JOURNAL OF BACTERIOLOGY, May 1983, p. 946-954 Vol. 154, No. 20021-9193/83/050946-09$02.00/0Copyright C 1983, American Society for Microbiology

Alkaline Phosphatase Secretion-Negative Mutant of Bacilluslicheniformis 749/C

R. KUMAR, A. GHOSH, AND B. K. GHOSH*

Department of Physiology and Biophysics, University ofMedicine and Dentistry ofNew Jersey, RutgersMedical School, Piscataway, New Jersey 08854

Received 21 October 1982/Accepted 10 February 1983

An alkaline phosphatase secretion-blocked mutant of Bacillus licheniformis749/C was isolated. This mutant had defects in the phoP and phoR regions of thechromosome. The selection procedure was based on the rationale that N-methyl-N'-nitro-N-nitrosoguanidine can induce mutations of closely linked multiplegenes. The malate gene and the phoP and phoR genes are located at the 260-minposition in the Bacillus subtilis chromosome; hence, the malate gene could beused as a marker for the mutation of the phoP and phoR regions of thechromosome. In a two-step selection procedure, strains defective in malateutilization were first selected with the cephalosporin C procedure. Second, thesemalate-defective strains were further screened in a dye medium to select strainswith defects in alkaline phosphatase secretion. One stable mutant (B. lichenifor-mis 749/C/NM105) had a total secretion block for alkaline phosphatase and hadthe following additional characteristics: (i) the amount of alkaline phosphatasesynthesized was comparable to that in the wild type; (ii) the alkaline phosphatasewas membrane bound; (iii) the mutant strain alkaline phosphatase, in contrast tothat of the wild type, could not be extracted with MgCl2, although the amounts ofprotein extracted from each strain were comparable; (iv) the sodium dodecylsulfate-polyacrylamide gel pattern of MgCl2-extracted proteins from the mutantstrain was different from that of the wild-type proteins; (v) the mutant, unlike thewild type, could not use malate as a sole source of carbon; and (vi) the outsidesurface of the wall of the mutant cells contained an additional electron-dense layerthat was not present on the wild-type cell wall surface.

Secretion is multistep cellular activity (2, 29,33). The secretory proteins are usually segregat-ed from the cytoplasmic proteins at a membranesite that is common for both biosynthesis andtransport across the membrane (27). It is nowbelieved that a unique structural feature of thenascent secretory protein molecule (e.g., the N-terminal hydrophobic amino acid signal se-quence) may be recognized by a specific mem-brane receptor (signal-specific membranereceptor [38-40]). This receptor-nascent secre-tory protein binding presumably initiates a mem-brane process, causing the movement of nascentsecretory protein molecules across the mem-brane permeability barrier. In procaryotic cells,the N-terminal signal sequence plays a crucialrole in the secretion process (5, 16, 31); howev-er, a variety of other factors are equally impor-tant (17).

It has been suggested that there are pluripo-tent secretion-specific regions in procaryotic cellmembranes that might have evolved, by a proc-ess of differentiation, into the specific secretoryorganelles (i.e., endoplasmic reticulum, Golgi

bodies, etc.) of eucaryotic cells (2). It is impliedin this concept that the plasma membrane ofprocaryotic cells may have secretion-specificregions containing components required for se-cretory activity (2, 29, 33). We showed earlierthat the secretory enzymes (i.e., penicillinaseand alkaline phosphatase) are distributed in afew discrete sites of the plasma membrane ofbacilli (10, 30). These specific membrane bindingsites could arise from membrane proteins, whichwe tentatively defined as receptors (30).

Preliminary results on the isolation of mem-brane receptors for the secreted proteins havebeen reported for animal cells (22, 24, 37). It isintriguing that the N-terminal signal sequencesof the nascent presecretory proteins are nothomologous (3). It is likely that the uniquestructure of a presecretory protein ensures itsbinding to the putative membrane receptor moi-ety, although the initial attachment to the mem-brane may be a nonspecific process dependentonly on the hydrophobic nature of the N-termi-nal signal sequence. The biosynthesis of a secre-tory protein and the putative receptor could be

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integrated if the genes for both of these proteinswere coordinately controlled. Thus, the putativereceptor gene and the alkaline phosphatasestructural or regulatory genes might be closelylinked.

Since 1965, several papers have been pub-lished on the genetic regulation of alkaline phos-phatase in Bacillus subtilis (23, 28). The chromo-somal map shows one structural gene (phoS)located in the 110-min position and the tworegulatory genes phoP and phoR located in the260-min position. However, the location andfunctions of these genes are not fully understood(20, 42). The malate utilization character is alsolocated in the 260-min position of the B. subtilischromosome (14). Interspecific variations ofthese landmark map positions are unlikely.Based on the observation that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces anumber of closely linked mutations within ashort segment of the Escherichia coli chrom-some, we developed a strategy to select PhoP orPhoR mutants (4, 13). Bacillus licheniformiscells defective in malate utilization were select-ed after MNNG mutagenesis. As these mutantswere likely to have a mutation within a shortsegment of the chromosome (i.e., at 260 min), asecond selection was made for defects in thePhoP or PhoR character. Thus, regulatory genemutants with altered alkaline phosphatase secre-tion were expected to be isolated.We report here one stable alkaline phospha-

tase secretion-blocked mutant, B. licheniformis749/C/NM105. This mutant and the wild-typestrain synthesized the same amount of the en-zyme; however, the rate of synthesis in themutant was faster than that in the wild-typecells. The enzymes of both strains were mem-brane bound, had the same molecular weight,and showed antigenic identity; but the mutantenzyme, unlike the wild type, could not beextracted with high concentrations of magne-sium salt.

This work was presented at the 82nd AnnualMeeting of the American Society for Microbiol-ogy, 7 to 12 March 1982, Atlanta, Ga.)

MATERIALS AND METHODSBacterial strains and media. B. licheniformis 749/C

was used in this study. The cells were grown in a low-Pi casitone (Difco Laboratories) and salt medium(CH/S medium) (15) and in a minimal medium contain-ing the following (in grams per liter): Tris, 12;K2HPO4, 0.87; amino acid mixture: cystine, asparticacid, asparagine, proline, and glutamine, 0.02 each;biotin, 0.001; cyanocobalamine, 0.001; glucose or ma-late, 2; and 10 ml of salt mixture [MgSO4 * 7H20, 1.2g; (NH4)2SO4, 20 g; Fe2SO4 * 7H20, 0.097 g; MnSO4,0.001 g in 100 ml of distilled water] per liter. The pHwas adjusted to 7.0. The Pi concentration was 5 mM.For alkaline phosphatase derepression, the P1 concen-

tration was lowered to less than 0.1 mM. Inocula wereprepared by growing a suspension of spores in saline ina high-phosphate medium (CH/S or minimal), andinocula grown for 18 h were used to study growth andenzyme synthesis in minimal or CH/S medium. Thecells were grown at 32°C with constant shaking.

Isolation of mutants. The bacterial colonies showingaltered characteristic for alkaline phosphatase werescreened in a low-Pi minimal medium containing aphosphorylated azo dye (5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine) at a concentration of 50 ,ug/ml(32); this dye was colorless and became blue afterdephosphorylation. The wild type and mutant strainswere maintained on two types of slants, sporulation (9)and low-phosphate minimal-glucose agars. The follow-ing screening protocol was used for the isolation ofmutants; (i) selection of malate-defective organisms;(ii) selection of organisms with altered alkaline phos-phatase characteristics from the malate-defective or-ganisms; and (iii) screening of the malate-defective andalkaline phosphatase-altered organisms for alkalinephosphatase synthesis and secretion in low-Pi liquidmedia. B. licheniformis 749/C cells were grown for 4 hin a low-Pi CH/S medium as described earlier (15).These cells were washed and treated with MNNG (1mg/ml) as described by Adelberg et al. (1). Thistreatment killed 99.95% of the cells; the survivorswere washed and suspended in a high-Pi minimalmedium containing malate as the sole carbon source.After 3 h of growth at 32°C, 200 ,ug of cephalosporin Cper ml was added, and the cells were grown for afurther 14 h. The cephalosporin C-selected cultureswere washed and enriched by 18 h of growth in a high-Pi minimal medium containing glucose. These cellswere diluted and plated on a low-Pi glucose-minimalmedium containing the azo dye. Discrete and distinctcolonies appeared on these plates after incubation at32°C for 96 h or longer. The colonies showed a range ofcolor variation (deep blue or white) and were frequent-ly encircled by blue halos of various size. Thesevariations of colony coloration could not be directlycorrelated with variations in alkaline phosphatase syn-thesis or secretion. The dye was used to guide ourselection of colonies with phenotypic variations. Or-ganisms from the selected colonies were maintainedon low-Pi glucose-minimal agar slants. Synthesis andsecretion were further examined in low-Pi CH/S-glu-cose liquid medium. Growth was assessed by turbidityas measured by a Klett-Summerson colorimeter with ano. 54 filter. Finally, the following species identifica-tion tests suggested by Gordon et al. (12) were per-formed: catalase, Voges-Proskauer, growth in anaero-bic agar, growth at 50°C, and growth in 7% sodiumchloride.

Extraction of alkaline phosphatase and SDS-PAGE.Extractions of the alkaline phosphatase by salt (MgCl2or NaCl), trypsin, and Triton X-100 from the mutantand wild-type cells were compared. The cells wereharvested when the cell-bound alkaline phosphatasecontent was highest, which was after 3 h for strain749/C/NM105 and 6 h for strain 749/C. The harvestedcells were washed in 0.01 M Tris-hydrochloride-sodi-um acetate (TNA buffer) (pH 7.4) (11). The washedcell residues were mixed with 1 M MgCl2 or NaCl inthe same buffer and incubated with constant shaking at37°C for 30 min. The treated cell suspensions werecentrifuged at 40,000 x g, and then the supernatants

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FIG. 1. Growth curve of B. licheniformis strains749/C (0) and 749/C/NM105 (U) in CH/S mediumsupplemented with salt mixture (see the text).

and residues were assayed for alkaline phosphataseand protein. A method described earlier (7) was fol-lowed for trypsin and Triton X-100 extractions. Toincrease the accessibility of trypsin to the membranesuface, we used protoplasts for this treatment insteadof whole cells.The salt-extracted material was dialyzed against

0.01 M TNA buffer (pH 7.4) containing 0.5 mM CoCl2in the cold (4°C) for 18 h, and the dialysate wasconcentrated with Aquacide II (Calbiochem). Theprotein and alkaline phosphatase contents of theseconcentrated materials were assayed (15, 21); in addi-tion, a sample was subjected to polyacrylamide gelelectrophoresis (PAGE) in a 10% sodium dodecylsulfate (SDS)-polyacrylamide slab gel (19). The pro-tein bands fixed in 15% trichloroacetic acid werestained with 0.1% methonolic Coomassie blue anddestained in a 5% methanol-10% acetic acid mixture.

Antigenicity. Membrane-bound enzyme was extract-ed and purified from washed membrane preparationsof B. licheniformis 749/C cells by the procedure usedfor alkaline phosphatase extraction and purificationfrom B. subtilis SB15 membrane (11). Antibody wasprepared against purified membrane-bound enzymefrom strain 749/C in rabbits (7, 11). The enzyme fromthe mutant was extracted with salt. Although only asmall amount of enzyme could be extracted, enoughcrude material was obtained from a large batch ofwashed cells for a preliminary study of antibody-enzyme interaction. There was no cross-reaction be-tween the B. licheniformis and B. subtilis alkalinephosphatase and anti-alkaline phosphatase antibody.

Electron microscopy. The wild-type and mutant cellsgrown for 3 h were fixed, dehydrated, embedded,sectioned, and stained for electron microscopic exami-nation (8). Ultrastructural cytochemistry of the alka-line phosphatase was performed by a method de-scribed previously (10). The thin sections wereexamined under a JEM IOOC (JEOL, Japan) electronmicroscope.

Construction of revertants. The method of Koyamaet al. (18) was followed to construct revertants of B.licheniformis 749/C/NM105. The inoculum of the mu-tant strain was grown in CH/S medium. After 18 h of

growth, the cells were washed in 0.155 M saline andinoculated into Pi-free CH/S medium to an opticaldensity of 25. After 3.5 h, the cells were again washedin 0.155 M saline and then treated with 1 mg ofMNNGper ml for 30 min at 37°C as described above. Thetreated cells were washed, diluted in 0.155 M saline,and plated on low-Pi glucose-minimal agar mediumcontaining dye. The colonies were selected on thebasis of color variation. Organisms from selectedcolonies were grown for 4 to 5 h in low-Pi CH/Smedium. Total and secreted alkaline phosphatase lev-els were determined as described above.

RESULTSGrowth and synthesis and secretion of alkaline

phosphatase. Growth of the mutant and wild-type strains was compared in CH/S medium(Fig. 1). The mutant grew much faster andreached the stationary phase much earlier; netgrowth was 40 to 45% higher than for the wildtype. The wild type exhibited poor growth,compared with the mutant, in minimal medium.In fact, growth of the mutant was twofold higherthan that of the wild type with glucose as thesole source of carbon (Fig. 2A); however, themutant did not shown any significant growthwhen malate was used as the sole carbon source(Fig. 2B).The data on the synthesis and secretion of

alkaline phosphatase for the mutant and wild

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FIG. 2. Growth curves of B. licheniformis strains749/C (0) and 749/C/NM105 in 0 in (A) glucose-minimal medium and (B) malate-minimal medium.

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FIG. 3. Synthesis (open symbols) and secretion(solid symbols) of alkaline phosphatase (APASE) bythe wild-type strain 749/C (0, *) and mutant strain749/C/NM105 (0, *).

type are shown in Fig. 3. The mutant and thewild type synthesized comparable amounts ofthe enzyme; however, the net amount of enzymeformed varied among different batches of medi-um. The enzyme yield was highly sensitive tominor variations of the Pi concentration in CH/Smedium. The striking difference between thesetwo strains was in secretion; the wild-type cellssecreted about 59% of the synthesized enzyme,but the amount secreted by the mutant cells wasalmost undetectable. Furthermore, the charac-teristics of alkaline phosphatase synthesis in themutant were significantly different. (i) Synthe-sis, once started, continued at a very rapid rate,reaching a peak value within 40 to 60 min (Fig.3). In contrast, in the wild-type culture thisrising phase of synthesis continued for severalhours (Fig. 3). (ii) After reaching the peak value,the enzyme in the mutant was degraded at a veryrapid rate, and nearly 50% of the total enzymeactivity was lost within 40 to 60 min (Fig. 3).By comparing growth (Fig. 1) and alkaline

phosphatase synthesis, it was evident that dur-ing this period of rapid alkaline phosphatasedegradation the mutant culture maintained thelogarithmic phase of growth. Such a rapid de-cline of enzyme activity during the logarithmicphase suggests that a specific control mecha-nism, rather than any autolytic process, wasrelated to this rapid degradation. In the wild-type cells, however, the enzyme activity de-clined slowly during the stationary phase ofgrowth (Fig. 3).As the selection procedure was elaborate, it

was important to examine whether the mutantretained the characteristics of typical B. licheni-formis cells. The key tests suggested by Gordon

et al. (12), described above, were all positive,confirming the authenticity of this Bacillus spe-cies.

Subcellular location of alkaline phosphatase. Instrain 749/C cells, 75 to 80% of the alkalinephosphatase was bound to the plasma mem-brane and cell wall (A. Ghosh, M. Smolensky,S. Vallespire, and B. K. Ghosh, Abstr. Annu.Meet. Am. Soc. Microbiol. 1980, J19, p. 83).The particle-bound nature of the enzyme wasunchanged in the mutant strain. The log-phasecells of the mutant were treated with lysozymein an osmotically unsupported medium to effectcomplete lysis of the cells. This lysate wascentrifuged in the cold (4°C) at 40,000 x g, andenzyme activity was assayed in the cytosol andparticle fractions. The results showed that about90% of the alkaline phosphatase was particlebound.The alkaline phosphatase of bacilli is salt

extractable, especially by salts of magnesium(11, 35). To gain insight into the possibility ofchanges in the nature of the binding of alkalinephosphatase to the membrane, we examined theextraction of the enzyme by different reagents.The data (Table 1) showed a clear and notablecontrast between the two strains. Magnesiumand sodium salts extracted 86 and 91%, respec-tively, of the alkaline phosphatase from the wildtype, but only 3 and 5%, respectively, from themutant. It was evident from the data (Table 2)that considerably more cell-bound proteinscould be extracted by magnesium or sodiumsalts from the mutant than from the wild type.The specific absence of alkaline phosphatase inextracts from the mutant was evident from com-paring the alkaline phosphatase-specific activi-ties of the magnesium and sodium salt extracts,which were 15- and 19-fold higher, respectively,for the wild type than for the mutant. Theamount extracted by Triton X-100 from the wildtype was three times higher than that from the

TABLE 1. Extraction of alkaline phosphatase byMgCl2, NaCl, trypsin, and Triton X-100

Alkaline phosphatase extracted (% oftotal enzyme content)' with:

Strain TritonMgCl NaCl Trypsin X-100(1 M) (1 M) (1%) (1%)

B. licheniformis 86 91 5 45749/C

B. licheniformis 3 5 1 14749/C/NM105a Note the remarkable differences in the salt extract-

ability of alkaline phosphatase. The data on enzymeactivity were consistently within the same range whenthe experiments were done under comparable condi-tions.

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TABLE 2. Protein extracted from whole cultures byMgCJ2, NaCl, trypsin, and Triton X-100

Protein extracted (% of total cellprotein)a with:

Strain TritonMgCl2 NaCl Trypsin TX100(1 M) (1 M) (1%) (1%)

B. licheniformis 1 1 7 13749/C

B. licheniformis 2 5 6 19749/C/NM105a Note that slightly more protein could be extracted

by the salts from the mutant (strain 749/C/NM105)than from the wild-type strain.

mutant cells. It should be noted, however, thatthe Triton X-100-extracted enzyme in these ex-periments included some membrane-bound par-ticulate enzyme. Centrifugation at 40,000 x gfailed to precipitate enzyme bound to smallmembrane particles present in the Triton X-100extract. However, prolonged centrifugation at100,000 x g showed that Triton X-100-solubi-lized enzyme, freed of particle-bound enzyme,represented about 20% of the total cell-boundenzyme. A detailed study on the comparativeextractability and properties of purified enzymesfrom the mutant and wild-type cells is in pro-gress. The data presented in this paper make itclear that the salt extractability of the alkalinephosphatase from the mutant is notably differentfrom that of the wild type. There was also anappreciable difference in the Triton X-100 ex-tractability. In both strains the intact protoplast-bound alkaline phosphatase was not availablefor trypsin release. A detailed study of theextraction of enzyme from this mutant strain bya variety of detergents is in progress.SDS-PAGE. The protein profile of the MgCl2-

extracted material was examined by SDS-PAGEon slab gels (Fig. 4). The protein band corre-sponding to pure membrane-bound alkalinephosphatase was absent from the extract of themutant but present in the wild-type cell extract.In addition to this major difference, the gelprofiles of both strains differed significantly;most conspicuous were bands A and C, whichwere only seen in the mutant extract. However,detailed analysis of the gel profile is beyond thescope of this paper. The most important pointwas that MgCl2 failed to extract alkaline phos-phatase from the mutant strain cell membrane.

Antigenicity. Detailed comparison of the enzy-matic properties is beyond the scope of thispaper; however, we have presented data on theaction of strain 749/C anti-alkaline phosphataseantibody on the mutant strain alkaline phospha-tase. The curves for the enzyme-antibody pre-cipitate (Fig. 5B) and for its residual catalytic

activity (Fig. 5A) were compared. The crudeenzyme preparations from both strains wereprecipitated with strain 749/C anti-alkaline phos-phatase (membrane bound) antibody. The resid-ual catalytic activity of the enzyme from bothstrains was stimulated. There were quantitativedifferences in the amount of the precipitatesformed and the residual activity stimulation. Itwas clear that the mutant and wild-type alkalinephosphatases were antigenically similar; a de-tailed study of the subtle quantitative differencesis in progress.

Ultrastructure. It was evident (Fig. 6A) thatstrain 749/C/NM105 produced endospores char-acteristic of bacilli. This is important morpho-logical information, supplementary to the spe-cies-identifying tests, that the mutant retainedthe properties of a typical B. licheniformis orga-nism. Both log-phase and stationary-phase cellshad a thin electron-dense layer on the outsidesurface of the cell wall (Fig. 6B). This layer wasnot detected on the wild-type cell wall surface(Fig. 6C).

Preliminary results from our study on theultrastructural cytochemistry of alkaline phos-phatase showed that the enzyme is bound to theinside surface of the plasma membrane (6D).

1 2 3

-A-

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FIG. 4. SDS-PAGE of the proteins extracted with1 M MgCl2 from the wild-type and mutant strains ofB.licheniformis. Lanes: 1, 50 jig of protein from themutant strain extract; 2, 50 jig of protein from thewild-type extract; 3, 10 p.g of purified alkaline phos-phatase protein from the wild-type. The band corre-sponding to the pure alkaline phosphatase (band B) ispresent in the wild-type extract but absent from themutant extract. In addition, the band patterns of otherproteins from the mutant and wild-type extracts dif-fered; bands A and C were seen only in the mutantextract.

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FIG. 5. (A) Residual catalytic activity of the wild-type (0) and mutant (A) strain alkaline phosphatasesafter interaction with the antibody prepared againstthe wild-type purified alkaline phosphatase. (B) Anti-gen (mutant or wild-type alkaline phosphatase)-anti-body (wild-type anti-alkaline phosphatase) precipita-tion curve.

Revertants. The alkaline phosphatase synthe-sis and secretion characteristics of the revertantstrains are shown in Table 3. The results showedthat strain 749/C/NM105 was totally blocked. Incontrast, 67% of the revertant strains secretedsignificant amounts of alkaline phosphatase. Infact, many of these strains secreted as much or

slightly more alkaline phosphatase as the wild-type strain did. This result is generally consis-tent with the information presented by Koyamaet al. (18) and Friedman and Huberman (6) thatMNNG may induce reversion by enhancing a

DNA repair phenomenon.

DISCUSSION

The basic assumption that led to this studywas that the movement of a secretory protein(alkaline phosphatase in this case) through thepermeability barrier of a membrane requires a

receptor function. The results of the studyshowed that MNNG mutation produced a secre-

tion-blocked mutant (B. licheniformis

749/C/NM105) with altered membrane-alkalinephosphatase association.There are three genes for alkaline phospha-

tase: phoS, phoP, and phoR (14). The phoS geneis located in the 110-min position of the B.subtilis chromosome. This gene regulates theconstitutive character of the enzyme (28). ThephoR and phoP genes are located in the 260-minposition of the B. subtilis chromosome. phoR isnot linked to phoP, and its activity regulates therate of alkalline phosphatase production. Anymutation in this gene causes a slower or fasterrate of enzyme synthesis (23). The phoP muta-tion, on the other hand, produces strains withlowered synthesis rates (23); in addition, thestructure of the enzyme molecule appears to beregulated by this gene (41).

B. licheniformis 749/C/NM105 synthesized al-most the same amount of enzyme as the wildtype did. The enzyme had the same molecularweight, and it was antigenically similar to thewild-type enzyme. Hence, based on these phys-iological properties, phoP mutation could beruled out. Enzyme synthesis in the mutant strainwas not constitutive; therefore, the possibility ofphoS mutation could be eliminated. The synthe-sis of the enzyme in the mutant, compared withthat in the wild type, however, started earlierduring growth, and the rate of synthesis wasmuch faster than the rate in the wild type.Furthermore, the amount of enzyme synthe-sized could not be retained at the peak level inthe mutant cells. In spite of rapid cell growth,

TABLE 3. Alkaline phosphatase synthesis andsecretion by revertant strains of B. licheniformis

749/C/NM105Amt of enzyme (U/mg % of

B. licheniformis [dry wt] of cells): enzymestrain

Synthesized Secreted secreted

749/C 409 197 48749/C/NM105 430 5 1749/C/NM105/R-29 413 260 62749/C/NM105/R-18 395 217 54749/C/NM105/R-33 382 195 51749/C/NM105/R-11 293 130 44749/C/NM105/R-37 200 86 43749/C/NM105/R-19 442 178 40749/C/NM105/R-28 55 22 40749/C/NM105/R-22 472 150 31749/C/NM105/R-1 245 65 26749/C/NM105/R-8 108 25 23749/C/NM105/R-16 270 57 21749/C/NM105/R-23 567 89 15749/C/NM105/R-10 172 21 12749/C/NM105/R-4 206 21 10749/C/NM105/R-38 281 27 9749/C/NM105/R-6 131 12 9749/C/NM105/R-13 337 8 2749/C/NM105/R-3 421 7 1

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C D

FIG. 6. Thin-section electron micrographs of mutant and wild-type cells. (A) Cross-section of a sporulatingcell from the mutant strain shows a typical endospore. (B) Mutant strain cell. (C) Wild-type strain cell. (D)Mutant strain cell. Note that the cytochemical reaction product is bound to a few discrete locations on the insidesurface of the plasma membrane (arrows). CW, Cell wall; OL, outer layer of cell wall (absent from wild-typestrain; cf. panel C; Me, mesosome; FP, forespore. Bars, 0.21-m.

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the enzyme content fell to 50% of the peak valuewithin a very short time. These physiologicalproperties suggest that strain 749/C/NM105 hadundergone a phoR gene mutation.The mutant strain showed three additional

changes: (i) alkaline phosphatase secretion wastotally blocked; (ii) the enzyme could not beextracted from the cell membrane with magne-sium or sodium salt; and (iii) the SDS-PAGE gelprofile of the magnesium salt extract of themembrane was different from that of the wild-type cell extract. It could be suggested fromthese data that the activity of the phoR generegion regulated the rate of alkaline phosphatasesecretion in addition to its rate of synthesis. Thiscontention is confirmed by the construction ofrevertant strains of the mutant with MNNG. Thesecretion block in strain 749/CINM105 was re-moved in 67% of these revertant strains. It hasbeen suggested that with some methylating mu-tagens, postreplication repair can cope with thelesions responsible for the mutation (6). Thus,the return of the wild-type secretion characteris-tics to the secretion-blocked mutant afterMNNG-induced reversion was likely a result ofthe lesion and repair of a specific region of thechromosome regulating alkaline phosphatase se-cretion.Magnesium salt extractability suggested that

the wild-type alkaline phosphatase was an ex-trinsic (peripheral) membrane protein that mightbind to a secretion-specific receptor. The latter,having affinity for the membrane, might haveformed a complex with an intrinsic membraneprotein(s) embedded through hydrophobic inter-action with the membrane interior (34, 36).Thus, a change in the structure of the putativereceptor protein brought about by mutationmight have caused an alteration in the bindingcharacteristic such that the alkalline phospha-tase formed an unusually tenacious complexwith the membrane and was resistant to saltextraction. Significant differences in the SDS-PAGE gel profiles of the mutant and wild-typemembrane extracts suggested a possible alter-ation in the protein profile after mutation. Pre-liminary evidence for the presence of salt-ex-tractable signal recognition protein in the animalsecretory cell membrane has been presented(40). It was not possible to infer from our dataany interrelationship between the altered alka-line phosphatase binding to the membrane andthe secretion block in this mutant. Such a rela-tionship, however, is likely, and study is pro-gressing in this direction. In fact, transformationexperiments are in progress, aimed at construct-ing transformants from B. licheniformis 749/Calkaline phosphatase-negative mutants with B.licheniformis 749/C DNA to produce alkalinephosphatase secretion-positive cells. Further-

more, a similar mutation technique was used tomutagenize B. subtilis SB15 cells that werephenotypically secretion negative. A large num-ber of these mutants secreted substantialamounts of alkaline phosphatase (R. Kumar, A.Ghosh, and B. K. Ghosh, Abstr. Annu. Meet.Am. Soc. Microbiol. 1983, H105, p. 123).

Finally, the results of our study are in generalagreement with the recently published data ofOliver and Beckwith (25, 26). They demonstrat-ed in E. coli cells a new gene (secA) product thatappears to be a 92,000-dalton protein. This prod-uct is likely to be involved in cell envelopeprotein secretion. It may be speculated that suchgene products are secretion receptors regulatingthe movement of protein molecules through themembrane.

ACKNOWLEDGMENTS

This work was supported by grant PCM8110613 from theNational Science Foundation.We thank J. 0. Lampen, Jan Tkacz, S. Palchaudhuri, and

Marian Glenn for helpful discussions. We appreciate GloriaTrechock's assistance in preparing the manuscript.

LITERATURE CITED

1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965.Optimal conditions for mutagenesis by N-methyl-N'-ni-tro-N-nitrosoguanidine in Escherichia coli K12. Biochem.Biophys. Res. Commun. 18:788-795.

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