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SUMMARY: When colicinogenic female bacteria are crossed with non-colicinogenic males, all the genes of the male chromosome can enter into the zygote without necessarily killing it. There is a great difference with regard to the number of certain recombinants tested, derived from crosses of the type Hfr col- x F-col+, as compared to their numbers in crosses of the type Hfr col- x F-col-. In certain cases this difference was reduced when the zygotes were incubated at 28° instead of the standard temperature of 37°. It was found that resistance to colicines is not always an all-or-none phenomenon. There are different degrees of colicine resistance and those mutants which show complete resistance by the standard tests are still found to be sensitive under more rigorous conditions. In the case of colicine B the sensitivity of these semi-resistant mutants is greater at 37° than at 28°.

It was found that cells of Escherichia coli K-12 strain C600, infected with phage λ, do not produce phage when the culture has been transferred from rich to minimal medium, whereas in induced cells such a shift-down does not prevent the development of the phage. The factors necessary for the infecting phage to develop in a shift-down culture were found to be the two amino acids valine and isoleucine. Neither of these amino acids is required for bacterial growth in minimal medium. After a transfer of the induced cells from minimal to rich medium, there is a longer lag in phage production than in the case of cells that have been growing all the time in minimal medium. In infected sensitive cells, a transfer from minimal to rich medium does not appear to prolong the lag of phage production.

Experiments are described with Escherichia coli K12 strains, the growth of which is inhibited in minimal media by the addition of 25 μg cysteine/ml. In such lysogenic (for λ) bacteria, induced by ultraviolet light, X-rays or thymine deprivation, the addition of 2·5 μg/ml. cysteine significantly decreased the number of induced cells. Cysteine at 2·5 μg/ml. did not have any effect on thymineless death of the corresponding nonlysogenic strains, nor did it decrease the number of infected sensitive bacteria that produced phage. Photoreactivation of ultraviolet-irradiated lysogenic bacteria had an approximately constant dose reduction factor for induction, while cysteine had not. It was concluded that cysteine reverses the induction by interfering at a step different from the one which is reversed by photoreactivation. This cysteine-sensitive step does not appear to be involved in the vegetative production of the phage.

The mode of action of pesticin, a bacteriocin produced by many strains ofPasturella pestis, was studied. Pesticin action on macromolecular synthesis of a sensitive strain ofEscherichia coli, strain φ, was found to have features similar to those of colicin E2-317 acting on the same strain. After exposure to pesticin, deoxyribonucleic acid synthesis was arrested and ribonucleic acid was degraded, but little effect was observed on protein synthesis. Pesticin, like colicin E2-317, induced lysogenicE. coliφ (P1), but, unlike the colicin, was active in the presence of dinitrophenol. Trypsin was found to reverse pesticin action up to 15 min after its addition at 40 C toE. coliφ. Pesticin action was studied on three sensitive bacterial strains,P. pestis2C,P. pseudotuberculosis, andE. colistrain φ, which vary widely in their optimal growth temperature.P. pestisgrows best at 29 C,P. pseudotuberculosisat 37 C, andE. coliφ at 40 C. It was found that pesticin action on all three strains was optimal at 40 C. Whereas the titer of pesticin was the same on all three strains when determined on agar,E. coliφ was the most sensitive to pesticin action in broth. No action of pesticin in broth onP. pseudotuberculosiswas observed unless Ca ions were added. The effect was not immediate; that is, the cells had to be grown in a medium containing Ca++before they displayed sensitivity to pesticin.

SUMMARY: Strains of Pasteurella pestis under certain conditions form a baeteriocin-like material, which has been named pesticin. Pesticin formation was induced by ultraviolet irradiation. A chemically denned medium composed of inorganic salts and amino acids supported the synthesis of pesticin by irradiated organisms. The minimal amino acid requirement for pesticin synthesis has been established. For the synthesis of pesticin at 37° the irradiated organisms required certain amino acids which they did not require at 27°. Bacteria in the stationary phase and actively dividing bacteria were equally able to produce pesticin upon irradiation. Concentrations of chloramphenicol lower than those required for bacteriostasis inhibited pesticin synthesis. Non-pesticin producing mutants of P. pestis were obtained by exposure of the bacteria to higher doses of u.v. irradiation than those required for the induction of pesticin formation. These mutants were sensitive to pesticin.

Strains of Pasteurella pestis under certain conditions form bacteriocin-like material, for which the name ‘pesticin’ is proposed; it inhibits the growth of P. pseudotuberculosis. The conditions necessary for induction of pesticin formation are similar to those which lead to induction of formation of some known bacteriocins. Of 24 strains of P. pestis tested, all but one produced pesticin. A mutant of P. pseudotuberculosis which was resistant to pesticin produced by one strain of P. pestis was found to be resistant to pesticin produced by all the other strains.

Plasmids of 44.4–46 Mdal were identified in conditional virulent Yersinia species. All virulent strains studied are unable to grow on oxalate-containing plates at 37 °C (OX− phenotype) which is a characteristic property of strains producing the essential virulence VW antigens. The phenotopic transition from OX− to OX+ in these strains is concomitant with loss of virulence and loss of this plasmid. The similarity in size and in the DNA fragmentation patterns, generated by HindIII, of the plasmids isolated from either Y. pseudotuberculosis or two conditional virulent Y. pestis strains, suggests that a common plasmid—pSB2—is carried by these strains. A plasmid of a similar size, ~42 Mdal, and function was recently identified ( P. Gemski, J. R. Lazere, and T. Casey, 1980 , Infect. Immunity27, 682–685; D. L. Zink, J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. Vickery, W. D. Roof, and G. A. O'Donovan, 1980 , Nature (London)283, 224–225) in virulent Y. enterocolitica. We conclude that pSB2 in Y. pseudotuberculosis and Y. pestis and its counterpart in Y. enterocolitica carry genetic information essential for virulence common to the Yersinia species, probably related to VW antigen production. Several additional plasmids were identified in several strains of Y. pestis. One of these plasmids, designated pSB3 (12.5 Mdal), appears to be associated with pesticin production.

Salmonella typhimurium LT2 excreted under certain conditions an antibiotic substance designated typhimuricin. It is suggested that the LT2 “cryptic” plasmid is involved in its production and in the immunity to it. Preliminary characterization of typhimuricin is presented.

A simple and rapid plate test is described for screening substances that induce colicine E2. By using chemicals activated with microsomal enzymes and a permeable (rfa) tester bacterium that is also deficient in DNA repair (uvrB), the range of inducing substances that can be detected has been extended. The possible correlation between colicine-inducing substances and carcinogens is discussed.

The antioxidants pyrogallol and its oxidative derivative, purpurogallin, both induce colicine E2 as well as base substitution and frameshift mutations. Because of the bactericidal effect of purpurogallin, its mutagenicity could be best demonstrated by short-term exposure followed by dilution on the test plates. The colicine-inducing potential of purpurogallin was also observed when tested directly on the plates.

A lysogenic, recombination-deficient bacterial mutant (rec−) can be induced to produce λind−C587 by a temperature shift, although it does not produce phage after u.v. irradiation when lysogenic for wild λ. When this mutant (rec−) was made colicinogenic for colicins E2 and I, colicin production was found not to be inducible.Superinfection of irradiated lysogenicrec−(λ) bacteria, at doses of irradiation that did not reduce their capacity to produce phage λ, was not productive. It seems therefore that a certain product(s) that usually arises following u.v. irradiation inrec+cells, and triggers the induction of lysogenic and colicinogenic cells by causing loss of immunity or repression, is not produced in therec−mutant. However, immunity is lost and the prophage is induced after a temperature shift inrec−cells which are lysogenic for λind−C857.

Addition of chloramphenicol or puromycin to colicinogenic bacteria carrying the colicinogenic factor E2 resulted, after resumption of protein synthesis, in the induction of colicin. An isogenic lysogenic strain carrying prophage λ was not induced by chloramphenicol.

When two lysogenic strains of Escherichia coli were grown in minimal media, washed, and irradiated with ultraviolet light for 1 min and then diluted again into their respective minimal media, with and without added cysteine, there was a marked difference in the titer of phages produced by the irradiated bacteria when compared with non-irradiated. When the bacteria were grown before irradiation in minimal media supplemented with cysteine, the production of phage was the same whether cysteine was added after irradiation or not. Results suggest that if protein synthesis is disturbed immediately after irradiation this disturbance can reverse the process of phage induction caused by ultraviolet light. Possible reaction mechanisms involved are discussed. (C.H.)

INDUCTION of lysogenic bacteria was first produced by the use of ultra-violet light1. It was shown later that other radiations2, nitrogen mustard, ethyleneimines, epoxides and organic peroxides3 can also induce lysogenic bacteria to produce phage. The process of induction is not yet clear. Several lines of evidence indicate that the primary lesion initiated by inducing agents could be an alteration in the nucleic acid economy of the host, an effect of unbalanced growth4.