Search

A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable of lysing cells of Staphylococcus aureus was purified approximately 500-fold by passage through diethylaminoethyl cellulose and chromatography on carboxymethyl-cellulose. The purified enzyme was shown to act as an endopeptidase, cleaving the pentaglycine cross-bridges of the cell wall peptidoglycan at d -alanyl-glycine and glycyl-glycine linkages with the release of di−, tri−, and tetraglycine fragments. Release of NH 2 -alanine indicated weak N -acetylmuramyl- l -alanine amidase activity, but most of the residual peptide remained attached to the glycan. No hydrolysis of the glycan occurred. The lytic spectrum of the enzyme toward a variety of other cell walls of known peptidoglycan composition indicated relatively high specificity for peptidoglycans with polyglycine bridges.

Detailed knowledge of bacterial cell wall peptidoglycan structures has expanded rapidly in the past few years, and very definite patterns have emerged which can be attributed to common biosynthetic pathways, and, more speculatively, to a common structural function. The glycan is β - 1, 4 linked and is therefore a substituted form of chitin, probably retaining its linear conformation. The peptide may be more flexible, and varied in its cross-linking, but all contain a sequence derived from a probable common precursor, UDP- N- acetylmuramy 1-(A)- D - glu -(B)- D -ala- D - ala, when (A) and (B) are amino acids with the carboxy 1 and a-amino groups of a center with L - configuration in the main chain. The conformation of this sequence is discussed.

The chemical nature and distribution of the peptidoglycan in Myxococcus xanthus at various stages of the cellular life cycle were investigated. Vegetative cells and microcysts contained approximately 0.6% by weight of peptidoglycan. The overall composition of the peptidoglycan was similar in both cell types and was approximately 1 glutamic acid, 1 diaminopimelic acid, 1.7 alanine, 0.75 N -acetylglucosamine, and 0.75 N -acetylmuramic acid. (We have assumed that all the hexosamines are N -acetylated.) The sizes of the subunits (estimated by gel filtration) solubilized by muramidases were considerably larger (tetramer and oligomer) in the microcysts than in the vegetative cells (mostly dimer). There was a transient decrease in cross-linking (measured as an increase in the amount of free amino group of diaminopimelic acid) during the stage of microcyst formation when the cells converted from ovoids to spheres. At the same time, there occurred a large and rapid increase in a galactosamine derivative which may have reflected the synthesis of capsular material. Immediately prior to this period of morphogenesis, the cells became resistant to penicillin but remained sensitive to d -cycloserine. The walls of vegetative cells were completely disaggregated by trypsin and sodium lauryl sulfate, suggesting a discontinuous peptidoglycan layer. This was no longer apparent after the ovoid-sphere stage of microcyst formation. The relationship to morphogenesis of the chemical changes in the cell wall is discussed.

Yeast ribonucleic acid (RNA) polymerase II, isolated after fractionation on diethylaminoethyl (DEAE)-cellulose (DE-52) or on DEAE-Sephadex (A-25), is 50% inhibited by 1.5 mug of alpha-amanitin. This inhibition is independent of the sequence of interaction of enzyme, template, nucleotides, and antibiotic and is expressed immediately on addition of alpha-amanitin to a preparation actively synthesizing RNA. Thus, alpha-amanitin's primary effect is inhibition of elongation of preinitiated RNA sequences in this system, as in others. A single peak of alpha-amanitin-resistant RNA polymerase activity (I) was eluted before enzyme II on either column. On A-25 but not on DE-52, a third peak of activity (III) was eluted after enzyme II. This activity was also resistant to alpha-amanitin. Enzymes I, II, and III were 50% inhibited by 3, 4, and 3 mug of thiolutin per ml, respectively. The extent of inhibition was independent of the nature of the template (native or denatured salmon sperm deoxyribonucleic acid or poly(dA-dT) or of the presence of 0.4 mM dithiothreitol, but this marked inhibition was only seen when enzymes were preincubated with thiolutin in the absence of template. Template protected the enzymes against thiolutin in the absence of nucleotides. Either the sensitive site on the polymerase is only accessible to thiolutin before interaction with template or thiolutin inhibits functional polymerase-template interaction but not elongation of preinitiated RNA chains.

Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone- and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.

An uncross-linked monomer (nascent peptidoglycan unit) accumulates in cells of Staphylococcus aureus treated with low concentrations of penicillin G or with ampicillin, methicillin, or cephalothin. The uncross-linked monomer has been isolated, and analyses indicate that it represents a prefabricated subunit of the wall bearing both of the d-alanine residues of its pentapeptide precursor as well as an open pentaglycine chain. Pulse labeling experiments indicate that this uncross-linked unit is a direct precursor of the cross-linked peptidoglycan and that its cross-linking is inhibited by penicillin G. At high concentrations of penicillin G, wall synthesis ceases abruptly and no accumulation of the nascent peptidoglycan units is observed. These data have been obtained in support of the hypothesis that penicillins are substrate analogues of the d-alanyl-d-alanine end of the nascent peptidoglycan units and that they acylate the transpeptidase which catalyzes the cross-linking reaction. The data may also provide an explanation of the paradoxical observation that the killing rate in S. aureus by penicillin G is higher at low concentrations than at high concentrations of the antibiotic. In the presence of low concentrations a weakened wall may be formed, thus rendering the organisms more susceptible to lysis than at high concentrations of penicillin where wall synthesis abruptly ceases.

WE have confirmed the findings of Okamoto and Suzuki1 that a cell harbouring an R factor which carries streptomycin resistance can inactivate the drug by an enzymatic process. The inactivation can be detected either by a microbiological assay (Fig. 1), or by a biochemical assay (Table 1) following incubation of the drug with a cell-free extract of the R factor strain. The responsible enzyme, streptomycin adenylate synthetase, can be partially purified from cell-free extracts by ammonium sulphate precipitation and DEAE-‘Sephadex’ chromatography, but a more convenient source is the cell-free supernatant from exponentially growing cells submitted to osmotic shock according to the procedure of Nossal and Heppel2, and a highly active enzyme preparation can be obtained in this way. The enzyme is almost completely released by a 25 min shocking of the treated cells (Table 1a). The shockate enzyme has been purified further and the properties of the purified enzyme will be published later.

The cell wall peptidoglycan of vegetative cells of Bacillus sphaericus 9602 contains l -lysine and d -isoasparagine and is devoid of diaminopimelic acid (Dap), whereas the peptidoglycan of its spore cortex is devoid of l -lysine and d -isoasparagine and contains meso -Dap. These two structures have a common biosynthetic precursor, uridine-diphospho- N -acetylmuramyl- l - alanyl- d -glutamic acid, which accepts either l -lysine or meso -Dap, the latter reaction being the first unique to the synthesis of the spore cortex peptidoglycan. l -lysine-adding activity decays at the end of vegetative growth to a level which is maintained until Dap-adding activity appears, when it declines rapidly again. Dap-adding activity is not detectable in refractile spores, in vegetative cells, or in sporulating cells until about 4 hr after the end of vegetative growth, when it increases rapidly for about 1.5 hr in a process dependent on continued protein and ribonucleic acid (RNA) synthesis. This process apparently involves transcription and translation during this period of a “sporulation-specific” gene whose product is essential for and unique to sporulation. It is closely followed by the acquirement of refractility. Another sporulation-specific gene, that for dipicolinate synthase, is apparently transcribed and translated in an overlapping period commencing about 0.5 hr later, although dipicolinate does not accumulate rapidly until 1.5 hr later, when about 75% of the cells are already refractile. Inhibition of protein synthesis with chloramphenicol or of RNA synthesis with streptolydigin inhibited accumulation of these enzymes in sporulating cells; this inhibition could be reversed by washing out the antibiotics after 1.5 hr. Sporulation recommenced with an unaltered sequence of events but with poorer synchrony. There was no evidence for a messenger RNA for either enzyme of lifetime greater than a small fraction of the period of enzyme accumulation, although dilution with 10 volumes of fresh medium failed to prevent synthesis of Dap-adding enzyme in cells which had become terminally swollen, a process preceding enzyme synthesis by about 1.5 hr. The synthesis of this enzyme in B. sphaericus is apparently dependent on programmed transcription of the appropriate gene.

Temperature-sensitive (ts) mutants of Staphylococcus aureus with defective cell wall biosynthesis have been differentiated from other ts mutants by their ability to grow at the restrictive temperature (43 C) in the presence of 1 m NaCl. Under all conditions they possess normal colonial and cellular morphology at the level of resolution of the light microscope and are, therefore, not protoplasts. However, differences between mutant and wild-type cells can be seen by scanning electron microscopy. Many of the mutants contained concentrations of nucleotide precursors of peptidoglycan synthesis in excess of those present in wild-type cells, at both 30 and 43 C. The types of peptidoglycan precursors accumulated by six of the mutants have been determined, and specific enzymatic defects in three of these have been identified.

Bacillus sphaericus strain P-1 has previously been shown to have a tetragonally arrayed (T layer) protein which forms the outer layer of the cell wall. The T layer was quantitatively extracted from whole cells by 6 M urea, and the T layer subunits were purified by electrophoresis of the extracts on acrylamide gels containing 0.1% sodium dodecyl sulfate or 6 M urea. Using ethylene diacrylate cross-linked gels, the T layer was found to make up 16% of the total cellular protein. A virulent bacteriophage which is inactivated by purified T layer was isolated from soil. Twenty-four phage-resistant mutants were isolated, of which 17 had T layer subunits of increased mobility on sodium dodecyl sulfate acrylamide gels. No mutants devoid of T layer were found. Mutants were grouped into six classes according to the molecular weight of their T layer subunits. These ranged from that of the wild type, 150,000 down to 86,000. Two mutants from different classes were examined in detail. Cells of the mutant strains did not adsorb phage nor did cell walls isolated from these mutants inactivate phage. The amino acid composition of the T layers from mutants differed little from that of the wild-type T layer.

Autolysis of isolated cell walls of Staphylococcus aureus strain Copenhagen was accompanied by the release of 1 mole of N-terminal alanine per mole of glutamic acid. No other N-terminal amino acids and no C-terminal amino acids were released. These observations indicated that complete hydrolysis of N -acetylmuramyl- l -alanine linkages (“amidase” action) had occurred. This was confirmed by fractionation and analysis of the products. Hydrolysis of 4- O -β- N -acetylglucosaminyl- N -acetylmuramic acid linkages also occurred to a variable extent; on one occasion, complete degradation to disaccharides and hexosamine-free polypeptides (with intact pentaglycine cross-bridges) occurred. In one other instance, hydrolysis within pentaglycine bridges also occurred. Analyses of intact cell walls indicated that, in vivo, glycine endopeptidase activity was negligible and amidase activity was low, but that endo-β- N -acetylglucosaminidase hydrolysed about 8% of the N -acetylglucosaminyl- N -acetylmuramic acid linkages. Autolysis of isolated cell walls was too slow for the enzymes isolated with them to have significant action during this isolation. The possible functions of these autolytic activities are discussed.

Thesulfur-containing antibiotic thiolutin hasbeenshowntobea potent, reversible inhibitor ofthegrowth ofSaccharomyces cerevisiae. Viability was unaffected overtheconcentration rangeof4to100ug/ml. Atconcentrations as lowas2 sg/ml, thedruginhibited ribonucleic acid(RNA)andprotein synthesis inwholecells andspheroplasts. Atthese lowconcentrations, protein synthesis continued for ashort period oftimeafter RNAsynthesis was completely stopped. Withhigher drugconcentrations (greater than20;g/ml) protein synthesis was inhibited; concentrations ofthiolutin up to100,ug/ml didnotaffect translocation orpeptide bondformation incell-free protein-synthesizing systemsfromyeast. Theeffect ofthiolutin on theactivity ofpartially purified deoxyribonucleic acid-dependent RNA polymerases was examined, andthedrug was found tobea potentinhibitor ofRNA synthesis invitro. Inhibition was greatest whenthe polymerase was preincubated withthiolutin. Several mechanisms arediscussed toexplain themultiple effects ofthiolutin onS.cerevisiae. Since theaction ofthe drugiseasily reversed, thiolutin may provetobeofuse instudies ofvarious stages ofyeastgrowth. Inbacteria, antibiotics haveplayed useful roles inproviding genetic markers foranumber ofmacromolecules andhavealso beenusedin dissecting theroles ofmacromolecules inconcerted reactions. Theuseofantibiotics inyeast hasnotbeenexploited tothesameextent. This isdue,inpart, tothefactthatmanyofthe better-characterized antibiotics donotaffect Saccharomyces cerevisiae, presumably because theyeastcell isimpermeable tothese compounds. Manyantifungal compounds havebeen isolated during theprocess ofscreening for antimicrobial agents, and,although theymay notbemedically useful because oftheir toxicity properties, itisquite possible that someofthese compounds mayhavebiochemical applications. Thisreport describes ourinitial studies onthe modeofaction ofonesuchantifungal agent, thiolutin.

R-factor strains which are streptomycin-resistant and spectinomycin-sensitive have been found to inactivate streptomycin by a new mechanism. The 3′-OH group of the 2-deoxy-2-methylamino-l-glucopyranosyl moiety is phosphorylated.

Theplasmid determinants ofkiller phenotypes intypeK1andK2killer yeast cells arethe1.9-kilobase (kb) Mland1.7-kb M2double-stranded RNAs(dsRNAs), respectively. Thesearedependent fortheir maintenance andencapsidation, inSaccharomyces cerevisiae virusScV-M, orScV-M2virus-like particles, onthecapsid provided byoneofagroupofmoderately related 4.7-kb dsRNAscalled LA.TheLIAandL2AdsRNAsfound innaturally isolated K1andK2killers encode 88-kilodalton VLlA-P1 and86-kilodalton VL2A-Pl capsids, respectively. Thesearecompetent forencapsidating homologous LAdsRNAsaswellasM dsRNAs.Most strains ofS.cerevisiae, including killers, contain oneofasecond groupofclosely related 4.7-kb dsRNAscalied LBC.Theseencode their own82-kilodalton capsid protein, VLBC-P1, which, atleast instrains containing only LBc, encapsidates homologous dsRNAinScV-Lnc virus-like particles. InaKlkiller strain containing bothLlA andLBC,ScV-M, particles contain onlyVLIA-Pl. Insuchstrains itisprobable thateachvirus-like particle contains asingle capsid typeandthateachLdsRNAisencapsidated byahomologous capsid. Moststrains oftheyeast Saccharomyces cerevisiae contain species ofdouble-stranded RNA (dsRNA) called L(ca. 4.7kilobases), encapsidated incytoplasmic virus-like parti

Erythromycin-resistant (Eryr) mutants of Bacillus subtilis 168 fail to sporulate at high temperature (47 degrees C) but sporulate normally at 30 to 35 degrees C. They also fail to sporulate at any temperature in the presence of 2.5 micrograms of erythromycin per ml. Neither of these nonpermissive conditions appears to affect vegetative growth, and the periods of sensitivity to both conditions extend from 40 to 90% of the sporulation period. At 47 degrees C, net incorporation of methionine and phenylalanine in postexponential Eryr and 168 cells was similar, and fractionation of the labeled products by polyacrylamide gel electrophoresis gave patterns in which many of the bands produced by mutant and parental cells coincided. However, distinct differences were seen, and since no spore-specific morphogenesis occurred in the Eryr cells at 47 degrees C, a selective defect in spore gene expression was inferred. At 35 degrees C plus erythromycin, spore morphogenesis proceeded normally until forespores were produced and then ceased, coincident with a marked increase in sensitivity of total protein synthesis to erythromycin. The effects seem to be nonspecific, therefore, and may indicate a change in cell permeability or ribosomal sensitivity to erythromycin.

Synthesis of enzymes functional in the synthesis of nucleotide precursors of peptidoglycan ceases upon initiation of sporulation in Bacillus sphaericus. During sporulation, two periods of synthesis of these enzymes occur. The first starts at spore septum formation and is conincident with forespore engulfment; it involves the synthesis of those enzymes required for making the precursor of vegetative-type peptidoglycan, including L-lysyl ligase but no mesodiaminopimelyl ligase. The second period occurs shortly before the appearance of cortex. It involves the synthesis of diaminopimelyl ligase and the other enzymes needed for making the precursor of cortical peptidoglycan, but not lysyl ligase. Both events are a consequence of derepression at the level of transcription. Neither period of synthesis occurs in asporogenous mutants whose morphological block is at the point of spore septum formation.

In vivo transcripts of the L (4.5 kb) and M (1.9 kb) dsRNA plasmids were examined in type I killers of Saccharomyces cerevisiae. Transcripts for both plasmids include full-length (l,m) and partial-length (la,ma) single-stranded species. Both L-dsRNA transcripts (l,la) have in vitro mRNA activity for L-P1, previously shown to be identical to ScV-P1, the 88,000 dalton major capsid protein of the virus-like particles containing L- and M1-dsRNAs. 1, but not 1a, is bound to poly(U)-sepharose and may be polyadenylated. Other L-dsRNA gene products and their transcripts may exist. For M1-dsRNA, both species (m, ma) have in vitro mRNA activity for M1-P1, the 32,000 dalton pre-protoxin encoded by M1-dsRNA. Both m and ma are bound to poly(U)-Sepharose and ma is probably a 5' terminal fragment of m. A functional model for M1-dsRNA killer plasmid structure is presented.

SUMMARY: A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA +) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo + recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.

Particulate preparations from sporulating cells of Bacillus sphaericus 9602 contained an endopeptidase activity that hydrolyzed the γ- d -glutamyl-( l ) meso -diaminopimelic acid linkages found in the spore cortical peptidoglycan of this organism. Diaminopimelic acid did not occur in the vegetative cell wall peptidoglycan, and the γ- d -glutamyl- l -lysine linkages found in this polymer were not hydrolyzed by the endopeptidase. The endopeptidase hydrolyzed (X)- l -alanyl-γ- d -glutamyl-( l ) meso -diaminopimelyl( l )- d -alanyl- d -alanine only after removal of the terminal d -alanine residue. The preparations contained an acyl- d -alanyl- d -alanine carboxypeptidase I activity which converted such pentapeptides into substrates for the endopeptidase and which was inhibited 50% by 4 × 10 −7 M benzylpenicillin. This activity also hydrolyzed the analogous pentapeptide substrates containing l -lysine. The preparations also contained an acyl- l -lysyl- d -alanine carboxypeptidase II activity that was not active on the meso -diaminopimelic acid-containing analogue. Neither this activity nor the endopeptidase was inhibited by 10 −3 M benzylpenicillin. The specificities of the carboxypeptidases were consistent with the exclusive presence of l -lysine C-termini in the vegetative peptidoglycan and of meso -diaminopimelyl- d -alanine C-termini in the spore cortical peptidoglycan of B. sphaericus 9602.

Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.

Several properties of the major proteins degraded during germination of spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis have been compared. All of the proteins had low molecular weights (6,000 to 13,000) and lacked cysteine, cystine, and tryptophan. The proteins could be subdivided into two groups: group I (B. megaterium A and C proteins, B. cereus A protein, and B. subtilis alpha and beta proteins) and group II (B. cereus and B. megaterium B proteins and B. subtilis gamma protein). Species in group II had lower levels of (or lacked) the amino acids isoleucine, leucine, methionine, and proline. Similarly, proteins in each group were more closely related immunologically. However, antisera against a B. megaterium group I protein cross-reacted more strongly with the B. megaterium group II protein than with group I proteins from other spore species, whereas antisera against the B. megaterium group II protein cross-reacted most strongly with B. megaterium group I proteins. Analysis of the primary sequences at the amino termini and in the regions of the B. cereus and B. subtilis proteins cleaved by the B. megaterium spore protease revealed that the B. cereus A protein was most similar to the B. megaterium A and C proteins, and the B. cereus B protein and the B. subtilis gamma protein were most similar to the B. megaterium B protein. However, amino terminal sequences within one group of proteins varied considerably, whereas the spore protease cleavage sites were more highly conserved.

The M species (medium sized) dsRNA (1.1–1.4 × 10 6 daltons) isolated from a toxin-producing yeast killer strain (K + R + ) and three related, defective interfering (suppressive) S species dsRNAs of the yeast killer-associated cytoplasmic multicomponent viral-like particle system were analyzed by in vitro translation in a wheat germ cell-free protein synthesis system. Heat-denatured M species dsRNA programmed the synthesis of two major polypeptides, M-P1 (32,000 daltons) and M-P2 (30,000 daltons). M-P1 has been shown by the criteria of proteolytic peptide mapping and cross-antigenicity to contain the 12,000 dalton polypeptide corresponding to the in vivo produced killer toxin, thus establishing that it is the M species dsRNA which carries the toxin gene. An M species dsRNA obtained from a neutral strain (K − R + ) also programmed the in vitro synthesis of a polypeptide identical in molecular weight to M-P1, thus indicating that the cytoplasmic determinant of the mutant neutral phenotype is either a simple point mutation in the dsRNA toxin gene or a mutation in a dsRNA gene which is required for functional toxin production. In vitro translation of each of the three different suppressive S dsRNAs resulted in the production of a polypeptide (S-P1) of approximately 8000 daltons instead of the 32,000 dalton M-P1 polypeptide programmed by M dsRNA. This result is consistent with the heteroduplex analysis of these dsRNAs by Fried and Fink (1978), which shows retention of M dsRNA ends, accompanied by large internal deletions in each of the S dsRNAs translated.

Biochemical investigations of the mode of action of beta-lactam antibiotics have focused on the interaction of these drugs with sensitive enzymes and penicillin-binding proteins in vitro and on the correlation of these data with physiological responses to the drugs. The classical response is inhibition of growth and cell death followed by lysis; however, the bacteriostatic response to penicillins, which is seen in certain bacterial species such as Streptococcus mutans and in mutants of species such as Streptococcus pneumoniae, is also described. The biochemical data remain consistent with the acyl-D-alanyl-D-alanine donor substrate analogue theory, but multiple targets with differential sensitivity to different beta-lactam antibiotics exist. The relationship of these targets to penicillin-binding proteins and their possible functions in cell growth and division are discussed.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.

The type 1 killer phenotype in S. cerevisiae, mediated by secretion of an 11.5 kilodalton (kd) protein toxin, is cytoplasmically determined by the 1.9 kb M1-dsRNA plasmid. Maintenance of M1-dsRNA is dependent on the 4.5 kb L1-dsRNA because L1 encodes the capsid protein of the virus-like particles that separately encapsidate both dsRNA species. We have shown that in vitro translation of denatured M1-dsRNA produces M1-P1, a 32 kd protein containing the toxin peptides. We now demonstrate the presence of an unstable, 42 kd, membrane-associated, glycosylated protoxin in killer cells, probably derived from M1-P1 by cotranslational processing, and glycosylation. In vitro cotranslational processing of M1-P1, derived both from in vivo mRNAs and from denatured M1-dsRNA, produces a product resembling protoxin. Processing involves loss of 1.6 kd of protein, presumably an N-terminal leader peptide, and glycosylation. This information, together with data on in vitro expression of suppressive deletion mutants of M1-dsRNA, allows construction of testable models for the functional sequence of M1-P1 and for its maturation to toxin.

The staphylolytic enzyme recently isolated from cultures of a Chalaropsis species by Hash is shown to be an acetylmurainidase that cleaves all the glycosidic linkages of N-acetylmuramic acid and N,O-diacetylmuramic acid in the cell wall of Staphylococcus aureus strain Copenhagen. It is similar in specificity to the "32 enzyme" from Streptomyces albus but it differs from egg-white lysozyme whose activity is inhibited by the presence of O-acetyl groups.

Publisher Summary This chapter focuses on the mechanism of enzymes that degrade bacterial cell walls. The cell walls of all bacteria are composed of two or more polymers. They are ubiquituous in the bacterial world, are responsible for the shape and strength of the walls, and allow the cells to live in environmental conditions which are hypotonic with respect to their high intracellular osmotic pressure. These polymers are a glycopeptide built up of polyacetylhexosamine and peptide chains, and the action of all bacteriolytic enzymes. The basic structure of the peptide subunits is probably similar to that found in uridine diphosphate (UDP)-acetylmuramyl-peptide cell wall precursors. Some or all of the peptide chains linked to adjacent polysaccharide backbones are in turn linked to each other, resulting in a network of at least two dimensions. The digestion of different bacterial walls by various bacteriolytic enzymes gives rise to fragments of highly diverse nature and molecular complexity. A lyric intrapeptide hydrolase induces the appearance of new terminal amino and carboxyl groups. The enzymes that degrade the glycopeptide of bacterial cell walls and of which the specificity is at least partially known, are tabulated in the chapter.

Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo- N -acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others.

Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis , an N -acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C- and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one l -alanine residue. In sphere cell walls, l -alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C- and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall.