Purification and properties of the exocellular beta-lactamase of Actinomadura strain R39.

The exocellular beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) of Actinomadura R39 consists of one single polypeptide chain of molecular weight about 15 200. It exhibits a highly asymmetrical shape, has a low isoelectric point (at pH 5.0) and contains about 9.3% (w/w) of a polydeoxyribonucleotide with which it forms a rather stable complex. Removal of a substantial amount of this deoxyribonucleotide by treatment with DNAase I has no effect on the enzyme activity. The beta-lactamase has a wide spectrum of activity. Penicillins and delta 3-cephalosporins can be either good or poor substrates. Oxacillin, which is a poor substrate of most beta-lactamases from Gram-positive bacteria, is a good substrate of the beta-lactamase of Actinomadura R39. Its best substrate, however, is nitrocefin (kcat/Km: 2300 000 M-1.s-1; catalytic centre activity: 210 s-1). The kcat/Km values observed with some penicillins and delta 3-cephalosporins are similar to the values of the bimolecular rate constants that govern the formation of the acyl-enzyme intermediates between these antibiotics and the serine D-alanyl-D-alanine peptidase that is also secreted by the same strain Actinomadura R39. Such a relationship, however, is not observed with all the beta-lactam compounds tested.

Characterization of iturin A in antibiotics from various strains of Bacillus subtilis.

Iturin A, an antifungal lipopeptide characterized by the presence of homologous liposoluble beta-aminoacids was found to be the active component to bacillomycin B, bacillomycin R and eumycin. Iturin A was identified by thin-layer chromatography, aminoacid analysis and by characterization of liposoluble aminoacids and peptides. Another two preparations: the antibiotic of Raubitschek and the bacillomycin of Landy et al. contain components of the same structural type but they are different from iturin A.

Antifungal activity upon Saccharomyces cerevisiae of iturin A, mycosubtilin, bacillomycin L and of their derivatives; inhibition of this antifungal activity by lipid antagonists.

The antifungal activity of three antibiotics of the iturin group: iturin A, mycosubtilin, bacillomycin L and of eleven methylated and acetylated derivatives of these antibiotics was tested upon Saccharomyces cerevisiae. The lowest MIC values were found for natural antibiotics. The substitution of polar groups diminished the antifungal activity. Various lipids, sterols, fatty acids, fatty acid methyl esters and phospholipids were tested as inhibitors of the antifungal activity of iturin A, mycosubtilin and bacillomycin L. Cholesterol was the strongest inhibitor upon the three antibiotics; ergosterol, oleic acid and cis-vaccenic acid were less potent inhibitors. Among phospholipids, phosphatidyl choline inhibited bacillomycin L and iturin A while diphosphatidyl glycerol inhibited bacillomycin L and mycosubtilin. The inhibitory effect appeared to be dependent on the nature of both the hydrophilic group and the fatty acid part of phospholipids.

Preparation and antibacterial activity upon Micrococcus luteus of derivatives of iturin A, mycosubtilin and bacillomycin L, antibiotics from Bacillus subtilis.

Methylated and acetylated derivatives of iturin A and mycosubtilin and methylated derivatives of bacillomycin L were prepared and their antibacterial activity on Micrococcus luteus was compared with the activity of the original substance. the results obtained show the importance of polar groups for the antibiotic activity of the substances of iturin group.

Identification of antibiotics of iturin group in various strains of Bacillus subtilis.

Thirty eight strains of B. subtilis were tested for the presence of antifungal antibiotics of the iturin group. The characterization of these antibiotics was made on the basis of: antifungal activity against P. chrysogenum, isolation and purification of the active substance, quantitative analysis of alpha-aminoacids and identification of the liposoluble dipeptide obtained by partial hydrolysis. The only antibiotics of the iturin group found were: iturin A, mycosubtilin and bacillomycin L.

Structures of bacillomycin D and bacillomycin L peptidolipid antibiotics from Bacillus subtilis.

The complete structures of bacillomycin D and bacillomycin L were revised by FAB mass spectrometry and by Edman degradation of the derivatives resulting from the N-bromosuccinimide reaction. The homologous components of both series of antibiotics were separated by HPLC and the beta-amino acids were identified by capillary gas chromatography.

Characterization of a new antibiotic of iturin group: bacillomycin D.

The characterization of an antibiotic isolated from a strain of Bacillus subtilis revealed that this compound is a new antifungal antibiotic of the iturin group. It contains a lipid moiety which is a mixture of 3-amino 12-methyl tridecanoic acid (40%) and 3-amino 12-methyl tetradecanoic acid (60%) and a peptide moiety: L-Asp1, D-Asp1, L-Glu1, L-Pro1, D-Ser1, L-Thr1 and D-Tyr1. These two moieties are joined by a threonyl-beta-aminoacid linkage.

[The structure of mycosubtilin, an antibiotic isolated from Bacillus subtilis (author's transl)]. / Structure de la mycosubtiline, antibiotique isolé de Bacillus subtilis

Mycosubtilin, an antifungal agent isolated from Bacillus subtilis is a mixture of homologous lipopeptides essentially C54H83N14O16 and C55H85N15O16. The differences in their structures was found in the lipid moiety which contains several beta-amino acids; the structure of these beta-amino acids was demonstrated by combined gas chromatography-mass spectrometry of the N-trifluoroacetyl n-butyl esters; a strong peak at m/e = 240 indicates a beta-amino group. The comparison of the derivatives of natural amino acids with synthetic 3-aminohexadecanoic and 3-aminoheptadecanoic acids indicates that natural beta-amino acids are a mixture of 3-amino-14-methylpentadecanoic acid (35%), 3-amino-14-methylhexadecanoic acid (59%), 3-aminohexadecanoic acid (6%) and a trace of C18 beta-amino acid. The peptide moiety contains 8 moles of amino acids, two of D-aspargine, two of L-asparagine, and one of L-glutamine, L-proline, D-serine and D-tyrosine. The peptide sequence was determinated by partial acid hydrolysis of mycosubtilin and isolation and structural determination of the peptides from the hydrolysates. Four liposoluble peptides and four hydrosoluble peptides were studied. The results gave the cyclic structure shown on Formula 1 for mycosubtilin.

[The structure of bacillomycin L, an antibiotic from Bacillus subtilis (author's transl)]. / Structure de la bacillomycine L, antibiotique de Bacillus subtilis.

The structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D-aspartic acid, L-aspartic acid, L-glutamine, L-serine, D-serine, L-threonine, and D-tyrosine. The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid (46%), 3-amino-12-methyltetradecanoic acid (38%, 3-amino-14-methylpentadecanoic acid (11%), and two minor homologues. The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis. The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.

Structure of bacillomycin D, a new antibiotic of the iturin group.

Bacillomycin D is an antifungal agent extracted from the culture medium of a strain of Bacillus subtilis. It is a mixture of two homologous lipopeptides: the lipid moiety consists of 3-amino-12-methyltridecanoic acid or 3-amino-12-methyltetradecanoic acid; the peptide moiety contains one residue of each of the following seven amino acid: D-asparagine, L-aspartate, L-glutamate, L-proline, D-serine, L-threonine and D- tyrosine. The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis and by cleavage of the molecule with N-bromosuccinimide.

The exocellular DD-carboxypeptidase-endopeptidase from Streptomyces albus G. Purification and chemical properties.

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500.

Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis.

A mixture of iturines extracted from Bacillus subtilis gave, on column chromatography, iturine A, iturine B, and iturine C. Iturine A has the entire antifungal activity. It is a mixture of two homologous peptidolipids C48,H74N12O14 and C49H76N12O14 (mp 177 degrees C, [alpha]D-1.7 degrees in methanol (c 0.05 g/mL); mol wt 1042 and 1056). The lipid moiety is a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid. The peptide moiety contains 7 mol of amino acids: D-Asn2, L-Asn, L-Gln, L-Pro, L-Ser, and D-Tyr. A cyclic structure for iturine A with the serine residue linked to the fatty amino acids through a peptide bond has been domonstrated. By mild HCl hydrolysis, lipid-soluble and water-soluble peptides were obtained. They were analyzed by chemical methods and by mass spectrometry. Permethylated and perdeuteriomethylated derivatives of iturine A were also subjected to mass spectrometric analysis. Both chemical analysis and mass spectrometry led to the cyclic structure I for iturine A.

Multiple forms of histone H2B from the nematode Caenorhabditis elegans.

The complete amino acid sequence of histone H2B from the nematode Caenorhabditis elegans was determined. The protein as obtained by us is a mixture of multiple forms. Approx. 90% of the molecules consist of a polypeptide chain of 122 amino acids with alanine as N-terminal residue and proline at the second position. In the remaining 10% alanine is lacking and the chain starts with proline. In addition to the heterogeneity of chain length, polymorphism occurs at the positions 7 (Ala/Lys), 14 (Ala/Lys) and 72 (Ala/Ser) of the major chain and at position 6 (Ala/Lys) of the shorter chain. In the N-terminal third of the molecule there is a high degree of sequence homology to the corresponding region in H2B from Drosophila (insect), Patella (mollusc) and Asterias (starfish). In contrast, this part of the molecule differs considerably from mammalian histone H2B.

The exocellular beta-lactamase of Streptomyces albus G. Purification, properties and comparison with the exocellular DD-carboxypeptidase.

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frère, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frère & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frère, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties.