Vol. 53, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1987, p. 379-3840099-2240/87/020379-06$02.00/0Copyright © 1987, American Society for Microbiology

Protease-Deficient Bacillus subtilis Host Strains for Production ofStaphylococcal Protein A

STEPHEN R. FAHNESTOCK* AND KATHRYN E. FISHER

Genex Corporation, Gaithersbuirg, Maryland 20877

Received 22 August 1986/Accepted 24 November 1986

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. Thesestrains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed invitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vectorwhich allowed the selection of chromosomal integrates and their subsequent excision by homologousrecombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insertwith a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin,no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as

0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strainsfor the production of staphylococcal protein A, accumulating higher levels of intact protein than do previouslyavailable B. subtilis strains.

Bacillus species produce large quantities of extracellularprotease during postexponential growth. With recent inter-est in the development of Bacillus systems for the synthesisand secretion of foreign proteins has come the realizationthat these proteases interfere with the goal of accumulatinghigh levels of foreign proteins, which are often proteasesensitive. For example, we have reported the construction ofBacillus subtilis strains which secrete staphylococcal proteinA encoded by a recombinant gene (3). Although the yields ofprotein A are less affected by protease than are someeucaryote proteins (20; unpublished data), yields of intactprotein A are significantly limited by proteolysis.The most abundant of the extracellular proteases are an

alkaline serine protease (subtilisin) and a neutral metal-loprotease. B. subtilis mutants devoid of extracellular metal-loprotease activity but without pleiotropic effects have beenisolated by conventional genetic means (13, 18). However,strains bearing mutations in the subtilisin structural gene aprare not among the protease-deficient mutants isolated byconventional means, and it is only more recently that suchmutants have been constructed by in vitro methods (11, 17).We independently constructed B. subtilis strains whichproduced no subtilisin or extracellular metalloprotease.Here we describe the methods, which we believe to be ofgeneral utility, used to construct the strains and the use ofthe strains as hosts for the production of staphylococcalprotein A.

MATERIALS AND METHODS

Bacterial strains and media. Escherichia coli SK2267 (F-gal thi Tlr hsdR4 recA endA sbcB15) was obtained from M.Nomura. B. subtilis BR151 (trpC2 lys-3 metBIO) was ob-tained from P. Lovett. B. subtilis 1S53 (spoOA677) wasobtained from the Bacillus Genetic Stock Center, Columbus,Ohio. B. subtilis 512 (Npr-) was obtained from J. Millet.

E. coli was grown in LB broth containing (per liter) 10 g oftryptone, 5 g of yeast extract, and 10 g of NaCl. LB agarcontained in addition 1.5 g of agar per liter. B. subtilis wasgrown in Penassay broth (Difco Laboratories, Detroit,

* Corresponding author.

Mich.) or on LB agar. Chloramphenicol and erythromycinwere used at 5 ,ug/ml, and ampicillin was used at 100 ,ug/ml.Medium A contained (per liter) 33 g of tryptone, 20 g of yeastextract, 7.4 g of NaCl, 8 g of Na2HPO4, 4 g of KH2PO4, 20 gof Casamino Acids (Difco), 10 g of glucose, 0.06 mM MnCl2,and NaOH to pH 7.5.

Construction of the vector pGX2945. pGX2945 is a shuttlevector derived from pE194 (9) and pGX145 (15). The pE194was linearized by digestion with AccI and blunted by briefBal 31 nuclease treatment. This material was ligated to aderivative of pGX145 which had been linearized with NruI.(The actual plasmid used was pGX324, which contains anEcoRI-HindIII fragment bearing a fused gene, the nature ofwhich is not relevant, which was then deleted from theresulting chimera by digestion with HindIII and religation.)The resulting plasmid pGX2945 had pBR322 sequences (29base pairs) between its unique EcoRI and HindIll sites,rather than the multisite linker sequence present in pGX145,but it was otherwise a chimera of pGX145 and pE194.

Subcloning of an apr-bearing XmnI fragment. DNA wasprepared from a bacteriophage X derivative identified (C. F.Rudolph, unpublished data) from a A Charon 4A library of B.subtilis sequences (4) on the basis of its cross-hybridizationwith a probe of Bacillus amyloliquefaciens apr[BamP] se-quences (22). The 6.6-kilobase-pair (kbp) EcoRI fragmentinsert present in this phage was purified by agarose gelelectrophoresis and electroelution. The purified fragmentwas then digested with XmnI, and a 1.5-kbp fragmentcontaining the apr gene was purified by agarose gel electro-phoresis and electroelution. This XmnI fragment was ligatedto Hindlll-cut pGX2945, the ends of which had been bluntedby filling in with DNA polymerase I. The ligated mixture wasused to transform E. coli SK2267 to ampicillin resistance. Aclone containing the correct plasmid (pGX2969; Fig. 1) wasidentified, and the orientation of the insert was determinedby restriction analysis (data not shown).

cat fragment insertion into apr. The plasmid pGX345 (15) isa pBR322-based vector which contains a fragment (1.03 kbp;MspI-MboI) derived from pC194 (10) and which carries theentire cat gene, which confers chloramphenicol resistance inB. subtilis and E. coli (10). A HindlIl site was placedupstream from this fragment by opening pGX345 at its

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380 FAHNESTOCK AND FISHER

(BamHl/Mspl) (Mbol/BamHl)

HindlIl Hindll

Icat

PstI

FIG. 1. Construction of the insertionally inactivated apr plasmid pGX2971.

EcoRI site, blunting with DNA polymerase, and reclosingwith a synthetic HindlIl-site linker. The cat gene wasobtained from this plasmid as a 1.08-kbp HindlIl fragment,which was purified by gel electrophoresis and electroelutionand was then inserted into the HindIll site of pGX2969. Theresulting ligated mixture was used to transform E. coliSK2267 to ampicillin resistance. Several transformantswhich were also resistant to chloramphenicol (5 [Lg/ml) wereidentified; among these, one in which the cat gene wasoriented in the same direction as the apr gene was identifiedby restriction analysis (pGX2971; Fig. 1).B. subtilis transformation. B. subtilis protoplasts were

transformed by the method of Chang and Cohen (1). Com-petent B. subtilis cells were prepared and transformed asdescribed previously (15).

Protease assays. Serine protease activity was determinedas follows. Cultures were grown at 370C in medium A withvigorous aeration. Culture supernatant (0.2 ml) was dilutedto 1 ml in a buffer containing 0.1 M Tris hydrochloride and0.05 M sodium EDTA (pH 8.0) in duplicate tubes. Phenyl-methane sulfonyl fluoride (PMSF) to 2 mM was added to onetube. Both tubes were then incubated for 10 min at 23°Cbefore the addition of the substrate (10 mg of hide powderazure [Calbiochem-Behring, La Jolla, Calif.]). After 20 to 40min of incubation at 37°C with shaking, the remainingsubstrate was removed by centrifugation; the A595 of thesupernatant was then determined after dilution with an equalvolume of water. Serine protease activity was expressed asAA595/min per A600 equivalent of culture supernatant minusthe value for the PMSF-containing blank and relative to thevalue for strain BR151, which was set at 100.

Metalloprotease activity was determined by a similarassay, except the assay buffer contained 0.1 M Tris hydro-chloride (pH 7.4), 1 mM CaCl2, and 2 mM PMSF. To one ofeach pair of duplicate tubes was added 0.05 M sodium EDTA(pH 7.4). The samples were then treated as described above.Metalloprotease activity was expressed as AA595/min perA600 equivalent of culture supernatant minus the value forthe EDTA-containing blank and relative to the value forstrain BR151, which was set at 100.

Gel electrophoresis. Cultures of strains 1S53 (see Fig. 3,host A) and GX4937 (host B) and protein A-producingderivatives of both strains (GX3362 and GX4999, respec-tively) carrying the apa-J plasmid pGX2939 were grown in

medium A at 37°C. Samples were taken at various times afterinoculation, and cells and extracellular supernatant wereseparated by centrifugation. The supernatant fraction wasimmediately made 50 mM in EDTA and 2 mM in PMSF. Thecell pellet was suspended in a buffer containing 50 mMEDTA (pH 8.0), 2 mM PMSF, and 2 mg of lysozyme per mland incubated for 15 min at 37°C. Samples (0.5 [L) of bothsupernatant and cellular fractions were subjected to sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, and pro-tein bands were then transferred to nitrocellulose. Bandswith immunoglobulin G (IgG)-binding activity were locatedimmunochemically (7) with normal rabbit serum and perox-idase-conjugated goat anti-rabbit IgG as first and secondreagents, respectively, and visualized with 4-chloro-1-naphthol and H202. Protein molecular weight standardswere purchased from Bethesda Research Laboratories, Inc.

RESULTS AND DISCUSSION

Inactivation of apr. Our strategy for inactivation of thesubtilisin gene (apr) in B. subtilis is illustrated in Fig. 2. Itinvolved replacing the chromosomal apr gene by one inac-tivated in vitro and used a two-step insertion and excisionprocedure similar in principle to that reported by Schererand Davis (16) in yeast cells and by Gutterson and Koshland(5) in Salmonella typhimurium. In vitro inactivation of aprwas accomplished by inserting into the DNA sequenceencoding the mature protein a sequence which encoded achloramphenicol acetyltransferase gene and conferred chlor-amphenicol resistance on B. subtilis. We chose this strategybecause it allowed us to monitor the procedure indepen-dently of any phenotype conferred by the mutant apr geneand it would clearly so indicate whether the desired mutationwere lethal, a desirable feature since we did not know at theoutset what physiological effects the mutation would have.The B. subtilis apr[Bsu] gene was identified in a phage

lambda library provided by J. Hoch (4), by hybridizationwith a cloned B. amyloliquefaciens apr[BamP] gene (22) as aprobe (C. F. Rudolph, unpublished data). The clonedapr[Bsu] gene was then inactivated by the insertion of afragment of pC194 bearing the cat gene at a HindIII sitelocated at the codon corresponding to amino acid residue 48of mature subtilisin (17). The insertion separated the pro-moter and secretion signal sequence from most of the mature

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apr

cat I' ,1I,/

I

pGX2971

rmC

cat

ermCVIIIAip

apr

apr

+cat

' cat

apr

FIG. 2. Two-step replacement of chromosomal apr by apr::cat.

protein-coding sequence and separated the active-site resi-due Asp-32 from other active-site residues His-64 andSer-221. The interrupted gene, therefore, could not yield anactive subtilisin product.The inactivated apr: :cat was introduced into B. subtilis by

using a plasmid shuttle vector, a chimera of pBR322 (E. coli)and pE194 (B. subtilis) sequences. Plasmids bearing se-

quences homologous to sequences present in the chromo-some of B. subtilis, but lacking an active replication func-tion, can readily become integrated into the chromosome atthe site of homology (6, 15). Furthermore, pE194 itself,integrated into the chromosome by a rare event not involvingchromosomal homology, can be stably maintained there,conferring erythromycin resistance (8). Such rare integratescan be selected by growth at 50°C in the presence oferythromycin, since the replication function of pE194 isinactive at temperatures above 45°C in B. subtilis. Theplasmid pGX2971 (Fig. 1) was therefore designed for thefollowing properties: (i) it could be propagated in E. coli, (ii)it could be established as an autonomous plasmid in B.subtilis, (iii) chromosomal integrates could be selected at50°C in the presence of an antibiotic (either erythromycin or

chloramphenicol), (iv) vector sequences could be monitored

via the erythromycin resistance marker, and (v) the inacti-vated apr gene could be monitored via the chloramphenicolresistance marker.

In the replacement strategy outlined in Fig. 2, the plasmidpGX2971, carrying the inactivated apr::cat, was first estab-lished in B. subtilis and then inserted into the chromosomeby homologous recombination in the apr sequences. Theintegration step should result in a tandem duplication inwhich one of the two copies of apr is unaltered. This wouldnot be expected to be lethal in any case, provided that bothends of the gene are complete on the plasmid and the aprmutation is not a dominant lethal. The plasmid sequenceswere then excised in a subsequent recombination event,leaving behind, in some fraction of such progeny, the inac-tivated gene. The second step (resolution) should yieldprogeny which retain in the chromosome either the alteredor unaltered version of apr in a proportion determined by thelength of chromosomal homology on either side of theinactivating insertion in the cloned gene (approximatelyequal in pGX2971). Lethality of the mutant apr gene wouldbe reflected in the absence of that class from among theresolution progeny.

Since the recE-independent chromosomal integration of

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382 FAHNESTOCK AND FISHER

TABLE 1. Extracellular protease production

% Protease production in 20 h"'

Strain (genotype) Serineprotease Metalloprotease

BR151 (trpC2 Ivs-3 inetBlO) 100 100GX4924 (trpC2 Iys-3 mnetBlO apr::(cat) 1.3 54

IS53 (spoOA677) 39 82GX4925 (spoOA677 apr: :cat) 1.3 85

512 (npr) 64 1.0GX4926 (npr apr::clat) 13 1.2

'8 Protease activity accumulating in the culture supernatant during growth at37°C on Penassay broth plus 1 mM CaCI2 for 20 h was assayed by using hidepowder azure as a substrate as described in Materials and Methods. Values ineach column are expressed relative to the value for BR151.

pE194 without chromosomal homology is a very rare event(frequency, approximately 3 x 10' [8]), the provision ofsubstantial chromosomal homology in the form of apr se-quences would be expected to result in integration predom-inantly at the site of homology. The excision of pE194integrated without homology is also rare (8), so progeny,which have experienced both the integration and excisionthat are required to generate the Cmr Ems phenotype, wouldbe expected to be even more predominantly the results ofinsertion at the site of homology.

Construction of mutant apr npr strains. We began withstrain 512, isolated by Michel and Millet (13), which pro-duces no detectable extracellular metalloprotease.Protoplasts of strain 512 were transformed with pGX2971DNA, and a transformant resistant to both chloramphenicol(Cm) and erythromycin (Em) was isolated. After passage at500C, first in the presence of chloramphenicol to select forintegration of the plasmid into the chromosome and then inthe absence of antibiotic to allow for excision, progeny werescreened for antibiotic resistance, and a Cmr Ems derivativewas designated GX4926. At the same time, analogousstrains, derived from BR151 (trpC2 lys-3 metBIO) and 1S53(spoOA677), were constructed. In the latter cases, the inte-gration-excision procedure proved unnecessary, because aCmr Ems derivative was identified among the progeny oftransformation of the parental strains with pGX2971. Thesestrains were designated GX4924 and GX4925. Southern blotanalysis of chromosomal DNA from the three putativeapr::cat strains verified that in all three cases the cat-bearingfragment had been inserted into the only chromosomal copyof the apr gene (data not shown). The strains contained noactive copy of apr.

Extracellular protease activity accumulated by the strainswas assayed by using hide powder azure as a substrate(Table 1). Among the three strains, GX4926, the apr: :cat nprderivative of strain 512, exhibited the highest level of resid-ual serine protease, although it accumulated no detectablemetalloprotease in the culture supernatant. This residualserine protease activity must have been due to an enzyme(s)other than subtilisin.To take advantage of the lower residual serine protease

levels exhibited by the apr::cat strain derived from BR151,we transferred the apr::cat and npr markers together fromGX4926 into BRI51. This was accomplished by transformingcompetent BR151 cells with chromosomal DNA fromGX4926, selecting for chloramphenicol resistance, andscreening among the Cmr transformants for acquisition ofthe npr marker (absence of a halo on a casein-agar plate).

Approximately 1% of Cm' transformants had also acquiredthe npr marker as a result of congression (2). One suchtransformant was designated GX4931 (apr::cat aipr trpC2lys-3 mnetB10). Under the conditions given in Table 1, theserine protease activity of GX4931 was 25% of that ofGX4926.

Finally, we introduced the spoOA marker from 1S53 intoGX4931 by congression, transforming competent cells with amixture of chromosomal DNA from 1S53 and a plasmidderived from pE194, selecting for erythromycin resistance,and screening among the Emr transformants for nonsporula-tors (unpigmented colonies). The plasmid was then cured bygrowth at 50°C. The resulting strain, designated GX4937(apr::cat nppr spoOA677 trpC2 lys-3 metBiC), was verified asSpo-.The extracellular protease levels accumulated by GX4931

and GX4937 in a medium which gave optimal protein Aproduction are shown in Table 2. The best of these strains,GX4937, accumulated no detectable extracellular metal-loprotease and less than 1% of the extracellular serineprotease of BR151. We have not yet identified the enzyme(s)responsible for the residual serine protease activity secretedby GX4931. One candidate is the bacillopeptidase F de-scribed by Roitsch and Hageman (14). The residual activitywas greatly reduced by the spoOA mutation in GX4937.

In addition to the apr::cat-bearing strains, we made deriv-atives of GX4931 and GX4937 in which the chromosomalinsertionally inactivated apr:: cat gene was replaced by adeletion. For this purpose, we constructed a plasmid similarto pGX2971. Instead of the apr::cat construction, this plas-mid carried a DNA sequence containing a deletion extendingfrom an HpaI site close to the sequence encoding the Nterminus of preprosubtilisin (17) to an HpaI site beyond theend of the apr' sequences; the plasmid still carried approxi-mately 1 kbp of chromosomal DNA sequences on either sideof the deletion. The plasmid (pGX2979) was established inGX4931, and chromosomal integrates were then selected bygrowth at 50°C in the presence of erythromycin. Aftersubsequent growth at 50°C in the absence of antibiotic, aderivative sensitive to both erythromycin and chloramphen-icol was identified and designated GX4935. This strain wasshown to accumulate extracellular protease at levels similarto those accumulated by its parent GX4931. The apr deletionderived from GX4937 was constructed similarly.

Effect on protein A accumulation. We have reported theconstruction of B. sutbtilis strains which synthesize andsecrete staphylococcal protein A, accumulating quantities ofup to several grams per liter (3). One such strain, GX3362

TABLE 2. Extracellular protease activities

% Protease production"

Strain (genotype) Serine protease Metalloprotease

12 h 24 h 12 h 24 h

BR151 (trpC2 Ivs-3 metBlO) 100 115 100 51S53 (spoOA677) 8 66 134 37GX4931 (tOpC2 IYs-3 mnetBIO 28 28 0.4 0

npr apr: :c at)GX4937 (trpC2 Iys-3 inetBIO 0.5 0.5 -0.6 -0.5

npr apr: :cat spoOA677)

" Cultures were grown in medium A at 37C under the conditions foroptimal protein A production (Fig. 3) and then assayed for protease activitiesas described in Materials and Methods. Samples were taken at 12 and 24 hafter inoculation. Serine protease or metalloprotease activity is expressedrelative to the value obtained for BR151 at 12 h.

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PROTEASE-DEFICIENT B. SUBTILIS 383

5 10

,SE* TE thj t> w e

XIi,00s w4~~~~

A B A B A B A B A B A B A B B A B Host- - + + + + + + + + + + + + + + + Protein A PlasmidC+ s S S S S S S S S S S S S C C C Fraction14145.55.57 7 9 911 11 13 13 24 24 9 13 13 Time(h)

FIG. 3. Gel electrophoretic analysis of protein A produced by GX3362 and GX4999. The positions of prestained protein molecular weightstandards are indicated on the right. Slot 18 (to the right) contains 0.2 ,ug of protein A from S. aureuis. (Pharmacia, Inc.). The size differencebetween this material and the B. subtilis product reflects a strain difference between S. aureus 8325-4, from which the cloned spa wasobtained, and the source of commercial protein A (19). Fractions: S, supernatant; C, cellular. B. subtilis host strains: A, 1S53; B, GX4937.

(3), is a derivative of B. subtilis 1S53, containing, on a

plasmid, a fused gene (apa-J) in which the protein A gene

(spa) from Staphylococcus aureus (12) is fused at its trans-lational initiation codon to the promoter and ribosome-binding sequences of a B. amyloliquefaciens a-amylase gene

(amyE[BamP]). Cultures of GX3362 accumulated protein Ain the growth medium at levels of up to approximately 1g/liter as determined by an enzyme-linked immunosorbentassay (12) based on the IgG-binding activity of the product.However, gel electrophoretic (Western blot) analysis re-

vealed that the full-length product predominated only atearly times and, at times of highest accumulation, themajority of IgG-binding activity resided in lower-molecular-weight forms generated by proteolysis (Fig. 3). This degra-dation was greatly reduced by the addition of the serineprotease inhibitor PMSF to the growth medium (3).We transformed both GX4931 and GX4937 with the same

apa-l-bearing plasmid (pGX2939) carried by GX3362. TheGX4931 derivative (designated GX4934) was found to accu-

mulate protein A at levels somewhat higher than GX3362did, up to >3 g/liter, but the product was more degraded thanthat accumulated by GX3362 (data not shown). This isconsistent with the relative serine protease levels of the hoststrains GX4931 and 1S53 (Table 2).The GX4937 derivative (designated GX4999), however,

proved superior to GX3362, providing a substantial improve-ment in the level of undegraded protein A accumulated forup to 24 h (Fig. 3). The bulk of IgG-binding activity in thesupernatant of GX4999 cultures remained in the form offull-length protein A for up to at least 24 h, although some

degradation was still observed.The protease-deficient strains described here represent

substantial improvements over previously available B. sub-tilis strains as hosts for the production of foreign proteins.Their reduced protease levels allowed the accumulation ofproduct with less degradation, whereas we observed no

disadvantages in growth properties or in the rate of synthesisand secretion of foreign proteins compared with the parentstrains. We believe these strains to be useful as startingpoints for the construction of the optimal B. subtilis host forthe production of foreign proteins.By the methods outlined here, an insertionally inactivated

and finally a deleted form of a cloned gene can be substitutedfor the wild-type allele without knowledge of or dependenceon any phenotype conferred by the desired mutation. Theselectable cat gene carried on the inactivating insert in apr

provides first a selection for replacement of the activechromosomal apr gene by the insertionally inactivated de-rivative and then a means by which to screen for itsreplacement by the deletion derivative. A novel feature ofthe method is the use of a vector which can first beestablished as an autonomous plasmid and then selected forchromosomal integration. This allows the operational sepa-ration of transformation and integration, which should facil-itate the selection of integrates obtained at low frequencywith short homology. These methods should prove useful as

well in the construction of the optimal host.

ACKNOWLEDGMENTS

We thank Charlie Saunders, Vasantha Nagarajan, Mark Guyer,and Ethel Jackson for suggestions and critical discussions; CathyRudolph for assistance in the cloning of apr[Bsu]; David Filpula andJames Nagle for sequencing the cloned gene; J. Hoch for the lambda

1 15 , Stds.

97K

68K

a43K

26K

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384 FAHNESTOCK AND FISHER

library; and J. Millet for the npr-deficient strain 512. We thankMartin Lindberg and his colleagues for the cloned spa gene.

The protein A work was supported in part by Pharmacia AB.

LITERATURE CITED1. Chang, S., and S. Cohen. 1979. High frequency transformation

of Bacillus sibtilis protoplasts by plasmid DNA. Mol. Gen.Genet. 168:111-115.

2. Dubnau, D. A. 1982. Genetic transformation in Bacillus subtiliis.p. 164. In D. A. Dubnau (ed.), The molecular biology of thebacilli, vol. 1. Bacillus siubtilis. Academic Press, Inc., NewYork.

3. Fahnestock, S. R., and K. E. Fisher. 1986. Expression of thestaphylococcal protein A gene in Bacillus subtilis by gene

fusions utilizing the promoter from a Bacillus ainvloliquefaciensQ-amylase gene. J. Bacteriol. 165:796-804.

4. Ferrari, E., D. J. Henner, and J. A. Hoch. 1981. Isolation ofBacillus sibtilis genes from a Charon 4A library. J. Bacteriol.146:430-432.

5. Gutterson, N. I., and D. E. Koshland. 1983. Replacement andamplification of bacterial genes with sequences altered in vivo.Proc. Natl. Acad. Sci. USA 80:4894-4898.

6. Haldenwang, W. G., C. D. B. Banner, J. F. Ollington, R. Losick,J. A. Hoch, M. B. O'Connor, and A. L. Sonenshein. 1980.Mapping a cloned gene under sporulation control by insertion ofa drug resistance marker into the Bacillus subHtilis chromosome.J. Bacteriol. 142:90-98.

7. Hawkes, R., E. Niday, and J. Gordon. 1982. A dot-immunobind-ing assay for monoclonal and other antibodies. Anal. Biochem.119:142-147.

8. Hofemeister, J., M. Israeli-Reches, and D. Dubnau. 1983. Inte-gration of plasmid pE194 at multiple sites on the Bacillus suibtilischromosome. Mol. Gen. Genet. 189:58-68.

9. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence

and functional map of pC194, a plasmid that specifies inducibleresistance to macrolide, lincosamide, and streptogramin type Bantibiotics. J. Bacteriol. 150:804-814.

10. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence

and functional map of pC194, a plasmid that specifies induciblechloramphenicol resistance. J. Bacteriol. 150:815-825.

11. Kawamura, F., and R. H. Doi. 1984. Construction of a Bacillus

subtilis double mutant deficient in extracellular alkaline andneutral proteases. J. Bacteriol. 160:442-444.

12. Lofdahl, S., B. Guss, M. Uhlen, L. Philipson, and M. Lindberg.1983. Gene for staphylococcal protein A. Proc. Nat]. Acad. Sci.USA 80:697-701.

13. Michel, J. F., and J. Millet. 1970. Physiological studies onearly-blocked sporulation mutants of Bacillus subtilis. J. Appl.Bacteriol. 33:220-227.

14. Roitsch, C. A., and J. H. Hageman. 1983. Bacillopeptidase F:two forms of a glycoprotein serine protease from Bacillussublilis 168. J. Bacteriol. 155:145-152.

15. Saunders, C. W., B. J. Schmidt, M. S. Mirot, L. D. Thompson,and M. S. Guyer. 1984. Use of chromosomal integration in theestablishment and expression of blaZ. a Staphylococcus aui-reuisf-lactamase gene, in Bacillus suibtilis. J. Bacteriol. 157:718-726.

16. Scherer, S., and R. W. Davis. 1979. Replacement of chromo-some segments with altered DNA sequences constructed invitro. Proc. Natl. Acad. Sci. USA 76:4951-4955.

17. Stahl, M. L., and E. Ferrari. 1984. Replacement of the Bacillussibtilis subtilisin structural gene with an in vitro-derived dele-tion mutation. J. Bacteriol. 158:411-418.

18. Uehara, H., K. Yamane, and B. Maruo. 1979. Thermosensitive,extracellular neutral proteases in Bacillus suibtilis: isolation,characterization, and genetics. J. Bacteriol. 139:583-590.

19. Uhlen, M., B. Guss, B. Nilsson, F. Gotz, and M. Lindberg. 1984.Expression of the gene encoding protein A in Staphylococcusaureis and coagulase-negative staphylococci. J. Bacteriol. 159:713-719.

20. Ulmanen, I., K. Lundstrom, P. Lehtovaara, M. Sarvas, M.Ruohonen, and I. Palva. 1985. Transcription and translation offoreign genes in Bacillus slbtilis by the aid of a secretion vector.J. Bacteriol. 162:176-182.

21. Vasantha, N., C. S. Rhodes, L. D. Thompson, C. D. B. Banner,and D. Filpula. 1984. Cloning of a serine protease gene fromBacillus amvlnoliquefaciens and its expression in Bacillus slbti-lis, p. 163. In A. T. Ganesan and J. A. Hoch (ed.), Genetics andbiotechnology of bacilli. Academic Press, Inc., New York.

22. Vasantha, N., L. D. Thompson, C. Rhodes, C. Banner, J. Nagle,and D. Filpula. 1984. Genes for alkaline protease and neutralprotease from Bacillus aInyloliquefaciens contain a large openreading frame between the regions coding for signal sequenceand mature protein. J. Bacteriol. 159:811-819.

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