Identification and partial purification of a dipeptidyl aminopeptidase from Streptococcus faecalis

A dipeptidyl aminopeptidase was identified in Streptococcus faecalis JH2SS and was partially purified (approximately 245-fold) by HPLC. Gel filtration chromatography indicated an Mr of 140 000. The partially purified enzyme exhibited a requirement for Co2+. The pH optimum for the hydrolysis of L-Val-L-Ala-p-nitroanilide was approximately 9.5. The apparent Km for this substrate was 0.22 mM. The enzyme preferentially hydrolysed X-Ala-Y substrates, but also utilized X-Pro-Y substrates, and therefore is most closely related to the mammalian dipeptidyl aminopeptidase II (EC 3.4.14.-). The enzyme was inhibited by p-chloromercuribenzoate, but not by iodoacetate, N-ethylmaleimide or the serine protease inhibitor phenylmethylsulphonyl fluoride.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Induction of autolysis in Enterococcus faecalis S-47 by peptide AS-48.

In addition to its bactericidal mode of action, the peptide antibiotic AS-48 exhibits a bacteriolytic effect on Enterococcus faecalis S-47 that is associated with autolysin activation. Bacteriolysis induced by the antibiotic can be modulated by addition of EDTA, divalent cations and autolysin activators (trypsin) or inhibitors (cardiolipin), suggesting that topologic regulation of the autolysins is involved in the process. In addition, inhibitors of protein and RNA synthesis interfere markedly with bacteriolysis, as do ionophores and the ATPase inhibitor DCCD, suggesting the participation of an internal messenger in autolysin activation in the presence of AS-48.     

Characterization and partial purification of plantaricin LC74, a bacteriocin produced by Lactobacillus plantarum LC74

Summary Plantaricin LC74, a bacteriocin produced during the growth of Lactobacillus plantarum LC74 isolated from crude goat's milk, inhibited some other mesophilic lactobacilli. It was sensitive to high temperature (95°C) but was relatively stable at 30°C in acidic conditions, and also in the presence of various organic solvents, several detergents, urea or -mercaptoethanol. It was destroyed by proteases. By ultrafiltration plantaricin LC74 showed an apparent molecular mass smaller than 5 kDa. Plantaricin LC74 bounded non specifically to both sensitive and resistant Gram-positive bacteria and displayed a bacteriostatic effect. Its partial purification was obtained by ammonium sulphate precipitation followed by cationic exchange chromatography and hydrophobic interaction chromatography.     

Characterization and partial purification of a non-interferon macrophage activating factor produced by human leukemic T cell line

Culture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-gamma, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-gamma, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-gamma monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40,000 to 60,000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1,300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-gamma.     

Bactericidal action of peptide antibiotic AS-48 against Escherichia coli K-12

Peptide antibiotic AS-48 exerts a bactericidal mode of action on exponential cultures of Escherichia coli K-12 through a multi-hit kinetics interaction. AS-48 causes a parallel and gradual cessation of all biosynthetic pathways monitored (protein, RNA, DNA, and cell wall synthesis), the rate of incorporation of labeled precursors, the rate of O2 consumption, and cell growth. These effects have been attributed to alterations of cytoplasmic membrane functions.     

Characterization of a bacteriocin produced by Streptococcus thermophilus ST134

A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins. Agar-diffusion tests with S. thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity. Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S.thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration. Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.     

Characterization of linear forms of the circular enterocin AS-48 obtained by limited proteolysis

AS-48 is a 70-residue circular peptide from Enterococcus faecalis with a broad antibacterial activity. Here, we produced by limited proteolysis a protein species carrying a single nicking and fragments of 55 and 38 residues. Nicked AS-48 showed a lower helicity by far-ultraviolet circular dichroism and a reduced stability to thermal denaturation, but it was active against the sensitive bacteria assayed. The fragments also partly retained the biological activity of the intact protein. These results indicate that circularization is not required for the bactericidal activity, but it is important to stabilize the native structure. Moreover, it is possible to reduce the sequence to a minimal AS-48 domain without causing inactivation of this bacteriocin.     

Identification and partial characterization of a pheromone-induced adhesive surface antigen of Streptococcus faecalis

A surface protein antigen that is produced only during the induction of aggregation of Streptococcus faecalis was shown to contribute to and perhaps be primarily responsible for aggregation. The antigen is an immunodominant surface component of induced cells. F(ab) fragments of immunoglobulins specific for this antigen prevented aggregation, providing direct evidence that the antigen is an adhesin. Consistent with this proposed association was the coincident timing of appearance of the antigen and the timing of aggregation after induction.     

Production and partial purification of a peptide antibiotic from Staphylococcus epidermidis

When grown on solid or in liquid Brain Heart Infusion at 37°C, Staphylococcus epidermidis NCIB 11536 produced antibiotic activity against a wide range of Gram positive bacteria. Production was influenced by aeration, pH, glucose concentration and specific growth rate. Inhibitory activity could be concentrated by ammonium sulphate precipitation (30–55% saturation). On Sephadex G50 using 0.05 mol/1 sodium phosphate buffer, pH 6.0, two peaks of antibiotic activity were detected. The first peak eluted with the void volume (K_d= 0) and the second peak was retained by the gel (K_d= 0.73–0.77). These two substances did not represent the monomeric and polymeric forms of a staphylococcal bacteriocin. The low mol. wt inhibitor, which was responsible for over 95% of the recovered activity on Sephadex G50, could be partially purified by a combination of gel filtration on Biogel P2 and ion-exchange chromatography on Sephadex C-25. Yields were increased by combining these two steps into a single procedure (duocolumn). The semi-purified inhibitor was desalted using Sep-pak C₁₈ cartridges. Biological activity was resistant to enzymic denaturation except by high concentrations of trypsin (50 units/μg, 3 h, 25°C). This peptide antibiotic is different from any previously described staphylococcal inhibitors.     

Purification and partial characterization of a cohaemolysin (CAMP-factor) produced by Streptococcus canis

Abstract A cohaemolysin from the culture supernate of a canine pathogenic group G streptococcus ( S. canis ) was purified to electrophoretic homogeneity. The purification procedure involved ammonium sulphate precipitation, ultrafiltration, gel filtration and preparative isoelectric focusing. The cohaemolysin consisted of a single polypeptide chain, 18.6 kDa, with an isoelectric point at pH 5.1. The protein reacted with an homologous anti-serum, appeared to be trypsin-sensitive and relatively heat-stable. The cohaemolysin did not show any non-specific IgG binding activities.     

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.     

Characterization of a new operon,as-48EFGH,from the as-48 gene cluster involved in immunity to enterocin AS-48

Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C(1), as-48D, and as-48D(1) genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T(2) and T(3), in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T(3) could be regulated, because in JH2-2(pAM401(EH)) transformants, T(3) was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D(1) gene for immunity against AS-48.