This invention relates to cells and in particular to cells of the Bacillus genus. The Bacillus cells of the present invention may be ^' transformed with heterologous genetic material and used for industrial fermentations and production of heterologous polypeptides,

Background to the Invention

Currently there is considerable interest in the development of expression systems for making recombinant products in Bacillus subtilis. The advantages of B.subtilis as a production host for recombinant products include its large secretory capacity, its regulatory acceptability, and its well developed fermentation and product recovery technologies. Like many Bacilli, B.subtilis can efficiently secrete large quantities of proteins directly into the growth medium. Such secretion has cost advantages in product recovery, since the protein is produced in a relatively pure form compared to an intracellular product. Secretion also permits the accumulation of product to very high levels in an active form, unlike the denatured protein recovered from insoluble inclusion bodies which usually result from insoluble high level expression and intracellular accumulation (Ruppen, Band and Henner, 1986, Bacillus Molecular Genetics and Biotechnology Applications, Ed. A. Ganesan and J. Hoch. Academic Press pρ423-432) .

A major problem in the use of B.subtilis as a production host for secreted recombinant products is the synthesis and secretion by this organism of a variety of proteases which degrade secreted heterologous proteins. About 90% of secreted protease activity is attributable to two major proteases, a neutral metalloprotease encoded by a gene "npr" and an alkaline serine protease (subtilisin) encoded by a gene "apr". Various approaches have been used in attempts to overcome or reduce the protease degradation problem, including : screening for low protease mutants which show reduced degradation of casein on skim milk plates (Palva et al, 1983, Gene 22:229-239); introducing mutations into the apr and npr genes

(Kawamura and Doi, 1984, J. Bacteriol 160:442-444; Yang et al, 1984, J. Bacteriol 160:15-21; European Patent Application EP-A-246678) ; using early-blocked sporulation mutations, especially spoOA. which confer reduced protease levels (Willimas et al, 1981, Gene 16:199-206; Fahnestock and Fisher, 1986, J. Bacteriol 165:796-804). Recently attempts have been made to produce a strain with extremely low levels of secreted protease by introducing a spoOA mutation into an Apr- Npr- strain (Fahnestock and Fisher, 1987, Appl. Env. Microbiol 53:379-384; International Patent Application No. WO 86/01825; Wang et al, 1988, Gene 69:39-47). None of these strains is completely defective for secreted proteases, and the residual minor proteases they produce still pose a major problem in the production of heterologous proteins, (Schein et al, 1986, Bio/Technology 4:719-725; Wang et al, 1988, Gene 69:39-47). When production and integrity of Staphylococcal Protein A for example, in Apr Npr and Apr Npr SpoOA strains were compared, degradation was reduced in the latter but was nevertheless still a very significant problem (Fahnestock and Fisher, 1987).

There is therefore a real need for a suitable strain for use in recombinant DNA technology which overcomes the problem of protease degradation of secreted heterologous proteins.

We have carried out further work to produce a strain which overcomes the above problem and have now found a strain which is better than the prior art strains.

The present invention provides a Bacillus species of cells with a significantly decreased level of proteases.

Summary of the Invention

According to the present invention there are provided Bacillus cells exhibiting, at least, the phenotype Apr , Npr , SpoOH .

In a first aspect the invention provides Bacillus cells exhibiting at least the phenotype Apr Npr and SpoOH .

The cells of the present invention include those carrying disabled apr, npr, and spoOH genes. The genes may be disabled by mutation and the mutations may be of any type capable of deactivating the gene such that functional protein is not produced and may each be naturally occurring or produced by the methods of genetic engineering. One or more of the genes may be deleted using techniques well known in the art.

It has been found that the Bacillus cells of the present invention exhibit significantly reduced levels of extracellular proteases and are therefore of considerable utility as host cells for the expression of recombinant heterologous polypeptides. In particular, it has surprisingly been found that the cells of the invention exhibit a lower level of extracellular proteases than the Bacillus strains known in the art having the phenotype Apr , Npr , SpoOA . The cells of the present invention may be prepared using the techniques described hereinafter e.g. by congression and using publicly available cell lines as starting materials. In congression a non-selectable marker (such as spoOH) is co-transferred with a selectable marker into a recipient cell by transformation of competent cells of the recipient at high concentration of DNA prepared from the donor strain. Under these conditions a high proportion of the transformants selected using the selectable marker (e.g. removal of a nutritional requirement) are found to have simultaneously required the non-selectable marker.

The cells are preferably of the species Bacillus subtilis.

The cells are most preferably Bacillus subtilis strain CIMB7, CIMB14 and CIMB15. The invention also provides B.subtilis CIMB7, CIMB14 and CIMB15.

Specific examples of Bacillus strains of the invention are the same as or similar to the strain CIMB7, CIMB14 or CIMB15 as hereinafter described. The cells of the present invention may in addition lack one or more of the other naturally occurring extracellular proteases

as a result of introducing other mutations e.g. spoOA. Furthermore, the cells may lack one or more of the naturally occurring intracellular protease genes, such as the gene encoding intracellular serine protease (isp) .

The cells may be transformed with a vector capable of expressing a gene coding for a heterologous polypeptide using techniques known in the art.

In a second aspect the invention provides Bacillus cells exhibiting at least the phenotype Apr Npr SpoOH transformed with a vector capable of expressing a gene coding for a heterologous polypeptide.

The vector may be any vector which replicates within the host cell, such as for example a vector containing a Staphyloeoceus aureus replicon, a Bacillus replicon e.g. pBClδ and derivatives; a Lactobacillus replicon, or any shuttle vectors derived therefrom, and is especially a Bacillus vector as described in International Patent Application No. WO/8806622.

The cells of the invention may especially be useful as host cells for the expression of heterologous genes coding for, for example, any prokaryotic polypeptide such as for example a bacterial polypeptide such as an enzyme, e.g. α-amylase, β-amylase or β-galactosidase or any eukaryotic polypeptide such as for exmaple a mammalian polypeptide such as an enzyme e.g. chymosin or gastric lipase; an enzyme inhibitor e.g. tissue inhibitor of metalloproteinase (TIMP) ; a hormone e.g. growth hormone; a lymphokine e.g. an interferon; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or prourokinase; or natural, modified or chimeric immunoglobulins or fragments thereof having dual acitivity such as antibody-enzyme or antibody-toxin chimeras.

In a third aspect the invention provides a process for the production of a polypeptide comprising culturing a Bacillus cell according to the second aspect of the invention and optionally recovering the polypeptide therefrom.

In a fourth aspect the invention provides a polypeptide produced by a process according to the third aspect of the invention.

The Bacillus cells of the invention have very low secreted protease ^' 5 . activities and are therefore of use as hosts for the expression of recombinant proteins.

Brief Description of the Drawings

10 The invention is now described by way of example only and with reference to the following figures in whieh:

Figure 1 is a photograph of a Coomassie stained gel of SPA incubated with supernatants of cultures of EMG50, CIMB12, CIMB13 and CIMB15 5 3 hrs and 15 hrs into stationary phase.

Figure 2 shows a graph of the results of a β-lactamase assay of samples of the supernatants of EMG50, CIMB12, CIMB13 and CIMB15, removed at time points throughout growth of the cultures. 0

Detailed Description of the Specific Embodiments Materials and Methods

1) Bacterial Strains 5

The B.subtilis strains used are listed in Table 1. Representative strains carrying the npr, spoOH and spoOA mutations can be freely obtained from the Bacillus Genetic Stock Centre at the Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA. 0 Strains which carry the apr mutation may be prepared by methods known in the art, as described for example in Kawamura and Doi, 1984 (J. Bacteriol 160:424-444). Strains were routinely stored in L broth (see below) containing 10% glycerol at -20 C.

Table 1 - strains used

Strain Genotype

EMG50 Prototroph

1A289 aroI906 metB5 sacA321

IS53 spoOAΔ677

CIMB5 lys-1 phe-1 trp C2 apr npr isp ΔCAT

0A34.1 SPQOA34 phe!2 rpe B2 v l-1

0A135 SPQOA135 trpC2

OB490 SPOQB 0 trρC2

OB492 SPQ0A43 SPQQB490 trpC2

OC 87 SPQ0C9V phe-12 trpC2

OF221 SPQ0F221 pheA12 trpC2

OH46 SPQOH 6 trpC2

2) Media

L broth. L broth or L broth solidified with 1.5% agar was used as the standard medium for cultivation of the strains. It contained per litre: lOgms tryptone: 5gms yeast extract; 5gms NaCl. Its pH was adjusted to 7 using concentrated NaOH.

M9. M9 minimal salts was used as the standard medium. M9 contained per litre: 12gms Na HPO ; 6gms KH PO ; Igm NaCl; 2gms NH Cl. Its pH was adjusted to 7.4. It was made up at twice final concentration, autoclaved, and mixed 1:1 (vol:vol) with 3% agar for use. After sterilisation separately sterilised glucose was added to a final

concentration of 1% as carbon source, and amino acids to satisfy a final concentration of 1% as carbon source, and amino acids to satisfy auxotrophic requirements to a final concentration of 50ug/ml except where otherwise stated.

LGA. LGA medium was used to distinguish sporulation-proficient

+ — +

(Spo ) from sporulation-defective (Spo ) strains, Spo strains appearing brown and opaque after 48hrs incubation at 37 C and Spo ^" strains appearing white and translucent (Piggot, 1973, J. Bacteriol 114:1241-1253). LGA contained per litre:2gms glutamic acid; 2.8gms sodium lactate; 15gms agar; lOOmgs L-alanine; 0.98mgs FeCl .6H 0;

8.3mgs MgCl .6H 0, 19.8mgs MnCl .4H 0; 535mgs NH Cl;. 106mgs Na SO ;

68mgs KH PO ; 96.5mgs NH NO ; 219mgs CaCl .

GM1 and GM2. GM1 and GM2 were the media used in the nutritional step down procedure employed for preparing competent cells for B.subtilis transformations (see below). They were based on a minimal salts medium containing per litre: 14gms K HPO ; 6gms KH PO ; Igm tri-sodium citrate;2gms (NH ) SO . GM1 was minimal salts with the following added after sterilisation: 0.25% glucose; 5mM MgSO ; 0.02% Difco casamino acids; 0.1% yeast extract; 50ug/ml L-tryptophan; 200ug/ml of any other amino acid supplements to satisfy auxotrophic requirements of the strains. GM2 was minimal salts with the following added after sterilisation: 0.25% glucose; lOmM MgSO ; 0.01% Difco casamino acids; 0.5mM CaCl ; 35ug/ml other amino acids needed to satisfy auxotrophic requirements.

3) Preparation of chromosomal DNA.

To prepare DNA for transformation of B.subtilis competent cells the donor strain was first grown overnight in 200mls L-broth in a 21 flask with shaking at 37 C. The cells were removed by centrifugation, washed in 80mls TES (comprising per litre 6gms T IS, 1.9gms EDTA, 2.9gms NaCl, pH8.0), then resuspended in 7mls TESS (TES containing 25% sucrose) . 20mgs of lysozyme were added and the cell suspension was mixed well, then incubated at 37 C for 20 mins.

After the addition of 1.5ml 0.5M EDTA and 2mls 12% SDS the mixture was incubated at 65 C for 7 mins. After cooling 2.5mls 5M sodium perchlorate were added and the mixture phenol extracted. Chromosomal DNA in the aqueous phase was then purified by caesium chloride density gradient centrifugation as described in Maniatis et al. 1982, Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory) .

4) Preparation and transformation of competent cells of B.subtilis

Competent cells were prepared by a modification of the method of Dooley et al. 1971, J. Bacteriol 108:668. A large single colony of the strain to be transformed was inoculated into 25mls L-broth and o grown overnight with shaking at 30 C. The cells from lOmls were collected by centrifugation and inoculated into 25mls in a 250ml flask to an optical density at 600nm (o.D600) of 0.4. The culture was incubated with shaking at 37 C and its growth monitored by measuring 0D600 of samples taken at hourly intervals. When the culture reached the end of the growth phase 1Orals were removed, the cells collected by centrifugation and resuspended in lOmls minimal salts. These cells were used to inoculate 25mls of GM2 to 0D600 of 0.2. This culture was grown with shaking at 37 C for 90 mins, the cells collected by centrifugation, resuspended in 2.5mls GM2 containing 10% glycerol, divided into 200ul aliquots and stored at -80 C until required for transformation.

For transformation 200ul aliquots of these competent cells were rapidly thawed at 37 C, mixed with chromosomal DNA at the required concentration, incubated at room temperature for 10 mins, washed in minimal salts, recentrifuged, resuspended in minimal salts and then spread on selective agar plates, which were incubated at 37 C. In strain construction experiments designed to transfer mutations by congression, i.e. contransformation with two separate DNA fragments, the donor chromosomal DNA was used at a final concentration of lOug/ml.

5. Protease assays by azocasein hydrolysis.

Protease activities in culture supernatants were assayed by a modification of the procedure described by Prestidge et al, 1971, J. Bacteriol 107:815-823. Cultures were grown in 25mls twice normal concentration L-broth in 250ml flasks with shaking at 37 C, and growth of the cultures monitored by measuring 0.D600. In this medium the cultures reached stationary phase at 0.D6O0 of about 4.5. 1.5ml samples were taken at various time points from late in ° the growth phase until IShrs into stationary phase. These samples were spun for 10 mins at 12000rpm in a bench microcentrifuge, the cells discarded and the supernatant respun for a further 10 mins to competely remove the cells. Protease activities in these supernatants were determined by the following azocasein assay, ⁵ 150ul of supernatant was mixed with 50ul 1M Tris/HCl pH8, 50ul H O and 250ul 2% azocasein (Sigma Chemical Company). This mixture was incubated at 30 C for 60 mins and the reaction stopped by the addition of cold 7% perchloric acid. The mixture was then centrifuged for 30 mins at 12000rpm in a bench microcentrifuge. 0 lOOOul of the supernatant was then removed and thoroughly mixed with

150ul 10M NaOH. The absorbance at 436nm of each mixture was measured on a spectrophotometer against a zero time blank prepared for each sample using the same procedure but adding perchloric acid before azocasein. 5

6) Estimation of protease production by skim milk hydrolysis

Protease levels of strains were crudely assessed by comparing the sizes of clearing zones after 2-5 days at 37 C on L-agar plates ⁰ containing 1% Difco skim milk (SMLA plates).

7) Staphylococcal Protein A (SPA) degradation test

20 ul of 0.75mg/ml Staphylococcal Protein A (of single band purity, 5 from Sigma Chemical Company) were mixed with 20ul of culture o supernatants and incubated at 37 C for 30 mins. Degradation of SPA was assessed by electrophoresing these samples on 10% SDS-containing

polyacrylamide gels and staining with Coomassie Blue as described in Maniatis et ajL, 1982, and observing integrity or disappearance of the SPA band.

Examples

Example 1 - Screening of strains for low protease production

Protease activities produced by all the sporulation-defective and other strains listed in Table 1 were assessd both by observing the size of clearing zones on SMLA plates and by performing the SPA degradation test as described in Materials and Methods. All these strains produced clearing zones on SMLA of similar size except CIMB5

(an Apr- Npr- strain), which produced no clearing zone, even after o several days incubation at 37 C. In the SPA degradation test, however, only supernatants of strains carrying mutations in the genes spoOA or spoOH showed reduced degradation compared to the control strains EMG50 and 1A289. Supernatants of all the other strains led to complete disappearance of the intact SPA band after 30 mins incubation, including that of CIMB5. Azocasein assays on culture supernatants were then performed (as described in Materials and Methods) for the strains identified by either test as showing reduced protease production, namely 0H46 (SpoOH ), 1S53 (SpoOA-), CIMB5 (Apr- Npr-) , and or a control strain 1A28 . The results are given in Table 2, expressed as %age of activity displayed by the control strain 1A289. The culture supernatants used in these assays were prepared from cultures two hours after their entry into stationary phase. At this time the strain carrying a mutation in the apr npr genes showed only 25% of the total protease activity found for 1A289. The SpoOH strain showed a significant reduction in protease activity, (68% of that found for 1A289). Unexpectedly the strain carrying a mutation in the spoOA gene showed a somewhat higher protease level than 1A289 (107%) . In similar experiments Fahnestock and Fisher, 1987, Appl. Env. Microbiol 53:379-384, found reduced levels of protease activity were produced by the same SpoOA- strain (1S53) compared to a control strain.

These results and other (see below) suggest that strain 1A289 itself is a low protease producer compared to most commonly used B.subtilis strains.

Table 2. Azocasein assays on culture supernatants

Strain Protease Activity

1A289 100 OH 6 68

1S53 107

CIMB5 25

(N.B. Assays performed on supernatants of cultures 2 hrs after entry into stationary phase, results expressed as %age 1A289 activity)

Although the strain CIMB5 carrying a mutation in the apr npr genes (i.e. apr- npr-) showed by far the lowest protease activities in the azocasein assays, the results of the SPA degradation tests suggested that both the SpoOA and SpoOH strains must have reduced levels of one or more minor proteases involved in SPA degradation. Attempts were therefore made to construct improved derivatives of the Apr Npr strain by introducing into it the spoOH and spoOA mutations.

Example 2 Construction of Apr. Npr SpoOA and Apr Npr SpoOH strains

The spoOH and spoOA mutations were transferred into CIMB5 by congression (see Materials and Methods). Competent cells of CIMB5 were prepared as described in Materials and Methods and transformed (separately) with chromosomal DNA of strains 0H46 and 1S53, at a DNA concentration of lOug/ml, also as described in Materials and Methods. The transformation mixtures were plated onto minimal salts agar supplemented with lysine and tryptophan to select for Phe transformants. 100 such transformants were restreaked onto LGA to test for simultaneous acquisition of the spoOA or spoOH mutations.

+

Representative Phe Spo strains were called CIMB7 in the case of the Apr- Npr- SpoOH- triple mutant and CIMB11 in the case of the Apr- Npr- SpoOA- triple mutant. When streaked on SMLA plates neither CIMB7 nor CIMB11 produced a clearing zone, indicating they had both retained the apr and npr mutations.

In addition to carrying the mutations affecting levels of secreted proteases CIMB5, CIMB7 and CIMB11 all carry an insertion mutation affecting the major intracellular serine protease, encoded by the gene isp. The fragment inserted in and inactivating isp carries the chloramphenicol resistance gene of plas id pC194. The Cm gene is also carried on the plasmid pPOD2400, a plasmid expressing Eschericia coli B-lactamase in B.subtilis which was later to be used to compare degradation of a secreted heterologous protein in these r low protease strains (see below). The presence of the same Cm gene in the chromosome of these strains would be likely to lead to integration of the plasmids following transformation. Derivatives

_< of CIMB5 lacking the Cm gene but carrying the mutations in the spoOA or spoOH genes were therefore constructed as follows. Competent cells of CIMB5 were tranformed with chromosomal DNA of strain 1S53 with selection on minimal salts agar supplemented with phenylalanine and tryptophan to select for Lys transformants. 100 such transformants were streaked onto L-agar containing chloramphenicol to identify those which had been simultaneously transformed to chloramphenicol sensitivity. One such strain was shown to have retained the apr npr mutations by SMLA plate tests and was designated CIMB12. The same procedures were then used to construct Apr Npr SpoOA and Apr Npr SpoOH strains from CIMB12 as are described above for constructing CIMB7 and CIMB11 from CIMB5. The chloramphenicol sensitive Apr Npr SpoOA and Apr- Npr ^" SpoOH strains were designated CIMB13 and CIMB14 respectively.

Example 3 Determination of protease activities for Apr Npr . Apr Npr- SpoOA- and Apr Npr SpoOH strains.

Cultures of CIMB5, CIMB7, CIMBll, CIMB13, CIMB14 and control strains were grown and azocasein assays performed to determine protease activities as described in Materials and Methods. The results are given in Table 3

Table 3 Protease activities in culture supernatants of Apr Npr-, Apr- Npr- SpoOA-, Apr- Npr- SpoOH- and control strains.

Strain Protease activit

(N.B. Assays performed on supernatants of cultures two hours after entry into stationary phase, results expressed as %age of 1A289 activity) .

The results show that introduction of the spoOH mutation reduced secreted protease activities of the Apr Npr strains by about half at the time point chosen, which was two hours into stationary phase.

The apr npr spoOA triple mutants (i.e. Apr , Npr ^" SpoOA-), however, showed unexpectedly high protease levels.

For many B.subtilis expression systems accumulation of the secreted product to high yields will probably require an extended period in

stationary phase. Protease activites in the supernatants of cultures grown for 4hrs and 18 hrs in stationary phase were therefore determined for strains CIMB5, CIMB7, 1A289 and a second control strain, this being EMG50 - a prototrophic strain more typical of commonly used B.subtilis strains in its levels of secreted proteases than 1A289. The results are given in Table 4.

Table 4 Protease activities in culture supernatants of Apr Npr-. Apr- Npr- SpoOH and control strains.

Strain Protease activit

EMG50 1A289 CIMB5 CIMB7

(N.B. Results expressed as %age of EMG50 activity)

These results show that the Apr Npr SpoOH triple mutant produces less than half the secreted protease activity of the apr npr double mutant 4 hrs into stationary phase, and only about a quarter after 18 hrs in stationary phase. The results also confirm that strain ^' 1A289 produces relatively low protease levels.

Example 4 Degradation of Staphylococcal protein A by Apr Npr~, Apr Npr SpoOA and Apr Npr SpoOH strains.

SPA degradation tests were performed using culture supernatants of strains EMG50, CIMB12 (Apr ^" Npr ^" ) _s CIMB13 (Apr- Npr ^" SpoOA-) and CIMB15 (a prototrophic derivative of the Apr- Npr ^" SpoOH- strain CIMB14). Supernatants of cultures 3 hrs and 15 hrs into stationary phase were used. Figure 1 is a photograph of the Coomassie stained SDS/acrylamide gel resulting from this experiment. The samples

electrophoresed were as follows: Lane 1, molecular weight markers; Lane 2, untreated SPA; Lane 3, SPA incubated with the 3 hr EMG50 supernatant; Lane 4 SPA incubated with the 3 hr CIMB12 supernatant; Lane 5, SPA incubated with the 3 hr CIMB15 supernatant; Lane 6, SPA incubated with the 3 hr CIMB13 supernatant; Lane 7, SPA incubated with the 15 hr EMG50 supernatant; Lane 8, SPA incubated with the 15 hr CIMB12 supernatant; Lane 9, SPA incubated with the 15 hr CIMB15 supernatant; Lane 10, SPA incubated with the 15 hr CIMB13 supernatant. Incubation with EMG50 supernatants leads to complete disappearance of the intact SPA band. Incubation with CIMB12 supernatants leaves only a trace of intact SPA, while both the CIMB13 and CIMB15 supernatant give less degradation that that of CIMB12, for both timepoints tested. The significant result is that the CIMB15 supernatants clearly cause less SPA degradation than those of CIMB13, at both timepoints. Gel scanning of lanes 4, 5, and 6 was performed using a Joyce/Loebel Chromoscan 3 gel scanner to quantify the extents of SPA degradation by the culture supernatant taken 3 hrs after entry into stationary phase. The results showed that incubation with the CIMB123 and CIMB13 supernatants had resulted in degradation of 93% and 64% of the SPA respectively, while incubation with the CIMB15 supernatant caused degradation of only 33% of the SPA.

Example 5 Production and degradation of β-lactamase by EMG50. CIMB12. CIMB13 and CIMB15.

In order to compare the performance of the Apr Npr , Apr Npr SpoOA- and Apr- Npr- SpoOH- strains in production and degradation of a heterologous secreted protein, namely E.coli beta-lactamase, plasmid pPOD2400 was transformed into strains CIMB12, CIMB13, CIMB15 and EMG50. pPOD2400 is derived from plasmid pCPP4 of Band et al, 1983, Gene 26:313-315, and carries the replication functions and kanamycin resistance of pUBHO, together with a hybrid gene comprising the mature coding region of the E.coli TEM beta-lactamase fused in frame with the signal sequence of a Bacillus alpha-amylase, such that the beta-lactamase is expressed in B.subtilis using the alpha-amylase transcription and translation initiation signals.

ρPOD2400 is thus very similar to plasmid pKTH78 described by Ulmanen et al, 1985, J. Bacteriol 162:176-182, who found that the beta-lactamase is efficiently secreted from B.subtilis using the alpha-amylase signal sequence but is rapidly degraded at the end of the growth phase by the secreted proteases of B.subtilis even in low protease strains. pPOD2400 was transformed into strains CIMB12, CIMB13, CIMB15 and EMG50 using the protoplast transformation procedure of Chang and Cohen, 1979, Molec. Gen. Genet 168:111-115, with selection on DM3 medium containing (in additon to the ingredients described by Chang and Cohen) 0.9% soluble starch, 0.4% gelatin, 0.04% bovine serum albumin and 300ug/ml kanamycin. Plasmid minipreparations were performed on representative kanamycin resistant transformants as described in Maniatis et al, 1982, and agarose gel electrophoresis was used to confirm the integrity of the plasmid in these transformants. One representative transformant of each strain carrying ρPOD2400 was then grown in a 25ml culture in a 200ml container in L-broth plus 3% glucose and lOug/ml kanamycin (to ensure retention of the plasmid), incubated with shaking at 37 C. 1ml samples were removed at various time points throughout growth of these cultures, OD600 measured as an indication of population density, the cells removed by centrifugation as described for the azocasein protease assays, and the supernatants assayed for beta-lactamase activity as described by Smith et al, 1986, J.Bacteriol 169:3321-3328. The results of the beta-lactamase assays were displayed graphically in Figure 2. All four strains reached the end of the growth phase after about 7 hrs, and Figure 2 shows that for all four strains β-lactamase activities reach their peak values around this time. EMG50 carrying ρPOD2400 produced the lowest β-lactamase activities. CIMB12 and CIMB13 carrying this plasmid produced significantly greater activities than EMG50, with activities being slightly greater for CIMB13 than CIMB12 up to the 9 hr time point. The presence of the spoOA mutation therefore conferred only a slight reduction in beta-lactamase degradation on the Apr Npr strain. In contrast to these results it is clear from Figure 2 that the presence of the spoOH mutation led to a dramatic decrease in beta-lactamase degradation : beta-lactamase activities were found to be dramatically higher in supernatants of CIMB15 than

in those of CIMB12 or CIMB13 at all time points tested. At the last time point, 24 hrs after inoculation and approximately 17 hrs into stationary phase, the beta-lactamase activity in the CIMB15 supernatant was approximately fourfold higher than in those of any of the other strains. At this time point dilutions of the cultures were plated out on both L-agar and L-agar containing lOug/ml kanamycin to determine the % of plasmid containing cells in each culture. The results indicated that the differences in beta-lactamase activities between CIM15 and the other strains cannot be explained by differences in plasmid stability.

Strains of B.subtilis with very low secreted protease activities have been constructed by introducing a spoOH mutation into an Apr- Npr double mutant, i.e. defective for both major secreted proteases. The new triple mutant strains carrying mutations in the genes apr npr spoOH have been shown to have very low secreted protease activities in three separate tests, namely azocasein hydrolysis, degradation of Staphylococcal protein A, and degradation of E.coli TEM beta-lactamase.