A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 106 'transformants per ,ug of DNA were consistently obta,ined within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid plP501 could transform S. faecalis at a high frequency (6.3 x 104 transformants per ,ug). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC1W4, we constructed a new E. coli-S. faecalis shuttle vector (pA,M401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.Genetic analyses of Streptococcus faecalis are largely impaired by the lack of an efficient transformation system. While conjugation of plasmids is common in S. faecalis (5, 7), neither transduction nor naturally occurring transformation has been previously described. Since protoplasts of S.faecalis could be reversibly generated by various groups (15,18,23), attempts were undertaken in this laboratory to develop a protoplast transformation system in this species. A recent report by Smith (29) showed that protoplast transformation could be achieved; however, that system worked only at a low frequency and, in our hands, was often difficult to reproduce. The primary objective of the work presented here was to optimize a protoplast transformation system for S. faecalis to a point where its efficiency was high enough that genetic manipulations such as shotgun cloning of chromosomal DNA would be possible.By optimizing various parameters (e.g., growth medium, transformation medium, types of plasmids used, etc.), we have developed a system which reproducibly results in up to 106 regenerated transformants per pug of DNA within 48 h.In addition we constructed a new Escherichia coli-S. faecalis shuttle vector, pAM401, which has nine unique restriction sites. By direct transformation of S. faecalis protoplasts, a chromosomal tetracycline resistance determinant from Streptococcus sanguis was cloned into pAM401, thus demonstrating the general utility of this system. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.Media. Unless otherwise noted, the growth medium for E. coli was L broth (26); for S. sanguis, S. faecalis, and Bacillus subtilis, we used brain heart infusion, Todd-Hewitt broth (THB), and Pennassay broth (all from Difco Laboratories), respectively. Other media tested were DM3 (4), nutrient broth no. 2 (Oxoid Ltd.), and methionine assay medium (Difco tetracycline, 10; chloramphenicol, 20; erythromycin, 20; and kanamycin was used at 50 ,ug/ml for E. coli and 500 ,ug/ml for S. faecalis. The concentra...

Background: Bacterocins are antimicrobial peptides produced by bacteria with a relatively narrow range of activity against closely related organisms. Subtilosin A is a bacteriocin produced by Bacillus subtilis that has activity against Listeria monocytogenes, which might indicate antimicrobial activity unusual for bacteriocins.Objectives: To examine the antimicrobial activity and factors affecting the activity of subtilosin A against a range of potentially pathogenic bacteria. Methods:The peptide was purified from cultures of B. subtilis and the MIC determined for 18 species of bacteria using a microdilution methodology. The extent of capsule formation was determined using microscopic examination of cells mounted in India ink. Protease mutants of a susceptible bacteria and mild heat shock were used to examine the effect of environmental stress on subtilosin A activity.Results: Subtilosin A proved to have antimicrobial activity against a wide range of bacteria including Gram-positive and Gram-negative bacteria and both aerobes and anaerobes. The peptide was less effective against capsulated forms of two Gram-negative bacteria than the non-capsulated strains of either. Heat shock but not protease activity also altered the effectiveness of the bacteriocin.Conclusions: Subtilosin A has limited antimicrobial activity against a number of human pathogens which, combined with its relative ineffectiveness against some capsulated pathogens, may limit its usefulness as a human therapeutic.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.