The action of tomicide on macromolecular synthesis in bacterial cells]

The study of the rate of incorporation of labeled precursors for nucleic acids and protein into Staphylococcus aureus 209 P cell fraction, insoluble in trichloroacetic acid, has revealed that in the presence of tomicide in the medium in a dose of 1 MCI (600 micrograms/ml) the synthesis of DNA in inhibited rapidly and almost completely (by 90%). The inhibition of the rate of incorporation of 3H-thymidine into the cells of staphylococcal culture by tomicide directly correlates with the concentration of the preparation within the range 100-600 micrograms/ml, the inhibition of the synthesis of RNA and protein being less pronounced than the inhibition of the synthesis of DNA.     

Effect of salvin on the synthesis of macromolecules in Staphylococcus aureus 209P

The effect of salvin and its active component, carnazolic acid, on the synthesis of macromolecular compounds in the cells of S. aureus 209P was studied. It was shown that the inhibitory action of salvin on the synthesis of peptidoglycane in the culture was defined by the presence of carnazolic acid in its composition. In the bactericidal concentration, carnazolic acid was twice as less active as salvin and inhibited incorporation of labeled precursors into RNA and protein. The findings grounded the conclusion that some nonidentified components of salvin with low antimicrobial activity contained in it in insignificant quantities had an additional inhibitory effect on the process. Comparative study of salvin and antibiotics with the known mechanisms of action such as benzyl penicillin or chloramphenicol revealed a certain similarity in the action of salvin and benzyl penicillin on incorporation of labeled precursors into the macromolecular compounds of S. aureus 209P.     

Effect of Ammonium, Sucrose and Light on the Regulation of Nitrate Reductase Level in Pisum sativum

In shoot apices of 7-day-old dark-grown peas the addition of ammonium along with the inducer nitrate resulted in a more than two-fold increase in nitrate reductase activity. Individual amino acids, amides and amino-acid mixture could not replace the ammonium effect. Ammonium also stimulated NADH-glutamate dehydrogenase but not glucose-6-phosphate dehydrogenase. Sucrose caused a marked stimulation of nitrate reductase induction and showed synergistic effect with light. In presence of cordycepin and cycloheximide, induction of nitrate reductase was inhibited more if ammonium or sucrose was supplied along with the inducer. With actinomycin D, α-amanitin or chloramphenicol, no differential inhibition took place in presence of ammonium. The inhibition of enzyme activity by chloramphenicol and 3-(3,4-dichlorophenyl)-l,dimethyl urea was completely relieved by sucrose. Incorporation of ¹⁴C-lysine was markedly stimulated by sucrose, but was not affected by ammonium. The effect of sucrose and light on ¹⁴C-lysine incorporation was additive. Cordycepin and cycloheximide did not have any differential effect on ¹⁴C-lysine incorporation in the presence of ammonium as well as sucrose. The inhibition of ¹⁴C-lysine incorporation caused by chloramphenicol was relieved by sucrose. Sucrose also caused a marked increase in ³H-uridine incorporation but ammonium had no effect. Actinomycin D and cordycepin blocked the sucrose dependent increase in ³H-uridine incorporation. The results suggest that ammonium mediated stimulation may depend on a regulatory protein(s) synthesized in response to ammonium, whereas sucrose acts mainly by an overall increase in RNA and protein synthesis. The effect of light does not seem to be dependent on photosynthetic light reactions.     

Studies on bacterial cell wall inhibitors III. 3-amino-3-deoxy-D-glucose,an inhibitor of bacterial cell wall synthesis

It was shown that 3-amino-3-deoxy-D-glucose, one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., inhibits the bacterial synthesis of cell wall. The antibiotic (100 μg/ml) significantly inhibits the growth of Straphylococcis aureus FDA 209P as well as the incorporation of DL-[¹⁴C]alanine into the acid-insoluble macromolecular fraction of its growing cells in the presence of chloramphenicol (100 μg/ml). In contrast, the antibiotic doed not affect the incorporation of [³H]thymidine, [³H]uridine and L-[¹⁴C]leucine. The other constituents of kanamycin, 6-amino-6-deoxy-D-glucose and deoxystreptamine do not inhibit the synthesis of bacterial cell wall peptidoglycan.     

Differential methicillin susceptibilities of peptidoglycan syntheses in methicillin-resistant Staphylococcus aureus.

The mechanism of staphylococcal resistance to methicillin is unknown. Peptidoglycan synthesis was studied in a methicillin-resistant and a derived methicillin-sensitive Staphylococcus aureus strain. Although the methicillin minimum inhibitory concentration for growth of the methicillin-resistant strain was 1,600 micrograms/ml, peptidoglycan synthesis by the organism incubated in a wall synthesis solution was inhibited about 90% by 5 micrograms of methicillin per ml. In contrast, high concentrations of methicillin added to actively growing cultures of the methicillin-resistant strain had little effect on growth or peptidoglycan synthesis. Peptidoglycan synthesis in chloramphenicol-treated cultures was more susceptible to methicillin than it was in actively growing cultures of the methicillin-resistant strain. It is proposed that in this strain cell wall thickening peptidoglycan synthesis which predominates in cell wall synthesis solution and chloramphenicol-treated cultures is methicillin sensitive, whereas peptidoglycan synthesis involved in cell division, primarily in the region of the septum, which predominates in actively growing cultures is methicillin resistant. Both cell wall thickening and septal peptidoglycan syntheses are methicillin sensitive in the methicillin-sensitive strain.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.     

The effect of salvin on the growth and ultrastructure of Staphylococcus aureus 209P

Antibiotic salvin obtained from Salvia officinalis has been studied for its effect on the growth and ultrastructure of Staphylococcus aureus 209P. The antibiotic in the sub-bacteriostatic concentration considerably elongates the lag-phase (up to 11-12 h) exerting no significant effect on the growth rate of the staphylococcus population as well as it prolongs duration of the exponential phase. The analysis of electronograms of staphylococcus cells subjected to the action of salvin in the concentrations similar to the minimal inhibitory concentration (MIC), has revealed the cell thinning, inhibition and destruction of the division. The introduction of 5MIC antibiotic into the exponentially grown culture made a cell wall considerably thinner, destructing its external layer; the number of lyzed cells sharply increased. The appearance of bodies not described previously with a membrane envelope and ribosomes as well as of mesosomal structures was observed.     

Penicillin-insensitive incorporation of D-amino acids into cell wall peptidoglycan influences the amount of bound lipoprotein in Escherichia coli.

Certain D-amino acids, such as D-methionine and D-cystine, were incorporated into cells of Escherichia coli under conditions inhibiting protein and cell wall synthesis. Part of the radioactivity of D-14C-amino acids incorporated into the cells was found in the isolated cell wall peptidoglycan. A covalent linkage between the amino group of the D-amino acids and the peptidoglycan was presumed to be the main cause of the binding of the D-amino acids to peptidoglycan, because the amino group of the D-amino acids in the incorporation product was substituted. Whether the carboxyl terminus was substituted was unknown. The formation of the D-amino acid-peptidoglycan linkage was insensitive to beta-lactam antibiotics such as benzylpenicillin and ampicillin (500 micrograms/ml) and therefore was not due to the reaction of DD-transpeptidation which is involved in the biosynthesis of peptidoglycan. The D-amino acids also strongly inhibited the formation of peptidoglycan-bound lipoprotein in the E. coli cells. The results may suggest the correlation between binding of D-amino acid to peptidoglycan and inhibition of formation of the bound form of lipoprotein.     

Biosynthesis of peptidoglycan in Gaffkya homari. The mode of action of penicillin G and mecillinam.

The effect of the beta-lactam antibiotics penicillin G and mecillinam on the incorporation of peptidoglycan into pre-formed cell wall peptidoglycan was studied with wall membrane enzyme preparations from Gaffkya homari. Using UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) as precursors the incorporation of peptidoglycan into the pre-existing cell wall of G. homari was inhibited to an extent of 50% (ID50 value) at a concentration of 0.25 mug of penicillin G/ml. With UDP-GlcNAc and UDP-MurNAc-tetrapeptide as precursors the ID50 value was about 2500-fold greater (630 mug/ml). The inhibition by penicillin G of the incorporation of peptidoglycan from UDP-MurNAc-[14C]Lys-pentapeptide could be overcome by addition of non-radioactive UDP-MurNAc-tetrapeptide to the incubation mixture. In the presence of 5 mug of penicillin G/ml the incorporation of peptidoglycan formed from the mixture of UDP-MurNAc-Ala-DGlu-Lys-D-[14C]Ala-D[14C]Ala and non-radioactive UDP-MurNAc-tetrapeptide proceeded virtually without release of D-[14C]alanine by transpeptidase activity. The enzyme preparation also exhibited DD-carboxypeptidase activity which was only slightly more sensitive to penicillin G and mecillinam than was the incorporation of peptidoglycan into the cell wall. Since the ID50 values for the beta-lactam antibiotics are similar to the concentrations required to inhibit the growth of G. homari to an extent of 50%, the DD-carboxypeptidase must be the killing site of both penicillin G and mecillinam.     

The site of inhibition of cell wall synthesis by 3-amino-3-deoxy-D-glucose in Staphylococcus aureus.

The inhibition of growth and cell wall synthesis by 3-amino-3-deoxy-D-glucose (3-AG), which is known to be one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., was almost completely overcome by glucosamine and N-acetylglucosamine in Staphylococcus aureus but scarcely affected by D-glucose and D-fructose. The antibiotic did not inhibit the incorporation of [14C]glucosamine and [3H]N-acetylglucosamine into the acid-insoluble fraction, but rather enhanced the incorporation of [14C]glucosamine. On the other hand, it inhibited the incorporation of D-[14C]fructose into the cell wall fraction but hardly affected the incorporation of D-[14C]fructose into the acid-insoluble fraction in the presence of pencillin G. Based on these results, it is suggested that the site of primary action of 3-AG is the formation of glucosamine-6-phosphate from D-fructose-6-phosphate, which is catalyzed by glucosamine synthetase [EC 2.6.1.16].     

Carbon Dioxide Fixation by Cells of Streptococcus faecalis var. liquefaciens

Fixation of NaH(14)CO(3) by a heavy cell suspension of Streptococcus faecalis var. liquefaciens was studied. Several nutrients, pyridoxal, riboflavine, adenine, uracil, and O(2) stimulated (14)CO(2) incorporation into cells only under conditions that were adequate for synthesis of cell macromolecules. Biotin increased CO(2) incorporation in the absence of extensive synthesis of macromolecules, whereas O(2) inhibited incorporation under these conditions. When (14)CO(2) fixation was occurring during synthesis of macromolecules, 71% of the (14)C was incorporated into cells and 29% occurred extracellularly. Ninety-three per cent of the cellular (14)C was in protein and 5.5% was in nucleic acid. Aspartic acid was the only amino acid in the protein fraction that was radioactive. Eighty-three per cent of the extracellular (14)C was resistant to precipitation by trichloroacetic acid. When (14)CO(2) fixation was occurring in cells that were not carrying on extensive synthesis of macromolecules, 38% of the (14)C was incorporated into cells and 59% occurred in the supernatant fluid. Sixty-nine per cent of the cellular (14)C was in protein, 21% was in low-molecular-weight compounds, and 9% was in nucleic acid. Addition of unlabeled aspartate to the medium inhibited incorporation of (14)CO(2). Based on studies of the rate of (14)CO(2) fixation, the cells fix CO(2) into a pool of intermediates which are either used for synthesis, primarily protein, or are excreted into the medium.     

Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

The Effect of Cycloheximide (Actidione) on Protein and Nucleic Acid Synthesis by Chlorella

Incorporation of ¹⁴C-phenylalanine, ¹⁴C-carbon dioxide, ¹⁴C-glucose,and ¹⁴C-glycine into the protein of Chlorella is inhibited bycycloheximide. A concentration of 2.5 µg per ml inhibitsincorporation by about 80 per cent; increasing the concentrationup to 10 µg per ml does not increase the degree of inhibition.The incorporation of ¹⁴C-adenine into ribonucleic acid (RNA)and deoxyribonucleic acid (DNA), and of ¹⁴C-glucose into polysaccharideis also inhibited. Unlike inhibition of protein synthesis, thatof nucleic acid and polysaccharide synthesis is observed onlyafter some delay. The delay is shortest for DNA synthesis andlongest for polysaccharide synthesis. Inhibition of ¹⁴C-glycineincorporation into DNA and RNA follows a similar pattern tothat obtained with ¹⁴C-adenine but the delay is much shorter.Cycloheximide also inhibits the formation of isocitrate lyasc(isocitrate-glyoxylate lyase, EC 4.1.3.1 [EC] ) when autotrophicallygrown cells are supplied with acetate.     

Action of tetracycline and sodium deoxycholate combinations on protein and nucleic acid synthesis in the cells of the NAG vibrio, Staphylococcus aureus and Escherichia coli]

The effect of tetracycline combination with sodium desoxycholate, a surface-active substance, on the synthesis of proteins and nucleic acids in the cells of NAG-vibrio, Staph. aureus and E. coli was studied by incorporation of 1-14C-glycine and 8-14C-adenine into proteins and nucleic acids. It was found that sodium desoxycholate suppressed the synthesis of proteins and nucleic acids in the cells of NAG-vibrio and Staph. aureus. Its combination with tetracycline resulted in summation or increase of the suppressive effects on proteins and nucleic acids as compared to the effect of the substances used alone. Sodium desoxycholate even in very high concentration, up to 12800 gamma/ml, had no effect on the synthesis of proteins and nucleic acids in the cells of E. coli and respectively it did not change the activity of tetracycline on combined use.     

The site of inhibition of bacterial cell wall peptidoglycan synthesis by azureomycin B, a new antibiotic

Azureomycin B (10 micrograms/ml), a new antibiotic from Pseudonocardia azurea nov. sp., caused the accumulation of lipid intermediate and inhibition of peptidoglycan synthesis in an invitro system using a particulate fraction from Bacillus megaterium KM with UDP-MurNAc-[3H]pentapeptide and cold UDP-GlcNac or cold UDP-MurNAc-pentapeptide and UDP-[3H]GlcNAc as substrates. At higher concentrations of azureomycin B (over 100 microgram/ml), lipid intermediate accumulation was also inhibited. When particulate fraction from Escherichia coli Y-10 and UDP-[14C[GlcNAc and cold UDP-MurNAc-pentapeptide were used, accumulation of lipid intermediate and inhibition of peptidoglycan synthesis were also observed. These results indicate that the primary target of azureomycin B is the transfer of the disaccharide peptide unit (GlcNAc-MurNAc-pentapeptide) from lipid-bound precursor to acceptor.     

Biosynthesis of proteins and ribonucleic acids in red and white rat skeletal muscles]

Protein biosynthesis is studied in red and white rat shank muscles in vitro. It is found that the incorporation rate of 14C-lysine in red muscle was 2-fold higher than that in white muscle. The difference in the lysine incorporation rate into muscle proteins studied increased with the increase of lysine molar concentration in the incubation medium, which was probably due to a selective protein synthesis activation in the red muscle. A higher level of 14C-lysine incorporation in red muscle proteins was found under similar uptake of the labelled amino acid in both red and white muscles. RNA synthesis rate was the same in both muscles and its inhibition with actinomycin D did not affect the ratio of protein synthesis rates in red and white muscles.     

The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, beta-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by beta-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of beta-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2-3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with beta-lactamase or by washing.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.