A composite plasmid has been constructed in vitro from colicin El factor (mass of 4.2 megadaltons [Mdl) and nontransmissible resistance factor RSF 1010 (mass, 5.5 Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin El, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (Sma R), which introduces a single scission in the colicin El factor but not in RSF 1010, converted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin El factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments-an RSF 1010 Eco RI linear and the two expected products from the colicin El factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin El, but colicin El itself was not synthesized. In contrast, colicin El was synthesized by cells containing simultaneously both colicin El factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h, whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin El factor is functional in the composite plasmid.
Sato, Ichiei
(School of Medicine, Gunma University, Maebashi, Japan),
Tokumitsu Tanaka, Kazuko Saito, and Susumu Mitsuhashi
. Inhibition of
Salmonella enteritidis
ingested in mononuclear phagocytes from liver and subcutaneous tissue of mice immunized with live vaccine. J. Bacteriol.
83:
1306–1312. 1962.—In our earlier studies it was shown that mice hyperimmunized with live vaccine of
Salmonella enteritidis
resisted intravenous challenge with 1,000 MLD of virulent strain 116–54 of
S. enteritidis
. Survivors of this challenge completely resisted additional intravenous challenge with 10,000 MLD of the same organism. Mononuclear phagocytes obtained from the abdominal cavity of mice immunized with live vaccine of
S. enteritidis
inhibited intracellular multiplication of virulent strain 116–54, regardless of the presence of antibody in the cell-culture medium. In the present study, mononuclear phagocytes were obtained in a nearly pure state from liver or subcutaneous tissue of mice and were maintained in vitro in good condition. These cells also resisted cellular degeneration caused by intracellular existence of virulent strain 116–54, regardless of the presence of antibody in the cell-culture medium. In contrast, cells obtained from normal mice or mice immunized with dead vaccine were subject to degeneration.
Many isolates belonging to the Enterobacteriaceae were collected in 1965 from the inpatients at geographically scattered hospitals in Japan. Among 2,650 Shigella strains examined, 58.4% were found to be drug-resistant; 95.0% of these resistant strains were multiply resistant. Among 434 resistant strains examined, 81 % carried R factors that were transferable by cell-to-cell contact. Of 160 isolates of other enteric bacteria, drug-resistant strains included 84.2% of the Escherichia coli, 93.0% of the Klebsiella, and 90.0% of the Proteus cultures. Among these resistant strains, 70.3% of the E. coli, 66.7% of the Klebsiella, and 52.0% of the Proteus were multiply resistant. Of these resistant strains, 84.0% of the E. coli, 88.0% of the Klebsiella, and 50.0% of the Proteus strains carried R factors. These results indicate that R factors are widespread among gram-negative bacteria of clinical significance.
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