Antonie van Leeuwenhoek 41 (1975) 309-318

309

A genetic study of tolerance and resistance to colicin A in

Citrobacter freundii A. MARIAN J. J. VAN VUGHT, J. DE GRAAFF AND A. H. STOUTHAMER Laboratory for Medical Microbiology, Postbus 7161, and Biological Laboratory, Microbiology Department, Free University, Amsterdam, The Netherlands

VAN VUGHT, A. M. J. J., DE GRAAFF, J. and STOUTHAMER,A. H. 1975. A genetic study of tolerance and resistance to colicin A in Citrobacterfi'eundii. Antonie van Leeuwenhoek 41 : 309-318. Colicin A-insensitive mutants of Citrobacter fi'eundii were isolated and grouped into six phenotypic classes characterized by sensitivity, insensitivity or partial insensitivity to the bacteriocins $6, D F 13 and colicin A, and sensitivity or insensitivity to deoxycholate (DOC) and ampicillin. Mapping by the gradient-of-transmission method revealed the chromosomal regions in which the responsible genes are situated. Res-3 mapped nearpur between pur and thr; Tol-5 mapped between aro and ilv and Tol-4 between gal and pyr; Tol-1, Tol-2 and Tol-3 are situated close to gal. All the mutations that mapped near gal rendered the bacteria more sensitive to D O C and ampicillin. Complementation analysis with E. coil plasmids showed that the three phenotypic groups that map near gal were complemented by E. eoli plasmids and fall into three complementation groups. Two of these are equivalent with the tol A and tol B genes in E. coll. INTRODUCTION The killing action of bacteriocins is a very complex phenomenon. Adsorption of the bacteriocin to a receptor on the bacterial cell surface initiates a lethal process (Nomura, 1964). These receptors are specific and limited in number. It is not yet known whether the bacteriocin penetrates the cell envelope to interact directly with the biochemical target or propagates a stimulus, which traverses the cell envelope. Insensitive mutants which do not adsorb the bacteriocin, due to ineffective receptors, are called resistant. Tolerant mutants though adsorbing the bacteriocin are not killed. In these mutants a post-adsorption step is possibly disturbed. Tolerant and resistant mutants have been isolated by several workers, because these mutants might give information not only on the mode of action of

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VAN VUGHT, J. DE GRUFF AND A. H. STOUTHAMER

the bacteriocin, but also on the composition and function of the cell envelope (Bernstein, Rolfe and Onodera, 1972; Foulds and Barrett, 1973; Cardelli and Koninsky, 1974). Indirect evidence that the cell envelope is altered in these mutants is given by the observation that the sensitivity of the cell to antibiotics, detergents and dyes is often increased (Nagel de Zwaig and Luria, 1967; de Graaf, van Vught and Stouthamer, 1973). Furthermore the protein composition of cell-wall and cell-membrane fractions of some of these mutants differ from that of the wild type (Samson and Hol[and, 1970; de G r a a f et al., 1973; Chai and Foulds, 1974). In E. coli several mutations leading to tolerance and resistance to bacteriocins have been described. With respect to colicin A, the tol PAB locus, near the gal locus, represents mutants, which are affected as to their sensitivity to this colicin (Bernstein et al., 1972). Tol F, mapping on 20 minutes of the E. coil chromosome, also provides tolerance to colicin A (Foulds and Barrett, 1973). Mutations leading to altered sensitivity to other bacteriocins have been mapped on various places on the E. coli chromosome (Hill and Holland, 1967; Buxton, 1971 ; Whitney, 1971 ; Cardelli and Koninsky, 1974). The present article describes the isolation and genetical characterization of colicin A-tolerant and -resistant mutants of C.fi'emldii. Since colicin A, originally produced by a C. fi'eundii strain, is adsorbed well to C. fi'eundii whereas it is not easily adsorbed to E. coil, we expected to find new classes of colicin A-insensitive mutants in C.fi'ezmdii

MATERIALS AND METHODS

StrahTs and media. The strains used are listed in Table 1. The preservation of C. freundii strains and 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). Colicin A was isolated as described by de G r a a f and Stouthamer (1970). Colichl A-insensitive mutants. Mutants, insensitive to colicin A, were isolated without mutagenic treatment from a multiply auxotrophic C.freundii strain G 502. l0 T stationary-phase cells were plated on NA containing 1 ml of an undiluted colicin A suspension. After 24 hours incubation at 37 C, colonies of colicin A-insensitive mutants had developed. Colicin A adsorption test. To distinguish between tolerant and resistant mutants the presence of colicin A receptors was determined (de G r a a f et al., 1973). Mutants were grown in NB containing 5 mm CaCI2 and 5 mM MgSO4. Patterns ofbacteriocin sensitivity. N A plates with colonies of the bacteriocin-

TOLERANT AND RESISTANTMUTANTSIN CITROBACTER FREUNDII

311

Table 1. Bacterial strains used. Collec- Biotype tion no.

Origin

Sex

G30 G497

Citrobacterfreundii Citrobaeterfi'eundii

CA31Fredericq de Graaffet al., 1974 Hfr 1

G499

Citrobacterfi'eundii

de Graaffet al., 1974 Hfr 3

G502

Citrobacterfi'eundii

de Graaffet al., 1974 F -

G527

Citrobacterfi'eundii

Hfr 5

$6 Klebsiella pneumoniae NTCC 9633 DF13 Enterobacter cloacae DF 13 BN37 Escherichia coil K12 A. Bernstein (1972)

F~

BN45 Eseherichia coli K12

A. Bernstein (1972)

F~

BN46 Escherichia coil K12

A. Bernstein (1972)

F~

G460

Citrobacterfi'etmdii

G538

Citrobacterfreundii

G331(de Graaff and F Stouthamer, 1971) BN45 • G460 F~

G539

Citrobacterfi'eundii

BN46 • G460

Properties

Production of colicin A arg-1 trp-1 tac-4 pyr-3 hsrI-~ hal-1 (: :Mu)" (transfers O-aro +-ilv 4 pur +-thr +-pro +) arg-i trp-1 lac-4 pyr-3 hsrI-1 nal-1 (transfers O-ilv +-aro +had +-his +-pro +) arg-1 trp-1 ilv-1 had-1 leu-3 thr-3 pro-4 aro-1 his-ll pur-6 bio-1 gal-4 hssI-1 hal-1 str-3 lac-5 ser-1 (::MU) (transfers O-had +-aro +-ilv +-put"+-thr +)

Production of bacteriocin $6 Production of cloacin DF 13

F~

gal, pro, met, ree A, Ftgal + tolA +B + thr leu bio gal tolA6a7 Flgal + tolA617 thr leu bio gal tolBsts Figal + tolB s 15 arg-1 trp-1 gal-3 nal-1 hsrI-1 arg-1 trp-1 gal-3 hal-1 hsrl-1 F ~gal +tolA6 ~7 arg-1 trp-1 gal-3 hal-1 hsrI-1 Ftgal+tolBsls

The nomenclature conforms to the recommendations of Demerec et al. (1966) and Taylor and Trotter (1972). a. (Mu) means lysogenic for bacteriophage Mu. producing strains G30, $6 and DF13 were treated with chloroform v a p o u r and overlaid with 3 ml of soft agar c o n t a i n i n g 108 cells of a m u t a n t strain. After 18 hr at 37 C, sensitivity was exhibited by inhibition zones a r o u n d the colonies of the bacteriocin-producing strain. NB was supplemented with 5 mM CaCI2 and 5 mM MgSO4. M a p p i n g b y g r a d i e n t o f transmission. M a p p i n g by the gradient-of-transmission method was performed using the membrane-filter technique as described for interrupted m a t i n g experiments (de Graaff et al., 1974). M a t i n g was continued for 1-3 hours a n d samples were plated on selective media, c o n t a i n i n g streptomycin (500 y/ml) or naladixic acid (50 y/ml) as contraselection. C o m p l e m e n t a t i o n e x p e r i m e n t s were performed by mixing l0 T donor- a n d 108 acceptor cells. After 1 hour at 37 C the mixture was plated on M M to which galactose (0.2 % 'V/v) a n d streptomycin (500 y/ml) had been added. The

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sexductants were tested for their Tol character by cross-streaking against colicin A on N A plates. RESULTS

Isolation of tolerant and resistant strains. Colicin A-insensitive mutants could be grouped into one resistant and five tolerant classes. The sensitivity patterns of these classes to colicin A, bacteriocin $6 and cloacin D F 13 are shown in Table 2. The classes Res-3, Tol-3, Tol-4 and Tol-5 were partially insensitive to one, two or all three of the bacteriocins tested i.e. high concentrations of the bacteriocin were able to kill part of the cells. The survival of wild type and mutant cells after incubation with an excess of colicin A is shown in Table 3. The survival of the C.freundii wild type strain was 0.0012 ~ indicating that the killing by colicin A was very effective in the concentration used. However in three of the six colicin A-insensitive mutant groups the survival was only 36-56 ~ . Similar partially insensitive phenotypes have also been described for the tol P and for some of the tol B mutants in E. eoli (Bernstein et al., 1972). Sensitivity to antibiotics and detergents. Bacteriocin-tolerant mutants often showed increased sensitivity to a variety of agents, including detergents, antibiotics, dyes and chelating agents. Table 4 shows the increased sensitivity of the different mutant classes to D O C (deoxycholate) and ampicillin. Furthermore sensitivity to EDTA (ethylenediaminetetraacetic acid), to the dyes methyleneblue and acriflavin, to the antibiotics polymixin B, novobiocin, Dcycloserin, to UV-irradiation and to the phages T3 and Tv was tested. No significant alteration of the mutants as compared to the wild type was found with respect to these agents. Mapp#lg of mutations by gradient of transmission. Tolerant and resistant markers of Citrobacter frem~dii mutants have been mapped by gradient of transmission, a useful method because direct selection on Tol + recombinants Table 2. Patterns of bacteriocin-sensitivity of colicin A-insensitive mutants of Citrobacter

fi'eundii Class

Colicin A

Wild type Res-3 Tol-1 Tol-2 Tol-3 Tol-4 Tol-5

S pR R R pR R pR

Bacteriocin $6

Cloacin DFI 3

S

S

S

S

S R pR pR pR

S R pR R R

Symbols: S, sensitive; R, insensitive; pR, partially insensitive.

TOLERANT AND RESISTANT MUTANTS IN CITROBACTER FREUNDII

3 13

Table 3. Survival of the six phenotypical classes of colicin A-insensitivemutants after incubation with colicin A t Class Wild type Res-3 Tol-1 Tol-2 Tol-3 To I-4 Tol-5

0.001270 38 70 10070 100 70 36 70 100 70 56

I Log-phase cells, grown in broth containing 5 mM MgSO4 were harvested, washed and resuspended in 0.9Yo NaCI at a concentration of l0 s cells/ml. This suspension was incubated with colicin A (100 KU/ml) at 37 C. After 30 min the cells were harvested, washed and resuspended in 0.970 NaC1. A viable count was made of the colicin A-treated cell suspension and a control cell suspension.

is not possible a n d because it eliminates the influence o f the lag in p h e n o t y p i c expression. Such a lag, d e m o n s t r a t e d by van Vught, de G r a a f f a n d S t o u t h a m e r (1974) to occur with Tol-2 mutants, m a y interfere with the d e t e r m i n a t i o n o f m a p position. Preliminary c o n j u g a t i o n experiments were p e r f o r m e d with C. freut~dii Hfr d o n o r strain G 497 a n d tolerant and resistant derivates o f a c c e p t o r strain G 502. Selection was m a d e for Ilv + Str R and r e c o m b i n a n t s were tested for inheritance o f the m o r e distal unselected m a r k e r s pur +, thr +, pro +, gal +, pyr- and and for colicin sensitivity. T a b l e 5 shows that res-55 (Res-3) and to1-128 (Tol-5) are situated in the ilv-pur region. The o t h e r t o l e r a n t m u t a t i o n s m a p p e d in the gal region. T o check the possibility that the res-55 a n d the to1-128 m u t a t i o n s are situated between ilv a n d the leading end o f the c h r o m o s o m e , a cross was p e r f o r m e d with Hfr d o n o r s t r a i n G 527. N a d + N a l R r e c o m b i n a n t s were tested on inheritance o f aro +, ih, +, put .+ a n d colicin sensitivity. Fig. 1 a n d T a b l e 5 show that res-55 is situated near put'. Tol-128 was m a p p e d between aro a n d ilv (Table 5). T h e tolerant m u t a t i o n s in the gal region were crossed with Hfr d o n o r strain G 497. T h r + S t r R r e c o m b i n a n t s were tested for cotransfer o f relevant markers. T a b l e 5 shows that tc/-122 (Tol-1), t o l l 10 (Tol-2) and tol-130 (Tol-3) are situated close to gal. Tol-lO1 (Tol-4) m a p p e d between gal and pyr. Several m u t a n t s o f the different m u t a n t classes have been studied by the m e t h o d o f g r a d i e n t o f transmission and they all gave the same results. This indicates that the different m u t a n t groups are h o m o g e n o u s . They are situated on four different regions o f the c h r o m o s o m e . Their a p p r o x i m a t e m a p p o s i t i o n is given in Fig. 2.

314

A.M.J.J.

VAN VUGHT, J. DE GRA.AFF AND A . H . STOUTHAMER

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A genetic study of tolerance and resistance to colicin A in Citrobacter freundii.

Colicin A-insensitive mutants of Citrobacter freundii were isolated and grouped into six phenotypic classes characterized by sensitivity, insensitivit...
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