Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis

Young, Frank E. (Western Reserve University, Cleveland, Ohio). Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis. J. Bacteriol. 92:839-846. 1966.-Autolysis of the cell wall of Bacillus subtilis by an indigenous autolytic enzyme results in solubilization of 90% of the cell wall. The solubilized cell wall (supernatant fraction) was fractionated by the combination of ion-exchange chromatography on diethylaminoethyl cellulose and gel filtration on Sephadex G-25 into polysaccharides (composed of N-acyl glucosamine and N-acyl muramic acid), mucopeptides, peptides, and teichoic acid. The chemical composition of the products of autolysis confirms the proposed mechanism of autolysis and establishes the autolytic enzyme as an N-acyl muramyl-l-alanine amidase. The heteropolymers in the cell wall are linked by peptide bridges. Two peptides which account for 70% of the peptides of the cell wall have a molar ratio of 1.0:0.9:1.3 for diaminopimelic acid, glutamic acid, and alanine, respectively. Other minor peptides contain diaminopimelic acid, glutamic acid, and alanine in molar ratios of 1.0:0.9:1.5, 1.0:0.5:1.0, and 1.0:1.5:1.7, respectively. The procedures employed in this study should be applicable to the fractionation of heteropolymers in cell walls of other gram-positive organisms and thereby aid in the study of the structure of antigenic determinants and endotoxins.     

Membrane particles from Escherichia coli and Bacillus subtilis, containing penicillin-binding proteins and enriched for chromosomal-origin DNA.

Rapid-sedimenting DNA-membrane complexes were obtained from both Bacillus subtilis and Escherichia coli by a method involving gentle lysis followed by restriction enzyme digestion and sucrose gradient fractionation. These complexes were substantially enriched in chromosomal origin DNA, and in B. subtilis, the complexes were enriched in penicillin-binding proteins relative to that of the total membrane. Such complexes may represent procaryotic membrane domains which are topographically and functionally distinct.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

A partial characterization of an autolytically solubilized cell wall glucan

Incubation of purified cell wall fragments from corn (Zea mays) coleoptiles results in solubilization of some of the wall dry matter. The portion of the weight loss due to enzymatic autolysis is due mainly to solubilization of a glucan and, to a small extent, to liberation of free glucose. No other carbohydrate wall components or sugars other than glucose are solubilized despite the high concentrations of, for example, galactans, arabans, and xylans in the walls. The glucan has been partially characterized and found to be a lichenan-like polymer composed of 1→3 and 1→4 linked glucosyl units.     

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.     

Autolysis in Staphylococcus aureus: Preferential Release of Old Cell Walls

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.     

In vitro autolysis of plant cell walls

Primary cell walls of Zea mays prepared in a glycerol medium are capable of autolysis in vitro. Autolysis results in solubilization of about 10% of the wall substance during an 8 hour incubation period. Approximately 10% of the solubilized material is glucose and the remainder consists of an unidentified polymer which yields only glucose upon hydrolysis. Cell wall autolysis is a linear function of time of incubation and of wall concentration. The autolytic process occurs optimally over the pH range of 5.5 to 6.5. The possible relationship between autolytic capacity and capacity for elongation is discussed.     

An antibiotic-inducible cell wall-associated protein that protects Bacillus subtilis from autolysis

In Bacillus subtilis, antibiotics that impair cell wall synthesis induce a characteristic stress response including the sigma(W) and sigma(M) regulons and the previously uncharacterized yoeB gene. Here we demonstrate that YoeB is a cell wall-associated protein with weak sequence similarity to a noncatalytic domain of class B penicillin-binding proteins. A yoeB-null mutant exhibits an increased rate of autolysis in response to cell wall-targeting antibiotics or nutrient depletion. This phenotype does not appear to be correlated with gross alterations in peptidoglycan structure or levels of autolysins. Promoter dissection experiments define a minimal region necessary for antibiotic-mediated induction of yoeB, and this region is highly conserved preceding yoeB homologs in close relatives of B. subtilis. These results support a model in which induction of YoeB in response to cell envelope stress decreases the activity of autolysins and thereby reduces the rate of antibiotic-dependent cell death.     

Asymmetric distribution of charge on the cell wall of Bacillus subtilis.

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.     

DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes.

A DNA-membrane complex extracted from Bacillus subtilis was studied further as a model system for initiation of bacterial DNA replication in vitro. Of three subcomplexes purified from the crude complex by a combination of CsCl and sucrose gradient centrifugation, the synthetic capability of only one was inhibited significantly by streptovaricin, a known inhibitor of RNA primer formation. A selective enrichment in the level of this subcomplex was obtained by manipulating a thymine-requiring mutant. The synthetic capabilities of an enriched and nonenriched DNA-membrane complex were compared in the presence and absence of streptovaricin. Although the rate and extent of DNA synthesis per unit of protein were approximately the same in the absence of the antibiotic, there was a much greater inhibition of synthesis shown by the enriched complex in the presence of streptovaricin. Although the amount of DNA present in the putative initiation subcomplex was less than 0.3 to 0.4% of the total DNA present in the crude complex, such DNA, except for a few quantitative differences, was still representative of genomic DNA. Newly synthesized DNA hybridized to specific origin- and non-origin-derived restriction fragments of the B. subtilis genome. However, when an elongation inhibitor (ddCTP) was added, hybridization of such DNA to almost all of the nonorigin fragments disappeared or was reduced drastically, whereas origin region hybridization patterns remained strong. The highest level of hybridization in the origin region occurred with a BamHI (B7) restriction fragment of 5.6 kilobases that has been implicated by others as one site initiation in vivo (N. Ogasawara, M. Seiki, and H. Yoshikawa, Nature (London) 281:702-704, 1979; S. J. Seror-Laurent and G. Henckes, Proc. Natl. Acad. Sci. USA 82:3586-3590, 1985).     

Deoxyribonucleic Acid-Membrane Complexes in the Bacillus subtilis Transformation System

The fate of transforming deoxyribonucleic acid (DNA) in Bacillus subtilis was studied by isolating the DNA-membrane complex on Renografin gradients. Soon after uptake, transforming DNA binds to the cell membrane and displays a greater resistance to shear than the recipient genome-membrane complex. Ten minutes after uptake, a portion of the donor DNA is released from the membrane. Most of the released donor radioactivity represents unintegrated, biologically inactive DNA. Recombinant or integrated DNA is enriched 1.5- to 1.7-fold in the membrane. This enrichment last at least 30 min after termination of DNA uptake, and probably much longer. The data suggest that transforming DNA may be integrated into the recipient genome on, or close to, the cell membrane.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Interaction of Concanavalin A with the Cell Wall of Bacillus subtilis

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.     

Autolytic Enzyme System of Streptococcus faecalis IV. Electron Microscopic Observations of Autolysin and Lysozyme Action

Cell walls (LOG walls) were isolated from cultures of Streptococcus faecalis ATCC 9790 in the exponential phase of growth. These walls were either allowed to undergo autolytic dissolution (in the presence or absence of trypsin) or wall autolysis was inactivated with sodium dodecylsulfate (SDS walls). Inactivated walls were treated either with lysozyme or with isolated, partially purified S. faecalis autolysin. During wall lysis, samples were removed, negatively stained with phosphotungstate, and examined in the electron microscope. Both lysozyme and isolated autolysin appeared to act over the entire surface of SDS walls. After partial dissolution, a fibrous network over the surface was revealed. Lysozyme digestion revealed the presence of prominent, highly-contrasted equatorial and subequatorial bands around the walls. After trichloroacetic acid extraction, the bands were seen less frequently and less distinctly in the partially lysozyme digested walls, suggesting that the bands contained nonpeptidoglycan polymers. In the absence of trypsin (which activates a latent form of the autolysin), autolysis of LOG walls appeared to start at the equatorial bands and to proceed back towards the apex of the coccus. Ribbons of wall material coming off the wide edge of the nearly hemispherical wall fragments were observed. Activation of latent autolysis resulted in lytic action over the entire wall surface. The results are consistent with the previously postulated location of active autolysin at the areas of new wall synthesis and the random location of latent autolysin in LOG walls.     

Energized membrane may regulate nucleoid conformation in Bacillus subtilis

Abstract It was shown in a previous study, using a temperature-sensitive initiation mutant of Bacillus subtilis that DNA synthesis at the origin and terminus of replication occurred at cell poles. In the current study, it is demonstrated that cells specifically labelled at the origin or near the terminus, show a re-distribution of radioactivity when treated with the uncoupling agent, sodium azide. Since it is most likely that the DNA-cell wall attachment is mediated through membrane interactions, we now propose that the maintenance of this attachment is dependent on the energized state of the membrane.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

Some Properties of the Autolytic N-Acetylmuramidase of Lactobacillus acidophilus

The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 mug/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.     

Effect of aerobic and anaerobic growth on the cell wall ofStaphylococcus aureus

Electron micrographs ofStaphylococcus aureus 7167 which had been grown anaerobically showed that the cell wall was approximately 5 times thicker than the wall of bacteria after aerobic growth. Cell walls prepared from anaerobically grownS. aureus were more sensitive to the bacteriolytic enzymes: lysostaphin, lysozyme, and the wall-associated autolytic enzyme ofB. subtilis 168 I^?. Our findings are interpreted as evidence that the cell wall or surface of anaerobically grownS. aureus 7167 is different from that of aerobically grownS. aureus 7167. The findings suggest that the cell wall peptidoglycan of the anaerobe is a more loosely formed network, resulting in a more rapid solubilization by the bacteriolytic enzymes.