The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate.

Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of beta-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillin-binding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillin-binding module of PBP3 is not a transglycosylase.

Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3.

The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP 1b or PBP 3. With PBP 1b, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP 1b activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP 1b could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.

Effects of moenomycin on Escherichia coli.

The antibiotic moenomycin is a valuable biochemical tool for studying the metabolism of peptidoglycan and the autolytic system in Escherichia coli, since as a specific inhibitor of peptidoglycan polymerases it can efficiently promote cell lysis. In liquid media the bacteriolytic effect on E. coli K12 was dependent on the concentration of moenomycin, on growth phase and on growth rate. Before lysis cells underwent major morphological alterations. In sucrose-containing medium complete transformation to osmotically sensitive spheroplasts was easily achieved by addition of moenomycin. The minimum inhibitory concentration of the antibiotic varied with the strain of E. coli and was highly dependent on the growth medium. A tritiated derivative of moenomycin, [3H]decahydromoenomycin A, was prepared and found to have the same inhibiting efficiency. Its binding to E. coli membranes and membrane proteins was investigated. The absence of irreversible binding suggested that moenomycin might be a competitive inhibitor of the peptidoglycan polymerases. Spontaneous moenomycin resistant variants were isolated at a frequency of about 10(-9).

Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K 12. Purification and properties of the enzyme.

N-Acetylmuramyl-L-alanine amidase activity was detected in Escherichia coli K 12 by usine N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)-meso-[3H]diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N-acetylmuramyl acid and L-alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K 12. The enzyme was purified to near homogeneity. A molecular weight of 39 000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N-acetylmuramyl-L-alanine amidase activity was carried out by investigating several of its properties.

[Action of microbial flora of the digestive tract on the metabolism of bile acids in the rat (author's transl)]. / Action de la flore microbienne du tractus digestif sur le metabolisme des acides biliaires chez le rat

An isotopic balance is established in rats receiving a regular feed intake of [4-14-C]cholesterol so that various chemical species of bile acids have the same specific activity. This property is used to study bile acids distribution in the rat liver, digestive tract and fecal excretion. Bile acids are separated by thin-layer chromatography, radioactivity is determined by liquid scintillation, and the mass by 3-hydroxysteroid dehydrogenase action. The resulting comparative study made between the germ-free rat (axenic rat) and the rat exposed to microbes ("holoxenic" or conventional rat) receiving a semi-synthetetic feed, shows the influence excercised on the metabolism by the microbial flora of the digestive tract. This study confirms that the axenic rat compared to its holoxenic homologue has a higher bile acids pool and a lower fecal excretion. At all levels of the digestive tract (small intestine and the whole caecum and large intestine), probably as well as in the liver, the total amount of bile acids which is observed in the axenic rat is about twice the amount observed in the holoxenic rat, but fecal excretion is decreased by 20%. Values obtained by this method are higher than those previously observed by other authors using gas-liquid chromatography or [14-C]cholic acid isotopic dilution. This study also confirms that cholic and beta-muricholic acids are the main bile acids in the axenic rat and in addition establishes that in this animal bile acids composition is complex and varies from the small intestine to the feces. Besides cholic, alpha- and beta-muricholic, chenodeoxycholic and ursodeoxycholic acids, unidentified chemical species constitute 21% of the whole in the feces. Comparing the compositions observed in axenic and holoxenic rats in this experiment, it could not be determined if the relative activity of the two pathways of bile acid biosynthesis is deeply or only slightly changed by the presence of microbial flora. This is because of a large fraction of unknown composants in the feces of the axenic rat and the extreme complexity in the feces of the holoxenic rat.