Antonie van Leeuwenhoek 43 (1977) 7-18
Genetics of coIicin E susceptibility in Citrobacterfreundii A. MARIAN J. J. VAN VUGHT AND ELLEN C. M . DELEMARRE
Laboratory for Medical Microbiology, Faculty of Medicine, Free University, Amsterdam, The Netherlands
VAN VuGmr, A. M. J. J. and D~L~MAggE,E. C. M. 1977. Genetics of colicin E susceptibility in Citrobacterfreundii. Antonie van Leeuwenhoek 43: 7-18. The insensitivity of Citrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants of C. freundii also lost their colicin E1 receptor function. Sensitivity to coticin E1 can be induced by F'gal+tol + plasmids, the tol A + gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by the E. coli F'14 bfe + plasmid, which is also able to enhance notably the receptor capacity for colicin El. The bfe + gene product ofE. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants of C. freundii. All results show that there is a remarkable difference between the E. coli bfe + gene product and the bfe + gene product of C. freundii and also between the toI A + gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F'14 bfe + plasmid. INTRODUCTION The group E colicins are particularly usefull for a study of the interaction of bacteriocins with bacterial cells, because the E receptor is shared by the coticins El, E2 and E3, which have quite distinct biochemical targets (Fredericq, 1949; Buxton, 1971; Nomura, 1964). Moreover this receptor also adsorbs colicin A and bacteriophage BF23. A mutation which confers simultaneously insensitivity to phage BF23 and colicins A, El, E2 and E3 (bfe) has been described by several authors (Hill and Holland, 1967; Buxton, 1971). This mutation is located at 79 minutes on the E. coli chromosome (Buxton, 1971 ; Jasper, Whitney and Silver, 1972). The cross-resistance studies suggested that common receptors exist for bacteriophage BF23 and the coticins A, El, E2 and E3. These observations are not fully in agreement with the properties of the purified receptor. Sabet and Schnaitman (1973) have purified the receptor for colicin E3 and it was characterized as a glycoprotein with a molecular weight
A. M. J. J. VAN VUGHT AND E. C. M. DELEMARRE
of 60 000 dalton. The purified receptor also adsorbs colicin E2, but not colicin El. Probably other components in addition to the glycoprotein are needed for reception of colicin El. The existence of a difference between the receptor for E1 on the one hand and for E2 and E3 on the other hand on the basis of the properties of purified receptor is also supported by genetic experiments, because Hill and Holland (1967) have isolated mutants, which were resistant to the colicins E2 and E3, but sensitive to colicin El. In previous studies we dealt with colicin A resistant mutants of C. freundii (van Vught, de Graaffand Stouthamer, 1975). Since C.freundii is not sensitive to the E. colicins and to phage BF23, it was not possible to compare the colicin A resistant mutants of C. freundii with the bfe nmtants of E. coli. Genetic analysis of our resistant mutants revealed that the mutation was situated in the same region as the bfe gene in E. coli (van Vught et al., 1975). In this study we report about our attempts to find a genetic basis for the difference in adsorption of colicins A, El, E2 and E3.
MATERIALS AND METHODS
Bacterial strains and media. The strains we used are listed in Table 1. The preservation of C. freundii strains, the composition of the minimal medium (MM), the nutrient broth (NB) and the nutrient agar (NA) are essentially as described by de Graaff and Stouthamer (1971). Tryptonagar (TA) contained 1 ~ Difco Tryptone, 0.5~ Difco Yeast Extract, 0.5~ NaC1 and 1 ~ Difco Bacto agar. Phage strains. Bacteriophage BF23 was obtained from S. K. Guterman and was propagated on E. coli strain ABt206. Preparation ofcolicins. Colicin A was isolated as described by de Graaf and Stouthamer (1970). Colicins El, E2 and E3 were prepared from colicinogenic cells, grown in aerated BHI broth at 37 C. At the early log-phase the colicin production was induced with 1 ~tg/ml of mitomycine C. The incubation was continued :for 10 min. The cells were harvested by centrifugation and resuspended in fresh, prewarmcd BHI broth; incubation was continued for 3 h at 37 C. After centrifugation the supernatant was sterilized by filtration through membrane filters and used as the colicin suspension. Patterns of colicin sensitivity were determined as described by van Vught et al. (1975). Colicin titers were estimated as described by de Graaf, Spanjaerdt Speckman and Stouthamer (1969). Colicin adsorption test. Adsorption of coticins by bacterial cells was determined by incubating an appropriate dilution of colicin (+ 45 KU/ml) with bacterial cells for 30 rain at 37 C. After centrifugation of the incubation mixture the unadsorbed colicin in the supernatant was titrated. The nutrient broth
FF FFF' F-
Escherichia coli K12 Escheriehia coli K12 Escherichia coli Citrobacterfreundii Escherichia coli Citrobacter freundii
CitrobacterJi'eundii Escherichia coli Escherichia coli Citrobacter ~'eundii Citrobacter Ji'eundii
TR1 TR2 CA38 G30 G385 G502
G502 res-55 AB1206 RB134 G538 G539
colicin E1 production colicin E2 production colicin E3 production colicin A production F'gal +to/+ arg-1 trp-2 ilv-1 had-1 leu-3 thr-3 pro-4 aro-1 his-11 pur-6 bio-1 gal-4 hssI-1 hal-1 str-3 colicin A resistant mutant of G502 F'14 bJe+ /pro his str V (ilv met B arg H) F'14 bfe/pro his str V (itv met B arg H) gal-3 F'gal + tol A617 gal-3 F'gal + tol B51~
Relevant characteristics
Fredericq Fredericq Fredericq Fredericq CA31 P. v. d. Putte KMBL1145
Origin
de Graaff et al., 1974 van Vught et al., 1975 R. S. Buxton R. S, Buxton van Vught et al., 1975 van Vught et al,, 1975
The nomenclature conforms to the recommendations of Demerec et al. (1966) and Taylor and Trotter (1972).
F F~ F' F' F'
Sex
Biotype
Collection no.
Table 1. Bacterial strains
Z
,q ,
Genetics of coIicin E susceptibility in Citrobacterfreundii A. MARIAN J. J. VAN VUGHT AND ELLEN C. M . DELEMARRE
Laboratory for Medical Microbiology, Faculty of Medicine, Free University, Amsterdam, The Netherlands
VAN VuGmr, A. M. J. J. and D~L~MAggE,E. C. M. 1977. Genetics of colicin E susceptibility in Citrobacterfreundii. Antonie van Leeuwenhoek 43: 7-18. The insensitivity of Citrobacter freundii to the E colicins is based on tolerance to colicin E1 and resistance to colicins E2 and E3. Spontaneous colicin A resistant mutants of C. freundii also lost their colicin E1 receptor function. Sensitivity to coticin E1 can be induced by F'gal+tol + plasmids, the tol A + gene product of which is responsible for this effect. Receptor function for colicins E2 and E3 is induced by the E. coli F'14 bfe + plasmid, which is also able to enhance notably the receptor capacity for colicin El. The bfe + gene product ofE. coli, which is responsible for these phenomena, also restores the receptor function for colicin A and E1 in colicin A resistant mutants of C. freundii. All results show that there is a remarkable difference between the E. coli bfe + gene product and the bfe + gene product of C. freundii and also between the toI A + gene products of these strains. The sensitivity to phage BF23 parallels the sensitivity to colicins E2 and E3 and is also induced by the F'14 bfe + plasmid. INTRODUCTION The group E colicins are particularly usefull for a study of the interaction of bacteriocins with bacterial cells, because the E receptor is shared by the coticins El, E2 and E3, which have quite distinct biochemical targets (Fredericq, 1949; Buxton, 1971; Nomura, 1964). Moreover this receptor also adsorbs colicin A and bacteriophage BF23. A mutation which confers simultaneously insensitivity to phage BF23 and colicins A, El, E2 and E3 (bfe) has been described by several authors (Hill and Holland, 1967; Buxton, 1971). This mutation is located at 79 minutes on the E. coli chromosome (Buxton, 1971 ; Jasper, Whitney and Silver, 1972). The cross-resistance studies suggested that common receptors exist for bacteriophage BF23 and the coticins A, El, E2 and E3. These observations are not fully in agreement with the properties of the purified receptor. Sabet and Schnaitman (1973) have purified the receptor for colicin E3 and it was characterized as a glycoprotein with a molecular weight
A. M. J. J. VAN VUGHT AND E. C. M. DELEMARRE
of 60 000 dalton. The purified receptor also adsorbs colicin E2, but not colicin El. Probably other components in addition to the glycoprotein are needed for reception of colicin El. The existence of a difference between the receptor for E1 on the one hand and for E2 and E3 on the other hand on the basis of the properties of purified receptor is also supported by genetic experiments, because Hill and Holland (1967) have isolated mutants, which were resistant to the colicins E2 and E3, but sensitive to colicin El. In previous studies we dealt with colicin A resistant mutants of C. freundii (van Vught, de Graaffand Stouthamer, 1975). Since C.freundii is not sensitive to the E. colicins and to phage BF23, it was not possible to compare the colicin A resistant mutants of C. freundii with the bfe nmtants of E. coli. Genetic analysis of our resistant mutants revealed that the mutation was situated in the same region as the bfe gene in E. coli (van Vught et al., 1975). In this study we report about our attempts to find a genetic basis for the difference in adsorption of colicins A, El, E2 and E3.
MATERIALS AND METHODS
Bacterial strains and media. The strains we used are listed in Table 1. The preservation of C. freundii strains, the composition of the minimal medium (MM), the nutrient broth (NB) and the nutrient agar (NA) are essentially as described by de Graaff and Stouthamer (1971). Tryptonagar (TA) contained 1 ~ Difco Tryptone, 0.5~ Difco Yeast Extract, 0.5~ NaC1 and 1 ~ Difco Bacto agar. Phage strains. Bacteriophage BF23 was obtained from S. K. Guterman and was propagated on E. coli strain ABt206. Preparation ofcolicins. Colicin A was isolated as described by de Graaf and Stouthamer (1970). Colicins El, E2 and E3 were prepared from colicinogenic cells, grown in aerated BHI broth at 37 C. At the early log-phase the colicin production was induced with 1 ~tg/ml of mitomycine C. The incubation was continued :for 10 min. The cells were harvested by centrifugation and resuspended in fresh, prewarmcd BHI broth; incubation was continued for 3 h at 37 C. After centrifugation the supernatant was sterilized by filtration through membrane filters and used as the colicin suspension. Patterns of colicin sensitivity were determined as described by van Vught et al. (1975). Colicin titers were estimated as described by de Graaf, Spanjaerdt Speckman and Stouthamer (1969). Colicin adsorption test. Adsorption of coticins by bacterial cells was determined by incubating an appropriate dilution of colicin (+ 45 KU/ml) with bacterial cells for 30 rain at 37 C. After centrifugation of the incubation mixture the unadsorbed colicin in the supernatant was titrated. The nutrient broth
FF FFF' F-
Escherichia coli K12 Escheriehia coli K12 Escherichia coli Citrobacterfreundii Escherichia coli Citrobacter freundii
CitrobacterJi'eundii Escherichia coli Escherichia coli Citrobacter ~'eundii Citrobacter Ji'eundii
TR1 TR2 CA38 G30 G385 G502
G502 res-55 AB1206 RB134 G538 G539
colicin E1 production colicin E2 production colicin E3 production colicin A production F'gal +to/+ arg-1 trp-2 ilv-1 had-1 leu-3 thr-3 pro-4 aro-1 his-11 pur-6 bio-1 gal-4 hssI-1 hal-1 str-3 colicin A resistant mutant of G502 F'14 bJe+ /pro his str V (ilv met B arg H) F'14 bfe/pro his str V (itv met B arg H) gal-3 F'gal + tol A617 gal-3 F'gal + tol B51~
Relevant characteristics
Fredericq Fredericq Fredericq Fredericq CA31 P. v. d. Putte KMBL1145
Origin
de Graaff et al., 1974 van Vught et al., 1975 R. S. Buxton R. S, Buxton van Vught et al., 1975 van Vught et al,, 1975
The nomenclature conforms to the recommendations of Demerec et al. (1966) and Taylor and Trotter (1972).
F F~ F' F' F'
Sex
Biotype
Collection no.
Table 1. Bacterial strains
Z
,q ,
