Incorporation of diaminopimelic acid into Escherichia coli membranes

Abstract Pulse-labeled, cell division-inhibited Escherichia coli incorporate [³H]diamino pimelic acid (DAP) into membrane fractions of light (= inner membrane) and intermediate densities and transfer the DAP rapidly into heavy membrane fractions (= outer membrane). The intermediate density fraction which has been shown to contain markers for membrane adhesion sites appears to serve as intermediary in the translocation.     

Activity of diaminopimelic acid decarboxylase in bacteria producing lysine

Activity of DAP-decarboxylase in lysine overproducing strains of Micrococcus luteus, M. varians and Arthrobacter globiformisLb reached the highest level during the end of the exponetial phase of growth and remained at the same high level during the stationary phase of growth when major bulk of lysine was accumulated. In comparison in a lysine non-producing strain of Arthrobacter globiformis I ₄ the activity of the same enzyme was low. DAP-decarboxylase of these three lysine overproducers has two specialities, persistence during the stationary phase and insensivity to repression by exogenous lysine.     

Two types ofPropionibacterium avidum with different isomers of diaminopimelic acid

Of 38 strains ofPropionibacterium avidum, most fell into one of two groups. One group (20 strains) hadLl-diaminopimelic acid as the diaminoacid of peptidoglycan, did not ferment inositol, and reacted with serum to strain VPI 0576; the other group (11 strains) hadDl-diaminopimelic acid as the peptidoglycan diaminoacid, fermented inositol, and reacted with serum to strain VPI 0670. DNA sequence similarity studies showed that, while overall intergroup similarity was about 80%, within each group the sequence similarities were over 90%. Seven strains were anomalous and did not fit exactly into either group.The results show that the isomer of diaminopimelic acid in peptidoglycan may differ in strains of a single species all of which show at least 80% DNA sequence similarity to each other.     

Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli.

In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine. The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used. Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid. Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi. For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed. The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values. Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis. The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides. HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.     

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.     

Turnover of murein in a diaminopimelic acid dependent mutant ofEscherichia coli

Escherichia coli 173-25, whose cell wall was labelled with¹⁴C-diaminopimelic acid, was found to lose about 15% radioactivity during growth in a fresh medium, two thirds or more being lost during the first two generations. Degradation products of the cell wall were mostly of low-molecular type. About 5% of the cells lyzed as a result of transfer associated with filtration, washing and resuspension of the bacterial population in a diaminopimelic acid (DAP) deficient medium. The degradation was very low during the first 20 min. The amount of wall material released from the cells increased between 20–30 min and a sudden decrease of viability of the population was observed. The degradation of murein triggered by starvation for DAP continued when supplementing the deficient medium with DAP and when growth was resumed. About one-half of the cell wall material released into the medium under these conditions was macromolecular. However, lysis of the cells and release of proteins into the medium were rapidly interrupted after DAP was added to the starving culture and the differential rate of synthesis of the cell wall increased. Turnover of murein was not associated with protein turnover.     

Lysine biosynthesis inMethanobacterium thermoautotrophicum is by the diaminopimelic acid pathway

Methanobacterium thermoautotrophicum, an archaebacterium, possesses the first and last enzymes of the diaminopimelic acid pathway for lysine biosynthesis, dihydrodipicolinate synthase, and diaminopimelate decarboxylase. It does not have saccharopine dehydrogenase, the last enzyme of the aminoadipate pathway for lysine biosynthesis. The dihydrodipicolinate synthase is inhibited but not repressed by lysine. We conclude that this microbe uses the diaminopimelate pathway for synthesis of lysine.Deceased.     

A simple chromatographic route for the isolation of meso diaminopimelic acid

Meso diaminopimelic acid is an important noncoded amino acid found in Gram‐negative bacterial peptidoglycan. In spite of its importance, this stereoisomer is not available commercially. A simple, economical procedure was developed for the isolation of pure meso diaminopimelic acid via an high‐performance liquid chromatography separation. In our new approach, the underivatized three isomers of diaminopimelic acid were separated on a crown ether‐based chiral stationary phase. For the structure identification, ¹H NMR spectroscopy was applied. Chirality, 2011. © 2010 Wiley‐Liss, Inc.