JOURNAL OF BACTERIOLOGY, JUlY 1975, p. 354-365 Copyright @ 1975 American Society for Microbiology
Properties of Bacillus
Vol. 123, No. 1 Printed in U.S.A.
cereus
Spore Coat Mutants
A. I. ARONSON* AND P. C. FITZ-JAMES Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,* and Department of Microbiology, University of Western Ontario Medical School, London, Ontario, Canada Received for publication 17 April 1975
Two classes of spore mutants have been selected in Bacillus cereus T, those producing lysozyme-sensitive spores, and those producing spores dependent upon lysozyme for germination. One mutant from each class was studied in detail and found to have defective packing of the spore coat layers. The major spore coat polypeptide appeared to be altered on the basis of gel electrophoretic profiles and/or peptide maps of half-cystine-containing peptides. The spores of the mutants of both classes were sensitive to lysozyme and failed to respond to the germinants L-alanine plus adenosine. The spores were also more sensitive to octanol than the parental strain, but contained the same amount of dipicolinic acid and were equally heat resistant. The reversion frequencies in both cases were consistent with an initial point mutation, suggesting that an alteration in the major coat polypeptide accounted for the phenotypic properties studied.
While the morphology of the bacterial spore coat layers varies considerably among species, the structural pattern in most cases may be resolved at least into inner (or under) and outer coats (2, 12, 14-16, 24, 30). Within each category there may be further morphological subdivisions, especially for the outer coat layers as discussed in this paper. These coats are comprised largely, if not exclusively, of protein (6, 21) with conditions for solubilization correlated with the disappearance of particular structural layers (6). Surprisingly, there is very little heterogeneity in the number of polypeptide species present in total coat extracts of Bacillus cereus spores. In fact, there is likely to be only one major species of coat polypeptide present in all of the coat layers despite the varying solubility properties (4, 6). This protein is "keratinlike" in that it is relatively rich in half-cystine residues (2, 29), has a low-angle X:ray diffraction pattern reminiscent of keratins (19), and is generally resistant to proteolytic enzymes with the notable exception of a keratinase isolated from Streptomyces fradiae (22, 23). Spore coats may be almost totally solubilized by treatment of intact spores with a mixture of dithioerythritol and sodium dodecyl sulfate (SDS) at alkaline pH (3, 6). As judged by the appearance of thin sections and freeze-etched preparations of treated spores, this treatment results in the removal of virtually all of the spore coat layers (3). The treated spores are still viable, heat resistant, and contain all their dipicolinic acid. They are very sensitive to
lysozyme (3), however, and respond relatively slowly to the germinants L-alanine plus adenosine after heat activation (unpublished observations). The sensitivity of treated spores to lysozyme may be exploited for isolating spore protoplasts (10) and for selecting mutants altered in coat appearance (4). In addition, a requirement for lysozyme to germinate mutant spores of Clostridium perfringens (8) was correlated with the altered appearance of the spore coat layers (9). We have exploited lysozyme sensitivity and a requirement for lysozyme to germinate to enrich for spores with altered coat structure. The primary alteration in many of these mutants is in the major coat polypeptide. The mutant spores differ from the wild type in their sensitivity to solvents and response to germinants, whereas heat resistance and dipicolinic acid content are not changed. MATERIALS AND METHODS Bacillus cereus strain T was grown in G tris(hydroxymethyl)aminomethane (Tris) or a synthetic medium, as previously described (1). Growth was followed in a Coleman 8 colorimeter employing a 650-nm filter. For labeling with 35SO., the G Tris medium was modified to reduce the sulfate content. Cations were present as the chloride salts (including ammonium ion as NH4Cl), and the yeast extract concentration was reduced to 0.05% (wt/vol). For mutagenesis, spores were treated with either nitrosoguanidine (7) or ethyl methane sulfate (17). After treatment, the spores were centrifuged, washed
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VOL. 123, 1975
three times with sterile medium, suspended in three times the original volume of medium, and incubated at 30 C for 36 to 48 h to permit growth and sporulation. For lysozyme-sensitive mutants, cells and spores were harvested by centrifugation as above, washed twice with 10 ml each of sterile 0.03 M Tris-hydrochloride (pH 7.8), and finally suspended in 0.5 ml of 0.03 M Tris-hydrochloride (pH 7.8) plus 50 ,g of lysozyme and incubated at 30 C for 90 min. After centrifugation, the pellet was suspended in 20% meglumine and sodium diatrizoate (Renografin) in 0.03 M Tris-hydrochloride (pH 7.8), and incubated at 0 C for 30 min. The suspension was layered over a linear 30 to 65% Renografin gradient in an SW25 tube, and centrifuged at 20,000 rpm for 40 min in an SW25 rotor (Spinco model L). There were three prevalent bands, one at approximately the density of intact spores at a density of 1.359 g/cm2 (3), one near the top consisting largely of cells, and a barely detectable band in between. This latter band was removed with a syringe and the spores were diluted into at least 10 volumes of G Tris. These cultures were incubated at 30 C for 48 h to permit germination, growth, and sporulation. The spores were harvested, washed, and treated with lysozyme as above. After centrifugation through Renografin, the intermediate band was diluted and plated on G Tris agar. After 36 h at 30 C, colonies were picked at random and suspended in 0.1 ml of 0.03 M Tris-hydrochloride (pH 7.6) plus 5 Mg of lysozyme. After incubation at 38 C for 2 to 3 h, each tube was examined for the presence of phase-dark spores. Any potential mutants were again streaked on G Tris and well-isolated colonies screened for lysozyme sensitivity of the spores. After two rounds of growth, lysozyme treatment, and separation in Renografin gradients, 10 to 80% of the colonies contained lysozyme-sensitive spores. To select for mutants dependent upon lysozyme for germination, spores produced after mutagen treatment and subsequent growth in G Tris were washed twice with 10-ml portions of 0.05 M sodium phosphate, pH 7.8. Spores (about 5 x 109) were suspended in 5 ml of phosphate and heat activated by incubating at 65 C for 30 min. L-Alanine was then added to 0.005 M and adenosine to 0.001 M and the suspension incubated at 30 C. After 40 min, the spores were pelleted, suspended in 20% Renografin, and fractionated on Renografin gradients as previously described. The density region corresponding to dormant spores was removed with a syringe and inoculated into 50 volumes of G Tris plus 5 Mg of lysozyme per ml to aid germination of presumptive mutants. After incubation at 30 C for 48 h, the germination and gradient separation procedures were repeated. The density region which should contain dormant spores was plated on G Tris agar containing 5 gg of lysozyme per ml. Colonies were replicated onto G Tris and those which grew slowly (presumably due to show germination or loss of viability of the sensitive spores on plates containing lysozyme) were selected from the original plates. About 10% of these were mutants dependent upon lysozyme for rapid germination. To measure reversion frequencies, a representative
SPORE COAT MUTANTS OF B. CEREUS
355
of each mutant class was selected for resistance to cycloserine by plating 10' spores on G Tris agar containing 300 Mg of D-cycloserine per ml. These resistant mutants were not altered in the lysozyme sensitivity of the spores nor on the dependence for lysozyme to germinate. The antibiotic resistance marker provided a means for eliminating any wildtype contaminants in the revertant studies. About 1010 spores of each mutant were treated with 50 Mg of lysozyme per ml in 5 ml of 0.03 M Tris-hydrochloride (pH 7.8) at 28 C for 2 h. The spores were then centrifuged through a Renografin gradient (SW25 rotor) as described above, and the density region corresponding to that containing intact spores was removed with a syringe, plated on G Tris containing 300Mg of cycloserine per ml, and incubated at 30 C for 78 h. To ensure that all sporulating colonies were revertants, 50 colonies were picked at random into 0.1 ml of 0.03 M Tris-hydrochloride (pH 7.8) plus 10 Ag of lysozyme. The frequency of lysozyme-resistant revertants could then be approximated. Isolation and characterization of spore coat proteins. Radioactive amino acids or 3"SO, was added to 10 ml of sporulating cultures at t,. 'H- and '4C-labeled reconstituted protein hydrolysate was added to G Tris cultures at 0.5 ,Ci/ml, respectively; 3"SO4 was added to cultures in low sulfate G Tris (supplemented with 3 Ag of methionine per ml at t,) to 200 MCi/ml. All cultures were incubated until free spores were present (36 to 40 h), and the spores were harvested, washed, and extracted by incubation in 0.01 M NaHCO8-0.05 M dithioerythritol with or without 0.5% SDS, pH 9.5 to 9.8, at 27 C for 3 h (6). For gel electrophoresis, 3H- and "4C-labeled spores were mixed so that the ratio of 3H/14C was 2 or 3 to 1 before extraction with 0.05 M dithioerythritol, pH 9.5, + 0.5% SDS at 28 C for 3 h. After rmoval of the extracted spores by centrifugation at 12,000 rpm for 15 min in a Sorvall RCIIB, a crystal of sucrose was dissolved in 20 Ml of the supernatant (5 to 20 Mg of protein). After addition of bromophenol blue, the total sample was placed on the top of 5 or 7% acrylamide gels (6 to 7 cm) and subjected to electrophoresis at 3 mA/tube for 2 to 3 h (until the dye marker had moved at least two-thirds of the way down the tube) in Tris (3 g/liter)-glycine (14.4 g/liter)-mercaptoethanol (0.04%), pH 9.0 (+0.1% SDS). The gel was removed from the tube, immediately frozen, divided into 1-mm slices with a Yeda macrotome, and processed for counting in Nuclear-Chicago solubilizer as previously described (6). For peptide analyses, the crude spore extract was either precipitated directly with a final concentration of 15% (wt/vol) trichloroacetic acid or purified before acid precipitation. For purification, the SDS in the crude coat extract was precipitated by addition of 0.05 ml of saturated KCl per ml of extract, and solid ammonium sulfate was added to the supernatant at fraction to 25% saturation (0 C). After incubation for 30 to 40 min at 0 C, the precipitate was collected by centrifugation at 12,000 rpm for 15 min. The precipitate was dissolved in 0.01 M NaHCO0-1% mercaptoethanol-0.4% SDS (pH 9.0), and passed over a Bio-Gel
356
ARONSON AND FITZ-JAMES
P-10 column (0.9 by 20 cm for up to 5 mg of protein) equilibrated with the same buffer. Radioactivity in the void volume (10 to 20% of the input) was discarded (impurities plus aggregated coat protein), and the tubes covering a major peak of radioactivity (80 to 90% of input) eluting at elution/void volume ratio of 2.1 were pooled and precipitated with trichloroacetic acid as above. Similar peptide maps were obtained with crude coat preparations, those purified on BioGel P-10 columns, or those purified on O-(triethylaminoethyl)-cellulose columns (5). After centrifugation at 10,000 rpm for 10 min in a Sorvall RCUB, the trichloroacetic acid precipitates were washed twice with 10-ml portions of 12% trichloroacetic acid and then ether. The pellets were air dried, dissolved in 0.2 to 0.5 ml of performic acid (20), and incubated at 0 C for 4 to 6 h. The solutions were diluted with water, dried in vacuo, and washed twice with distilled water. The pellets were dissolved in 0.2 to 0.4 ml of 0.01 M NaHCO, and adjusted to pH 9.0 A purified keratinase preparation (22, 23) was added to 5% of the protein concentration and the tubes incubated at 37 C. After 5 h, the pH was readjusted to 9.0 and an identical amount of keratinase was added. After incubation for a total of 12 h, the contents of the tubes were dried in vacuo. Peptides were separated first by electrophoresis at pH 1.9 (25 ml of 90% formic acid plus 87 ml of glacial acetic acid per liter) on a Whatmann 3MM at 35 V/cm for 110 min. The strips were air dried and a 1 by 9 inch segment sewn onto a 8 by 9 inch piece of Whatman 3MM for ascending chromatography in isobutanol-pyridine-water 87.5:87.5:75) for 12 to 16 h. Autoradiograms of the peptides were developed by exposure to Kodak RP54 medical X-ray film for varying periods. Peptides were also developed by ninhydrin (0.4% wt/vol in acetone) and spots cut out and plated into scintillation vials for counting in 10 ml of Omnifluor. Electron microscopy. Samples for freeze etching were frozen in Freon 22 onto scored copper disks and stored in liquid nitrogen until their transfer to the precooled (-150 C) stage of the Balzers apparatus. After cleavage, the surfaces were etched for 90 s at -100C, shadowed with platinum-carbon and then stabilized with more carbon. Replica pieces were cleaned by sequential floatation on concentrated H,SO,, 6% (wt/vol) sodium hypochlorite (Javex) and distilled water before being picked up on a copper grid for microscopy in a Philips 200. Thin section electron microscopy was conducted by standard methods already described (10). Spore coats were prepared as previously described (2).
J. BACTERIOL.
Analysis of spore properties. Heat resistance was based on the percentage of survival after heating at 80 C for 20 min. Dipicolinic acid was determined colorimetrically (18). For measurements of lysozyme sensitivity, washed spores were suspended in 0.03 M Tris-hydrochloride (pH 7.8) and 10 to 100 gg of lysozyme per ml added. The tubes were incubated 27 C and the change in absorbancy at 650 nm was followed in a Zeiss spectrophotometer. Germination rates were measured by washing and suspending spores in 0.05 M sodium phosphate or 0.03 M Tris-hydrochloride, pH 8.0. After heat activation at 65 C for 30 min (or 70 C for 20 min). L-alanine and adenosine were added to 0.01 M and 0.001 M final concentration, respectively. The change in absorbancy at 650 nm was followed at 27 C. Chemicals and isotopes. 31SO4 (carrier free) was purchased from Amersham-Searle; 3H-labeled reconstituted protein hydrolysate, algal profile, and 14C' labeled reconstituted protein hydrolysate were purchased from Schwarz/Mann. Omnifluor was from New England Nuclear Corp., SDS from British Drug House, Ltd., and dithioerythritol from Pierce Chemical Co. Twice recrystallized lysozyme (EC 3.2.1.17) was purchashed from Worthington Biochemicals Corp; Renografin as a 76% sterile solution was purchased from E. R. Squibb and Sons, Inc; D-cycloserine was from Sigma Chemical Co. A keratinase produced by Streptomyces fradiae (22, 23) was generously provided by W. Nickerson.
RESULTS Since there is no evidence for extensive differences among the mutants isolated by each procedure, only one from each class designated JOLD and 13LS will be described. In fact, there is overlap in the properties of mutants since all those selected for lysozyme-dependent germination are lysozyme sensitive, and many of those selected on the basis of lysozyme sensitivity are dependent upon lysozyme for germination. Morphology. Freeze-etch preparations of untreated spores of B. cereus exhibited an outer surface spore coat almost completely covered with the cross-patch array of protein fibers (Fig. 1). This coat-exosporium cleavage plane was encountered in a good proportion of spores. In a few small areas, the underlying pitted layer could be seen. In the lysozyme-dependent germination mutant, 1OLD, two defects appeared in
FIG. 1. A Freeze-etch preparation of a B. cereus spore. The knife has sheared off the exosporium (Ex), revealing the underlying outer cross-patch (CP) layer of the spore coat. In a few areas, the underlying pitted-coat layer (P) may be seen. Bar represents 0.1 gm in all electron micrographs. FIG. 2. Spore of the lysozyme-dependent mutant JOLD. The exosporium has been cleaved, revealing the ragged application of the spore coat (Ct) to the cortex (Cx). In some regions the coat appears thickened and folded under (see also Fig. 7) and generally the cross-patch (CP) layer is greatly reduced. FIG. 3. Higher magnification of mutant JOLD showing the incomplete cortical (Cx) coverage by the spore coat and the exposed pitted layer (P). The rods of protein in the remaining patches of cross-patch (CP) layer appear longer than those of the wild-type spores.
Ift~~~~~~~~~~~~~~t
357
358
ARONSON AND FITZ-JAMES
the spore coats. Both could be detected in thin sectioning and in freeze-cleave-etch preparations. In the latter, the defects were perhaps more striking; the spore coat presented a ragged and incomplete covering of the lysozyme-sensitive cortex (Fig. 2) and the coat itself, normally well endowed with the cross-patch layer (Fig. 1), showed a variable deficiency. This latter defect mimics the appearance of the coat of spores in which the uptake of cysteine has been inhibited by 10 mM sulfite (2). Like the sulfitetreated spores, those strands of cross-patch material which have been deposited are longer than the control and reveal extensive regions of the underlying pitted layer (Fig. 2 and 3). The pitted layer of mutant JOLD and control show similar dimensions: 6 to 7 nm of the hexogonally packed pits or holes, 8.9 to 9 nm apart in rows spaced 7.5 to 8 nm (Fig. 3). In some sections, the coat deficiency appeared more marked and large segments of cortex were uncovered (Fig. 8). Regardless of the amount of coat, however, a slackness usually resulted in a space between undercoat and cortex. This slackness of the coat may account for the apparent folding of the coat layers seen in sections (Fig. 7) and for the somewhat rolled edge seen in both sections (Fig. 8) and freeze etchings (Fig. 2 and 3). In wild-type spores, the coat is closely applied to the cortex and the undercoat is usually poorly stained (2). The undercoat is more readily seen in sections of free-spore coats (Fig. 4). The double-dense line, the backbone of the spore coat, has an overall thickness of 6 to 6.5 nm and corresponds to the pitted layer of freeze-etch preparations. In the sections of mutant 10LD, this layer is only partly covered by the outer-coat (cross-patch layer, Fig. 5 and 6), confirming the freeze-etch observations. The thickness of the normal spore coat profiles where covered with the cross-patch varies from 9 to 14 nm depending on the amount of cross-patch material. The addition of the less dense undercoat brings the overall spore coat thickness to 25 to 35 nm (Fig. 4). Reduced amounts of undercoat and the absence or thinning of the CP layer can reduce the overall mutant coat thickness to 16 to 18 nm (Fig. 6). Lysozyme-sensitive mutant 13LS had a simi-
J. BACTERIOL.
lar overall defect of the coat coverage (Fig. 9 and 10) and hence coat slackness as did the tOLD mutant. Where coat was deposited, all layers were present, the overall coat being some 35 nm thick (Fig. 10 and 11). In no cleavages was any extensive exposure of the pitted layer encountered; the cross-patch coverage was usually adequate if not heavier than normal. In both mutants exosporium was always complete. Occasionally (less than 10%) there were spores totally lacking coat material, but when these were purified on Renografin (higher density than intact spores) and cultured in G Tris, a mixed population of spores was produced, i.e., they do not breed true. Apparently, an incomplete outer coat results in total coat loss in some cases. Biochemical properties of mutants. Spore coat protein was extracted from a mixture of wild-type and lysozyme-sensitive spores labeled with either 14C or 3H-labeled reconstituted protein hydrolysate as described above. The gel electrophoretic profiles of the radioactive proteins from the wild type and mutant 13Ls are shown in Fig. 12. Because of the restricted conditions for solubility and ease of aggregation of the coat protein, it was necessary to use a high pH system and run fresh extracts. Under these conditions, one principle peak is found in each preparation (6) with a slight difference in mobility. Storage, fractionation, or dialysis of the extracts results in aggregation with considerable smearing of radioactivity across the gel. Reversal of the radioactive labels or use of 5% gels results in essentially the same profile. Coat extracts of mutant JOLD had the same mobility as the wild type at pH 9.0. If SDS is added (or the spores are extracted directly in the presence of SDS), then a peak with the same mobility is found for both preparations (Fig. 12). The rapid mobility in SDS gels indicates a low-molecular-weight which was more accurately determined to be 12,000 on agarose columns (6). While these results suggest an altered polypeptide, the electrophoretic mobility difference may be due to changes in bound components such as polysaccharide or secondary modifications of the proteins. More direct evidence for
FIG. 4. Section of a normal spore coat of B. cereus showing the under-coat (UC) and double-track layer (or pitted [PI layer of freeze-etch preparations). The outer covering material corresponds to the cross-patch layer shown in Fig. 1. The exosporium encircles the entire structure. FIG. 5-8. Sections of spores of lysozyme-dependent mutant B. cereus JOLD. The major profile changes are the breaks in spore coat (Ct) continuity, minimal in some sections (Fig. 5) while in others leaving a major part of the cortex (Cx) unprotected (Fig. 8). The exosporium (Ex) was always intact. The coat itself shows a thinning or absence of the outer cross-patch (CP) layer over the locally intact double track (P) and undercoat (UC) layers (Fig. 6). In some sections, the free edge of the loose spore coat appears duplicated or more probably folded under (Fig. 7).
.-C P
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7 359
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Cx
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WLAL.
6
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AN
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luc 4 p
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FIG. 9-11. Electron micrographs of lysozyme-sensitive spores of B. cereus 13LS. FIG. 9-10. Stage VII spore, undergoing the final stages of development of refractility, showing incomplete coverage of cortex (Cx) by spore coat. The curved edges of the loose coat were common in this mutant. The coat itself appeared to have the normal undercoat (UC), double track (P), and outer covering (CP). FIG. 11. Freeze-cleaved preparation showing the curled edge of the incomplete coat over a bare patch of cortex (Cx). No deficiency of cross-patch (CP) layer was encountered where there was coat. 360
VOL. 123, 1975
differences among the major coat structural proteins was provided by examination of the sulfur containing peptides of the various mutants and wild type. Coat protein is insensitive to virtually all proteolytic enzymes (partial digestion by Pronase) but is rendered about 70% soluble in 12% trichloroacetic acid by treatment with this particular keratinase. Surprisingly, another keratinase (32) does not digest the coat protein extensively (about 10% rendered soluble in 12% trichloroacetic acid). The specificity of this enzyme has not been determined. A highly reproducible pattern of about 12 peptides is found which is unique to coat preparations (Fig. 13). Very similar peptide maps were obtained with solubilized coat protein from spores of B. megaterium KM or B. subtilis 168. Since the half-cystine residues are believed to be critical for the formation of the coat layers (2, 4, 6), 35S-labeled peptides were compared (Fig. 14). The inclusion of excess unlabeled methionine ensured that most of the radioactivity (>90% on the basis of amino acid analyses) was incorporated into half-cystine residues. In the wild type (+ in Fig. 14), two major labeled peptides were found with a 2:1 ratio of radioactivity. This value is consistent with the presence of three half-cystine residues per monomer (assuming a molecular weight of 12,000) and supports other evidence for the presence of only one principal species of coat polypeptide (4,5; A. I. Aronson and P. C. Fitz-James, manuscript in preparation). The major cystine-containing peptide in mutant JOLD has an altered mobility in the chromatographic solvent. There are three 35S-labeled peptides in digests of coat protein from spores of 13LS. It appears that the major 35S-labeled peptide has been cleaved resulting in three peptides with a counts/minute ratio of 1.2:1.2:0.9.
Several properties of the mutant and wildtype spores are summarized in Table 1 and Fig. 15 and 16. The mutant spores contain the normal amount of dipicolinic acid and are heat resistant. As anticipated from the enrichment procedures, they are sensitive to lysozyme (Fig. 15). They are also more sensitive to octanol than the parental strain but are equally resistant to chloroform, toluene, and 1% deoxycholate. They respond poorly to L-alanine plus adenosine as germinants (Fig. 16), and also did not respond to 0.04 M calcium dipicolinic acid (25). No other potential germinating agents have been tested although the mutant spores do germinate slowly in G Tris medium after heat activation. It should be mentioned, however, that of six lysozyme-sensitive mutants tested to date, only
SPORE COAT MUTANTS OF B. CEREUS
361
100+ 600 -i"' 200
900
A L
300 ft -I_j_
SDS
a
1500
100C 600
900
200
300
il.,-
L--
'0
-I-
20
-.30
40
50
FIG. 12. Gel electrophoretic profiles of mixtures of 3H- and "4C-labeled coat extracts prepared from wild-type (x) and mutant 13LS (0) spores. In the top half, the spore mixture was extracted with 0.01 M NaHCO0-0.05 M dithioerythritol (DTE), pH 9.5, at 27 C for 3 h, and 30 jAl of the extract run on a 7% gel at pH 9.0 (see text). In the bottom half, the spores were extracted with 0.01 M NaHCO3-0.05 M DTE-0.5% SDS and run on a 7%o gel in pH 9.0 buffer containing 0.1% SDS. Slices (1 mm) were processed and counted. Recoveries in both cases were greater than 95% of input counts/minute.
four behaved as did strain 13L8. Two others germinated more rapidly albeit somewhat more slowly than did the wild type in L-alanine plus adenosine (about 40% of the wild-type rate). Revertants of both classes of mutants selected as described above occurred with a frequency consistent with initial point mutations. These revertants germinated in the presence of L-alanine plus adenosine were octanol resistant, and the pattern of 35S-labeled peptides was as in the wild type (Fig. 14). The properties and frequency of these revertants indicate that an alteration of the coat protein accounts for the phenotypic behavior of these mutants.
DISCUSSION The ability of lysozyme to penetrate the spore coat layers and the dependence upon lysozyme for germination (8, 9) have been exploited to enrich for coat mutants. The lysozyme sensitivity appears to be due to defects in coat coverage rather than the absence of specific coat layers. Since the same principal polypeptide is present in all coat layers, the mutation must affect the packing and interaction of all the layers in a given region. Areas where the coat is deposited (probably the innercoat initially) can apparently undergo secondary modifications to form the outercoat layers (4). As was anticipated from previous studies involving coat extraction (3, 26, 28), the spores were heat resistant and
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FIG.
J. BACTERIOL.
ARONSON AND FITZ-JAMES
13.
Peptide
protein (top) and bovine serum albumin Peptides were stained with 0.4% Arrows in the top figure indicate peptides containing half-cystine residues.
maps
of keratinase digest of total B.
(bottom). Procedures for digestion and separation
ninhydrin
in acetone.
are
contained all their dipicolinic acid. Of course, any lethality due to a coat alteration would not have been detected although we have recently isolated conditional mutants which are lysozyme sensitive only at 38 C. These mutants grow as well as the wild type at all tempera-
cereus
T coat
described in text.
tures. It should also be mentioned that there are strains of B. megaterium which produce lysozyme-sensitive spores (27) although it is not known whether a coat alteration is involved. Abnormal coat deposition does result in less stable spores in that they autogerminate during
SPORE COAT MUTANTS OF B. CEREUS
VOL. 123, 1975
363
IA f.,
e-
I
FIG. 14. Autoradiograms of keratinase digests of 3"S-labeled coat protein preparations. Peptides separated as in Fig. 13. Radioactive peptides were aligned with the origin and an [3"S]cysteic acid marker.
storage at 4 C and have the cortex and apparently the inner membrane (octanol sensitivity) more exposed. The coat layers, therefore, appear to have primarily a protective function although a role in spore maturation is not excluded. Formation of the outer coat is correlated with a change in appearance of the endospore in the phase microscope (31) so the deposition of these layers may be important in forming a phase-bright spore and other properties related to dormancy. The appearance of heat resistance is apparently independent of coat formation since inhibition of coat completion by addition of chloramphenicol does not
prevent formation of heat-resistant structures (11). Of particular interest is the loss of ability to respond to germinants. A similar loss is not observed when the coats are chemically removed from B. megaterium (28) although stripped B. cereus spores do respond to L-alanine plus adenosine more slowly than unstripped spores (unpublished data; T. Hashimoto, personal communication). Since a spore lytic activity may be involved in germination (13), this enzyme may normally be localized beneath or associated with the coat layers. Heat treatment and germinants may be neces-
364
J. BACTERIOL.
ARONSON AND FITZ-JAMES TABLE 1. Summary of properties of mutant and wild-type spores Property
J10LD
Wild type
DPA (g x 10-I "/spore)a Resistance to 80 C/20 min Response to germinants: L-Alanine + adenosine 0.04 M calcium-DPA Lysozyme sensitivityd % Survival after treatment with octanole Storage in distilled water or 0.03 M Tris-hydrochloride, pH 7.6, at 4 C
102-118 95-100%
13LS
85-1056 85-95%
98-115 88-100%
e c
c
60-80 100% phase bright after 2 weeks
c
5-8 Less than 50% phase bright after 48 h
5-8
1/100-1/107 t
Reversion frequency
1/10-1/107 t
a DPA,
Dipicolonic acid. Original mutant was also a leaky lysine auxotroph and contained only 40% of the wild-type DPA. Prototrophic revertants were selected and studied here. c See Fig. 16. d See Fig. 15; similar results obtained for spores produced in G Tris or a synthetic medium (1). e One-twentieth of the volume of octanol was added to a spore suspension in 0.03 M Tris-hydrochloride (pH 7.6) and mixed on a Vortex mixer for 40 s at 25 C. Spores were diluted and plated on G Tris agar. J Ranges for three separate experiments. Frequencies were determined as described in the text with 30 to 50 colonies picked for examination. Of these, 10 to 20% were lysozyme resistant.
1.0 0.
0.
E
c0 0.6
g 0.6
0.4~
0.2
° D
0.4
0.2
0
8
16
24
32
40
48
56
64
Minutes
FIG. 15. Lysozyme sensitivity of mutant and wildtype spores. Spores were washed and suspended in 0.03 Tris-hydrochloride (pH 7.8) plus 10 usg of lysozyme per ml. Symbols: A, wild type; 0, mutant JOLD;
0,
mutant 13LS.
2
4
6 8 Minutes
10
"
20
40
FIG. 16. Germination of mutant and wild-type spores. Spores were heat activated at 65 C for 30 min in 0.03 M Tris-hydrochloride (pH 8.0) and germinated in the presence of L-alanine plus adenosine as described in the text. Symbols: A, wild type; 0, mutant JOLD; 0, mutant 13LS.
of B. cereus spores are comprised primarily of one species of polypeptide. The altered coat protein is found as the principal component when total coat is extracted or when extraction techniques are employed which result in enrichment for the inner or outer-coat layers (6). This altered polypeptide must be a major component the packing or maturation of one or more spore of all the coat layers and it is probably the layers. Alterations of these components may inability account for the inability to respond to L-alanine such as to undergo secondary modifications disulfide interchange reactions (4, 6) and adenosine. Because there are lysozyme-sen- which accounts for the abnormal appearance. sitive mutants which still respond to these germinants, a comparison of the two classes ACKNOWLEDGMENTS may provide further insights into the role of the The assistance of J. Tjepkema, Doryth Lowey, and Leah spore coats in germination. Mitchell is appreciated as is the provision of the keratinase by As previously reported (4, 6), the coat layers W. Nickerson. Research was supported by grants from the Na-
to activate this enzyme. The resulting hydrolysis of a few mucopeptide bonds in the cortex may be sufficient to break the dormant state. Alternatively, coat deposition may influence
sary
VOL. 123, 1975 tional Science Foundation and the National Research Council of Canada, and by Public Health Service research grant GM20606 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Aronson, A. I., N. Angelo, and S. C. Holt. 1971. Regulation of extracellular protease production in Bacillus cereus T: characterization of mutants producing altered amounts of protease. J. Bacteriol. 106:1016-1025. 2. Aronson, A. I., and P. C. Fitz-James. 1968. Biosynthesis of bacterial spore coats. J. Mol. Biol. 33:199-212. 3. Aronson, A. I., and P. C. Fitz-James. 1971. Reconstitution of bacterial spore coat layers in vitro. J. Bacteriol. 108:571-578. 4. Aronson, A. I., and P. C. Fitz-James. 1973. The formation of spore coat layers in Bacillus cereus T, p. 275-296. In R. Markham, J. B. Bancroft, D. R. Davies, D. A. Hopwood, and R. W. Home (ed.), The generation of subcellular structures. American Elsevier, New York. 5. Aronson, A. I., and D. Horn. 1969. Synthesis and regulation of the bacterial spore coat, p. 72-81. In L. L. Campbell (ed.), Spores IV. American Society for Microbiology. Bethesda, Md. 6. Aronson, A. I., and D. Horn. 1972. Characterization of the spore coat protein of Bacillus cereus T, p. 19-27. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology. Washington, D.C. 7. Balassa, G. 1969. Biochemical genetics of bacterial sporulation. I. Unidirectional pleiotropic interactions among genes controlling sporulation in Bacillus subtilis. Mol. Gen. Genet. 104:73-103. 8. Cassier M., and M. Sebald. 1969. Germination lysozymedependente des spores de Clostridium perfringens ATCC 3624 apres traitement thermique. Ann. Inst. Pasteur Paris 117:312-324. 9. Cassier M., and A. Ryter. 1971. Sur un mutant de Clostridium perfringens donnant des spores sans tuniques a germination lysozyme-dependente. Ann. Inst. Pasteur Paris 121:717-732. 10. Fitz-James, P. C. 1971. Formation of protoplasts from resting spores. J. Bacteriol. 105:1119-1136. 11. Fitz-James, P. C., and E. Young. 1969. Morphology of sporulation, p. 39-72. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., New York. 12. Freer, J. H., and H. S. Levinson. 1967. Fine structure of Bacillus megaterium during microcycle sporogenesis. J. Bacteriol. 94:441-457. 13. Gould, G. W., and W. L. King. 1969. Action and properties of spore germination enzymes, p. 276-286. In L. L. Campbell (ed.), Spores IV. American Society for Microbiology. Bethesda, Md. 14. Hashimoto, T., and S. F. Conti. 1971. Ultrastructural changes associated with activation and germination of Bacillus cereus T spores. J. Bacteriol. 105:361-368. 15. Hoeniger, J. F. M., P. F. Stuart, and S. C. Holt. 1968.
SPORE COAT MUTANTS OF B. CEREUS
16. 17.
18.
19. 20. 21.
22.
23. 24.
25. 26.
27.
28. 29.
30. 31.
32.
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Cytology of spore formation in Clostridium perfringens. J. Bacteriol. 96:1818-1834. Holt, S. C., and E. R. Leadbetter. 1969. Comparative ultrastructure of selected aerobic spore-forming bacteria: a freeze etching study. Bacteriol. Rev. 33:346-378. Ito, J., and J. Spizizen. 1971. Increased rate of asporogenous mutations following treatment of Bacillus subtilis spores with ethyl methanesulfonate. Mutat. Res. 13:93-96. Janssen, F. W., A. J. Lung, and L. E. Anderson. 1958. Colorimetric assay for dipicolinic acid in bacterial spores. Science 127:26-29. Kadota, H., K. Iijima, and A. Uchida. 1965. The presence of keratin-like substance in the spore coat of Bacillus subtilis. Agric. Biol. Chem. 29:870-875. Moore, S. 1963. On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235-237. Murrell, W. G. 1969. Chemical composition of spores and spore structures, p. 216-273. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., New York. Nickerson, W. J., J. S. Noval, and R. J. Robison. 1963. Keratinase. I. Properties of the enzyme conjugate elaborated by Streptomyces fradiae. Biochim. Biophys. Acta 77:73-86. Nickerson, W. J., and S. C. Durand. 1963. Keratinase. II. Properties of the crystalline enzyme. Biochim. Biophys. Acta 77:87-99. Ohye, P. R., and W. G. Murrell. 1973. Exosporium and spore coat formation in Bacillus cereus T. J. Bacteriol. 115:1179-1190. Riemann, H., and Z. J. Ordal. 1961. Germination of bacterial endospores with calcium and dipicolinic acid. Science 133:1703-1704. Somerville, J. J., F. P. Delafield, and S. C. Rittenberg. 1969. Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species. J. Bacteriol. 101:551-560. Suzuki, Y., and L. J. Rode. 1969. Effect of lysozyme on resting spores of Bacillus megaterium. J. Bacteriol. 98:238-245. Vary, J. C. 1973. Germination of Bacillus megaterium spores after various extraction procedures. J. Bacteriol. 116:797-802. Vinter, V. 1958. Sporulation of bacilli. VmI. The participation of cysteine and cystine in spore formation by Bacillus megaterium. Folia Microbiol. (Prague) 4:216-221. Walker, P. D. 1970. Cytology of spore formation and germination. J. Appl. Bacteriol. 33:1-12. Young, E., and P. C. Fitz-James. 1959. Chemical and morphological studies of bacterial spore formation. I. The formation of spores in Bacillus cereus. J. Biophys. Biochem. Cytol. 3:467-782. Yu, R. J., S. R. Harmon, P. E. Wachter, and F. Blank. 1969. Amino acid composition and specificity of a keratinase of Trichophyton mentagrophytes. Arch. Biochem. Biophys. 135:363-370.
Properties of Bacillus
Vol. 123, No. 1 Printed in U.S.A.
cereus
Spore Coat Mutants
A. I. ARONSON* AND P. C. FITZ-JAMES Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907,* and Department of Microbiology, University of Western Ontario Medical School, London, Ontario, Canada Received for publication 17 April 1975
Two classes of spore mutants have been selected in Bacillus cereus T, those producing lysozyme-sensitive spores, and those producing spores dependent upon lysozyme for germination. One mutant from each class was studied in detail and found to have defective packing of the spore coat layers. The major spore coat polypeptide appeared to be altered on the basis of gel electrophoretic profiles and/or peptide maps of half-cystine-containing peptides. The spores of the mutants of both classes were sensitive to lysozyme and failed to respond to the germinants L-alanine plus adenosine. The spores were also more sensitive to octanol than the parental strain, but contained the same amount of dipicolinic acid and were equally heat resistant. The reversion frequencies in both cases were consistent with an initial point mutation, suggesting that an alteration in the major coat polypeptide accounted for the phenotypic properties studied.
While the morphology of the bacterial spore coat layers varies considerably among species, the structural pattern in most cases may be resolved at least into inner (or under) and outer coats (2, 12, 14-16, 24, 30). Within each category there may be further morphological subdivisions, especially for the outer coat layers as discussed in this paper. These coats are comprised largely, if not exclusively, of protein (6, 21) with conditions for solubilization correlated with the disappearance of particular structural layers (6). Surprisingly, there is very little heterogeneity in the number of polypeptide species present in total coat extracts of Bacillus cereus spores. In fact, there is likely to be only one major species of coat polypeptide present in all of the coat layers despite the varying solubility properties (4, 6). This protein is "keratinlike" in that it is relatively rich in half-cystine residues (2, 29), has a low-angle X:ray diffraction pattern reminiscent of keratins (19), and is generally resistant to proteolytic enzymes with the notable exception of a keratinase isolated from Streptomyces fradiae (22, 23). Spore coats may be almost totally solubilized by treatment of intact spores with a mixture of dithioerythritol and sodium dodecyl sulfate (SDS) at alkaline pH (3, 6). As judged by the appearance of thin sections and freeze-etched preparations of treated spores, this treatment results in the removal of virtually all of the spore coat layers (3). The treated spores are still viable, heat resistant, and contain all their dipicolinic acid. They are very sensitive to
lysozyme (3), however, and respond relatively slowly to the germinants L-alanine plus adenosine after heat activation (unpublished observations). The sensitivity of treated spores to lysozyme may be exploited for isolating spore protoplasts (10) and for selecting mutants altered in coat appearance (4). In addition, a requirement for lysozyme to germinate mutant spores of Clostridium perfringens (8) was correlated with the altered appearance of the spore coat layers (9). We have exploited lysozyme sensitivity and a requirement for lysozyme to germinate to enrich for spores with altered coat structure. The primary alteration in many of these mutants is in the major coat polypeptide. The mutant spores differ from the wild type in their sensitivity to solvents and response to germinants, whereas heat resistance and dipicolinic acid content are not changed. MATERIALS AND METHODS Bacillus cereus strain T was grown in G tris(hydroxymethyl)aminomethane (Tris) or a synthetic medium, as previously described (1). Growth was followed in a Coleman 8 colorimeter employing a 650-nm filter. For labeling with 35SO., the G Tris medium was modified to reduce the sulfate content. Cations were present as the chloride salts (including ammonium ion as NH4Cl), and the yeast extract concentration was reduced to 0.05% (wt/vol). For mutagenesis, spores were treated with either nitrosoguanidine (7) or ethyl methane sulfate (17). After treatment, the spores were centrifuged, washed
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three times with sterile medium, suspended in three times the original volume of medium, and incubated at 30 C for 36 to 48 h to permit growth and sporulation. For lysozyme-sensitive mutants, cells and spores were harvested by centrifugation as above, washed twice with 10 ml each of sterile 0.03 M Tris-hydrochloride (pH 7.8), and finally suspended in 0.5 ml of 0.03 M Tris-hydrochloride (pH 7.8) plus 50 ,g of lysozyme and incubated at 30 C for 90 min. After centrifugation, the pellet was suspended in 20% meglumine and sodium diatrizoate (Renografin) in 0.03 M Tris-hydrochloride (pH 7.8), and incubated at 0 C for 30 min. The suspension was layered over a linear 30 to 65% Renografin gradient in an SW25 tube, and centrifuged at 20,000 rpm for 40 min in an SW25 rotor (Spinco model L). There were three prevalent bands, one at approximately the density of intact spores at a density of 1.359 g/cm2 (3), one near the top consisting largely of cells, and a barely detectable band in between. This latter band was removed with a syringe and the spores were diluted into at least 10 volumes of G Tris. These cultures were incubated at 30 C for 48 h to permit germination, growth, and sporulation. The spores were harvested, washed, and treated with lysozyme as above. After centrifugation through Renografin, the intermediate band was diluted and plated on G Tris agar. After 36 h at 30 C, colonies were picked at random and suspended in 0.1 ml of 0.03 M Tris-hydrochloride (pH 7.6) plus 5 Mg of lysozyme. After incubation at 38 C for 2 to 3 h, each tube was examined for the presence of phase-dark spores. Any potential mutants were again streaked on G Tris and well-isolated colonies screened for lysozyme sensitivity of the spores. After two rounds of growth, lysozyme treatment, and separation in Renografin gradients, 10 to 80% of the colonies contained lysozyme-sensitive spores. To select for mutants dependent upon lysozyme for germination, spores produced after mutagen treatment and subsequent growth in G Tris were washed twice with 10-ml portions of 0.05 M sodium phosphate, pH 7.8. Spores (about 5 x 109) were suspended in 5 ml of phosphate and heat activated by incubating at 65 C for 30 min. L-Alanine was then added to 0.005 M and adenosine to 0.001 M and the suspension incubated at 30 C. After 40 min, the spores were pelleted, suspended in 20% Renografin, and fractionated on Renografin gradients as previously described. The density region corresponding to dormant spores was removed with a syringe and inoculated into 50 volumes of G Tris plus 5 Mg of lysozyme per ml to aid germination of presumptive mutants. After incubation at 30 C for 48 h, the germination and gradient separation procedures were repeated. The density region which should contain dormant spores was plated on G Tris agar containing 5 gg of lysozyme per ml. Colonies were replicated onto G Tris and those which grew slowly (presumably due to show germination or loss of viability of the sensitive spores on plates containing lysozyme) were selected from the original plates. About 10% of these were mutants dependent upon lysozyme for rapid germination. To measure reversion frequencies, a representative
SPORE COAT MUTANTS OF B. CEREUS
355
of each mutant class was selected for resistance to cycloserine by plating 10' spores on G Tris agar containing 300 Mg of D-cycloserine per ml. These resistant mutants were not altered in the lysozyme sensitivity of the spores nor on the dependence for lysozyme to germinate. The antibiotic resistance marker provided a means for eliminating any wildtype contaminants in the revertant studies. About 1010 spores of each mutant were treated with 50 Mg of lysozyme per ml in 5 ml of 0.03 M Tris-hydrochloride (pH 7.8) at 28 C for 2 h. The spores were then centrifuged through a Renografin gradient (SW25 rotor) as described above, and the density region corresponding to that containing intact spores was removed with a syringe, plated on G Tris containing 300Mg of cycloserine per ml, and incubated at 30 C for 78 h. To ensure that all sporulating colonies were revertants, 50 colonies were picked at random into 0.1 ml of 0.03 M Tris-hydrochloride (pH 7.8) plus 10 Ag of lysozyme. The frequency of lysozyme-resistant revertants could then be approximated. Isolation and characterization of spore coat proteins. Radioactive amino acids or 3"SO, was added to 10 ml of sporulating cultures at t,. 'H- and '4C-labeled reconstituted protein hydrolysate was added to G Tris cultures at 0.5 ,Ci/ml, respectively; 3"SO4 was added to cultures in low sulfate G Tris (supplemented with 3 Ag of methionine per ml at t,) to 200 MCi/ml. All cultures were incubated until free spores were present (36 to 40 h), and the spores were harvested, washed, and extracted by incubation in 0.01 M NaHCO8-0.05 M dithioerythritol with or without 0.5% SDS, pH 9.5 to 9.8, at 27 C for 3 h (6). For gel electrophoresis, 3H- and "4C-labeled spores were mixed so that the ratio of 3H/14C was 2 or 3 to 1 before extraction with 0.05 M dithioerythritol, pH 9.5, + 0.5% SDS at 28 C for 3 h. After rmoval of the extracted spores by centrifugation at 12,000 rpm for 15 min in a Sorvall RCIIB, a crystal of sucrose was dissolved in 20 Ml of the supernatant (5 to 20 Mg of protein). After addition of bromophenol blue, the total sample was placed on the top of 5 or 7% acrylamide gels (6 to 7 cm) and subjected to electrophoresis at 3 mA/tube for 2 to 3 h (until the dye marker had moved at least two-thirds of the way down the tube) in Tris (3 g/liter)-glycine (14.4 g/liter)-mercaptoethanol (0.04%), pH 9.0 (+0.1% SDS). The gel was removed from the tube, immediately frozen, divided into 1-mm slices with a Yeda macrotome, and processed for counting in Nuclear-Chicago solubilizer as previously described (6). For peptide analyses, the crude spore extract was either precipitated directly with a final concentration of 15% (wt/vol) trichloroacetic acid or purified before acid precipitation. For purification, the SDS in the crude coat extract was precipitated by addition of 0.05 ml of saturated KCl per ml of extract, and solid ammonium sulfate was added to the supernatant at fraction to 25% saturation (0 C). After incubation for 30 to 40 min at 0 C, the precipitate was collected by centrifugation at 12,000 rpm for 15 min. The precipitate was dissolved in 0.01 M NaHCO0-1% mercaptoethanol-0.4% SDS (pH 9.0), and passed over a Bio-Gel
356
ARONSON AND FITZ-JAMES
P-10 column (0.9 by 20 cm for up to 5 mg of protein) equilibrated with the same buffer. Radioactivity in the void volume (10 to 20% of the input) was discarded (impurities plus aggregated coat protein), and the tubes covering a major peak of radioactivity (80 to 90% of input) eluting at elution/void volume ratio of 2.1 were pooled and precipitated with trichloroacetic acid as above. Similar peptide maps were obtained with crude coat preparations, those purified on BioGel P-10 columns, or those purified on O-(triethylaminoethyl)-cellulose columns (5). After centrifugation at 10,000 rpm for 10 min in a Sorvall RCUB, the trichloroacetic acid precipitates were washed twice with 10-ml portions of 12% trichloroacetic acid and then ether. The pellets were air dried, dissolved in 0.2 to 0.5 ml of performic acid (20), and incubated at 0 C for 4 to 6 h. The solutions were diluted with water, dried in vacuo, and washed twice with distilled water. The pellets were dissolved in 0.2 to 0.4 ml of 0.01 M NaHCO, and adjusted to pH 9.0 A purified keratinase preparation (22, 23) was added to 5% of the protein concentration and the tubes incubated at 37 C. After 5 h, the pH was readjusted to 9.0 and an identical amount of keratinase was added. After incubation for a total of 12 h, the contents of the tubes were dried in vacuo. Peptides were separated first by electrophoresis at pH 1.9 (25 ml of 90% formic acid plus 87 ml of glacial acetic acid per liter) on a Whatmann 3MM at 35 V/cm for 110 min. The strips were air dried and a 1 by 9 inch segment sewn onto a 8 by 9 inch piece of Whatman 3MM for ascending chromatography in isobutanol-pyridine-water 87.5:87.5:75) for 12 to 16 h. Autoradiograms of the peptides were developed by exposure to Kodak RP54 medical X-ray film for varying periods. Peptides were also developed by ninhydrin (0.4% wt/vol in acetone) and spots cut out and plated into scintillation vials for counting in 10 ml of Omnifluor. Electron microscopy. Samples for freeze etching were frozen in Freon 22 onto scored copper disks and stored in liquid nitrogen until their transfer to the precooled (-150 C) stage of the Balzers apparatus. After cleavage, the surfaces were etched for 90 s at -100C, shadowed with platinum-carbon and then stabilized with more carbon. Replica pieces were cleaned by sequential floatation on concentrated H,SO,, 6% (wt/vol) sodium hypochlorite (Javex) and distilled water before being picked up on a copper grid for microscopy in a Philips 200. Thin section electron microscopy was conducted by standard methods already described (10). Spore coats were prepared as previously described (2).
J. BACTERIOL.
Analysis of spore properties. Heat resistance was based on the percentage of survival after heating at 80 C for 20 min. Dipicolinic acid was determined colorimetrically (18). For measurements of lysozyme sensitivity, washed spores were suspended in 0.03 M Tris-hydrochloride (pH 7.8) and 10 to 100 gg of lysozyme per ml added. The tubes were incubated 27 C and the change in absorbancy at 650 nm was followed in a Zeiss spectrophotometer. Germination rates were measured by washing and suspending spores in 0.05 M sodium phosphate or 0.03 M Tris-hydrochloride, pH 8.0. After heat activation at 65 C for 30 min (or 70 C for 20 min). L-alanine and adenosine were added to 0.01 M and 0.001 M final concentration, respectively. The change in absorbancy at 650 nm was followed at 27 C. Chemicals and isotopes. 31SO4 (carrier free) was purchased from Amersham-Searle; 3H-labeled reconstituted protein hydrolysate, algal profile, and 14C' labeled reconstituted protein hydrolysate were purchased from Schwarz/Mann. Omnifluor was from New England Nuclear Corp., SDS from British Drug House, Ltd., and dithioerythritol from Pierce Chemical Co. Twice recrystallized lysozyme (EC 3.2.1.17) was purchashed from Worthington Biochemicals Corp; Renografin as a 76% sterile solution was purchased from E. R. Squibb and Sons, Inc; D-cycloserine was from Sigma Chemical Co. A keratinase produced by Streptomyces fradiae (22, 23) was generously provided by W. Nickerson.
RESULTS Since there is no evidence for extensive differences among the mutants isolated by each procedure, only one from each class designated JOLD and 13LS will be described. In fact, there is overlap in the properties of mutants since all those selected for lysozyme-dependent germination are lysozyme sensitive, and many of those selected on the basis of lysozyme sensitivity are dependent upon lysozyme for germination. Morphology. Freeze-etch preparations of untreated spores of B. cereus exhibited an outer surface spore coat almost completely covered with the cross-patch array of protein fibers (Fig. 1). This coat-exosporium cleavage plane was encountered in a good proportion of spores. In a few small areas, the underlying pitted layer could be seen. In the lysozyme-dependent germination mutant, 1OLD, two defects appeared in
FIG. 1. A Freeze-etch preparation of a B. cereus spore. The knife has sheared off the exosporium (Ex), revealing the underlying outer cross-patch (CP) layer of the spore coat. In a few areas, the underlying pitted-coat layer (P) may be seen. Bar represents 0.1 gm in all electron micrographs. FIG. 2. Spore of the lysozyme-dependent mutant JOLD. The exosporium has been cleaved, revealing the ragged application of the spore coat (Ct) to the cortex (Cx). In some regions the coat appears thickened and folded under (see also Fig. 7) and generally the cross-patch (CP) layer is greatly reduced. FIG. 3. Higher magnification of mutant JOLD showing the incomplete cortical (Cx) coverage by the spore coat and the exposed pitted layer (P). The rods of protein in the remaining patches of cross-patch (CP) layer appear longer than those of the wild-type spores.
Ift~~~~~~~~~~~~~~t
357
358
ARONSON AND FITZ-JAMES
the spore coats. Both could be detected in thin sectioning and in freeze-cleave-etch preparations. In the latter, the defects were perhaps more striking; the spore coat presented a ragged and incomplete covering of the lysozyme-sensitive cortex (Fig. 2) and the coat itself, normally well endowed with the cross-patch layer (Fig. 1), showed a variable deficiency. This latter defect mimics the appearance of the coat of spores in which the uptake of cysteine has been inhibited by 10 mM sulfite (2). Like the sulfitetreated spores, those strands of cross-patch material which have been deposited are longer than the control and reveal extensive regions of the underlying pitted layer (Fig. 2 and 3). The pitted layer of mutant JOLD and control show similar dimensions: 6 to 7 nm of the hexogonally packed pits or holes, 8.9 to 9 nm apart in rows spaced 7.5 to 8 nm (Fig. 3). In some sections, the coat deficiency appeared more marked and large segments of cortex were uncovered (Fig. 8). Regardless of the amount of coat, however, a slackness usually resulted in a space between undercoat and cortex. This slackness of the coat may account for the apparent folding of the coat layers seen in sections (Fig. 7) and for the somewhat rolled edge seen in both sections (Fig. 8) and freeze etchings (Fig. 2 and 3). In wild-type spores, the coat is closely applied to the cortex and the undercoat is usually poorly stained (2). The undercoat is more readily seen in sections of free-spore coats (Fig. 4). The double-dense line, the backbone of the spore coat, has an overall thickness of 6 to 6.5 nm and corresponds to the pitted layer of freeze-etch preparations. In the sections of mutant 10LD, this layer is only partly covered by the outer-coat (cross-patch layer, Fig. 5 and 6), confirming the freeze-etch observations. The thickness of the normal spore coat profiles where covered with the cross-patch varies from 9 to 14 nm depending on the amount of cross-patch material. The addition of the less dense undercoat brings the overall spore coat thickness to 25 to 35 nm (Fig. 4). Reduced amounts of undercoat and the absence or thinning of the CP layer can reduce the overall mutant coat thickness to 16 to 18 nm (Fig. 6). Lysozyme-sensitive mutant 13LS had a simi-
J. BACTERIOL.
lar overall defect of the coat coverage (Fig. 9 and 10) and hence coat slackness as did the tOLD mutant. Where coat was deposited, all layers were present, the overall coat being some 35 nm thick (Fig. 10 and 11). In no cleavages was any extensive exposure of the pitted layer encountered; the cross-patch coverage was usually adequate if not heavier than normal. In both mutants exosporium was always complete. Occasionally (less than 10%) there were spores totally lacking coat material, but when these were purified on Renografin (higher density than intact spores) and cultured in G Tris, a mixed population of spores was produced, i.e., they do not breed true. Apparently, an incomplete outer coat results in total coat loss in some cases. Biochemical properties of mutants. Spore coat protein was extracted from a mixture of wild-type and lysozyme-sensitive spores labeled with either 14C or 3H-labeled reconstituted protein hydrolysate as described above. The gel electrophoretic profiles of the radioactive proteins from the wild type and mutant 13Ls are shown in Fig. 12. Because of the restricted conditions for solubility and ease of aggregation of the coat protein, it was necessary to use a high pH system and run fresh extracts. Under these conditions, one principle peak is found in each preparation (6) with a slight difference in mobility. Storage, fractionation, or dialysis of the extracts results in aggregation with considerable smearing of radioactivity across the gel. Reversal of the radioactive labels or use of 5% gels results in essentially the same profile. Coat extracts of mutant JOLD had the same mobility as the wild type at pH 9.0. If SDS is added (or the spores are extracted directly in the presence of SDS), then a peak with the same mobility is found for both preparations (Fig. 12). The rapid mobility in SDS gels indicates a low-molecular-weight which was more accurately determined to be 12,000 on agarose columns (6). While these results suggest an altered polypeptide, the electrophoretic mobility difference may be due to changes in bound components such as polysaccharide or secondary modifications of the proteins. More direct evidence for
FIG. 4. Section of a normal spore coat of B. cereus showing the under-coat (UC) and double-track layer (or pitted [PI layer of freeze-etch preparations). The outer covering material corresponds to the cross-patch layer shown in Fig. 1. The exosporium encircles the entire structure. FIG. 5-8. Sections of spores of lysozyme-dependent mutant B. cereus JOLD. The major profile changes are the breaks in spore coat (Ct) continuity, minimal in some sections (Fig. 5) while in others leaving a major part of the cortex (Cx) unprotected (Fig. 8). The exosporium (Ex) was always intact. The coat itself shows a thinning or absence of the outer cross-patch (CP) layer over the locally intact double track (P) and undercoat (UC) layers (Fig. 6). In some sections, the free edge of the loose spore coat appears duplicated or more probably folded under (Fig. 7).
.-C P
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FIG. 9-11. Electron micrographs of lysozyme-sensitive spores of B. cereus 13LS. FIG. 9-10. Stage VII spore, undergoing the final stages of development of refractility, showing incomplete coverage of cortex (Cx) by spore coat. The curved edges of the loose coat were common in this mutant. The coat itself appeared to have the normal undercoat (UC), double track (P), and outer covering (CP). FIG. 11. Freeze-cleaved preparation showing the curled edge of the incomplete coat over a bare patch of cortex (Cx). No deficiency of cross-patch (CP) layer was encountered where there was coat. 360
VOL. 123, 1975
differences among the major coat structural proteins was provided by examination of the sulfur containing peptides of the various mutants and wild type. Coat protein is insensitive to virtually all proteolytic enzymes (partial digestion by Pronase) but is rendered about 70% soluble in 12% trichloroacetic acid by treatment with this particular keratinase. Surprisingly, another keratinase (32) does not digest the coat protein extensively (about 10% rendered soluble in 12% trichloroacetic acid). The specificity of this enzyme has not been determined. A highly reproducible pattern of about 12 peptides is found which is unique to coat preparations (Fig. 13). Very similar peptide maps were obtained with solubilized coat protein from spores of B. megaterium KM or B. subtilis 168. Since the half-cystine residues are believed to be critical for the formation of the coat layers (2, 4, 6), 35S-labeled peptides were compared (Fig. 14). The inclusion of excess unlabeled methionine ensured that most of the radioactivity (>90% on the basis of amino acid analyses) was incorporated into half-cystine residues. In the wild type (+ in Fig. 14), two major labeled peptides were found with a 2:1 ratio of radioactivity. This value is consistent with the presence of three half-cystine residues per monomer (assuming a molecular weight of 12,000) and supports other evidence for the presence of only one principal species of coat polypeptide (4,5; A. I. Aronson and P. C. Fitz-James, manuscript in preparation). The major cystine-containing peptide in mutant JOLD has an altered mobility in the chromatographic solvent. There are three 35S-labeled peptides in digests of coat protein from spores of 13LS. It appears that the major 35S-labeled peptide has been cleaved resulting in three peptides with a counts/minute ratio of 1.2:1.2:0.9.
Several properties of the mutant and wildtype spores are summarized in Table 1 and Fig. 15 and 16. The mutant spores contain the normal amount of dipicolinic acid and are heat resistant. As anticipated from the enrichment procedures, they are sensitive to lysozyme (Fig. 15). They are also more sensitive to octanol than the parental strain but are equally resistant to chloroform, toluene, and 1% deoxycholate. They respond poorly to L-alanine plus adenosine as germinants (Fig. 16), and also did not respond to 0.04 M calcium dipicolinic acid (25). No other potential germinating agents have been tested although the mutant spores do germinate slowly in G Tris medium after heat activation. It should be mentioned, however, that of six lysozyme-sensitive mutants tested to date, only
SPORE COAT MUTANTS OF B. CEREUS
361
100+ 600 -i"' 200
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50
FIG. 12. Gel electrophoretic profiles of mixtures of 3H- and "4C-labeled coat extracts prepared from wild-type (x) and mutant 13LS (0) spores. In the top half, the spore mixture was extracted with 0.01 M NaHCO0-0.05 M dithioerythritol (DTE), pH 9.5, at 27 C for 3 h, and 30 jAl of the extract run on a 7% gel at pH 9.0 (see text). In the bottom half, the spores were extracted with 0.01 M NaHCO3-0.05 M DTE-0.5% SDS and run on a 7%o gel in pH 9.0 buffer containing 0.1% SDS. Slices (1 mm) were processed and counted. Recoveries in both cases were greater than 95% of input counts/minute.
four behaved as did strain 13L8. Two others germinated more rapidly albeit somewhat more slowly than did the wild type in L-alanine plus adenosine (about 40% of the wild-type rate). Revertants of both classes of mutants selected as described above occurred with a frequency consistent with initial point mutations. These revertants germinated in the presence of L-alanine plus adenosine were octanol resistant, and the pattern of 35S-labeled peptides was as in the wild type (Fig. 14). The properties and frequency of these revertants indicate that an alteration of the coat protein accounts for the phenotypic behavior of these mutants.
DISCUSSION The ability of lysozyme to penetrate the spore coat layers and the dependence upon lysozyme for germination (8, 9) have been exploited to enrich for coat mutants. The lysozyme sensitivity appears to be due to defects in coat coverage rather than the absence of specific coat layers. Since the same principal polypeptide is present in all coat layers, the mutation must affect the packing and interaction of all the layers in a given region. Areas where the coat is deposited (probably the innercoat initially) can apparently undergo secondary modifications to form the outercoat layers (4). As was anticipated from previous studies involving coat extraction (3, 26, 28), the spores were heat resistant and
362
~~~'I
|
FIG.
J. BACTERIOL.
ARONSON AND FITZ-JAMES
13.
Peptide
protein (top) and bovine serum albumin Peptides were stained with 0.4% Arrows in the top figure indicate peptides containing half-cystine residues.
maps
of keratinase digest of total B.
(bottom). Procedures for digestion and separation
ninhydrin
in acetone.
are
contained all their dipicolinic acid. Of course, any lethality due to a coat alteration would not have been detected although we have recently isolated conditional mutants which are lysozyme sensitive only at 38 C. These mutants grow as well as the wild type at all tempera-
cereus
T coat
described in text.
tures. It should also be mentioned that there are strains of B. megaterium which produce lysozyme-sensitive spores (27) although it is not known whether a coat alteration is involved. Abnormal coat deposition does result in less stable spores in that they autogerminate during
SPORE COAT MUTANTS OF B. CEREUS
VOL. 123, 1975
363
IA f.,
e-
I
FIG. 14. Autoradiograms of keratinase digests of 3"S-labeled coat protein preparations. Peptides separated as in Fig. 13. Radioactive peptides were aligned with the origin and an [3"S]cysteic acid marker.
storage at 4 C and have the cortex and apparently the inner membrane (octanol sensitivity) more exposed. The coat layers, therefore, appear to have primarily a protective function although a role in spore maturation is not excluded. Formation of the outer coat is correlated with a change in appearance of the endospore in the phase microscope (31) so the deposition of these layers may be important in forming a phase-bright spore and other properties related to dormancy. The appearance of heat resistance is apparently independent of coat formation since inhibition of coat completion by addition of chloramphenicol does not
prevent formation of heat-resistant structures (11). Of particular interest is the loss of ability to respond to germinants. A similar loss is not observed when the coats are chemically removed from B. megaterium (28) although stripped B. cereus spores do respond to L-alanine plus adenosine more slowly than unstripped spores (unpublished data; T. Hashimoto, personal communication). Since a spore lytic activity may be involved in germination (13), this enzyme may normally be localized beneath or associated with the coat layers. Heat treatment and germinants may be neces-
364
J. BACTERIOL.
ARONSON AND FITZ-JAMES TABLE 1. Summary of properties of mutant and wild-type spores Property
J10LD
Wild type
DPA (g x 10-I "/spore)a Resistance to 80 C/20 min Response to germinants: L-Alanine + adenosine 0.04 M calcium-DPA Lysozyme sensitivityd % Survival after treatment with octanole Storage in distilled water or 0.03 M Tris-hydrochloride, pH 7.6, at 4 C
102-118 95-100%
13LS
85-1056 85-95%
98-115 88-100%
e c
c
60-80 100% phase bright after 2 weeks
c
5-8 Less than 50% phase bright after 48 h
5-8
1/100-1/107 t
Reversion frequency
1/10-1/107 t
a DPA,
Dipicolonic acid. Original mutant was also a leaky lysine auxotroph and contained only 40% of the wild-type DPA. Prototrophic revertants were selected and studied here. c See Fig. 16. d See Fig. 15; similar results obtained for spores produced in G Tris or a synthetic medium (1). e One-twentieth of the volume of octanol was added to a spore suspension in 0.03 M Tris-hydrochloride (pH 7.6) and mixed on a Vortex mixer for 40 s at 25 C. Spores were diluted and plated on G Tris agar. J Ranges for three separate experiments. Frequencies were determined as described in the text with 30 to 50 colonies picked for examination. Of these, 10 to 20% were lysozyme resistant.
1.0 0.
0.
E
c0 0.6
g 0.6
0.4~
0.2
° D
0.4
0.2
0
8
16
24
32
40
48
56
64
Minutes
FIG. 15. Lysozyme sensitivity of mutant and wildtype spores. Spores were washed and suspended in 0.03 Tris-hydrochloride (pH 7.8) plus 10 usg of lysozyme per ml. Symbols: A, wild type; 0, mutant JOLD;
0,
mutant 13LS.
2
4
6 8 Minutes
10
"
20
40
FIG. 16. Germination of mutant and wild-type spores. Spores were heat activated at 65 C for 30 min in 0.03 M Tris-hydrochloride (pH 8.0) and germinated in the presence of L-alanine plus adenosine as described in the text. Symbols: A, wild type; 0, mutant JOLD; 0, mutant 13LS.
of B. cereus spores are comprised primarily of one species of polypeptide. The altered coat protein is found as the principal component when total coat is extracted or when extraction techniques are employed which result in enrichment for the inner or outer-coat layers (6). This altered polypeptide must be a major component the packing or maturation of one or more spore of all the coat layers and it is probably the layers. Alterations of these components may inability account for the inability to respond to L-alanine such as to undergo secondary modifications disulfide interchange reactions (4, 6) and adenosine. Because there are lysozyme-sen- which accounts for the abnormal appearance. sitive mutants which still respond to these germinants, a comparison of the two classes ACKNOWLEDGMENTS may provide further insights into the role of the The assistance of J. Tjepkema, Doryth Lowey, and Leah spore coats in germination. Mitchell is appreciated as is the provision of the keratinase by As previously reported (4, 6), the coat layers W. Nickerson. Research was supported by grants from the Na-
to activate this enzyme. The resulting hydrolysis of a few mucopeptide bonds in the cortex may be sufficient to break the dormant state. Alternatively, coat deposition may influence
sary
VOL. 123, 1975 tional Science Foundation and the National Research Council of Canada, and by Public Health Service research grant GM20606 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Aronson, A. I., N. Angelo, and S. C. Holt. 1971. Regulation of extracellular protease production in Bacillus cereus T: characterization of mutants producing altered amounts of protease. J. Bacteriol. 106:1016-1025. 2. Aronson, A. I., and P. C. Fitz-James. 1968. Biosynthesis of bacterial spore coats. J. Mol. Biol. 33:199-212. 3. Aronson, A. I., and P. C. Fitz-James. 1971. Reconstitution of bacterial spore coat layers in vitro. J. Bacteriol. 108:571-578. 4. Aronson, A. I., and P. C. Fitz-James. 1973. The formation of spore coat layers in Bacillus cereus T, p. 275-296. In R. Markham, J. B. Bancroft, D. R. Davies, D. A. Hopwood, and R. W. Home (ed.), The generation of subcellular structures. American Elsevier, New York. 5. Aronson, A. I., and D. Horn. 1969. Synthesis and regulation of the bacterial spore coat, p. 72-81. In L. L. Campbell (ed.), Spores IV. American Society for Microbiology. Bethesda, Md. 6. Aronson, A. I., and D. Horn. 1972. Characterization of the spore coat protein of Bacillus cereus T, p. 19-27. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed.), Spores V. American Society for Microbiology. Washington, D.C. 7. Balassa, G. 1969. Biochemical genetics of bacterial sporulation. I. Unidirectional pleiotropic interactions among genes controlling sporulation in Bacillus subtilis. Mol. Gen. Genet. 104:73-103. 8. Cassier M., and M. Sebald. 1969. Germination lysozymedependente des spores de Clostridium perfringens ATCC 3624 apres traitement thermique. Ann. Inst. Pasteur Paris 117:312-324. 9. Cassier M., and A. Ryter. 1971. Sur un mutant de Clostridium perfringens donnant des spores sans tuniques a germination lysozyme-dependente. Ann. Inst. Pasteur Paris 121:717-732. 10. Fitz-James, P. C. 1971. Formation of protoplasts from resting spores. J. Bacteriol. 105:1119-1136. 11. Fitz-James, P. C., and E. Young. 1969. Morphology of sporulation, p. 39-72. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press Inc., New York. 12. Freer, J. H., and H. S. Levinson. 1967. Fine structure of Bacillus megaterium during microcycle sporogenesis. J. Bacteriol. 94:441-457. 13. Gould, G. W., and W. L. King. 1969. Action and properties of spore germination enzymes, p. 276-286. In L. L. Campbell (ed.), Spores IV. American Society for Microbiology. Bethesda, Md. 14. Hashimoto, T., and S. F. Conti. 1971. Ultrastructural changes associated with activation and germination of Bacillus cereus T spores. J. Bacteriol. 105:361-368. 15. Hoeniger, J. F. M., P. F. Stuart, and S. C. Holt. 1968.
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