Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis

Abstract

Minicells produced by Bacillus subtilis CU403 (divIVBl) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and n-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of >-and Lalanine N-acetylglucosamine at concentrations in excess of 10 ,tg of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, n-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acidsoluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.

Mucopeptide Biosynthesis by Minicells of Escherichia coli

“…L-Methionine is, however, converted to a product that is not solubilized by lysozyme and is therefore most likely converted to protein, as previously described (6, 7). It is clear from the results presented that minicells produced by E. coli X925 do synthesize mucopeptide and, indeed, on a mass basis (absorbance of 660 nm of 0.5 is equivalent to 4 x 109 B. subtilis minicells and 5 x 109 E. coli minicells), the amount synthesized is very similar to that synthesized by minicells produced by B. subtilis (8). The results indicate that careful choice of precursor and inhibitors must be made in order to interpret incorporation curves de-picting synthesis in minicells of macromolecules from amino acid precursors.…”

“…An explanation for this phenomenon in Escherichia coli minicells was provided by the identification of stable messenger ribonucleic acids that segregate into minicells and that permit the synthesis of cell membrane proteins (3,7). We recently investigated the phenomenon with minicells produced by a strain of Bacillus subtilis (8) and found that these minicells synthesized teichoic acid and mucopeptide from precursor amino acids. The polymerization of amino acids by minicells of B. subtilis was inhibited by vancomycin, D-cycloserine (CYC), and penicillin G (PEN).…”

“…Incorporation of the mixture of amino acids is inhibited 20% by CYC, whereas incorporation of the direct precursors of mucopeptide, i.e., D-alanine and N-acetyl-D-glucosamine, is inhibited by over 80%. The composition of the amino acid mixture has been previously described (8), and the possibility that the inhibition observed in Fig. 1A was due to inhibition of incorporation of the known mucopeptide precursor L-alanine was further tested.…”

“…Minicells (0.1 ml; absorbance of 660 nm = 0.4) were incubated for 45 min at 37°C in supplemented M9 medium plus 0.5 ,Ci of L-"4C-labeled amino acid mixture (8) or 0.1 ,uCi of L-[U-14C]alanine plus 0.0025% (wt/ vol) pancreatic casein hydrolysate (Merck, Germany) (final concentration of L-alanine = 1.80 jig/ml) or 0.09 ,uCi of L-[U-'4C]methionine (250 mCi/mmol) plus 25% (vol/vol) methionine assay medium (Difco) (6). Two milliliters of 5% (wt/vol) trichloroacetic acid containing 0.1% (wt/vol) casein hydrolysate was added to each sample, and the hot trichloroacetic acid-precipitable material was measured as previously described (8).…”

“…Portions of these suspensions were placed on 20 volumes of 5% trichloroacetic acid either directly (0 min) or after incubation in buffer with or without lysozyme (2 mg of lysozyme per ml, final concentration) for 15 min at 37°C. The hot trichloroacetic acid-precipitable material in each sample was determined as previously described (8).…”