Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

Abstract

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 degrees C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 degrees C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.

Monolayer Characteristics of Bacteriocin AS-48, pH Effect and Interactions with Dipalmitoyl Phosphatidic Acid at the Air–Water Interface

“…At pH 5.0 and 7.0, no relevant deviation from the linearity predicted by Eq. [1] is found for the compression range. Short positive deviations are observed only at a low proportion of AS-48 in the mixed film, indicating slightly repulsive interactions between AS-48 and DPPA.…”

“…• C for 7.30 h in CM-G broth [1], consisting of BHI (0.1%), casaminoacids (0.1%), yeast nitrogen base (YNB; 0.14%), MgSO 4 (40 mM), and glucose (1%) dissolved in 0.1 M sodium phosphate buffer, pH 7.2. After the cultivation period, the cells were removed by centrifugation and the supernatant containing AS-48 was brought to pH 6.5 with 0.1 M NaOH and mixed (20 : 1, v : v) with carboxymethyl-Sephadex CM-25 (Pharmacia, Uppsala, Sweden) under stirring for 1 h. The mixture was allowed to settle, the supernatant was decanted and discarded, and the gel was loaded on a 2 × 50 cm glass column.…”

“…The material retained in the gel was eluted with 1.5 M NaCl in water. The different fractions obtained were tested for antimicrobial activity as described elsewhere (1).…”

“…The bacteriocin AS-48 produced by Enterococcus faecalis S-48 is a ribosomally synthesized cyclic peptide (7.4 kDa) of broad inhibitory spectrum against Gram-positive and Gramnegative bacteria (1)(2)(3). AS-48 is a strongly basic molecule (isoelectric point 10.5) that contains a high proportion (49%) of hydrophobic amino acids and uncharged hydrophilic residues which form a hydrophobic helical peptide (4,5).…”

“…The comparative analysis of the amino acid sequence deduced from the as-48 gene and that of a fragment of AS-48 peptide obtained by endoproteinase digestion has demonstrated unambiguously that the mature peptide AS-48 has a cyclic structure in which a peptide bond links the amino group of the Nterminal initial amino acid methionine to the carboxyl group of the final triptophane (Chart 1). Therefore, AS-48 appears to be the first example of a new type of ribosomally synthesized 1 To whom correspondence should be addressed. E-mail: mjgalvez@ugr.es.…”