Induction of Colicin la at High Temperature

Rosa H. Gromkova
1971 Journal of Bacteriology  
Induction of colicin la at a high temperature (42 C) was demonstrated in a wild strain of Shigella sonnei. After transfer of the Col factor, a similar effect was observed in those recipient cells acquiring colicinogeny. The induction is suggested to be due to the presence of a thermosensitive colicin repressor. The wild-type strain segregated sensitive cells and showed heterogeneity in colicin production. Colicins may be induced by certain physical and chemical agents such as ultraviolet or
more » ... ultraviolet or X-irradiation, mitomycin C, hydrogen peroxide, etc. Colicin E2 induction in deoxyribonucleic acid (DNA) temperature-sensitive mutants of Escherichia coli K-12 at high temperature was shown in 1965 by Kohiyama and Nomura (7) . Strains producing colicin E2 as well as colicin El which are inducible at elevated temperature were recently described by Kennedy (Bacteriol. Proc., p. 61, 1970). In the present paper, induction of colicin la at high temperature in a wild strain of Shigella sonnei is described. MATERIALS AND METHODS Bacterial strains. S. sonnei 35, isolated from a child ill with dysentery, had typical morphological, cultural, biochemical, and serological properties. E. coli Row, E. coli B, E. coli K-12 CSH-2, and S. sonnei 3793 were used as colicin-sensitive indicators. Identification of the colicin was carried out by using the 16 type-specific strains from the Fredericq collection (2). Media. Nutrient agar (Difco) at concentrations of 1.5 or 0.7% and nutrient broth (Difco) were used for the propagation of all organisms employed in these investigations. Colicin production. Colicinogeny was tested on solid nutrient medium by the "stab" method (2). In liquid medium the colicin was examined after 48 hr of incubation followed by centrifugation of broth cultures. The supernatants were treated with chloroform, serial dilutions were spotted on agar previously seeded with the indicator strain, and the colicin titer was determined after 18 hr of incubation at 37 C. The number of colicin units per milliliter was defined as the greatest dilution which gave a clear zone of inhibition of growth of the indicator bacteria. Cultures not treated with chloroform were tested in the same manner. The number of colicin-producing bacteria was deter-' Present address: mined by the method of Ozeki et al. (10) by using 18hr broth cultures at 2 x 108 cells/ml. Induction assays. Ultraviolet irradiation was achieved by using a 15-w germicidal lamp (wavelength 25.37 pm). Samples (5 ml) of cells from 3-hr cultures at 2 x 108 cells per ml in 0.5 M phosphate buffer (pH 7.6) were irradiated for 60 sec at 50 cm with an intensity of 5 ergs per mm2 per sec. For mitomycin C induction, nutrient broth cultures containing 0.05 Ag of mitomycin C per ml were incubated for 18 hr at 37 C; the number of lacunae was then determined. Thermal induction was achieved by incubating the colicinogenic strains for 18 hr either on nutrient agar or in broth at 32 or 42 C, whereas incubation with the indicator strain was at 37 C. Transfer experiments. S. sonnei 35 was used as a donor, with colicin-and streptomycin-resistant mutants of E. coli Row, E. coli B, E. coli CSH-2, and S. sonnei 3793 as recipients. Three-hour broth cultures at 2 x 108 cells of donor and recipient per ml were mixed in equal volumes, incubated for 2, 4, 6, and 18 hr at either 32 or 42 C, and then seeded on streptomycin-agar plates at a dilution designed to obtain about 100 colonies of the recipient strain per plate. The colicinogeny of these colonies was tested by replica-plating. RESULTS The colicin testing of strain S. sonnei 35 in mass culture by the stab method showed a very small, almost filiform inhibition zone less than I mm at 37 C. No colicin was detectable in undiluted liquid nutrient medium, whether chloroform was used or not. No significant differences in colicin production were found with different indicator strains. In a standard inoculum of 2 x 108 cells of S. sonnei 35 per ml, 2 x 103 barely visible lacunae were formed on incubation at 37 C. No increase in the number of lacunae was observed after ultraviolet irradiation, whereas with mitomycin C a 100-fold increase in the number of lacunae was observed. Upon incubation at high temperature (42 C), there was an in-720 on May 9, 2020 by guest
doi:10.1128/jb.106.3.720-723.1971 fatcat:tuswtl2hjfe4hfdxseadrfhgwm