JOURNAL OF BACTERIOLOGY, July 1978, p. 190-197 Vol. 135, No. 1 0021-9193/78/0135-0190$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Functional Interaction of the tonA/tonB Receptor System in Escherichia coli K. HANTKE AND V. BRAUN* Lehrstuhl Mikrobiologie II, Universitat Tubingen, 7400 Tubingen, West Gernany Received for publication 7 February 1978 Host range mutants of phage Ti (Tlh), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is appar- ently only required for the irreversible adsorption of the phage Ti, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages Ti and Tlh, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of Tlh (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA+ cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferri- chrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 uM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100,M ferrichrome with the tonB+ parent strain in the presence of glucose as energy source, and 90% inhibition by 1 ,uM ferrichrome with partially starved parent cells. We conclude that there exist different func- tional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved. Strains of Escherichia coli with a mutated requires energy. Infection can be inhibited by tonA gene are resistant to the phages T5, Ti, arsenate in respiratory-deficient hemA mutants 4)80 and to colicin M and albomycin, and they or by uncouplers in uncA mutants lacking a are unable to take up ferric iron as ferrichrome functional ATPase (11). These data have led to complex (5). Mutants in the tonB region show a the conclusion that the tonB function may cou- similar resistance pattern, except that they re- ple the energized state of the cytoplasmic mem- main T5 sensitive. The tonA protein is localized brane to the receptor-dependent translocation in the outer membrane (6) and serves as receptor steps across the outer membrane. In this hypo- for the phages and colicin M. It is also required thetical view, the functional state ofthe receptor for the translocation of ferric iron as a ferri- protein is dependent on the energized state of chrome complex across the outer membrane. the cytoplasmic membrane. The tonB function, so far not identified bio- To obtain further insights into tonA/tonB chemically, is also necessary for the lethal action interactions we isolated mutants of Ti which of colicins B and I and in the energy-dependent productively infect tonB mutants of E. coli. uptake of ferric enterochelin (4, 5, 12, 15, 16, 22), These phage mutants are apparently altered in ferric citrate (9, 12), and vitamin B12 (2). Under such a way that they bind to the tonA protein experimental conditions which allow bypassing and release their DNA independent of the tonB of the outer membrane receptor protein in ferric function. In addition we found that starvation or enterochelin (8, 12) and in vitamin B12 uptake uncoupling of the tonA/tonB interaction is a (1), the tonB function is also bypassed. In addi- prerequisite for ferrichrome to inhibit adsorp- tion, irreversible binding of phages Ti and 480 tion of T5 to whole cells. 190 VOL.VOL.135,135,19781978~~~~tonA/tonB INTERACTION IN E. COLI 191 In view of the specificity problem which arises ance were transduced by P1 (19) either with the trp from binding of very different substances to one marker (for tonB) into E. coli A 33 trp or with the we also studied tonA mutants dapD marker (for tonA) into E. coli AT982 dapD receptor protein, two transductants of each donor were stud- normal amounts of an altered (Table 1); which contained ied in more detail (numbered 7/111, 7/112, 9/411, receptor in the outer membrane. The mutated 9/412). To test albomycin sensitivity in the recipients, protein served as a receptor for a newly isolated E. coli AT982, originally albomycin resistant, was host range mutant of T5. made sensitive by isolating pan mutants into which the pan' gene was reincorporated by P1 transduction MATERIALS AND METHODS by using E. coli AR 2847 pan' albomycin sensitive as Strains and culture conditions. The parent donor. Albomycin (50 iLg/ml) and the strains to be strain for most of the colicin and phage-resistant mu- tested were cross-streaked. T5-resistant derivatives of tants was E. coli K-12 AB 2847 (aroB tsx thi malA) B9/4i and B9/71 were isolated by seeding i09 phage described previously (4, 15). The derivatives are listed particles and i09 cells in 2.6 mnl of TY overlay agar in Table 1. The gene symbols used for colicin B- onto TY plates. Colonies of resistant cells were puri- resistant mutants is feuB, and cir for colicin I-resistant fied by streaking repeatedly on TY plates (strains 41/2 mutants. The feuB mutants were originally character- and 71/i). The tonA marker was transduced into E. ized as being deficient in ferric enterochelin uptake coli AT982 as described above (transductants 41/56 (12, 15). feuB mutants lack the outer membrane re- and 71/71). ceptor protein, as do cbr mutants (21). For the isolation of Ti-sensitive tonB derivatives, tonA mutants of cir and feuB mutants were isolated solutions of 0.1 mnl of colicin B (reciprocal titer, 128) on tryptone-yeast (TY) agar plates (8 g of tryptone, 5 and 0.i mnl of albomycin (0.5 mg/ml) were added to 0.8 g of yeast extract, 5 g of NaCl, and 15 g of agar per ml ofcellsofE. coliAB 2847 (4 x 108 cellspermrl) in liter [Difco L-aboratories] supplemented with 50 t&M TV medium and incubated for 30 rnn at 3700. Sam- FeSO4) seeded with i09 cells and 108 Ti phages in 2.5 ples of 0.1 ml were spread with overlay agar onto TV ml of TY overlay agar (0.3%). After 4 days of incuba- plates and incubated at 370C for 1 day. Colonies were tion at 370C, small colonies were picked and grown in cloned several timnes. Resistance to colicin B and lb 1 ml of TY medium overnight. Samples were cross- and to phage Ti was tested by cross-streaking on streaked on TY plates against Ti and T5. T5-sensitive, plates, resistance to colicin V was tested by cross- Ti-resistant derivatives were cloned and used further. streaking samples of the colonies over plates onto From strain B7 (Table 1) we obtained 1 isolate which the colicinogenic strain had been grown in a (B7/11), and from strain B9 we obtained 10 T5-sensi- streak and killed by exposure to chloroform vapor, and tive, Ti-resistant derivatives (B9/9, B9/15, B9/17, resistance to albomycin was tested by applying a filter B9/24, B9/35, B9/41, B9/66, B9/71, B9/77). The genes paper disk imnpregnated with a solution of albomycin of two strains, B7/11 and B9/41 conferring Ti resist- (50 ,ug/ml) to the surface of TV plates. TABLE i. E. coli K-12 strains used Strain' Relevant markers and properties Source or reference AR 2847 aroB; parental; only produces enterochelin when supplied with the 15 precursor dihydroxybenzoate R7 AB 2847 cir feuB derived from VR42 12 R9 AB 2847 cir feuB derived from VR42 12 B7/11 AB 2847 cir feuB tonA; partially T5 sensitive This study B9/9 AR 2847 cir feuB tonA; partially T5 sensitive This study B9/41 AR 2847 cir feuB tonA; partially T5 sensitive This study B9/71 AR 2847 cir feuB tonA; partially T5 sensitive This study 41/2 tonA; T5-resistant derivative of B9/41 This study 71/i tonA; T5-resistant derivative of B9/7i This study 41/56 dapD' tonA transductant of strain AT982 using phage P1 grown on This study strain B9/41 71/71 dapD' tonA transductant of strain AT982 using phage P1 grown on This study strain B9/71 BR158 AR 2847 tonB; isolated as colicin B resistant is BRR185 AR 2847 tonB; isolated as colicin B resistant 15 IR112 AR 2847 tonB; isolated as colicin I resistant 15 1R114 AR 2847 tonB; isolated as colicin I resistant 15 Hi1-5, 7, 8, ii AR 2847 tonB; Ti resistant isolated as simnultaneously resistant to This study colicin R and albomycin; also resistant to colicin Ib, V, phage Ti H31, 32, 35 AR 2847 tonB but Ti sensitive This study AW405/7 tonE derivative of AW405 thr leu his obtained from J.