Morphological and physiological study of autolytic-defective Streptococcus faecium strains.

Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.     

pH induced damage and repair in E. coli

Escherichia coli lost its colony-forming ability when suspended in Tris/NaOH or Tris/Mg2+ buffers of pH 10.0 and 4.0, respectively. A significant decrease in the survival of radiation-sensitive mutants recA, polA, res, rer and lexA was observed as compared to their wild-type counterpart under these conditions. The alkali-injured cells were found to recover when incubated at 37 degrees C for 2 h in 0.05 M phosphate buffer of pH 8.0, whereas no such liquid holding recovery was observed in recA and lexA mutants. Recovery in phosphate buffer was not affected by metabolic inhibitors. As a result of alkali treatment, the sensitivity of bacteria to ultraviolet light (UV) was enhanced. However, on incubation for 2 h in recovery buffer at 37 degrees C, the bacteria regained partial UV resistance. Bacteria exposed to alkaline environment exhibited an enhanced level of mutagenesis. Contrary to the treated wild-type, the mutants recA and lexA did not exhibit any increase in the mutation frequency. Alkali treatment to GC----AT transition mutants of Salmonella typhimurium, TA102 and TA104 resulted in the highest number of revertants per plate.     

A mutant of Streptococcus pneumoniae that exhibits thermosensitive penicillin tolerance and the paradoxical effect

Mutants of Streptococcus pneumoniae that contain active autolysin and yet cannot be induced to lyse during treatment with penicillin (Lyt+Tol+ mutants) have been described. We have now shown that these mutants are temperature dependent (32 degrees C); at 37 degrees C these bacteria underwent penicillin-induced lysis. In addition, mutants at the lysis-permissive temperature showed the so-called 'paradoxical response' to penicillin. Temperature shift experiments indicated that the change from tolerant to lytic response or vice versa is a fast process. No differences were detected in autolysin specific activity or in the kinetics of inhibition of protein, peptidoglycan and teichoic acid syntheses in cells treated with penicillin at 32 and 37 degrees C. The results of genetic crosses indicated that the thermosensitivity of penicillin-induced autolysis in the Lyt+Tol+ mutants is not a property of the autolytic enzyme itself. The observations suggest that the thermosensitive process in the mutants represents either a step(s) in autolysin regulation or involves some difference in the structure of the cell walls produced at 32 degrees C versus 37 degrees C.     

Autolysis of psychrophilic bacteria from marine fish.

Two psychrophillic bacterial isolates of marine fish origin unable to grow at 20 degrees C or above were found to be distinguishable on the basis of autolysis at elevated temperature in various buffer systems. Isolate OP2 exhibited autolysis at 30 degrees C and above, while isolate OP7 underwent autolysis only at 35 degrees C and above. Tris buffer at pH 7.0 and 8.0 and at 35 degrees C significantly protected isolate OP2 from autolysis and failed to do so with isolate OP7. At pH 5.0, suspension phosphate buffer resulted in significantly greater autolysis of both isolates than did suspension in succinate buffer.     

Autolysis of psychrophilic bacteria from marine fish

Two psychrophillic bacterial isolates of marine fish origin unable to grow at 20 degrees C or above were found to be distinguishable on the basis of autolysis at elevated temperature in various buffer systems. Isolate OP2 exhibited autolysis at 30 degrees C and above, while isolate OP7 underwent autolysis only at 35 degrees C and above. Tris buffer at pH 7.0 and 8.0 and at 35 degrees C significantly protected isolate OP2 from autolysis and failed to do so with isolate OP7. At pH 5.0, suspension phosphate buffer resulted in significantly greater autolysis of both isolates than did suspension in succinate buffer.     

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.     

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.     

The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol

Treatment of Streptococcus faecium ATCC 9790 with 3.5 micrograms/ml of dodecylglycerol produces a nonwall entity found in the 25,000 X g supernatant cell fraction which activates the autolysin activity of S. faecium. The stimulation of the autolysin activity by dodecylglycerol mimics the activation of the autolysin from a latent to an active form by trypsin and other proteolytic enzymes. This stimulation of autolytic activity by dodecylglycerol can be reversed by specific proteinase inhibitors. Dodecylglycerol also markedly stimulates the proteinase activity endogenous to S. faecium, and this stimulation can be reversed by several proteinase inhibitors. It is concluded that one primary antibacterial mode of action of dodecylglycerol is to stimulate the proteinase of S. faecium which activates the cell's autolysin and thereby prevents bacterial growth.     

Programmed death in bacteria.

Programmed cell death (PCD) in bacteria plays an important role in developmental processes, such as lysis of the mother cell during sporulation of Bacillus subtilis and lysis of vegetative cells in fruiting body formation of Myxococcus xanthus. The signal transduction pathway leading to autolysis of the mother cell includes the terminal sporulation sigma factor Esigma(K), which induces the synthesis of autolysins CwlC and CwlH. An activator of autolysin in this and other PCD processes is yet to be identified. Autolysis plays a role in genetic exchange in Streptococcus pneumoniae, and the gene for the major autolysin, lytA, is located in the same operon with recA. DNA from lysed cells is picked up by their neighbors and recombined into the chromosome by RecA. LytA requires an unknown activator controlled by a sensory kinase, VncS. Deletion of vncS inhibits autolysis and also decreases killing by unrelated antibiotics. This observation suggests that PCD in bacteria serves to eliminate damaged cells, similar to apoptosis of defective cells in metazoa. The presence of genes affecting survival without changing growth sensitivity to antibiotics (vncS, lytA, hipAB, sulA, and mar) indicates that bacteria are able to control their fate. Elimination of defective cells could limit the spread of a viral infection and donate nutrients to healthy kin cells. An altruistic suicide would be challenged by the appearance of asocial mutants without PCD and by the possibility of maladaptive total suicide in response to a uniformly present lethal factor or nutrient depletion. It is proposed that a low rate of mutation serves to decrease the probability that asocial mutants without PCD will take over the population. It is suggested that PCD is disabled in persistors, rare cells that are resistant to killing, to ensure population survival. It is suggested that lack of nutrients leads to the stringent response that suppresses PCD, producing a state of tolerance to antibiotics, allowing cells to discriminate between nutrient deprivation and unrepairable damage. High levels of persistors are apparently responsible for the extraordinary survival properties of bacterial biofilms, and genes affecting persistence appear to be promising targets for development of drugs aimed at eradicating recalcitrant infections. PCD in unicellular eukaryotes is also considered, including aging in Saccharomyces cerevisiae. Apoptosis-like elimination of defective cells in S. cerevisiae and protozoa suggests that all unicellular life forms evolved altruistic programmed death that serves a variety of useful functions.     

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.     

Effects of different buffers on the thermostability and autolysis of a cold-adapted protease MCP-01

A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913. The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied. The autolysis process of MCP-01 was studied by capillary electrophoresis. The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate. The optimum temperature for casein hydrolysis also increased in the same order. This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species. The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products. In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different. These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface. In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.     

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.     

Effect of incubation media on the recovery of Escherichia coli K12 heated at 52 degrees C.

The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.     

Autolytic Enzyme System of Streptococcus faecalis III. Localization of the Autolysin at the Sites of Cell Wall Synthesis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.     

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.     

Factors affecting malate dehydrogenase activity in freezing-thawing processes

Malate dehydrogenases from several sources show different behaviour when frozen-thawed in 100 mM sodium phosphate buffer, pH 7.4, containing chaotropic ions. The effects produced by the addition of various metabolites, protein concentration and buffer medium used on the loss of activity induced by the freezing-thawing process are reported. The major part of the loss of activity is caused by the formation of "wrong" aggregates of high mol. wt.     

Cell wall autolysis in log phase cells of Micrococcus lysodeikticus (luteus).

Log phase cells of Micrococcus lysodeikticus (luteus) IFO 3333 autolyzed when incubated at 37 C in 0.01 M sodium-phosphate buffer pH 7.5. The enzyme involved in the autolysis was recovered mainly in an aqueous phase from cytoplasmic membranes and cytoplasmic materials treated with n-butanol, and proved to be an N-acetylmuramyl-L-alanine amidase. The autolysis of log phase cells suspended in autolyzing buffer was depressed by the addition of trypsin to the buffer.     

Increased production of PGI2-like activity by frozen-thawed rat aorta.

The effects of storage conditions, temperature, and time on the ability of the rat thoracic aorta to produce a platelet aggregation inhibitor were investigated. Aortic fragments were incubated in Tris buffer, aliquots of which were then tested for their ability to inhibit ADP-induced human platelet aggregation. The incubation fluid of samples that had been soaked in Tris buffer at 4 degrees C for 24 hours contained no inhibitor activity, whereas the incubation fluid of similar samples that had been kept at 4 degrees C but not soaked in buffer contained comparable inhibitor activity as that of fresh samples. The incubation fluid of samples that had been kept at -20 degrees C or -80 degrees C contained greater inhibitor activity than that of fresh samples, and was maintained in -20 degrees C samples for 7 days, and -80 degrees samples for 28 days. The aortic inhibitor had similar properties as PGI2.     

Programmed Death in Bacteria

Programmed cell death (PCD) in bacteria plays an important role in developmental processes, such as lysis of the mother cell during sporulation of Bacillus subtilis and lysis of vegetative cells in fruiting body formation of Myxococcus xanthus. The signal transduction pathway leading to autolysis of the mother cell includes the terminal sporulation sigma factor Eς^K, which induces the synthesis of autolysins CwlC and CwlH. An activator of autolysin in this and other PCD processes is yet to be identified. Autolysis plays a role in genetic exchange in Streptococcus pneumoniae, and the gene for the major autolysin, lytA, is located in the same operon with recA. DNA from lysed cells is picked up by their neighbors and recombined into the chromosome by RecA. LytA requires an unknown activator controlled by a sensory kinase, VncS. Deletion of vncS inhibits autolysis and also decreases killing by unrelated antibiotics. This observation suggests that PCD in bacteria serves to eliminate damaged cells, similar to apoptosis of defective cells in metazoa. The presence of genes affecting survival without changing growth sensitivity to antibiotics (vncS, lytA, hipAB, sulA, and mar) indicates that bacteria are able to control their fate. Elimination of defective cells could limit the spread of a viral infection and donate nutrients to healthy kin cells. An altruistic suicide would be challenged by the appearance of asocial mutants without PCD and by the possibility of maladaptive total suicide in response to a uniformly present lethal factor or nutrient depletion. It is proposed that a low rate of mutation serves to decrease the probability that asocial mutants without PCD will take over the population. It is suggested that PCD is disabled in persistors, rare cells that are resistant to killing, to ensure population survival. It is suggested that lack of nutrients leads to the stringent response that suppresses PCD, producing a state of tolerance to antibiotics, allowing cells to discriminate between nutrient deprivation and unrepairable damage. High levels of persistors are apparently responsible for the extraordinary survival properties of bacterial biofilms, and genes affecting persistence appear to be promising targets for development of drugs aimed at eradicating recalcitrant infections. PCD in unicellular eukaryotes is also considered, including aging in Saccharomyces cerevisiae. Apoptosis-like elimination of defective cells in S. cerevisiae and protozoa suggests that all unicellular life forms evolved altruistic programmed death that serves a variety of useful functions.     

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).