Purification and Genetic Determination of Bacteriocin Production inEnterobacter cloacae

Abstract

Enterobacter cloacae (strain DF1 3) was found to produce a bacteriocin which could be induced by mitomycin C. In the supernatant fluid of the induced culture phagelike particles were found. The bacteriocin was partially purified from induced cultures by ammonium sulfate precipitation and gel-filtration on Sephadex G-150. Ultraviolet-absorbing material was eluted from the Sephadex column in three fractions. The biological activity was mainly present in the second fraction and is associated with a protein with a molecular weight of about 61,000. The phagelike particles were found in the first fraction and show no biological activity. Upon conjugation of E. cloacae strain DFI 3 with another strain of the same species and with Escherichia coli K-12S, the ability to produce bacteriocin was transferred. The new bacteriocinogenic strain produced bacteriocin, which could not be distinguished from that produced by E. cloacae strain DF13. Although transfer of the bacteriocinogenic factor often occurred together with transfer of the ability to produce phagelike particles, it was shown that these two factors are two separate genetic entities. In addition to a bacteriocinogenic factor, E. cloacae strain DF13 was found to carry two other transferable plasmids: one determining resistance against streptomycin and sulfanilamide and another determining resistance against penicillin.

Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli

“…Plasmid pABN1 DNA (4) was purified by cesium chloride-ethidium bromide ultracentrifugation, and a 6.8-kilobase-pair fragment was digested with restriction endonucleases Hindlll and EcoRI (Genofit, Geneva, Switzerland) used in accordance with the recommendations of the manufacturer. bla Susceptible to cloacin DF13 Former S15 strain (14) incubation at 37°C, the colonies were lysed, and the filters were dried, baked, and hybridized with the radioactive probe as described previously (6). Strains KH576 and BM21 were used as positive and negative controls, respectively (see Fig.…”

“…Cloacin DF13 is a bacteriocin that attaches to the same outer membrane protein that is involved in the transport of aerobactin, and cloacin sensitivity is indicative of the presence of a functional aerobactin transport system (3,23). The method used to examine susceptibility to cloacin DF13 was derived from De Graaf et al (14). Mitomycin C (Sigma) was added at 1 jig/ml to an exponentially growing culture of E. coli N5160 (Table 1) in brain heart infusion broth (Difco) incubated under aerated conditions for 10 min at 37°C.…”

Role of aerobactin in systemic spread of an opportunistic strain of Escherichia coli from the intestinal tract of gnotobiotic lambs

“…assayed by the cloacin sensitivity test (7). Cloacin DF13 was prepared by adding mitomycin to a growing nutrient broth culture of Enterobacter cloacae S458 as described by De Graff et al (13), except that the crude bacteriocin preparation was neither concentrated nor purified. Cells were pelleted by centrifugation, and the supernatant fluid containing cloacin activity was sterilized by membrane filtration and used as a crude cloacin DF13 preparation.…”

Differences in excretion and efficiency of the aerobactin and enterochelin siderophores in a bovine pathogenic strain of Escherichia coli

“…The presence of the aerobactin outer membrane receptor was assayed by the cloacin sensitivity test (7). Cloacin DF13 was prepared by adding mitomycin C to a growing nutrient broth culture of Enterobacter cloacae S458 as described by De Graaf et al (11), except that the crude bacteriocin preparation was neither concentrated nor purified. XA/CV30 and H646 were used as sensitive control strains, and DB6433 and IR20/111N were used as resistant control strains.…”