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 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 expressing normal amounts of outer membrane receptor were isolated and found to be partially sensitive to phage T5 and resistant to the phages Ti and Tlh, 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 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 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 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. Adler This study E. coli 83-24 Derivative of E. coli W (ATCC 9637) 14 E. coli A33 tip U. Henning AT982 dapD4 thi rel-1 7

Representative strains are given. For additional details, see text. 192 HANTKE AND BRAUN J. BACTERIOL. Isolation of a T5 host range mutant (T5h). of Ti were isolated from two plaques formed on Cross-streaks of phage T5 against the missense tonA plates seeded with 108 cells of the E. coli tonB mutants 41/2 and 71/1 as well as the tonA transduc- mutant BR158 and 109 phage particles. Lysates tants 41/56 and 71/71 revealed partial lysis. Samples with titers of 2 x 1010 plaque-forming units were of the lysis zones were plated with 109 cells by the agar overlay technique. Turbid plaques, which were most prepared by growth on BR158. The plating ef- clearly recognizable on strain 41/2, were obtained. ficiency of the Tlh on various tonB derivatives When plated on E. coli AB 2847 tonA+, a mixture of of different parent strains isolated either as col- clear and turbid plaques appeared. Only the turbid icin B, colicin I, Ti, or albomycin resistant (Ta- plaques could be propagated on 41/2, and after several ble 1) was the same as on tonB+ strains. tonA passages a stable T5h was obtained. mutants were absolutely resistant against Tlh. Isolation of mutants. Isolation of mutants by To be sure that we were dealing with a Ti treatment with N-methyl-N'-nitro-N-nitrosoguani- derivative, we infected E. coli AB 2847 and dine (NTG) or ethyl methane sulfonate (EMS) and determined the pattern of radioactively labeled other genetic techniques, if not described specifically, proteins synthesized under direction of the followed the methods of Miller (19). Inhibition of phage infection by ferrichrome. phage genome. Since the pattern formed by Cells to be tested (1 x 108) were first suspended in 2.5 approximately 20 protein bands was identical for ml of TY overlay agar, ferrichrome was added to final both phages, and T5 infection led to a very concentrations of 5, 10, 50, 100, 200, 500, and 16,000 different band pattern, Tlh should be consid- ,uM, and the suspension was poured over 20-ml TY ered a derivative of Ti. No detailed analysis was agar plates. Suspensions of the phages Tl, Tlh, T5, performed to identify a possibly altered minor and T5h with 20, 100, and 2,000 (in the case of T5h, (phage tail) protein in the Tlh mutants. also 10,000) plaque-forming units in 10 pl were spotted Presence of the tonA-coded protein is appar- on the plates. In some experiments the phages were sufficient for the host range mutants to mixed with the cell-ferrichrome suspension before ently plating. After incubation for 14 h at 37°C the plates infect cells. The wild-type phages are not inac- were inspected for plaques. tivated by isolated outer membranes (11). We Inactivation of phages by outer membranes. therefore studied inactivation of Tlh by outer Purified outer membrane of E. coli W 83-24 (5 ,tg) membranes of the tonA+ strain of E. coli W83- dissolved in 100 t,l of 0.117 M sodium phosphate buffer 24 and found that, under conditions where the (pH 7.2) was mixed with 200,ul of Tlh (200 plaque- titer of the wild-type phage was reduced by 5 to forming units) and 90 ,ul of nutrient broth. After incu- 15%, the host range mutant was inactivated by bation for 2 h at 370C, the mixture was plated on tonB 86 to 93%. The same concentration of outer strain BR158. Wild-type phage was treated accord- membranes from a tonA mutant ingly and plated on strain AB 2847. (K176) lacking Preparation of outer membranes and poly- the receptor protein inactivated the Tlh only by acrylamide gel electrophoresis. Outer membranes 15%. were prepared according to the method of Osborn et Inhibition of phage infection by ferri- al. (20). Presence of the tonA-specific protein was chrome. Since T5 adsorption and ferrichrome tested by polyacrylamide gel electrophoresis per- uptake require the receptor protein specified by formed by the technique ofLugtenberg et al. (18), with the tonA gene, we tested whether ferrichrome the modification that stock solution 1 contained only protects cells against T5 infection. With T5 0.3% instead of 0.8% (wt/vol) N,N-methylenebisacryl- plated on tonA+ tonB+ strains, up to 16 mM amide. ferrichrome caused neither a reduction in the Protein synthesis during Ti infection. E. coli AB 2847 was grown in M9 medium to a density of 5 number of plaques nor a reduction in their size. x 108 cells per ml, irradiated with UV light (survival, However, when T5 was plated on tonB strains, 0.1%), incubated for 15 min at 37°C, spun down, and 5 ,iM ferrichrome already reduced the number then suspended in 0.1 volume of M9 medium (6). Ti of 2,000 clear plaques to 50 small turbid plaques. was added to 10,ul of cells at a multiplicity of 5, and At 50 ,tM ferrichrome, plaque formation on all the mixture was incubated for 5 min at 37°C. The tonB strains tested was completely abolished. mixture was diluted 1:10 into M9 medium containing Growth of T5h was not inhibited at all by ferri- 5 ,uCi of 3H-labeled amino acid mixture (NEN, Chi- chrome on tonB+ and tonB cells. The number cago). After incubation for 8 min at 37°C, the mixture and size of the plaques formed by Ti and Tlh was diluted 1:10 into TY medium. The cells were spun on tonB+, and by Tlh on tonB cells, was reduced down, suspended in 30 A1 of sodium dodecyl sulfate containing solubilization buffer (18), boiled for 2 min, at 10 ,uM ferrichrome. Ti infection was slightly and then subjected to gel electrophoresis (18). Radio- more affected by ferrichrome than was Tlh in- actively labeled proteins were visualized by fluorog- fection. For example, 100 ttM ferrichrome re- raphy. duced the Ti plaque number from 2,000 to 200 small turbid plaques and, in the case of Tlh, to RESULTS 30. Host range mutants ofphage Ti infecting The sharp discrimination between tonB+ and tonB mutants ofE. coli Spontaneous mutants tonB cells in the inhibition of T5 infection by VOL. 135, 1978 tonA/tonB INTERACTION IN E. COLI 193 ferrichrome, although the plating efficiency of t T5 is the same on both cell types, prompted a I~ ~~~~*BR 158/±G more careful study in liquid culture. We mea- .o2100- sured the inhibition of T5 adsorption by ferri- chrome in the concentration range of from 0.001 a to 100 ,uM (Fig. 1). The results obtained were basically the same as those with the plate test. o 50- O The adsorption rate of the T5 strain that we I- - °WT/G used from the collection of the late W. Weidel .2 V /~~~~~A6SA6^W /M9 was low in rich media and best in M9 salts .C2X medium. T5 adsorption on the tonB strain BR158 was completely inhibited by 0.1 ,uM fer- 0'001 0.'01 0;1 i 10 100 richrome. At this ferrichrome concentration, T5 [PM] ferrichrome adsorption to the tonB+ parent was inhibited by FIG. 1. Inhibition ofT5 adsorption by ferrichrome. 48%. More than 10-times-higher concentrations Designations: (x) WT, T5 adsorption to the wild-type of fermchrome inhibited the parent not more tonA+ tonB+ parent strain E. coli K-12 AB 2847 in than 90% (Fig. 1). However, when the adsorption M9 salt solution; (0) WT/G, adsorption to AB 2847 was measured in the presence of glucose as in M9 salt solution containing 0.4% glucose; (A) energy source or in a complete miniimal growth WT/M9, adsorption to AB 2847 in complete M9 medium with glucose and the required aromatic growth medium containing 0.4% glucose and the re- inhibition of remained quired aromatic amino acids; (a) BR158/± G, ad- amino acids, adsorption sorption to the tonB derivative ofAB 2847 in M9 salt low, even at 100 ,uM ferrichrome. Inhibition of solution with or without glucose added. Cells were T5 adsorption to the tonB strain was not influ- grown in TY medium to the late logarithmic growth enced by added glucose and amino acids. phase. They were spun down, washed with M9 salt E. coli mutants containing nonfunctional solution and then taken up in M9 salt solution-10 tonA protein in the outer membrane. Spon- mM MgSO4-0.5 mM CaC12 to a concentration of 2 x taneous, EMS-, or NTG-induced tonA mutants 109 cells per ml. T5 (108 plaque-forming units per ml) previously selected as resistant to T5, Ti, or 480 were added and incubated at 37°C for 13 min. The or to colicin M or albomycin lost all tonA-related ferrichrome concentrations present in the incubation functions. Later it was found out that they were samples are listed on the abscissa. Adsorption was stopped by 106)-fold dilution. Cells were spun down, all deficient in the tonA protein (4, 14). Revert- and the phages remaining in the supernatant were ants had regained all tonA related functions to titrated on E. coli K-12 AB 2847. the extent the protein was expressed. After many attempts, in a search for tonB mutants, we ob- tained 11 tonA mutants which contained the M and supported fully the growth of T5h de- tonA protein in wild-type amounts. In Fig. 2 the scribed below. Strain B7/11 was also Ti resist- electropherograms ofseven strains are presented ant; however, the plating efficiency of Tlh was as examples. We started from cir feuB double as high as that on a tonA+ tonB+ strain, it was mutants lacking the receptor proteins for colicin partially albomycin and colicin M sensitive, and B and I to avoid interference with identification T5h showed the same plating efficiency as that of the tonA protein, since the three proteins on the host 41/2 where it was originally isolated. have similar electrophoretic mobilities. Mutants The transductant B7/111 showed similar prop- of E. coli B7 and B9 selected for Ti resistance erties as the donor strain. and T5 sensitivity and considered according to With the four mutants tested (B7/11, B9/9, this test as being mutated on the tonB gene B9/41, B9/71), plaque formation of T5 was not turned out to be tonA derivatives. The mutation inhibited by ferrichrome; thus, there was no could be cotransduced in the four strains tested indication of an impaired coupling between the (B9/9, B9/41, B9/71, B7/11) with a frequency altered protein and the tonB system. of 81 to 96% with the dapD marker at 4 min From the partial T5-sensitive tonA mutants close to tonA in the 100-min linkage map of E. of strain B9 we isolated completely T5-resistant coli, but not with the trp marker close to tonB. derivatives. By gel electrophoresis ofouter mem- The physiology of all 10 of the B9 derivatives branes we identified two strains designated 41/2 and of two transductants of B9/41 into E. coli and 71/1, which still contained the tonA protein AT982 was the same: although they appear to (Fig. 2). To verify the similarity to the tonA be T5 sensitive in the cross-streak test, the plat- protein, we isolated T5h, a host range mutant of ing efficiency of T5 was only 0.5 to 4% on the T5 that was able to grow on strain 41/2. T5h various mutants compared with that ofthe wild- grew on all tonA mutants containing the tonA type AB 2847 or the cir feuB derivatives. They protein with the same or a slightly decreased were resistant to Ti, Tlh, albomycin, and colicin (30%) plating efficiency as on the tonA+ strains. 194 HANTKE AND BRAUN J. BACTERIOL.

feu B/citt =1 _ = _ _ _-,_.- -4.m-wmmk ton A -- C _ - - - cir-- 2 3 - -8 9 1 1 2 ton A 1 2 3 A 5 6 7 8 9 10 71 12

FIG. 2. Sections oftwo electropherograms showing outer membraneproteins in the molecular-weight range of 80,000 obtained after polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. Designations: (1) AB 2847 containing the receptor proteins for colicin B, designated feuB, for colicin I, designated cir, for phage T5 designated tonA; (2-4) three Ti-resistant, partially T5-resistant derivatives of AB 2847, B9/9, B9/24, B9/71 cir, feuB tonA mutants containing tonA protein, lacking the receptor proteins of colicins B and I; (5 and 6) two tonA derivatives ofB9 lacking the tonA protein; (7) 71/1, T5-resistant cir feuB, tonA mutant containing the tonA protein; (8) Kl 64, tonA derivative ofAB 2847 without tonA protein; (9-11) B9/41, B9/66, B9/67, Ti resistant, partially T5 sensitive with tonA protein. (12) 41/2, T5-resistant tonA mutant with tonA protein.

All mutants lacking the tonA protein were com- 5 8 13 pletely T5h resistant. T5h adsorbed to strain 41/2 in contrast to T5 (Fig. 3). T5h adsorbed 100 - T5 on 4112 faster to the tonA+ parent strain than did T5. The enhanced binding to the wild-type protein apparently allows strong enough binding to the T5h on 4112 altered protein to infect the mutant. 50 - In the mutant described, the tonB function could not be determined (except ferric citrate 820 and vitamin B12 uptake, which were not mea- Ts on WT sured) since the strains were also colicin B and I resistant. We therefore transduced the tonA gene of four strains into a tonB+ strain (AT982). 20- The transductants remained colicin B and I sensitive, and they were also impaired in all the TSh on WT tonA-related functions. Apparently not only do the various phages and colicin M bind to the same area of the receptor protein, but also the FIG. 3. Inactivation ofphages T5 and T5h by ad- uptake of the small molecule albomycin has the sorption to E. coli AB 2847 (tonA+) or E. coli 41/2 same structural requirements. We then tested (tonA "missense"). Cells (2 x 109 per ml) and phages whether transport of ferric iron as ferrichrome (1 x 109 per ml) were incubated in M9 salts medium complex was to be found in these mutants. No (5) supplemented with 10 mM MgS04-0.5 mM CaCl2 at 37°C. Samples of50 ,Il were withdrawn at the times uptake occurred (Fig. 4). Two examples are indicated on the abscissa and diluted into 5 ml ofice- many more given for tested. Iron uptake into cold TY medium. Cells were spun down, and the the parent wild-type strain, a tonB mutant, and phages in the supernatant were titrated on E. coli AB a revertant of the tonB mutant are presented for 2847 or E. coli 41/2, respectively. P0 denotes the input comparison. These mutants containing appar- phage number; P denotes the number ofphages re- ently an altered tonA receptor complex are of maining in the supernatant after adsorption has been the same functional phenotype as the mutants allowed to the cells. lacking the tonA protein. E. coli tonB mutants remaining Ti and colicin B- and albomycin-resistant mutants, 14 480 sensitive. We briefly reported on two tonB remained Ti and 480 sensitive, with the same mutants which were Ti sensitive but resistant plating efficiency as in wild-type cells. Five of to colicins B, I, V, and albomycin and which the phage-sensitive mutants were also colicin V were unable to take up ferric iron in the form of insensitive, nine remained colicin V sensitive, all the ferrichrome, enterochelin, and citrate com- were colicin I resistant, and all remained T5 plexes, all functions known to be tonB depend- sensitive. All the tonB mutations mapped near ent (15). In light of the results described in this trp such that the P1-mediated cotransduction paper it was desirable to extend these observa- frequency was between 45 and 60%. Of all the tions to obtain further insight into functional mutants tested for reversion either sponta- requirements of tonB-related processes. To neously or induced by NTG, only one, originally avoid selection of tonB mutants from the more Ti resistant, that had regained all functions was frequent mutants missing outer membrane re- found. ceptor proteins, we used colicin B and albomycin The tonB mutants remaining Ti and 480 sen- simultaneously to isolate tonB mutants. Of 46 sitive were unable to take up ferric iron as fer- VOL. 135, 1978 tonA/tonB INTERACTION IN E. COLI 195 richrome complex, as exemplified by mutant 8- H36 (Fig. 4). The only tonB mutant, H19, which spontaneously reverted with high frequency, re- gained upon reversion all the tonB-related func- tions, including transport offerrichrome (Fig. 4). DISCUSSION A previous paper (11) showed that the energy 6- required for the irreversible adsorption ofphages Ti and )80 could be provided either by the electron transport chain or by ATP hydrolysis. The phages bind only reversibly to tonB mu- tants or to isolated outer membranes containing the tonA receptor protein. It was concluded that 0 the functional state of the tonA receptor protein x might be directed by the energized state of the -WC 4- cytoplasmic membrane and that the tonB func- tion could serve as a coupling device between E the two membranes. It is shown that phage 0L. mutants, which infect cells independently of the CL tonB function, can be obtained. The important a finding is that these phage mutants can be in- -ft0 activated by the tonA gene product alone. We u 2- interpret this to mean that the structural re- quirements for binding and triggering of phage DNA release have changed in the phage mu- tants. Mere contact with the receptor complex 0 in tonA+ strains apparently is sufficient to inac- -0* . -e tivate the mutants. The fact that the mutant phages plate on tonB strains with the same efficiency as they do on tonB+ strains shows that 10 20 the translocation of the phage DNA across the Time (min) outer and the cytoplasmic membrane into the cell occurs independently of tonB+ function. FIG. 4. Uptake of 55Fe3' as ferrichrome complex This further strengthens the conclusion that by E. coli. Uptake by wild-type strain AB 2847 (A), by tonB is required only for the processes occurring tonA mutants 41/56 (A) and 71/71 (E), by tonB mu- in concert with the outer . tant H36, which was Tl and 080 sensitive (0), and by Further steps subsequent to the receptor protein a tonB revertant, H19, which was originally Tl and p080 resistant (0). Cells were grown in TY medium to take place independently of tonBE function. a density of 5 x 108 per ml, spun down, washed once That this may be true for other receptor/tonB with an M9 medium containing 0.4% glucose, 50 WM systems has become apparent by binding con- deferri-ferrichrome, and 10 mM nitrilot-iacetate. ditions under which an outer membrane protein They were suspended in the same medium, except and the requirement for the tonB function were that the concentration ofdeferri-ferrichrome was re- bypassed (13). In addition, the type of energy duced to I ILM and shaken for 5 min at 37°C. Then required for the process was altered. Dihydrox- 0.1 ml ofa solution of 'Fe3" (1.1 ,uCi corresponding to ybenzoate-promoted iron uptake via the enter- 5 nmol of Fe3+) in deferri-ferrichrome (final concen- tration, 0.735 pM) was added. Samples (1 ml) were ochelin transport system is independent of the taken at time intervals as indicated on the abscissa, feuB outer membrane receptor protein. No filtered through Selectron filters (Schleicher and tonB+ function was required, and the uptake Schull; type BA85; pore size 0.45 ,um), washed twice was little affected by dinitrophenol in contrast with the M9 medium without glucose, dried, and to ferric enterochelin uptake, which depends on counted in a liquid scintillation counter. tonB+ function and is strongly inhibited by di- nitrophenol (22). Enterochelin synthesized in- comes dispensable under conditions where re- side the cell probably takes over ferric iron in ceptor proteins are bypassed. P. Wookey and H. the periplasm so that the uptake bypasses the Rosenberg (personal communication) showed outer membrane and the tonB+ function. that spheroplasts of tonA and fepA (probably The tonB function appears to be related to synonymous with feuB) mutants showed a binding and translocation processes in which greatly enhanced rate of uptake of ferrichrome outer membrane proteins participate. It be- and ferric enterochelin compared with that of 196 HANTKE AND BRAUN J. BACTERIOL. whole cells. However, they observed no stimu- tonA receptor protein and thus prevents T5 lation of ferric enterochelin uptake when tonB adsorption. When the cells are energized and the cells were converted to spheroplasts, implicating tonB gene product functions, ferrichrome is a function for the tonB+ gene product in the taken up so that the local concentration at the translocation of iron across the cytoplasmic receptor may be low. But since the high ferri- membrane. It seems that spheroplasts promise chrome concentration had no inhibitory effect further insights into the tonB+ function when on plates and only a low inhibitory effect in their properties are studied in greater detail. liquid medium, we favor the idea that the func- The relation of tonB function with various tional state of the receptor protein is dependent receptor-dependent uptake systems was further on the energized state of the cell and the tonB studied by isolating tonB mutants, which re- function. Binding of ferrichrome thus differs in mained fully sensitive to the wild-type phages partially starved cells and tonB mutants and Ti and 080 but which had lost all other tonB- does interfere with T5 infection. In isolated outer related functions tested. The cotransduction fre- membranes and tonB mutants, tonA is uncou- quency with the trp marker fell into the same pled from tonB so that ferrichrome directly in- range as the phage-resistant tonB mutants. The terferes with T5 adsorption. In this context it is same type of mutants was apparently also ob- of interest that the plating efficiency of T5h was tained by Gratia (10) and Franklin et al. (8). No not reduced by ferrichrome. Whether the host spontaneous or mutagen-induced revertants range mutant binds to a site which is not coin- were obtained; thus, one is possibly dealing with cident with the binding site of ferrichrome at various types of deletions. It is presently un- the receptor protein remains to be tested. known whether one gene or several linked genes Interaction of apparently very different sub- are affected. If the tonB+ function is specified by strates with the receptor protein presents a prob- one gene, one could imagine that products of lem of specificity (4). The various tonA mutants certain tonB mutants still can fulfill partial func- containing presumably altered protein in normal tions. Bassford et al. (3) isolated temperature- amounts in the outer membrane showed a lim- sensitive amber suppressor mutants of tonB, ited range of T5 sensitivity which, measured as which argues for a single gene. They showed plating efficiency, amounted to 0.5 to 4% of that that the tonB product is functionally unstable, of tonA+ cells. One mutant, B7/11, remained as indicated by the rapid loss of B12 uptake and Tlh sensitive and slightly albomycin and colicin decreased sensitivity to colicin D upon cessation M sensitive. The other mutants, as well as of tonB expression. strains into which the altered tonA gene were The tonA/tonB system is multifunctional in transduced, were completely Ti, Tlh, albomy- that it is involved in the uptake of several struc- cin, and colicin M resistant. The size of the tonA turally and functionally unrelated substances. protein was indistinguishable from that of the By competition for the same receptor protein, wild-type protein. This does not exclude small ferrichrome inhibits killing of the cells by colicin deletions giving rise to shortened proteins since M (14) and by phage 480 (23). It was also found the accuracy of molecular-weight determina- that inactivation of T5 by outer membranes was tions on sodium dodecyl sulfate-polyacrylamide prevented by ferrichrome (17). In this paper we gels is to within 5%. Regardless of whether the showed that ferrichrome did not inhibit T5 in- mutated proteins are altered by amino acid re- fection of growing tonA+ tonB+ cells on plates. placements or by short deletions, small changes Growth apparently was not an essential condi- affect the interactions with all substrates. A tion since, in liquid culture, addition of glucose limited region of the protein may be exposed at alone without the required amino acids was suf- the cell surface to which all substrates necessar- ficient to obtain a marked reduction of ferri- ily bind. chrome inhibition. During the adsorption assays no measurable growth took place; thus, we con- ACKNOWLEDGMENTS clude that glucose served as energy source and We thank Sonja Holzhoffer for providing us with the H changed the action of ferrichrome. T5 adsorp- strains, Ursula Holzwarth and Helga Wolff for excellent tech- tion to tonB mutants under all conditions was nical assistance, and James Coulton for reading the manu- low concentrations of ferrichrome. script. inhibited by This work was supported by the Deutsche Forschungsge- Partially starved wild-type cells without glucose meinschaft (Sonderforschungsbereich 76). in the medium behaved in a manner similar to that of tonB mutants because ferrichrome, al- LITERATURE CITED at a 10-times-higher concentration, in- though 1. Bassford, P. J., and R. J. Kadner. 1977. Genetic anal- hibited T5 adsorption. It is possible that, in ysis of components involved in vitamin B,2 uptake in isolated outer membranes, partially starved Escherichia coli. J. Bacteriol. 132:796-805. cells, and tonB mutants, ferrichrome stays at the 2. Bassford, P. J., C. Bradbeer, R. J. Kadner, and C. A. VOL. 135, 1978 tonA/tonB INTERACTION IN E. COLI 197 Schnaitman. 1976. Transport of vitamin B,2 in tonB 13. Hancock, R. E. W., K. Hantke, and V. Braun. 1977. mutants of Escherichia coli. J. Bacteriol. 128:242-247. Iron transport in Escherichia coli K-12. 2,3-Dihydrox- 3. Bassford, P. J., C. A. Schnaitman, and R. J. Kadner. ybenzoate-promoted iron uptake. Arch. Microbiol. 1977. Functional stability of the bfe and tonB gene 114:231-239. products in Escherichia coli. J. Bacteriol. 130:750-758. 14. Hantke, K., and V. Braun. 1975. Membrane receptor 4. Braun, V., R. E. W. Hancock, K. Hantke, and A. dependent iron transport in Escherichia coli. FEBS Hartmann. 1976. Functional organization of the outer Lett. 49:301-305. membrane ofEscherichia coli: phage and colicin recep- 15. Hantke, K., and V. Braun. 1976. A function common to tor as components of iron uptake systems. J. Supramol. iron-enterochelin transport and action of colicins B, I, Struct. 5:37-58. V in Escherichia coli. FEBS Lett. 59:277-281. 5. Braun, V., and K. Hantke. 1977. Bacterial receptors for 16. Ichihara, S., and S. Mizushima. 1977. Involvement of phages and colicins as constituents of specific transport outer membrane proteins in enterochelin-mediated iron systems, p. 101-130. In J. L. Reissig (ed.), Microbial uptake in Escherichia coli. J. Biochem. 81:749-756. interactions (receptors and recognition, series B, volume 17. Luckey, M., R. Wayne, and J. B. Neilands. 1975. In 3). Chapman and Hall, London. vitro competition between ferrichrome and phage for 6. Braun, V., K. Schaller, and H. Wolff. 1973. A common the outer membrane T5 receptor complex of Esche- receptor protein for phage T5 and colicin M in the outer richia coli. Biochem. Biophys. Res. Commun. membrane of Escherichia coli B. Biochim. Biophys. 64:687-693. Acta 323:87-97. 18. Lugtenberg, B., J. Meijers, R. Peters, P. van der 7. Bukhari, A. T., and A. L. Taylor. 1971. Genetic analysis Hoek, and L. van Alphen. 1975. Electrophoretic res- of diaminopimelic acid- and lysine-requiring mutants of olution ofthe "major outer membrane protein" ofEsch- Escherichia coli. J. Bacteriol. 105:844-854. erichia coli into four bands. FEBS Lett. 58:254-258. 8. Franklin, N. C., W. F. Dove, and C. Yanofsky. 1965. 19. Miller, J. E. 1972. Experiments in molecular genetics. The linear insertion of a prophage into the chromosome Cold Spring Harbor Laboratory, Cold Spring Harbor, of E. coli shown by deletion mapping. Biochem. Bio- N.Y. phys. Res. Commun. 18:910-923. 20. Osborn, M. J., J. E. Gander, E. Parisi, and J. Carson. 9. Frost, G. E., and H. Rosenberg. 1975. Relationship 1972. Mechanism of assembly of the outer membrane between the tonB locus and iron transport in Esche- of Salmonella typhimurium: isolation and characteri- richia coli. J. Bacteriol. 124:704-712. zation of cytoplasmic and outer membrane. J. Biol. 10. Gratia, J.-P. 1964. Resistance a la colicine B chez Chem. 247:3962-3972. Escherichia coli. Relations de specificite entre colicine 21. Pugsley, A. P., and P. Reeves. 1977. Role of colicin B, I. et V et phage Tl. Etude genetique. Ann. Inst. receptors in the uptake of ferrienterochelin by Esche- Pasteur (Paris) 107:132-151. richia coli K-12. Biochem. Biophys. Res. Commun. 11. Hancock, R. E. W., and V. Braun. 1976. Nature of the 74:903-911. energy requirement for the irreversible adsorption of 22. Pugsley, A. P., and P. Reeves. 1977. Uptake of ferrien- TI and @80 to Escherichia coli. J. Bac- terochelin by Escherichia coli: energy-dependent stage teriol. 125:409415. of uptake. J. Bacteriol. 130:26-36. 12. Hancock, R. E. W., K. Hantke, and V. Braun. 1976. 23. Wayne, R., and J. B. Neilands. 1975. Evidence for Iron transport in Escherichia coli K-12: involvement of common binding sites for ferrichrome compounds and the colicin B receptor and of a citrate-inducible protein. 4080 in the cell envelope of Escherichia J. Bacteriol. 127:1370-1375. coli. J. Bacteriol. 121:497-503.