Incorporation Of Diaminopimelic Acid Research Articles

Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid

Peptidoglycan synthesis rate in nonconstricting filaments of Escherichia coli dnaX(Ts) has been studied by autoradiography of incorporated [3H]diaminopimelic acid. Analysis of autoradiograms of whole cells and sacculi showed that peptidoglycan is synthesized at a reduced rate in the nucleoid-containing parts of these filaments. The lower rate of peptidoglycan synthesis in the cell center coincides with a higher local rate of protein synthesis. DNA-less cell formation in dnaX(Ts), dnaX(Ts) sfiA, and the minB minicell-forming mutant is accompanied by a local increase in peptidoglycan synthesis at the constriction site.

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan ofSalmonella typhimurium

Diaminopimelic acid is incorporated into the peptidoglycan of Salmonella typhimurium in an efficient and quantitative manner. The amount of DAP incorporated is similar to the number of molecules estimated to exist in the Salmonella cell wall. In contrast, strains of E. coli, including those most used for studies of cell wall synthesis, are much less efficient in the incorporation of diaminopimelic acid. The lysine-requiring strains of E. coli appear to excrete diaminopimelic acid related material during growth and this accounts, in part, for the inefficient incorporation of radioactive diaminopimelic acid into Escherichia strains. In addition, the Escherichia strains are much less permeable to DAP than Salmonella strains. Cysteine and cystine inhibit the incorporation of DAP into the cell and this result suggests that Salmonella uses the cystine uptake system to allow DAP into the cell.

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Efficient and quantitative incorporation of diaminopimelic acid into the peptidoglycan of Salmonella typhimurium

Incorporation of diaminopimelic acid into the old poles of Escherichia coli.

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.

Mode of action of Myxococcus xanthus antibiotic TA

Antibiotic TA inhibited incorporation of diaminopimelic acid and uridine diphosphate-N-acetylglucosamine into Escherichia coli cell walls without altering the ratio of cross-linked to uncross-linked peptidoglycan. Formation of the lipid intermediate was not blocked by TA, suggesting that TA interferes with polymerization of the lipid-disaccharide-pentapeptide.

Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation.

Furazlocillin (1 microgram/ml) and piperacillin (5 microgram/ml) bound specifically to penicillin-binding protein 3 (PBP-3) and not to the other major PBPs in intact Escherichia coli cells. The effect of this specific binding to PBP-3 on murein synthesis of elongating and synchronously septating cells was investigated in two thermosensitive division mutants, E. coli BUG6 and E. coli JE10730, the latter possessing a thermolabile PBP-3. Synchronous cell division was induced by shifting the cultures from the nonpermissive temperature (42 degrees C) to 30 degrees C. Both [14C]diaminopimelic acid incorporation into murein of intact cells and [14C]N-acetylglucosamine incorporation into murein of cells permeabilized with ether was inhibited by an average of 42% in septating cells. In filaments growing at the nonpermissive temperature, we detected no inhibition and, frequently, a 10 to 15% stimulation of murein synthesis. The two drugs, at concentrations used in the above experiments, bound exclusively to PBP-3 both in elongating and septating intact cells and in ether-treated cells. These results support the hypothesis that PBP-3 activity is exclusively required for septal murein synthesis.

Epoxypeptide antibiotic tetaine mimics peptides in transport to bacteria.

Tetaine induced the lysis of Escherichia coli cells. Several di- and tripeptides were found to protect this cells against tetaine action. Certain peptides are able to diminish the inhibition by tetaine of diaminopimelic acid incorporation into peptidoglycan and the extent of this corresponds to the protection of the cells against the tetaine-induced lysis. The data indicate that tetaine enters E. coli cells predominantly by dipeptide permease and in part by one or more oligopeptide permease system. A number of di- and tripeptides diminished the inhibitory effect of tetaine on the incorporation of lysine into peptidoglycan of Staphylococcus aureus Oxford. In contrast to E. coli, tetaine seems to be transported into S. aureus by a single transport system.

Lysine Synthesis in Rust Infected Flax Cotyledons

Pathways of lysine biosynthesis in rust infected flax cotyledons have been investigated. Non infected cotyledons were shown to possess enzymes of the diaminopimelic acid pathway of lysine synthesis, but these enzymes could not be detected in axenic cultures of the flax rust fungus. Feeding experiments demonstrated the incorporation of diaminopimelic acid into lysine by the host but not by the fungus. Conversely, enzymes of the aminoadipic acid pathway of lysine synthesis are present in the rust fungus, but, with the exception of aminoadipate transaminase, not in non-infected cotyledons. Aminoadipic acid stimulated lysine production by the fungus but not by the host. Cotyledons infected for 7 days were shown to possess enzymes of both pathways, and seemed able to convert both diaminopimelic acid and aminoadipic acid into lysine. We consider these studies to demonstrate that infected flax cotyledons possess the ability to synthesize lysine by either pathway. The fungus may thus be capable of synthesizing at least some of the lysine it requires.

Peptidoglycan synthesis in cocci and rods of a pH-dependent, morphologically conditional mutant of Klebsiella pneumoniae.

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.

Study of cell wall growth in Bacillus megaterium by high-resolution autoradiography.

Growth of the cell wall of Bacillus megaterium was studied by pulse-labeling the cell wall of a DAP- Lys- mutant for a very short time with tritium-labeled diaminopimelic acid. The distribution of radioactivity along the cell wall was examined by high-resolution autoradiography on isolated cell walls and thin sections of bacteria. The results indicate that cell wall elongation occurs by diffuse intercalation of newly synthesized murein into the expanding cell wall during exponential growth, as well as during germination, and that the only zone of highly localized diaminopimelic acid incorporation is found at the cross wall during its synthesis. This zone contains about 30% of the radioactivity incorporated into the cell wall. Analysis of autoradiographs of thin sections of bacteria shows that the total radioactivity incorporated per bacterium doubles during the life cycle. This doubling occurs in the cylindrical part of the cell wall but not in the polar caps. This seems to indicate that elongation of the bacterium is not constant during the life cycle but increases with the length of the cell.

Reconstitution of cell wall synthesis in toluene- and LiCl-treated Bacillus megaterium cells by addition of a soluble protein extract.

Proteins required for the synthesis of peptidoglycan and incorporation of diaminopimelic acid into cell walls have been solubilized from Bacillus megaterium toluene-treated cells. Some of these proteins might have been removed from the cytoplasmic membrane through the cell wall by extraction with LiCl. The solubilized proteins have molecular weights in the range of 40,000 to 70,000 and can be added back to B. megaterium toluene-treated cells to reconstitute the synthetic reactions.

Penicillin-resistant temperature-sensitive mutants of Escherichia coli which synthesize hypo- or hyper-cross-linked peptidoglycan.

A group of Escherichia coli mutants which are ampicillin resistant at 32 C and which either are unable to grow or lyse at 42 C has been selected. These mutants have been classified by a number of characteristics: total peptidoglycan synthesis measured by [(14)C]diaminopimelic acid incorporation, extent of cross-linking of the peptidoglycan which is synthesized, growth characteristics at the two temperatures, and morphology. Two especially interesting groups of mutants have been described. In one of these, a hypo-cross-linked peptidoglycan was synthesized at the nonpermissive temperature. Most of these organisms lysed at 42 C. In another group, the peptidoglycan synthesized at 42 C was hyper-cross-linked. Many of these organisms were spherical. Studies of revertants indicated that ampicillin resistance, temperature sensitivity, cross-linking, growth characteristics, and morphological changes may be related to a single mutational event in both of these groups.

Penicillin-sensitive Transpeptidation during Peptidoglycan Biosynthesis in Cell-free Preparations from Bacillus megaterium: I. INCORPORATION OF FREE DIAMINOPIMELIC ACID INTO PEPTIDOGLYCAN

Abstract A particulate enzyme system from Bacillus megaterium QMB1551 is described which catalyzes the utilization of the uridine nucleotides, UDP-N-acetylmuramyl-l-Ala-d-Glumeso-Dap-d-Ala-d-Ala and UDP-N-acetylglucosamine, for peptidoglycan synthesis. Unlike systems previously studied in gram-positive microorganisms, this particulate enzyme preparation catalyzed the terminal cross-linking reaction in cell wall biosynthesis. This system could also incorporate free diaminopimelic acid dependent on the formation of peptidoglycan polymer from the uridine nucleotide substrates but independent of ATP. Furthermore, the incorporation of diaminopimelic acid was inhibited by penicillins, and the diaminopimelic acid appeared to be incorporated onto a terminal position of the peptide of a disaccharide-pentapeptide peptidoglycan unit with the release of alanine. The disaccharide-peptide products formed during the incorporation of free diaminopimelic acid were isolated from peptidoglycan and analyzed. A reaction sequence for incorporation of free diaminopimelic acid into peptidoglycan is proposed.

Penicillin-sensitive Transpeptidation during Peptidoglycan Biosynthesis in Cell-free Preparations from Bacillus megaterium: II. EFFECT OF PENICILLINS AND CEPHALOSPORINS ON BACTERIAL GROWTH AND IN VITRO TRANSPEPTIDATION

Abstract The effect of penicillins and cephalosporins on the various transpeptidation reactions presented in the preceding paper is discussed. Inhibition by cloxacillin and penicillin G of [14C]diaminopimelic acid incorporation during peptidoglycan synthesis appeared to be partially reversible after the antibiotic was destroyed by treatment with penicillinase. Inhibition of the cross-linking reaction by cloxacillin appeared to be fully reversible. These and other results suggested the presence of more than one transpeptidase. In addition, a d-alanine carboxypeptidase activity present in the particulate enzyme preparation was examined and found to be irreversibly inhibited by penicillin G and relatively insensitive to cloxacillin. It is suggested that the transpeptidases(s) and the d-alanine carboxypeptidase are separate enzymes.

Bacterial Cell Division Regulation: Physiological Effects of Crystal Violet on Escherichia coli lon + and lon − Strains

The Escherichia coli lon(-) mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon(+) strains. In enriched broth, the lon(+) strain continued to grow and divide in the presence of CV, but lon(-) cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon(-) cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon(+) and lon(-) strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon(+) and lon(-) strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon(-) cells than by lon(+) cells.

Sporulation of a cortexless mutant of a variant of Bacillus cereus.

A stage 4 sporulation mutant of a strain of Bacillus cereus var. alesti fails to synthesize a cortex although all other structural components appear normal. With terminal lysis the spore core as well as the sporangium is lysed. Both the uptake of (45)Ca and the synthesis of dipicolinic acid (DPA) are similar to these activities in the parent strain, but these components (DPA and Ca) are lost to the medium with the drastic lysis. The first stage of diaminopimelic acid incorporation, that into germ cell wall mucopeptide, is intact in the mutant; the second stage, that into cortical mucopeptide, is absent. These biochemical studies as well as phospholipid metabolism and freeze-etch analysis suggest the lesion lies in the outer forespore membrane.

BIOCHEMICAL EFFECTS OF ETHIDIUM BROMIDE IN MICRO-ORGANISMS.

SUMMARY Growth of Escherichia coli was partially inhibited by 1·2 × 10−4 m-ethidium bromide, a phenanthridinium trypanocide. In the presence of manganese the drug’s effect was decreased. During growth in the presence of ethidium, RNA and protein contents were relatively unaffected when comparison was made between experimental and control cultures at similar turbidities; DNA content, on the other hand, was considerably decreased. A differential effect of ethidium on the formation of polynucleotide pyrimidines from labelled uracil and orotic acid was observed. Oxygen uptake continued almost unchanged during growth whether in the presence or absence of drug. Bacillus cereus was extremely sensitive to the growth-inhibitory action of ethidium (10−5 m) and morphological changes were observed. Manganese protected the organisms from the drug’s actions. RNA and DNA biosynthesis were both suppressed during inhibition of growth to a greater extent than was total protein formation, whereas diaminopimelic acid incorporation into cell wall and oxygen uptake continued almost unaffected. Some evidence was obtained that the pattern of protein synthesis was disturbed. It was concluded that the drug’s actions were species dependent, and that the effect on Escherichia coli resembled that described for a flagellate, while that on Bacillus cereus did not. Evidence for compartmentation of nucleic acid synthesis, as obtained with the drug in tumour cells, was not shown for either micro-organism.