the recombinant plasmid pSB660 obtained in this manner carries the bac genes within a 7.5 kb-PstI insert (embodiment 2). The PstI insert of pSB660 is, based on DNA homologies and complying restriction data (Fig. 1), identified as 7471 bps DNA sequence (Fig.2), localized in the region of bp 3 867 455 up to bp 3 874 925 of the genomic DNA sequence (SubtiList database, Institut Pasteur, Moszer et al., 1995; Moszer, 1998) of the genome Bacillus subtilis 168 (embodiment 3). This position lies within the 90 kb region, in which the bac-1 locus was mapped by means of transduction (Hilton, H., et al., 1988b); the localization and function of the bac genes have thus been confirmed. In accordance with the invention, the recombinant plasmid pSB660 encodes in Bacillus subtilis GSB298 an increased, or at least with the gene donor strain Bacillus subtilis 168 comparable, bacilysin formation (Fig. 3) so that the defect in the ligase reaction caused by the bac-1 mutation (Hilton et al., 1988b) is fully complemented functionally in GSB298 by pSB660. Contrary to a comparable growth and comparable maximum values of bacilysin formation, a different course of the bacilysin formation can be observed, which commences in the recombinant strain GSB298(pSB660) immediately upon start of the growth. The chromosomally encoded bacilysin formation, however, shows a clear lag phase and adjusts itself to the level of the plasmid-encoded bacilysin formation only in the stationary phase. This adjustment of the maximum values is evaluated as a sign of the end product inhibition through accumulated bacilysin. The PstI insert contains 6 open reading frames (ORFs) with previously unknown function and the original designations (see SubtiList database) ywfA to ywfF, which are renamed, due to their described complementation and additional findings (see below) relating to their function in the bacilysin biosynthesis, as bacA to bacF (Fig. 1) and claimed in accordance with the invention. But this does not exclude additional genes from also participating in an optimum bacilysin biosynthesis. An essential function of the genes bacB to bacE for the bacilysin biosynthesis is evidenced on the basis of inactivating mutations in the individual bac genes, which more or less reduce or eliminate the formation of extracellular bacilysin. For this purpose, deletion (A) and insertion (::) mutations are generated in cloned bac genes in recombinant plasmids (embodiment 4) and in chromosomal bac genes (embodiment 5) in Bacillus subtilis 168, and their influence on the bacilysin formation is examined. An essential role of the genes bacB to bacE is evidenced through the fact that a deletion derivative pSB672 = pSB660 AbacA AbacF containing these genes encodes in GSB298 a bacilysin formation comparable with that of pSB660 (Fig. 3), while the mutants pSB676 = pSB672 bacB* , pSB677 = pSB672 AbacC and pSB678 = pSB672 AbacD derived from pSB672 only encode in GSB298 approximately 1/10 of the bacilysin formation compared to pSB660 and pSB672 (Fig. 4). A deletion derivative pSB674 = pSB672 AbacE leads in GSB298 to a complete failure of the formation of extracellular bacilysin and a complete inhibition of the growth as well as cell lysis, correlated with distinct swellings of the naturally rod-shaped cells to protoplast-similar cell forms, as are typical for the antibiotic 6 effect of anticapsin. This leads to the conclusion that bacE encodes an enzyme required for the ligase reaction of bacilysin formation and that in GSB298(pSB674) with inactive bacE gene both in pSB674 and in GSB298 bac-1 (such as evidenced for the bac-1 mutation; Hilton et al., 1988b), a concentration of anticapsin lethal for the host cell occurs intracellularly. But it cannot be excluded that an additional gene has mutated in GSB298, e.g. a transport gene so that the excretion of bacilysin and/or anticapsin is inhibited. This assumption is proven by the fact that pSB674 also causes the same aberrant cell forms and cell lysis, including in a multiple insertion mutant GSB322, but not in insertion mutants, in which only individual chromosomal bac genes were inactivated (embodiment 7). Evidences for the transport function of a bac gene in connection with the self-protection of the bacteria cells against bacilysin formed and excreted by themselves cannot be obtained, since all chromosomal insertion mutants, including multiple ones, do not have a negative effect on their resistance to extracellular bacilysin (embodiment 6). An essential function of the genes bacB to bacE for the bacilysin biosynthesis can also be proven by insertion mutations (embodiment 5) of the chromosomal genes bacB in GSB319, bacC in GSB320, bacD in GSB321 or bacE in GSB315, since these lead to the loss of the chromosomally encoded bacilysin formation. Under these conditions, no essential function can be proven for the genes bacA and bacF, since the inactivation of the chromosomal genes bacA to bacF in GSB322 can be complemented through pSB672 with the active genes bacB to bacE (embodiment 7). The essential function of the genes bacB to bacE, their coupling within a gene cluster with same orientation of the ORFs (see Fig. 1) and the activation through joint regulatory sequences of the transcription upstream of bacB (embodiment 3) suggest a transcription unit of bacB to (at least) bacE. The experimental findings and conclusions relating to the bac genes in bacilysin biosynthesis are substantiated by a comparison of the putatively encoded proteins BacB, BacE and BacF with proteins of the "SWISS-PROT database" in the ExPASY Molecular Biology Server (embodiment 3). Strong homologies to a prephenate dehydratase for BacB, to D-alanine-D-alanine ligases for BacE or to an efflux protein for BacF can be proven that show a clear relationship to the bacilysin biosynthesis. A relationship of the bac-gene-encoded proteins to prephenate as primary precursor of the anticapsin biosynthesis is substantiated through the experimental findings that the chromosomal bac insertion derivatives derived from Bacillus subtilis 168 (see embodiment 5) GSB319, GSB320, GSB321, GSB315 and GSB322 do not cause auxotrophy for aromatic amino acids (embodiment 5) and that therefore the reactions catalyzed by bac gene products, starting from prephenate, act in the biosynthesis for anticapsin or bacilysin (but not in the biosynthesis for prephenate). In a further variant (embodiment 8) of the procedure, various recombinant plasmids are transformed into heterologous host strains of different Bacillus species, and, surprisingly, the recombinant strains obtained show a quite different plasmid-encoded bacilysin formation.

7 Even after the transformation of pSB660, which contains bacA to bacF in an active form, no extracellular bacilysin can be evidenced for Bacillus coagulans (pSB660), Bacillus lichinformis ATCC9789 (pSB660) and Bacillus megaterium PV361 (pSB660). But it cannot yet be excluded, in respect of these strains, that plasmid-encoded bacilysin is synthesized intracellularly, which is not excreted and which, therefore, cannot be detected extracellulary. But for Bacillus pumilus ATCC12140, as for GSB298 (see above.), a high plasmid-encoded bacilysin formation can be detected, alone through pSB674, which only contains bacB to bacE in active form. This again confirms the essential function of these bac genes for bacilysin biosynthesis. A particularly high plasmid-encoded bacilysin formation is identified for the host strain Bacillus amyloliquefaciens GSB272 so that GSB272 (pSB660) and GSB272 (pSB672) show an approximately 5 times higher bacilysin formation compared to the donor strain Bacillus subtilis 168. An especially surprising result is that a recombinant plasmid pSB679 = pSB660 A(bacA bacD bacE bacF), which only contains bacB and bacC in active form, causes an approximately equally high bacilysin formation in GSB272. These findings prove the essential gene function of bacB and bacC in the bacilysin formation and suggest in addition that in GSB272 the remaining essential bac genes are present in a functioning form in the bacterial chromosome. This suggestion is quite obvious, since most of the wild type strains of Bacillus amyloliquefaciens show a chromosomally encoded bacilysin formation (see above). Due to their effects on a specific target, the limited stability of bacilysin and anticapsin as well as the ubiquitous occurrence of inhibitors in eukaryotes, toxicity in human beings, animals and plants is not to be expected from their application against microbial pathogens and putrefactive agents. The invention is explained in detail below on the basis of the following figures and embodiments. The figures show in Fig. 1: the restriction map of the PstI insert of pSB660 corresponding to the DNA sequence in Exhibit 1 with the genes bacA, bacB, bacC, bacD, bacE and bacF of Bacillus subtilis 168. Only those restrictase sites are indicated that are used for the construction of plasmid derivatives 8 Fig. 2: growth and bacilysin formation in the recombinant strain Bacillus subtilis GSB298(pSB672) compared to the gene donor strain Bacillus subtilis 168 and the bacilysin negative host strain GSB298 bac-1 at 30*C in PA minimal medium Fig. 3: growth and bacilysin formation in the recombinant strains Bacillus subtilis GSB298(pSB672), GSB298(pSB674) und GSB298(pSB676) at 30*C in PA minimal medium DNA sequence of the 7471 bps PstI insert of pSB660, localized from bp 3 867 455 to bp 3 874 925 in the DNA sequence (SubtiList database) of the genome of Bacillus subtilis 168 according to sequence protocol Embodiments Commonly used materials and methods are not described in detail in the following. Knowledge about the cultivation of bacteria, strain selection, isolation of chromosomal DNA, plasmid DNA and DNA fragments, restrictase cleavage of DNA, ligation of DNA fragments, plasmid transformation, electrophoresis and DNA sequence analysis is taken for granted and is generally accessible from publications (Sambrook et al., 1989; Harwood and Cutting, 1990; Nicholl, 1994). When commercially available kits, enzymes and other materials are used, the procedures indicated by the manufacturers or suppliers are complied with. The following specific materials and methods are used more often: # Bacterial strain Bacillus subtilis GSB285 hip (Steinborn, 1996/98) as recipient strain for subclonings of bac genes, derived as spontaneous, plasmid-stabilizing (for recombinant plasmids with large insert) mutant from GSB26 sacA321 aroI906 metB5 amyE strl of the strain line of Bacillus subtilis 168. The aroI906 mutation prevents the biosynthesis of anticapsin and bacilysin so that these cannot impair the plasmid transformation (especially into protoplasts). # Plasmid vector pPS15 (15,4 kb; MLSr) as palindrome, unipositive-selection vector with high cloning capacity and stability (Steinborn, 1996/98) so that in clonings almost all plasmid transformants contain a recombinant plasmid and even recombinant plasmids with a large insert are maintained in a stable form. PIasidvector pSB595 (7 kb; MLSr) as non-palindrome cloning vector with high cloning capacity and stability, derived from a plasmid pSB472 (Steinborn, 1996/98) through the introduction of a multiple cloning site. Both plasmid vectors belong to the incompatibility group inc 18, and their high structural and segregative stability is based on the Theta type of their replication and a partition gene parS, respectively.

9 # DNA isolations from strains of Bacillus and Escherichia coli: - Plasmid DNA and high-molecular chromosomal DNA on a large scale using the QIAGEN Plasmid Kit and Genomic Kit, respectively - Plasmid DNA on a small scale (Minipreps) based on an alkaline method (Sambrook et al., 1989) # Isolation of DNA fragments from gels using theQIAGEN QIAEX II Kit and electro elution for fragments <10 kb and >10 kb, respectively # DNA sequencings using an A.L.F. DNA Sequencer (Pharmacia LKB) # Plasmid transformation into Bacillus, in most cases, using the PEG protoplast method (Chang and Cohen,1979) or (embodiment 5) into competent cells (Dubnau and Davidoff Abelson, 1971) of Bacillus subtilis 168 # Plasmid transformation into competent cells of Escherichia coli according to Hanahan (1983) # Selection by plasmid-encoded chloramphenicol resistance (Cmr) or MLS antibiotics resistance (MLSr), including erythromycin resistance (Emr), through the addition of 10 pg/ml chloramphenicol (Cm) or 5ptg/ml erythromycin (Em), respectively # Insertion mutagenesis (Niaudet et al., 1982) for the inactivation of chromosomal bac genes of the gene donor Bacillus subtilis 168 through chromosomal insertion. # Cultivation, in most cases (see below), at 37*C in a liquid TBY complete medium (1 % bactotryptone, Difco - 0.5 % yeast extract, Difco - 0.5 % NaCl; pH 7.0) or on TBY agar (1.8 % agar-agar) # Cultivation for the formation of bacilysin at 30'C in liquid PA minimal medium (optimized for the biosynthesis of bacilysin; Perry and Abraham, 1979) - 0.2 % glucose - 50 pg/ml essential amino acids or on PA agar (1,5 % agar-agar) # Screening for bacilysin-forming clones through the replica method or spreading onto PA minimal agar containing Staphylococcus aureus, Proteus vulgaris or preferably Escherichia coli B as bacilysin-sensitive indicator organisms by inhibition zones around growing colonies. Test plates: 25 ml or 50 ml PA agar in round plates, d = 10 cm, or square plates, 14x14 cm, per 100 ml agar with 1 ml washed suspension of the indicator strain, E 4 70 nm = 1, suspended at 50 *C # half-quantitative determination of bacilysin formation in culture supernatants using the hole agar diffusion assay and the described (see above) PA agar test plates. Hole diameter: 5 mm, sample volume: 30 pl. Bacilysin activity is presented through relative units (Ure) that are determined using a calibration curve (linear coat of the inhibition zone diameter to the logarithmic coat of dilutions of a representative bacilysin-containing culture supernatant). # The plasmid-encoded synthesis of bacilysin, for the differentiation from the chromosomally encoded bacilysin synthesis and synthesis of other inhibitors, is confirmed biologically by positive results to all of the following tests: 10 - Bacilysin synthesis in correlation to the presence of the recombinant plasmid in a bacilysin-negative host strain - no bacilysin synthesis in Aro-auxotrophic (Aro) host strains, such as Bacillus subtilis GSB285 aroI906 - competitive inhibition of the antibiotic activity through N-acetylglucosamine - inhibition of the bacilysin synthesis through supplements (L-alanine, casamino acids) in the PA culture medium and cultivation at 37"C - antibiotic activity (insofar as tested) against known bacilysin-sensitive indicator strains (Escherichia coli B, Escherichia coli K12, Proteus mirabilis, Proteus vulgaris, Staphylococcuc aureus, Salmonella typhimurium, Saccharomyses cerevisiae) # biochemical proof of anticapsin and bacilysin through thin-layer chromatography and bioauthography, such as described by Sakajoh et al. (1987) Embodiment 1: Primary cloning of the bac genes of Bacillus subtilis 168 Primary cloning is effected through "shot gun" after partial restrictase cleavage of the chromosomal donor DNA and preselection of large DNA fragments (> 10 kb) for ligation. For this, a positively selective cloning vector pPS15 is used, transformed into a stabilizing bacilysin-negative (Bac~) recipient strain Bacillus subtilis GSB298 bac-1, and screening for bacilysin-positive (Bac*) clones on PA agar plates with Escherichia coli B as bacilysin sensitive indicator organism is carried out. After partial BamHI cleavage, a highly expressing Back clone is found among 2944 recombinant transformants that is termed "GSB298 (pSB657)". In pSB657, a 21.5 kb-BamHI insert is identified, and the plasmid-encoded formation of bacilysin is confirmed through all (see above) biological and biochemical tests. pSB657 complements in GSB298 the bac-1 mutation so that through GSB298(pSB657), compared to the donor strain Bacillus subtilis 168, an at least comparable or increased bacilysin formation ((as for GSB298(pSB672) in Fig. 3)) occurs. With comparable growth, a strong time-delayed bacilysin formation is observed for the donor strain. Embodiment 2: Localization of the bac genes in the primary insert of pSB657 For this, subclonings are effected after cleavage of the insert of pSB657 using different restrictases and again pPS15 and GSB298 are used as the host vector system. Highly expressing clones, comparable to GSB298(pSB657), are obtained after PstI cleavage, which are designated "GSB298(pSB660)". PSB660 contains a 7.5 kb-PstI insert, which after recloning to pPS15 and retransformation into GSB298 again encodes reproducibly a high bacilysin formation (Fig. 3). Embodiment 3: Localization of the bac genes in the genomic DNA sequence of Bacillus subtilis 168 11 The 7.5 kb-PstI insert contained in pSB660 (embodiment 2) is recloned into pUC18, the recombinant strain Escherichia coli XL1 (pSB661) thus being obtained. Sequences are taken from both ends of the PstI inserts, and a homology comparison of the DNA sequences (429 bps and 226 bps) with the genomic DNA sequence (SubtiList database) of the gene donor Bacillus subtilis 168 is carried out. A complete DNA homology is found for the region from bp 3 867 455 to bp 3 874 925 of the genomic DNA sequence so that the PstI insert contains 7 471 bps. Through restrictase cleavages of the PstI insert, restrictase sites (Fig. 1) can be identified, which are all confirmed through the restriction pattern of the DNA sequence of the database and are used for further plasmid constructions (embodiments 4 and 5). In addition, the PstI insert is localized within the approximately 90 kb region between the known genes ctrA and sacA through this homology comparison, in which region a bac-1 locus has been mapped according to the literature.. In accordance with the database data, the PstI fragment contains 6 complete, functionally unknown ORFs ywfA to ywfF that are renamed due to their function in bacilysin biosynthesis as bacA to bacF (Fig. 1). First indications of their functions are obtained through a homology comparison of the proteins BacA to BacF, putatively encoded through the genes bacA to bacF, with known proteins of other organisms using the "SWISS-PROT database" (ExPASY Molecular Biology Server, Fasta 3): High homologies for BacB to a prephenate hydratase of Streptomyces coelicolor (28 %), for BacE to D-alanine-D-alanine ligases of Haemophilus influenzae (29 %) and of Bacillus subtilis (21.5 %) as well as for BacF to a macrolid efflux protein (24.8 %) suggest functions in the synthesis from prephenate to anticapsin, ligation of anticapsin with L-alanine to bacilysin and in transportation, respectively. Through the search for putative operon structures, promoter motifs (acattg/taatat) are identified upstream of bacB (from bp 3874425 to bp 3874360 of the genomic DNA sequence), whereas a transcription terminator probably only occurs downstream of bacA (from bp 3873120 to bp 3873061). Embodiment 4: Mutants of pSB660 For identifying the biological functions of the individual bac genes, first the flanking genes bacA and bacF are deleted, by cloning from the insert of pSB660 (see Fig. 1) a subfragment of PvuII (bp 1680) to SalI (bp 5642) into a plasmid vector pSB595 and isolating a recombinant derivative pSB672 = pSB660 AbacA AbacF. pSB672 also complements the bac-1 mutation in GSB298 and encodes a high bacilysin formation comparable to that of pSB657 and pSB660 (Fig. 3). Also through subcloning from the insert of pSB660, pSB679 = pSB660 A(bacA bacD bacE bacF) is generated, by cloning a subfragment of Pvull (bp 1680) to Sac (bp 3558) into the plasmid vector pSB595. Further mutants pSB676 = pSB672 bacB*, pSB677 = pSB672 AbacC, pSB678 = pSB672 AbacD and pSB674 = pSB672 AbacE are derived from pSB672 (position of the following restrictase sites see Fig. 1) through "Klenow-fill-in" (*;Kit by Stratagene) in BglII (bp 2236), deletion in EcoRV (bp 2863) to NruI (bp 3144), deletion in 12 EheI (bp 3539) to Ecl136I (bp 3558) and deletion in PvuI (bp 4816) to PvuI (bp 5242), respectively. pSB676, pSB677 and pSB678 encode in GSB298 a bacilysin formation that is reduced to 1/10 compared to the original plasmids (Fig. 4). But the deletion in bacE has very drastic effects: GSB298(pSB674) does not form bacilysin, cannot grow when cultivated in the PA medium (under the conditions of bacilysin formation), forms non-typical, swollen cell forms and finally lyses. Embodiment 5: Insertion mutagenesis of chromosomal bac genes in Bacillus subtilis 168 through double cross-over Other than in embodiment 4, chromosomal bac genes of the gene donor are inactivated through insertion in order to identify their function in bacilysin biosynthesis. Insertion is effected through general recombination via double cross-over and using integration plasmids. In the integration plasmids, a selection marker is inserted within the bac gene to be inactivated, thus being flanked by fragments of the bac gene, which lead to the double cross over in an homologous host strain. In the most cases (exceptions: Klenow-fill-in at pSB676 or pSB682, see below), an internal part of the bac gene is deleted first so that a reversion for reactivating the bac gene is excluded after the double cross-over. Because of the double cross over, only one chromosomal DNA fragment with the active bac gene is exchanged recombinatively with a homologous fragment with the inactivated bac gene derivative of the integration plasmid so that the vector portion is excluded from the integration. Integration plasmids are constructed in accordance with the mutants pSB674 to pSB678 (embodiment 4), and a cmlR gene for chloramphenicol resistance is also introduced as selection marker into the mutated site. A plasmid pSB523 and pSB573, in which the cmlR gene is contained within multiple cloning sites, serve as gene donor for the cmlR selection marker. The integration plasmids pSB682 = pSB672 bacB*::cmlR, pSB683 = pSB672 AbacC::cmlR, pSB684 = pSB672 bacD::cmlR, pSB680 = pSB672 AbacE::cmlR and pSB685 = pSB660A(bacA to bacF)::cmlR are obtained. pSB685 is derived from pSB660 (see Fig. 1) after the deletion of PvuII (bp 1680) to EcoRV (bp 5681). For the chromosomal integration, the integration plasmids are linearized (as a prerequisite for double cross-over) and transformed into competent cells of Bacillus subtilis 168. The selection is effected via chloramphenicol resistance, and the double cross-over (to exclude simple cross-over and mutations) is evidenced through the absence of plasmid-encoded MLS antibiotics resistance (encoded by the plasmid-inherent selection marker of the integration plasmid) and of free plasmid DNA of the integration vector, through Southern hybridization with the cmlR gene as a marked sample as well as through cloning and restriction analysis of the relevant chromosomal region. GSB319 = Bacillus subtilis 168 bacB*::cmlR, GSB320 = Bacillus subtilis 168 AbacC::cmlR, GSB321 = Bacillus subtilis 168 AbacD::cmlR, GSB315 = Bacillus subtilis 168 AbacE::cmlR und GSB322 = Bacillus subtilis 168 A(bacA to bacF)::cmlR are isolated as insertion mutants, which all prove to be bacilysin-negative.

13 For the examination for auxotrohpy, the insertion mutants are spread out on PA minimal agar and identified as prototrophic for the amino acids phenylalanine and tyrosine, whose biosynthesis (as for anticapsin) branches from prephenate in the biosynthesis pathway of aromatic amino acids. Embodiment 6: Function of the bac genes for the protection of cells of Bacillus subtilis 168 against extracellular bacilysin. The insertion mutants are tested for bacilysin sensitivity in a hole-agar diffusion assay with different chromosomal insertion mutants (see embodiment 5) as indicator strains and a bacilysin-containing culture supernatant as sample. All insertion mutants, including GSB322 with deletion in bacA to bacF, show no inhibition zones and thus prove to be, as before, bacilysin-resistant (as GSB298 and Bacillus subtilis 168). Embodiment 7: Effect of deletion plasmids on chromosomal insertion mutants Different deletion plasmids (see embodiment 4) are transformed into different chromosomal insertion mutants (see embodiment 5), and the plasmid transformants thus constructed are examined for effects relating to bacilysin formation or correlated features. The following results are remarkable: # GSB322(pSB672) shows a high plasmid-encoded bacilysin formation, comparable to that of GSB298(pSB660) or GSB298(pSB672) so that bacB to bacE can be evaluated as essential or sufficient, bacA and bacF, however, as non-essential or not relevant to a high bacilysin formation. # GSB322(pSB674) (but not plasmid transformants of pSB674 with other chromosomal insertion mutants) behaves, when cultivated in PA minimal medium, with respect to defects in the bacilysin formation, aberrant cell forms and cell lysis like GSB298(pSB674) so that this mutant phenotype (such as with NG79 or GSB298) cannot be based on the defect in only one bac structure gene. Embodiment 8: Plasmid-encoded bacilysin formation in heterologous Bacillus species pSB660 and various deletion plasmids (see embodiment 4) are transformed into Bacillus amyloliquefaciens GSB272, Bacillus coagulans, Bacillus lichenformis ATCC9789, Bacillus megaterium PV361 and Bacillus pumilus ATCC12140, and the plasmid transformants obtained are tested for plasmid-encoded bacilysin formation. Plasmid transformants of Bacillus coagulans, Bacillus lichen'formis ATCC9789 and Bacillus megaterium ATCC12140 do not form extracellular bacilysin, while in Bacillus pumilus ATCC12140 (as in Bacillus subtilis GSB298) pSB672 with bacB to bacE as active genes encodes a high bacilysin formation. A particularly high plasmid-encoded bacilysin formation, up to five times as high compared to that of GSB298(pSB660) or GSB298(pSB672), is achieved in Bacillus 14 amyloliquefaciens GSB272, whereby the deletion plasmid pSB679 (embodiment 4) with bacB and bacC is already sufficient.

15 Literature Baldwin, J. E., et al., Tetrahedron 51, 5193-5206 (1995) Chang, S., and S. N. Cohen, Molec. Gen. Genet. 168, 111-115 (1979) Chmara, H., et al., Drugs under Experimental and Clinical Res. 6, 7-14 (1980) Chmara, H., et al., J. Antibiotics 34, 1608-1612 (1981) Chmara, H., J. Gen. Microbiol. 131, 265-271 (1985) Diddens, H., et al., J. Antibiotics 32, 87-90 (1979) Dubnau, D., and R. Davidoff-Abelson, J. Mol. Biol. 56, 209-221 (1971) Hanahan, D., J. Mol. Biol. 166, 557- (1983) Hilton, M. D., et al., J. Bacteriol. 170, 482-484 (1988a) Hilton, M. D., et al., J. Bacteriol. 170, 1018-1020 (1988b) Katzer, W., Dissertation, Universitat Tubingen, 1991 Kenig M. D., et al., J. Gen. Microbiol. 94, 46-54 (1976) Kenig, M. D., and E. P. Abraham, J. Gen. Microbiol. 94, 37-45 (1976) Loeffler, W., et al., J. Phytopathology 115, 204-213 (1986) Loeffler, W., et al., Forum Mikrobiologie 3, 156-163 (1990) Milewski, S., Biochim. Biophys. Acta 1161, 269 (1993) Moszer, I., FEBS Letters 430, 28-36 (1998) Moszer, I., et al., Microbiology 141, 259-289 (1995) Neuss, N., et al., Biochem. J. 118, 571-575 (1970) Niaudet, B., et al., Gene 19, 277-284 Nicholl, D. S. T., Gentechnische Methoden. Spektrum Akademischer Verlag Heidelberg, Berlin, Oxford (1995) Ozcengiz, G., and N. G. Alaeddinoglu, Folia Microbiol. 6, 522-526 (1991) Perry, D., and E. P. Abraham, J. Gen. Microbiol. 115, 213-221 (1979) Rogers, H. J., et al., Biochem. J. 97, 579-586 (1965) Sambrook, J., et al., Molecular cloning. A laboratory manual. Cold Spring Laboratory Press (1989) Steinborn, G., DE-Al 196 54 841 (1996) Walker, J.E., and E. P. Abraham, Biochem. J. 118, 557-561 (1970a) Walker, J. E., and E. P. Abraham, Biochem. J. 118, 563-570 (1970b) Walton, R. B., and E. L. Rickes, J. Bacteriol. 84, 1148-1151 (1962) Whitney, J. G., and S. S. Funderburk, Abstracts for the X. International Congress for Microbiology, Mexico City, p 101 (1970) Whitney, J. G., et al., Bacteriological Proceedings 7 (1970) Wild, H., J. Org. Chem. 59, 2748-2761 (1994) Zuber, P., et al., in Bacillus subtilis and other Gram-positive bacteria. Sonenshein, A. L., et al. (eds); American Soc. of Microbiol.. Washington, DC, pp. 897-916 (1993)

Claims (4)

1. bac genes for the biosynthesis of anticapsin and bacilysin as well as their fragments, mutants and variants.

2. bac genes according to claim 1, wherein they have been isolated from a bacterial gene donor strain, in particular Bacillus.

3. bac genes according to claims 1 or 2, wherein they preferably stem from Bacillus subtilis

168. 4. DNA sequence according to one of the claims 1 to 3, wherein it relates to the sequence ID No. 1 as well as fragments, mutants and variants of this sequence. 5. RNA sequences according to one of the claims 1 to 4, wherein they are fully or partially complementary. 6. DNA sequences according to one of the claims 1 to 5, wherein they are fully or partially homologous. 7. Recombinant vectors that contain for anticapsin or bacilysin encoding genes. 8. Recombinant plasmids that contain for anticapsin or bacilysin encoding genes. 9. A recombinant plasmid pSB657 according to claim 8, wherein it is obtained by shot-gun cloning of bac genes of Bacillus subtilis 168 and encodes extracellular bacilysin in host strains of Bacillus. 10. Plasmid according to claim 9, wherein the bac genes are contained within a 21.5 kb BamHI insert in the recombinant plasmid pSB657, generated through primary cloning. 11. A recombinant plasmid pSB660 according to claims 8, 9 or 10, wherein it is obtained through PstI subcloning from the 21.5 kb-BamHI insert of pSB657 and contains the bac genes within a 7.5 kb PstI insert. 12. Plasmid according to claim 11, wherein the 7.5 kb PstI insert of pSB660 is identified, through DNA sequencing and comparison of the DNA homology, as 7471 bps Pstl fragment, localized from bp 3 867 455 to bp 3 874 925 in the DNA sequence (SubtiList database) of the genome of Bacillus subtilis 168. 25 13. Open reading frames ywfA, ywJB, ywfC, ywfD, ywfE and ywfF (SubtiList database) in the insert of the recombinant plasmid pSB660, which encode functions participating in the bacilysin biosynthesis in Bacillus host strains and are therefore renamed as bacA to bacF in accordance with Fig. 1, which is therefore an object of the claims. 14. A recombinant plasmid pSB672 according to claims 8, 11 or 13, wherein it is obtained through subcloning from PvuII, bp1680, to SalI, bp5642, from the PstI insert of pSB660 and contains the genes bacB to bacE within a 3 968 bps insert. 15. A recombinant plasmid pSB679 according to claims 8, 11 or 13, wherein it is obtained through subcloning from PvuII, bp1680, to Sac, bp 3558, of the PstI insert of pSB660 and contains the genes bacB and bacC within a 1878 bps insert. 16. Use of the recombinant plasmids pSB657, pSB660, pSB672, pSB679 or of derivatives of these plasmids according to one of the claims 9 to 15 in bacterial host strains, preferably in Bacillus subtilis and also in heterologous Bacillus host strains, for bacilysin formation. 17. Use according to claim 16 in the heterologous host strains Bacillus amyloliquefaciens GSB272 and Bacillus pumilus ATCC12140. 18. Procedure for isolating bac genes according to one of the claims 1 to 7 and 13, wherein these genes are isolated utilizing genetic methods and amplified in suitable host strains and wherein their function in the biosynthesis of anticapsin and bacilysin is characterized. 19. Procedure according to claim 18, wherein palindrome, positive-selection plasmid vectors and non-palindrome plasmid vectors with high cloning capacity and stability are used for the isolation of gene libraries with large chromosomal DNA inserts or for subcloning. 20. Procedure according to claim 19, wherein a palindrome, unipositive-selection plasmid vector pPS 15 and a non-palindrome plasmid vector pSB595 are used. 21. Procedure according to claims 19 to 20, wherein bacilysin-negative host strains, preferably of Bacillus subtilis 168, which do not show a chromosomally encoded bacilysin formation because of a chromosomal mutation, are used for primary cloning, and wherein the cloning of bac genes is detected because of the complementation of the chromosomal mutation. 22. Procedure according to claim 21, wherein a mutant NG79 bac-1 or derivatives from NG79 of the strain family Bacillus subtilis 168 are used as bacilysin-negative host strains. 26 23. Procedure according to claim 22, wherein a stabilizing strain Bacillus subtilis GSB298 or a comparable strain is used as bacilysin-negative host strain, which has an increased transformation capacity and a stabilizing effect on recombinant plasmid derivatives with a large insert. 24. Procedure according to claim 23, wherein GSB298 is derived as spontaneous mutant from NG79 and isolated because of a stabilizing effect on palindrome, dipositive-selection plasmid vectors. 25. Procedure according to claims 18 to 24, wherein gene libraries with large chromosomal DNA inserts are isolated preferably utilizing partial restrictase cleavage of the donor DNA and insertion of preselectable, large (> 10 kb) DNA fragments. 26. Procedure according to claims 18, 20, 23 and 25, wherein after partial bamHI cleavage a gene library is obtained and a recombinant strain Bacillus subtilis GSB298 (pSB657) with cloned bac genes of Bacillus subtilis 168 is isolated, which encode extracellular bacilysin. 27. Procedure according to claim 26, wherein the plasmid-encoded extracellular bacilysin is detected on the basis of the inhibition of a bacilysin-sensitive indicator strain, preferably of Escherichia coli B or Proteus vulgaris, and the inhibition of bacilysin through N-acetyl glucosamine and is not formed in host strains such as Bacillus subtilis GSB285 aroI906 that are defective in the synthesis of aromatic amino acids. 28. Procedure according to claim 16, wherein pSB660, pSB672 or pSB679 or derivatives of these recombinant plasmids in bacterial host strains, preferable of Bacillus, lead to an overproduction of anticapsin or bacilysin due to the gene amplification and wherein the recombinant strains are used for the industrial production of the agents. 29. Procedure according to claim 16 and 28, wherein pSB660, pSB672 or pSB679 leads to a several-times increased bacilysin formation in the host strain Bacillus amyloliquefaciens GSB272. 30. Procedure according to claims 28 and 29, wherein the plasmid-encoded bacilysin formation commences immediately at the start of growth, an advantage compared to the chromosomally encoded bacilysin formation of the donor strain thus being achieved, which occurs with a considerable delay in time at comparable growth.