Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

Physicochemical Effects of Long Chain Fatty Acids on Bacterial Cells and their Protoplasts

S ummary . Fatty acids of chain length > C₁₀ induced lysis of protoplasts at pH 7·4 when the concentration was nearly bactericidal. At pH 6, lauric and linoleic acids produced lysis above bactericidal concentrations but, at pH 8, lysis was produced by the same acids below bactericidal concentrations. The lysis was immediate at pH 8, but at pH 6 the effect was preceded by contraction of protoplasts. At pH 7·4 the order of lytic activity between individual fatty acids was similar to that of bactericidal activity and the response of protoplasts of Bacillus megaterium relative to those of Micrococcus lysodeikticus reflected differences in bactericidal sensitivity though whole cells were much less sensitive to fatty acid-induced leakage effects than protoplasts. Reversal agents antagonized the lysis of protoplasts by fatty acids. A physicochemical basis for the action of fatty acids and reversal agents on protoplasts and whole cells is discussed.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

The involvement of protein kinase A in the immune response of Galleria mellonella larvae to bacteria

The role of protein kinase A (PKA) in the humoral immune response of the greater wax moth Galleria mellonella larvae to live gram-positive bacteria Micrococcus lysodeikticus and gram-negative bacteria Escherichia coli was investigated. The immune challenge of larvae with both kinds of bacteria caused an increase in fat body PKA activity depending on the injected bacteria. Gram-positive M. lysodeikticus was a much better inducer of the enzyme activity than gram-negative E. coli. The PKA activity was increased about 2.5-fold and 1.5-fold, after M. lysodeikticus and E. coli injection, respectively. The in vivo inhibition of the enzyme activity by a cell permeable selective PKA inhibitor, Rp-8-Br-cAMPS, was correlated with considerable changes of fat body lysozyme content and hemolymph antimicrobial activity in bacteria-challenged insects. The kinetics of changes were different and dependent on the bacteria used for the immune challenge of G. mellonella larvae.     

Effect of polyene antibiotics on bacterial protoplasts

The carbonyl-conjugated pentaenes flavofungin, nigrofungin and flavopentin exhibit considerable lytic activity toward Micrococcus lysodeikticus and Bacillus megaterium protoplasts. The antibiotics at concentrations of 5 to 14 microgram/ml cause lysis of 50% of the protoplasts within 15 min of their incubation. The antibiotics inhibit the activity of NADH oxidase and malate oxidase by 50% in the lysates of Micrococcus lysodeikticus and Bacillus megaterium protoplasts at concentrations of 30 to 50 microgram/ml; preincubation of the lysates with the antibiotics intensify the inhibiting action of the polyenes. Growth of the bacteria is inhibited when the minimal concentration of the polyenes is 75 to 100 microgram/ml. Interaction of the polyenes with bacterial membranes lacking sterols indicates that resistance of at least some bacteria to polyenes is caused by impermeability of the cell wall for these substances rather than by the absence of sterols in the membranes.     

Effect of gramicidin S and its derivatives on protoplasts of Bacillus subtilis

The lysis of Bacillus subtilis protoplasts by gramicidin S, a membrane active antibiotic, and its derivatives was studied according to free amino groups of the ornithine residue. The initial antibiotic and guanylgramicidin , a positive charge-preserving derivative, had a high lytic activity. Succinylgramicidin , a gramicidin S derivative with acid properties, and carbomoylgramicidin , a neutral derivative, actively lysed B. subtilis protoplasts suspended in 1/15 M phosphate buffer solution with sucrose . No lytic activity of succinylgramicidin was observed with respect to B. subtilis protoplasts suspended in an aqueous solution of sucrose. Comparative study on the sensitivity of the protoplasts of Micrococcus lysodeikticus, B. megaterium and B. subtilis to the lytic action of gramicidin S and its derivatives showed in the main a similar character of their interaction with the membranes of the protoplasts of the taxonomically close species (B. megaterium and B. subtilis). It is likely that the specificity of the action of the above substances on the protoplasts of M. lysodeikticus, i. e. a complicated character of the dependence of the lytic action of gramicidin S on its concentration, manifestation of the lytic activity of the neutral and acid derivatives in the presence of phosphates or other salts and in sucrose aqueous solution was mainly defined by the properties of the micrococcal membranes.     

Bactericidal activities of rat defensins and synthetic rabbit defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (mucoid and nonmucoid strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS mutants) and Escherichia coli.

Rat defensins were purified and tested for in vitro bactericidal assay against gram-positive and gram-negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram-negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20 min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40 min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins. Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.     

Antibacterial nitroacridine, Nitroakridin 3582: effects on bacterial growth and macromolecular biosynthesis in vivo

The antibacterial drug Nitroakridin 3582 inhibited the growth of selected grampositive bacteria more strongly than it inhibited the growth of gram-negative bacilli. Nitroakridin at concentrations of the order of 5 x 10(-5)m induced lysis of Bacillus licheniformis and Micrococcus lysodeikticus. At concentrations less than 10(-4)m, Nitroakridin 3582 reduced the exponential growth rate of Escherichia coli C-2; at 10(-4)m the drug was bacteriostatic, and, at concentrations greater than 10(-4)m, it was bactericidal. Prolonged bacteriostasis resulted in the formation of long filaments by E. coli, Serratia marcescens, Shigella sonnei, and Proteus mirabilis. The reversible effects of Nitroakridin 3582 on the growth of E. coli correlated with partial inhibitions of deoxyribonucleic acid biosynthesis; ribonucleic acid and protein syntheses were inhibited less strongly. Nitroakridin 3582 at concentrations greater than 2 x 10(-4)m, which block deoxyribonucleic acid biosynthesis, produced an accelerated bactericidal action.     

STABILIZATION OF PROTOPLASTS AND SPHEROPLASTS BY SPERMINE AND OTHER POLYAMINES.

Tabor, Celia W. (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.). Stabilization of protoplasts and spheroplasts by spermine and other polyamines. J. Bacteriol. 83:1101-1111. 1962.-Spermine (10(-3)m) or spermidine prevents lysis of lysozyme-produced protoplasts of Escherichia coli W, E. coli B, and Micrococcus lysodeikticus in hypotonic media. Spheroplasts prepared by the action of penicillin are also stabilized by these concentrations of spermine and spermidine, but the protection is not as complete. Streptomycin, polylysine, and Ca(++) are also effective or partially effective stabilizers, but 1,4-diaminobutane, 1,5-diaminopentane, ornithine, Mg(++), and monovalent cations have no protective action at 10(-3)m concentration, and only a slight effect at higher concentrations. The osmotic stability conferred on protoplasts by spermine is irreversible. However, the protective effect of polyamines against lysis is not accompanied by restoration of viability to lysozyme protoplasts. There is a marked reduction in the loss of ultra-violet-absorbing material from the protoplasts to the medium when 10(-3)m spermine is present.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.     

Effect of salts on the lytic activity of gramicidin S and its derivatives

Potassium and sodium chlorides, sulfates, acetates and phosphates activated the lytic action of gramicidin S and its derivatives on protoplasts of M. lysodeikticus. The derivatives used were positively charged and neutral by the free amino groups in the ornithine moieties. The salts had no effect on lysis of the bacillar protoplasts by gramicidin S and its positively charged derivatives. The lytic effect of the neutral derivative on the bacillar protoplasts markedly increased in the presence of the salts, activation of the lysis by the phosphates being more pronounced than that by the other salts. Increased membrane activity of gramicidin S in the presence of the salts was not connected with association of the substance molecules in solution. Probably it was due to increased destruction of the membranes at the account of activated detergent effect of the antibiotic and its derivatives.     

Study of the adaptation resistance in bacteria to membrane active polypeptide antibiotics

Variants of Micrococcus lysodeikticus resistant to 100 micrograms/ml of gramicidin S with preserved resistance in subcultures on media without the antibiotic were isolated as a result of prolonged adaptation on a solid medium with increasing concentrations of gramicidin. The sensitive and resistant cells did not differ by their ability to bind gramicidin. Under the antibiotic effect permeability of the cytoplasmic membranes of the intact cells in the sensitive bacteria appeared to be impaired to a greater extent than that of the membranes of the cells in the resistant variant. Comparison of the lytic activity of gramicidin and its derivatives with respect to the protoplasts prepared with the cells of the initial and resistant variants of M. lysodeikticus revealed much higher resistance of the resistant variant protoplasts to the membrane-disorganizing effect of the preparations. Malate dehydrogenase and NADH-oxidase in the membrane preparations of the resistant variant cells differed from analogous enzymes from the membranes of the initial strain by the levels of their activity and sensitivity to gramicidin. It is likely that during adaptation of M. lysodeikticus to gramicidin significant changes in the cell cytoplasmic membranes occurred.     

Lysis of bacterial protoplasts and spheroplasts and suppression of their dehydrogenase activity by neotehomycin]

Neotelomycin induced lysis of the protoplasts of Bac. megaterium and inhibited their succinate dehydrogenase activity. Direct correlation between the lytic activity of the antibiotic and its effect on succinate dehydrogenase was found. Neotelomycin had no effect on the dehydrogenase activity of the protoplast lysates. Possibly, suppression of the protoplast succinate dehydrogenase of Bac. megaterium under the effect of neotelomycin was due to significant structural changes caused by the antibiotic in the protoplast membranes and leading to their lysis and not to the direct effect on the enzyme. Neotelomycin had practically no effect on the spheroplast dehydrogenase activity of E. coli resistant to the antibiotic and did not induce their lysis. Resistance of E. coli to neotelomycin must be associated not with the presence of the antibiotic non-permeable cell wall but the peculiar properties of the membrane cytoplasm.     

Comparative effectiveness of antimicrobial action of antiseptics against pathogens of chronic purulent otitis media]

Comparable antimicrobial and disinfecting action of decamethoxine and silver preparations on pathogens of chronic purulent otitis media (CPOM) was studied. The clinical isolates of staphylococci proved to be most sensitive to decamethoxine whose MBcC conformed to 16.5 micrograms/ml. The antimicrobial action on Proteus spp. and Pseudomonas aeruginosa was less pronounced. The required concentrations for bactericidal action on these pathogens were 69 and 93.5 micrograms/ml, respectively. The antimicrobial activity of the silver preparations such as poviargol, collargol and protargol was low. Depending on the microbial species, the bactericidal effect of the silver preparations was 12-235 times lower than that of decamethoxin. It was also shown that decamethoxin had a high disinfecting action on CPOM pathogens. It was noted that decamethoxin had a marked ability to increase the bactericidal action of poviargol (by 2-14 times) and its disinfecting action (by 2 times) on Proteus spp., E. coli and Ps. aeruginosa.     

Variation of cell membrane lipid composition by means of lipid transfer proteins. Properties of the protoplast membrane of Micrococcus lysodeikticus after incorporation of phosphatidylcholine

After incorporation of phosphatidylcholine (PC) into the protoplast membrane of M. lysodeikticus by protein mediated transfer from PC liposomes, the activity of some membrane bound respiratory chain enzymes was studied. It was found that incorporation of PC decreases the rates of oxidation of exogenous substrates (NADH, malate) but the level of endogenous respiration was not changed. Ferricyanidreductase activity of ghosts of M. lysodeikticus was not dependent upon the PC content of protoplasts. PC containing protoplasts showed a higher osmotic stability than unmodified protoplasts. It is concluded that the incorporation of PC into the protoplasts results in resealing, i. e. in the repair of local defects in the protoplast membrane.     

Immunological Properties of Micrococcus lysodeikticus Membranes

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.     

Effect of Lytic Enzymes of Acanthamoeba castellanii on Bacterial Cell Walls

Extracts of Acanthamoeba castellanii (Neff) contain alpha- and beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, amylase, and peptidase. All of these activities are optimal between pH 3 and 4. These extracts also were found to clarify suspensions of cell walls from nine different gram-positive bacteria, including Micrococcus lysodeikticus. The pH optimum for the lytic activity was between 3 and 4. The extent of lysis of the various cell walls did not correlate with the release of free amino groups and of free N-acetylated sugars from the walls during digestion with these extracts. Suspensions of cell walls of Escherichia coli (a gram-negative bacterium), Cordiceps militaris (a fungus), and Acanthamoeba cysts, as well as of colloidal chitin, were not clarified by incubation with these extracts, although reducing sugars were released from each of these materials. Exhaustive digestion of M. lysodeikticus walls by lysozyme released no free N-acetylglucosamine. The products of exhaustive digestion of this cell wall with Acanthamoeba extracts were free N-acetylglucosamine, free N-acetylmuramic acid, glycine, alanine, glutamic acid, lysine, and N-acetylmuramic acid peptide fragments. These results suggest that the amoeba extracts contain endo- and exo-hexosaminidases, in addition to beta-hexosaminidase and peptide hydrolases.     

Biotinylation facilitates the uptake of large peptides by Escherichia coli and other gram-negative bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin.

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.     

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.