Ultrastructural Studies on a Mutant of Bacillus subtilis Whose Growth is Inhibited Due to Insufficient Autolysin Production

The growth of Bacillus subtilis mutant betaA177 can be inhibited under special conditions in which not enough autolytic enzymes are produced for optimal growth. Electron microscopy studies show that during growth inhibition there is localized thickening of the cell wall at positions where cells bend. A model is proposed to explain this result. Rapid growth can be restored by adding lysozyme or a B. subtilis autolysin mixture to a growth-inhibited betaA177 culture. Such addition reduces the localized wall thickening and causes other changes in surface morphology which are described and discussed. Septum formation seems to be relatively less inhibited than cell elongation when lytic enzyme levels are reduced. Measurements were made demonstrating that walls at ends of cells are morphologically different from walls at sides of cells in cultures of betaA177 growing at 51 C.     

Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases

The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.     

New centrifugation technique for isolating enzymes from large cell structures: isolation and characterization of two Bacillus subtilis autolysins

Bacillus subtilis cell walls can be centrifuged through a linear gradient of 0 to 2 m LiCl and 10 to 25% sucrose so that different autolysins are removed by different salt concentrations and banded in separate positions as the walls pass through the gradient. Using this technique we have found that B. subtilis cell walls are isolated with two autolytic enzymes attached. One autolysin, a glycosidase, can be eluted from walls with 0.5 m LiCl, has a pH optimum between 5 and 8, is relatively heat-sensitive, and has a molecular weight of 60,000. The other autolysin, an alanine amidase, can be eluted from walls with 1.5 m LiCl, has a pH optimum around 8, is relatively heat-stable, has a molecular weight of 35,000, and is present in quantities ten times greater than the glycosidase.     

Autolytic defective mutant of Streptococcus faecalis.

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.     

Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis; location of active and latent autolysin.

Over 80% of the active and porteinase-activatable, latent forms of the autolytic N-acetylmuramide glycanhydrolase of Streptococcus faecalis ATCC 9790 were released to the supernatant buffer during the autolytic formation of protoplasts (autoplasts) in the presence of absence of trypsin. Autolysin activity was not found in association with released mesosomal vesicles and had little affinity for binding to membranes or to the outer surface of the wall. Isolated walls were able to bind over four times as much autolysin activity as that present on wall exponential-phase cells. Using a rapid technique for wall isolation, evidence was obtained that the latent form (as well as the active form) was wall bound in intact cells. In addition, isotope labeling and ultrastructural studies were able to show that latent autolysin was concentrated in the newer, septally associated portion of the wall.     

Optical tweezers cause physiological damage to Escherichia coli and Listeria bacteria

We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited more-pronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.     

Isolation and purification of a Campylobacter upsaliensis autolysin.

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS)-PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Studies on the competence-inducing factor of Bacillus subtilis

1. Aqueous extracts of competent cells of Bacillus subtilis 168I(-) were shown to contain a competence-inducing factor. The aqueous extracts were fractionated on DEAE-cellulose columns. 2. Those fractions from DEAE-cellulose columns containing the competence-inducing factor were shown to exhibit a powerful lytic effect on isolated cell walls of B. subtilis 168I(-). Little or no lytic activity was exhibited by the other fractions. 3. The kinetics of the lytic enzyme were investigated and found to be first-order. Treatment of cell wall lysates with 1-fluoro-2,4-dinitrobenzene suggested that the enzyme may be identical with N-acetylmuramyl-l-alanine amidase. The amino acid composition of the partially purified enzyme was determined. 4. It is suggested that competence-induction may be dependent on the limited action of the autolytic amidase.     

The energized membrane and cellular autolysis in Bacillus subtilis

Lysis of exponential cultures of B. subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes. Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium. Autolysis occurs after brief starvation for a carbon source. Protoplasts are unaffected by azide or other lysis-inducing agents. Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis. The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis. These results suggest that cellular lysis in B. subtilis and energized membrane are tightly coupled. The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis. Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation. Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity. Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces. We interpret the results to suggest that the in vivo control of autolysin activity in B. subtilis is related to the energized membrane     

Relationship between the Latent Form and the Active Form of the Autolytic Enzyme of Streptococcus faecalis

A 10-hr starvation of Streptococcus faecalis ATCC 9790 for the amino acids methionine and threonine results in cells which are resistant to autolysis and which contain greatly reduced quantities of both active and latent (proteinase activable) forms of the autolytic enzyme (an N-acetyl-muramide glycanhydrolase). Cell walls were isolated from cells harvested at various times during the recovery from such starvation and were assayed for active and latent forms of the autolysin. Within 10 min of recovery the latent enzyme began to increase. Only after 30 to 60 min did the active enzyme begin to increase; after a similar lag, the cells' proneness to lysis markedly increased. The intracellular localization of both forms of the autolysin was examined, using as an experimental tool the ability of added cell wall to bind autolysin. (14)C-lysine-labeled, inactivated cell walls were added to exponential-phase cells, which were then disrupted, and the mixed wall population was isolated. Measurement of the (14)C release during wall autolysis indicated that the active enzyme in the cells was not available for binding to the added (14)C-labeled walls and was therefore wall-bound in vivo. In contrast, up to 85% of latent autolysin activity was found to have been efficiently bound to the added (14)C walls. The results obtained suggest (i) cellular autolysis is a reflection of the level of active enzyme and not of latent enzyme, and (ii) autolysin is synthesized and mainly located in the cytoplasm as an inactive latent precursor (proenzyme) which is transported to sites on the cell wall associated with wall biosynthesis, where it becomes activated.     

Sporangila autolysis in Clostridium perfringens type A

The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of Clostridium prefringens was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co²⁺ (0.3 · 10⁻³ M CoCl₂) and trypsin (48 μg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N-Acetylmuramyl-l-alanine amidase activity which released N-terminal alanine, and endopeptidase activity which hydrolysed the d-alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not known if one or two enzyme are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to that of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporilated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation medium or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.     

Cell Wall Turnover at the Hemispherical Caps of Bacillus subtilis

Cell walls made by Bacillus subtilis bacteria grown in D(2)O medium have buoyant densities in CsCl which are different from walls made by cells grown in H(2)O medium. Cell wall turnover was studied by measuring the change in wall buoyant density after a B. subtilis culture was shifted from growth in D(2)O medium to aeration in H(2)O medium. Walls from the hemispherical caps were isolated after preferential digestion of wall from the cylindrical regions using the B. subtilis autolytic amidase. The walls from the polar regions were found to turn over extensively.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.     

Possible involvement of bacterial autolytic enzymes in flagellar morphogenesis.

Autolytic enzymes were found to be required for flagellar morphogenesis in Bacillus subtilis 168 and Bacillus licheniformis 6346. Two previously characterized, poorly lytic, chain-forming mutants of B. subtilis 168, strains FJ3 (temperature conditional) and FJ6, each 90 to 95% deficient in the production of N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase, were observed to be nonmotile at 35 degrees C in a variety of liquid and semisolid meida. In contrast, cells of the isogenic wild-type strain were motile and fully separated. Electron microscopy revealed the complete absence of flagella on the mutant cells. Similar observations were made with another poorly lytic strain of B. subtilis 168 (Nil5) and with two poorly lytic, phosphoglucomutase-deficient mutants of B. licheniformis 6346 (MH-3, MH-5). In minimal media lacking galactose (restrictive conditions), the B. licheniformis mutants failed to form flagella, or had serious abnormalities in flagellar morphogenesis and motility. Under permissive conditions, mutants FJ3 (grown at 17 degrees C) and MH-5 (grown with addend galactose) showed increased autolytic activities, grew in the dechained form, and regained their capacities to synthesize functional flagella. Examination of several classes of spontaneous revertants derived from the various mutant strains further demonstrated a close relationship between autolysin acttivity and flagellation in the two Bacillus spp.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.     

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis.

The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands A1 to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin profile was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands A1 and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.     

Site of Initiation of Cellular Autolysis in Streptococcus faecalis as Seen by Electron Microscopy

Low concentrations of glutaraldehyde (0.1% or higher) blocked cellular and wall autolysis. The site of autolytic activity was studied by allowing cell autolysis to proceed for very short periods (0 to 15 min) before addition of glutaraldehyde. Electron microscopy of ultrathin sections showed that the primary site of autolytic activity was the leading edge of the nascent cross wall. The base of the cross wall seemed more resistant than the tip. Evidence supporting the involvement of autolysin activity in continued wall extension and in cell separation as well as in the initiation of new sites of wall extension was obtained. In cells exposed for 10 min to chloramphenicol, wall dissolution was very much slower but occurred at the same cross wall site.