Abstract
The peptide pheromone, cCF10, which induces aggregation and high frequency plasmid transfer in Streptococcus faecalis cells carrying the tetracycline resistance plasmid, pCF10, was isolated and its structure determined. The molecular weight of cCF10 is 789, and its amino acid sequence is H-Leu-Val-Thr-Leu-Val-Phe-Val-OH. Pheromone activity, as determined by a clumping induction assay, was detectable at a concentration of 2.5 x 10(-11) M. A peptide of the same sequence as that of the cCF10 produced by S. faecalis cells was synthesized by the liquid-phase method. The synthetic pheromone showed biological activity and chromatographic behavior that was identical to that of the cCF10 of bacterial origin. When the response of S. faecalis cells to various concentrations of synthetic cCF10 was monitored by measuring both the frequency of plasmid transfer and the synthesis of pheromone-inducible antigens, an excellent correlation was observed between donor ability and the appearance of a 150-kilodalton protein that appears to be involved in formation of mating aggregates. The dose-response data in the range of concentrations where the amount of pheromone became limiting (10(-11)-10(-12) M) were consistent with the notion that as few as one or two molecules per donor cell may be sufficient to induce a mating response.
Highlights
Most of the genetic and biochemical analyses of pheromone-inducible plasmid transfer systems to date supi P D l : H-Ala-Leu-Ile-Leu-Thr-Leu-Val -Ser-OH A i Dl: H-Leu-Phe-Val-Val-Thr-Leu-Val-Gly-OH (9, 10, 13-15).the pheromone induction process as measured by different criteria.Pronaseor chymotrypsin abolished its biological activity We exposed identical cultures of S. fuecalis responder cells,we assumed that, like the other S. faecalis to various concentrations
We compare the sequence of cCFlO to those of previously analyzed pheromones and inhibitors, we demonstrate directly that thesynthetic pheromone induces clumping, mating, and synthesis of surface antigens, and we provide evidencethat a singlepheromone molecule may be sufficient to induce a mating response in a S.faecalis cell carrying pCF10
Purification, Structural Analysis,and Synthesis ofcCF10The S. faecalis sex pheromone cCFlO was purified from 60 liters of culture filtrate by the eight steps shown in Table I and described under "Materials and Methods." We achieved a 1.45 X 108-foldpurification and obtained 4.1 pgof pure compound
Summary
Bacterial Strains, Media, and Bioassays for Pheromone ActivityThe responder strain used for the assay of cCFlO activity was OGlSSp (pCFlO), which carries the 58-kilobase tetracycline resistance plasmid, pCFlO (3). The active fractions eluted at approximately 28% acetonitrile, and material from the two aliquots described above, each representing 10liters of culture supernatant, was recombined and diluted to 10 ml and 10%acetonitrile. This sample was reapplied to thesame column, washed with 10% acetonitrile in 0.1% trifluoroacetic acid, and eluted with 10-26% acetonitrile for 5 min, and 26-31% acetonitrile for 25 min at 1ml/min. The purity was checked by HPLC and TLC (precoated Silica Gel 60FZX, Merck)T. he analogs of cCFlO discussed in the textwere synthesized by very similar methods
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