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A method involving direct affinity chromatography of undialysed bovine seminal plasma on a calmodulin-agarose column was developed for the purification of seminalplasmin. Seminalplasmin thus obtained was further purified from any contaminants left by ion-exchange chromatography on a short CM-Sepharose column. The method gave an excellent yield of seminalplasmin (0.7 mg per ml seminal plasma) that showed 100% inhibition of bacterial growth at a concentration of 10 micrograms/ml. The purified seminalplasmin was found to be homogeneous as tested by HPLC on a reverse-phase column and SDS-PAGE.

A pair of relA+ and relA E. coli strains, otherwise isogenic, were studied with regard to the susceptibility of starved cells to lysis induced by the natural peptide seminalplasmin. Starved relA cells were more sensitive to seminalplasmin-induced lysis when compared to starved relA+ cells. Nevertheless, pronounced lysis of starved relA+ cells was observed with increase in the concentration of seminalplasmin. In contrast, ampicillin could not lyse starved relA+ cells even at very high concentrations. Further, seminalplasmin could cause loss of viability and degradation of peptidoglycan in starved relA+ cells. These observations suggest that, unlike many other antibiotics, seminalplasmin can induce autolysis under the conditions of a stringent response.

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, inhibited peptidoglycan synthesis in Escherichia coli in a concentration-dependent manner. The inhibition of peptidoglycan synthesis appears to be a cause rather than a consequence of growth inhibition as it was observed soon after the addition of the antibiotic even in E. coli cells whose growth was totally inhibited by chloramphenicol.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.

Seminalplasmin, an antimicrobial protein from bovine seminal plasma, lysed both Gram-positive and Gram-negative bacteria but not Candida albicans. The lytic activity was not lysozyme-like and was not affected by inhibitors of RNA or protein synthesis or by azide; it was strongly inhibited by divalent cations like Ca2+, Mn2+ and Mg2+ at millimolar concentrations. Maximum lysis of Escherichia coli was obtained at 37 degrees C; heat treatment of E. coli drastically reduced its susceptibility to lysis by seminalplasmin. E. coli cells in the stationary phase of growth were lysed much less than those in the exponential phase, and those grown in an enriched medium were lysed much more than those grown in a minimal medium. It appears that the lytic activity of seminalplasmin is due to the activation of an autolysin.

Seminalplasmin, a 6,000 dalton antimicrobial protein present in bovine seminal plasma, is shown to inhibit growth and/or RNA synthesis in several bacterial species. In only one strain out of twenty one belonging to fourteen species, did both RNA synthesis and growth appear to be resistant to seminalplasmin. The antibacterial activity of seminalplasmin, in the case of E. coli, was also studied as a function of its concentration and of time; the minimal concentration of the protein required for 100% bactericidal activity was only about twice that required for 100% bacteriostatic activity. The killing of E. coli cells proceeded in two phases, a slow phase and then a rapid one, and required several hours for completion. Several bacterial species tested secreted proteases into the medium that destroyed seminalplasmin.