Quick viewing(Text Mode)

Siderophore Protection Against Colicins M, B, V, and Ia in Escherichia Coli RUSH WAYNE, KEVIN FRICK, and J

Siderophore Protection Against Colicins M, B, V, and Ia in Escherichia Coli RUSH WAYNE, KEVIN FRICK, and J

JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 7-12 Vol. 126, No. 1 Copyright © 1976 American Society for Microbiology Printed in U-SA.

Siderophore Protection Against Colicins M, B, V, and Ia in RUSH WAYNE, KEVIN FRICK, AND J. B. NEILANDS* Department ofBiochemistry, University of California, Berkeley, California 94720 Received for publication 17 December 1975 A variety of natural and synthetic capable of supporting the growth of Escherichia coli K-12 on -limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protec- tion against colicin B and ferrichrome protection against colicin M, has proper- ties of a specific receptor competition between the and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake ofenterobactin (Wayne and Neilands, 1975). Similarly, ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption ofcolicin Ia to cells in vivo or to colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None ofthe siderophores tested protect cells against the killing action of colicin El or K, or against the energy poisons azide, 2,4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ta may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption. A variety of microorganisms produce low- tonB gene and required for the transport of molecular-weight iron carrier molecules called several siderophores, since mutants in tonB are siderophores, which solubilize iron and make it resistant to 4)80, tolerant to colicins B, V, and I, available to iron-deficient cells (20). Although and defective in most siderophore uptake (1, 4, Escherichia coli and Salmonella typhimurium 10, 24). synthesize only one siderophore, enterobactin Ferrichrome prevents adsorption of (180 to (enterochelin), they possess efficient uptake cells (24). As a result, nanomole quantities of systems for a broad spectrum of siderophores ferrichrome will prevent the formation of produced by other species (4, 17). In certain plaques of (180 on E. coli K-12, and this effect cases, bacteriophages and colicins have parasi- provides a useful assay for the interaction be- tized cell surface components of these sidero- tween ferrichrome and its receptor. In a paral- phore uptake systems. Thus, phages 4)80, lel fashion, siderophores might be expected to 4)80h, T5, and colicin M all appear to bind to a affect the activity of the tonB-dependent coli- specific surface receptor for the siderophore, cins. If such an interaction could be detected, it ferrichrome, and mutants lacking the receptor would offer a valuable system for probing both (tonA-) are both phage resistant and unable to colicin action and siderophore transport. utilize ferrichrome (10, 18, 24). Similarly, In this paper we report that a wide variety of phage 1)80 and colicins B, V, and I appear to siderophores and iron compounds can protectE. interact with a cell component specified by the coli K-12 against colicins B, V, and Ia. We find 7 8 WAYNE, FRICK, AND NEILANDS J. BACTZRIOL. that siderophore-mediated insensitivity to were evaluated by the serial dilution method of Gu- these colicins can occur by at least two mecha- terman (8). nisms. The first has characteristics of a specific Colicin protection test. To evaluate the protec- receptor competition between the siderophore tive potency of a given compound, a filter paper disk and the colicin involved, whereas the second containing 20 or 100 nmol of the compound was applied to the surface of a nutrient broth plate that requires the availability of siderophore iron. had been overlayered with molten soft agar contain- ing 2 drops of the strain to be tested and 1 to 3 drops MATERIALS AND METHODS ofcolicin preparation. Plates were incubated for 20 h Chemicals. Hydroxamate and sidero- at 37 C, and the radius of the zone of growth around phores were obtained as described previously (24; for the disk was then measured. structures, see 17). Traces of ferrichrome were re- Colicins B, V, Ia, and M were used at approxi- moved from recrystallized ferrichrome A by dissolv- mate titers of 102 for the protection test. ing the latter in dilute neutral phosphate buffer and Siderophore growth tests. Disk growth tests were extracting the former with benzyl alcohol. Deferri- performed as described previously (17) on NB-apo- ferrichrome A was prepared in the usual way (3). ferrichrome A or M9 minimal plates. Crude colicin V, B, M, K, and El lysates were Preparation of colicin Ia receptor. Membrane prepared by the method ofNagel de Zwaig and Luria material containing solubilized reqeptor activity (19). Colicin Ia was prepared by the method of Kon- against colicin Ia was prepared from strain JK1 by isky and Richards (15), carried through the ammo- the methods of Konisky et al. (13) and Konisky and nium sulfate fractionation step. Liu (14). Bacterial strains. The bacterial strains used in Colicin adsorption tests. Adsorption of colicin Ia this study are shown in Table 1. Cultures were to whole cells or to solubilized receptor material was grown at 37 C with aeration. assessed by a modification of the triple-layer plate Mutant isolation. The tonA+ strain, RW193, was test of Davies and Reeves (1). Ferrichrome (100 derived from AN193 by P1 transduction (6) with nmol) was incorporated into the second layer of tonA + donor phage and selection with colicins V and agar, which was streaked with colicin-sensitive cells Ia in the presence of ferrichrome. The SidA mutant, or receptor preparation. Cells or cell envelope mate- RW1935, was selected by albomycin resistance (17) rial capable of adsorbing colicin allowed growth of from RW193. indicator cells in the third layer above the streaks. Media. All used in this study were grown A tonA indicator strain was used to allow detection in Difco nutrient broth (21) for overnight cultures of colicin killing without ferrichrome protection of and storage. NB-apoferrichrome A plates were nu- the indicator strain itself. trient broth plates supplemented with ultrapure de- Chemical uncoupler protection tests. Chemical ferriferrichrome A to a concentration of 10-4 M. uncouplers or energy poisons were added to soft Addition of this compound specifically renders iron agar, and strain RW193 was then plated with and in the medium unavailable to E. coli strains that without siderophore also in the soft agar on nutrient cannot produce enterobactin, since ferrichrome A broth plates. Growth was observed after 20 h. has a high affinity for iron (III) (log K, = 29.6; J. B. Neilands, Pacific Conf. Chem. Spectroscopy, 28-30 RESULTS Oct. 1975, Los Angeles, Calif.), yet iron bound by the ligand cannot be utilized for growth by E. coli (see Siderophore protection against colicins B, Table 3). V, Ia, and M. Strain RW193 was exposed to M9 medium has been described previously (8) and colicin B, V, Ia, or M in the presence of sidero- was supplemented with the appropriate nutrients. phores (Table 2). All ofthe siderophores capable Titration of colicin. Approximate colicin titers of supporting growth of strain RW193 on NB-

TABLE 1. Bacterial strains Strain Relevant characteristics Source E. coli K-12 AN193 pro-,leu-,trp-,lac-,strr,tonA-,entA I. G. Young RW193 tonA+ from AN193 This paper RW1935 SidA from RW193 This paper AN92 pro-,arg-,phe-,tyr-,trp-,aroB-,thi- I. G. Young AN272 fesB351 from AN92 I. G. Young W3110str-r(JK1) Prototroph J. Konisky Colicinogenic strains 32T19/VT5 (col M) B. A. D. Stocker R2.1/VX (col B) S. Guterman W3110str-r(colIa-CA53) (col Ia) J. Konisky a178 (col V) D. Elseviers K235 (col K) J. Campbell K33 (col El) J. Campbell VOL. 126, 1976 SIDEROPHORE PROTECTION IN E. COLI 9 apoferrichrome A plates also protected this TABLE 3. Protection ofE. coli K-12 against colicin Ia strain against colicins B, V, and Ia on nutrient Concn Protectione Nutrition"be broth plates. By contrast, only ferrichrome and (MM) (mm) (mm) its chromium analogue protected against coli- Compound' 1 10 11 cin M (Table 2), and none of the siderophores Enterobactin Ferrichrome ..... 1 20 16 inhibited colicins K or El (data not shown). The Ferrichrome A ... 1 0 0 potency of protection by siderophores roughly Chromium (III) de- corresponds to the ability ofthe siderophores to ferriferrichrome 1 0 0 support growth in the absence of colicin. Schizokinen ...... 1 8 11 Chromium (III) deferriferrichrome did not Rhodotorulic acid 1 5 8 block the action of colicins B, V, and Ia, but it Ferrioxamine B 1 0-1 3-7 did protect against colicin M. Iron (III) citrate 5 7 7 Table 3 presents the results of a more de- Iron (III) EDTA . 5 2 10 tailed investigation of siderophore protection Na2EDTA ...... 5 0 0 Iron (III) NTA" ... 5 4 4 against colicin Ia, showing the range of poten- Na2NTA ...... 5 0 0 cies involved. The synthetic aminocarboxylic Iron (II) 2,2-dipyri- acid chelating agents, nitrilotriacetic acid and dyl ...... 5 9 9 ethylenediaminetetraacetic acid, gave moder- FeCl3 .5...... 6 1 4 ate protective activity when supplied as the chelates with iron but showed no a Unless otherwise specified, compounds were (III), they added as the iron (III) complex, 20 dLl per test. activity when added as the free ligands. b Numbers refer to the radius ofgrowth about the Siderophore protection against colicins in siderophore disk. siderophore utilization mutants. Although a ""Nutrition" refers to siderophore-dependent broad spectrum ofiron compounds could protect growth in the absence of colicin, observed on NB- strain RW193 against colicins B, V, and Ia apoferrichrome A plates. (Tables 2 and 3), mutant strains with defects in d EDTA, Ethylenediaminetetraacetic acid. hydroxamate utilization were not protected e NTA, Nitrilotriacetic acid. from these colicins by the affected siderophores (Table 4). The tonA - strain, AN193, which was defective in growth on ferrichrome, was not capable ofgrowth on hydroxamate siderophores protected from colicin Ia (Table 4) or from coli- to varying degrees when NB-apoferrichrome A cins B and V (data not shown) by ferrichrome. plates were used (Table 4). Thus, although the The SidA mutant, RW1935, which was unable SidA strain was no longer protected by hydrox- to grow on any of the hydroxamates on M9 amates, it retained the ability to grow on these minimal plates, was likewise not protected by compounds in conditions similar to those used any of the hydroxamates on nutrient broth for the protection tests. Additionally, ferri- plates against the action ofcolicins B, V, and Ia. chrome, and only ferrichrome, rendered RW- We note, however, that the Sid A mutant proved 1935 insensitive to colicin M. The fes- mutant, AN272, which is able to TABLE 2. Siderophore nutrition and protection ofE. accumulate but not utilize ferric enterobactin coli K-12 strain RW193 (16), was not protected against colicins Ia or V by enterobactin (Table 5), whereas the parent Protective potencyb of fes+ strain, AN92, became insensitive to these Siderophore" colicin: Nutrition' c colicins in the presence of enterobactin. How- M B V Ia ever, strain AN272 was protected against coli- cin B by enterobactin and against all three Enterobactin ...... 0 + + + + + + + + Ferrichrome ...... + + + + + + + + + + colicins by ferrichrome. Schizokinen 0...... + + + + + + + + Effect of ferrichrome on colicin Ia adsorp- 0 Rhodotorulic acid . + + + + tion and receptor activity. Using a modified Iron (III) citrate ... 0 + + + + Chromium (III) de- triple-layer plate test (1), we investigated the ferriferrichrome . + 0 0 0 ability of cells of strain RW193, grown in the presence of ferrichrome, to adsorb colicin Ia in All compounds were added as the iron (III) complex, with the exception of chromium (IH) deferriferrichrome. the presence of ferrichrome (1 ;tM). Although Symbols indicate growth relative to growth of cells in the strain was rendered fully insensitive to the the absence of siderophore. ++, About a 10- to 20-mm- colicin, substantial adsorption was still ob- radius zone of growth about the disk; +, about a 1- to 9-mm served. Additionally, the colicin Ia receptor was zone; 0, no growth. in "Nutrition" refers to siderophore-dependent growth in partially purified and its activity was tested the absence of colicin, observed on NB-apoferrichrome A the presence of ferrichrome (about 3 ,uM). No plates. reduction in receptor activity was detected. 10 WAYNE, FRICK, AND NEILANDS J. BACTZRIOL. TABLE 4. Nutrition and colicin protection ofE. coli K-12 mutants in siderophore utilization Strain AN193 (tonA-) RW1935 (SidA) Siderophore° Protectionl Nutrition" e Protection Nutrition (mm) (Col (mm) (NB) ClI o B M Ia) Cll o B M Enterobactin .1 10 11 10 0 11 15 Ferrichrome ...... 0 4 0 10 2 0 Schizokinen ...... 5 11 0 0 5 0 Rhodotorulic acid 2 7 0 0 10 0 Ferrioxamine B 0 10 0 0 3 0 Iron (III) citrate 6 4 4 0 8 5 a All compounds were added as the iron (III) complex. Twenty nanomoles ofsiderophore was used for each test with the exception of citrate, for which 100 nmol was used. Numbers refer to the radius of growth about the siderophore disk. "Nutrition" refers to siderophore-dependent growth in the absence of colicin, observed on NB-apoferri- chrome A plates (NB) or on M9 minimal glucose plates (M9).

TABLE 5. Protection against colicins B, V, and Ia in recent investigations (2, 10, 11, 12, 18, 22, 23), it AN272 (fes-) and AN92 (fes+)a was tempting to believe that the phage and Protective potency colicin receptors altered by the tonB mutation Strain Siderophore are involved in iron uptake. Thus, Frost and Coli- Coli- Coli- Rosenberg (4) have recently suggested that cin B cin V cinIa tonB- strains lack an outer-membrane compo- AN272 Enterobactin + + 0 0 nent necessary both for the uptake of ferric Ferrichrome + + + + + + enterochelin and for the adsorption of phage AN92 Enterobactin + + + + + + 080, since tonB mutants are defective in both of Ferrichrome + + ++ ++ these functions. However, the tonB mutation a Symbols and terms are as in Table 2. results in several changes involving major outer-membrane proteins (1) and it brings about Effect of siderophores on the action of en- reduction ofcellular utilization ofseveral structurally dissimilar ergy poisons. Since siderophores block the ac- siderophores (4). Since tion of colicin Ia and this colicin mimics the the mutational alteration is not specific, it is effects of a number of chemical agents that impossible to distinguish the functions of the block energy metabolism (5), the possibility ex- individual tonB-dependent receptors by study- isted that siderophores might be able to prevent ing the in vivo defects of tonB mutants. Data the action of the energy poisons. Cells of strain capable of distinguishing the action of specific RW193 were therefore exposed to azide (2.4 cell components must be obtained by other ap- mM), 2,4-dinitrophenol (0.8 mM), or carbonyl- proaches. cyanide m-chlorophenylhydrazone (30 AM) in We therefore surveyed the capacity of several the presence and absence of 3 ,uM enterobactin, siderophores to protect E. coli K-12 from the B- ferrichrome, schizokinen, or iron (II) 2,2-dipyri- group colicins M, B, V, and Ta. For this purpose dyl. No protection against the poisons by the we used strain RW193 (tonA+ entA-), which siderophores was observed. cannot produce its native siderophore, entero- bactin, and which possesses all of the sidero- phore uptake systems known to exist in E. coli DISCUSSION K-12. Using a disk protection test that can de- Mutants carrying lesions in the tonB region tect picomole quantities of a potent inhibitor, of the E. coli chromosome are resistant to we found that all of the siderophores that effi- phages 4'80 and Ti and insensitive to colicins M, ciently supported growth of strain RW193 on B, V, and I, and are simultaneously defective in iron-limited nutrient broth medium also pro- iron transport (1, 4, 10, 23). Since the existence tected this strain against colicins B, V, and Ia, of cell surface binding sites that serve both as whereas only ferrichrome protected against col- phage or colicin receptors and as components of icin M. the uptake systems for low-molecular-weight In studies ofenterobactin inhibition ofcolicin compounds has been demonstrated in several B (7-9), Guterman did not detect enterobactin VOL. 126, 1976 SIDEROPHORE PROTECTION IN E. COLI 11 protection against colicin V or ferrichrome pro- the affected siderophores are no longer able to tection against colicin B. In our hands both protect against colicins Ia and V and, with the colicins B and V were inhibited by enterobactin exception of enterobactin, against colicin B. and ferrichrome, a difference that may be due Additionally, the synthetic chelating agents to the high sensitivity of our disk protection ethylenediaminetetraacetic acid and nitrilotri- test. However, Guterman would have obtained acetic acid are active in protection but only as a negative result for ferrichrome protection the iron complexes. Moreover, although ferri- against colicin B if the colicin indicator strain chrome is a potent inhibitor of colicins B, V, she used had been tonA- (see below). and Ia, the chromium (III) analogue of ferri- The finding that siderophores of widely dif- chrome was without protective activity against fering structures could protect against colicins colicins. Finally, ferrichrome did not impair B, V, and Ia but not M suggested that receptor the ability of colicin Ia to adsorb to its receptor competition, as has been implicated in the pro- either in vivo or in vitro. Thus siderophore pro- tection by ferrichrome against phages 080 and tection against the B-group colicins can occur T5 (18, 24), might not be involved in all cases. without specific receptor competition. Instead, resistance might be due to metabolism The mechanism ofthe noncompetitive protec- of siderophore iron or direct chemical inactiva- tion against colicins afforded E. coli by sidero- tion ofthe colicins by specific compounds. These phores is not known. However, we have shown possibilities ultimately have to be distin- that mutational alterations in the colicin indi- guished for each combination of colicin and cator strain can reduce or abolish the protective siderophore, since the generality of any par- activity of specific compounds, ruling out a di- ticular response cannot be guaranteed. rect chemical inactivation of the colicins by We elected to resolve the alternative mecha- those compounds. Additionally, we have ob- nisms by comparing the protective response in served that in the SidA strain, specific sidero- conditions ofcellular availability ofsiderophore phores that efficiently supply iron for growth iron to the response in situations where iron are unable to protect against colicins B, I, and V. was not available to cells from the sidero- This indicates that the availability ofiron is not phores. With this approach we were able to dis- sufficient for protection to occur and that the tinguish two kinds of siderophore protection pathway by which iron is provided to cells is a against colicins. The first kind has the prop- significant determinant of protective activity. erties of specific receptor competition between Finally, none of the siderophores active in pro- siderophore and colicin, allowing protection in tection against colicin Ia protect E. coli from the absence of siderophore utilization. Thus, the killing action of colicins K or El, or from chromium (III) deferriferrichrome specifically the energy poisons 2,4-dinitrophenol, azide, or protected against colicin M, and only colicin M, carbonylcyanide m-chlorophenylhydrazone, al- in strain RW193, and ferrichrome protected though colicin Ia mimics the effects of these against colicin M, and only colicin M, in the agents (5). Thus, the siderophores do not gener- hydroxamate utilization mutant RW1935. Simi- ally protect against substances that disrupt en- larly, enterobactin protected against colicin B, ergy metabolism in E. coli. and only colicin B, in the enterobactin utiliza- The noncompetitive protection against coli- tion mutant AN272 (fes-). These observations cins B, V, and Ia may be due to an induction of agree with our previous proposal that ferri- membrane alterations in response to sidero- chrome and colicin M compete for a common phore iron metabolism, since there is evidence receptor involved in the uptake of ferrichrome, that some siderophore transport systems are and that enterobactin and colicin B compete for induced by the siderophore involved, whereas a common receptor involved in the uptake of others are repressed by iron (4, 16). Alterna- enterobactin (24). As predicted by this scheme, tively, protection may be due to a direct inter- colicin M receptor mutants (tonA-) are specifi- ference by siderophore iron with some un- cally defective in the utilization of ferrichrome known step in colicin action subsequent to ad- (Table 4) and colicin B receptor mutants are sorption. Further work will be necessary to specifically defective in the utilization of enter- distinguish these possibilities. obactin (R. Wayne, Ph.D. thesis, Univ. of Cali- fornia, Berkeley, 1976). ACKNOWLEDGMENTS The second kind ofprotective response, previ- We thank Mary Luckey, John Leong, and James Patras ously undetected, is less specific and requires for helpful discussions, and the several people who contrib- siderophore utilization. Thus virtually all of uted chemicals and bacterial strains. This research was supported by Public Health Service the natural siderophores inhibit killing by coli- grants Al 04156 from the National Institute of Allergy and cins B, V, and Ia. Yet when siderophore utiliza- Infectious Diseases and AM 17146 from the National Insti- tion mutants (SidA, tonA, and fes-) are used, tute of Arthritis, Metabolism and Digestive Diseases. 12 WAYNE, FRICK, AND NEILANDS J. BAcTzRIoL. LITERATURE CITED Colicin Ia and lb binding to Escherichia coli envelopes and partially purified cell walls. J. Supramol. Struct. 1. Davies, J. K., and P. Reeves. 1975. Genetics of resist- 1:208-219. ance to colicins in Escherichia coli K-12: cross-resist- 14. Konisky, J., and G. T. Liu. 1974. Solubilization and ance among colicins of group B. J. Bacteriol. 123: partial characterization of the colicin I receptor of 96-101. Escherichia coli. J. Biol. Chem. 249:835-840. 2. DiMasi, D. R., J. C. White, C. A. Schnaitman, and C. 15. Konisky, J., and F. M. Richards. 1970. Characteriza- Bradbeer. 1973. Transport of vitamin B,2 in Esche- tion of colicin Ia and colicin Ib: purification and some richia coli: common receptor sites for vitamin B12 and physical properties. J. Biol. Chem. 245:2972-2978. the E colicins on the outer membrane of the cell 16. Langman, L., I. G. Young, G. E. Frost, H. Rosenberg, envelope. J. Bacteriol. 115:506-513. and F. Gibson. 1972. Enterochelin system of iron 3. Emery, T., and J. B. Neilands. 1960. Contribution to transport in Escherichia coli: mutations affecting fer- the structure of the ferrichrome compounds: charac- ric enterochelin esterase. J. Bacteriol. 112:1142-1149. terization of the acyl moieties of the hydroxamate 17. Luckey, M., J. R. Pollack, R. Wayne, B. N. Ames, and functions. J. Am. Chem. Soc. 82:3658-3662. J. B. Neilands. 1972. Iron uptake in Salmonella typhi- 4. Frost, G. E., and H. Rosenberg. 1975. Relationship murium: utilization of exogenous siderochromes as between the tonB locus and iron transport in Esche- iron carriers. J. Bacteriol. 111:731-738. ,ichia coli. J. Bacteriol. 124:704-712. 18. Luckey, M., R. Wayne, and J. B. Neilands. 1975. In 5. Gilchrist, M. J. R., and J. Konisky. 1975. Effects of vitro competition between ferrichrome and phage for colicin Ia on transport and respiration in Escherichia the outer membrane T5 receptor complex of Esche- coli. J. Biol. Chem. 250:2457-2462. richia coli. Biochem. Biophys. Res. Commun. 64:687- 6. Goldberg, R. B., R. A. Bender, and S. L. Streicher. 693. 1974. Direct selection for P1-sensitive mutants of en- 19. Nagel de Zwaig, R., and S. E. Luria. 1967. Genetics and teric bacteria. J. Bacteriol. 118:810-814. physiology of colicin-tolerant mutants ofEscherichia 7. Guterman, S. K. 1971. Inhibition of colicin B by entero- coli. J. Bacteriol. 94:1112-1123. chelin. Biochem. Biophys. Res. Commun. 44:1149- 20. Rodgers, G. C., and J. B. Neilands. 1973. Microbial iron 1155. transport compounds, p. 823-830. In A. Laskin and H. 8. Guterman, S. K. 1973. Colicin B: mode of action and Lechevalier (ed.), Handbook of microbiology. CRC inhibition by enterochelin. J. Bacteriol. 114:1217- Press, Cleveland. 1224. 21. Roth, J. R. 1970. Genetic techniques in studies of bacte- 9. Guterman, S. K., and L. Dann. 1973. Excretion of en- rial metabolism, p. 3-35. In H. Tabor and C. W. Tabor terochelin by exbA and exbB mutants of Escherichia (ed.), Methods in enzymology, vol. 17A. Academic coli. J. Bacteriol. 114:1225-1230. Press Inc., New York. 10. Hantke, K., and V. Braun. 1975. Membrane receptor 22. Szmelcman, S., and M. Hofnung. 1975. Maltose trans- dependent iron transport in Escherichia coli. FEBS port in Escherichia coli K-12: involvement of the bac- Lett. 49:301-305. teriophage lambda receptor. J. Bacteriol 124:112-118. 11. Hazelbauer, G. L. 1975. Role of the receptor for bacteri- 23. Wang, C. C., and A. Newton. 1971. An additional step ophage lambda in the functioning of the maltose in the transport of iron defined by the tonB locus of chemoreceptor of Escherichia coli. J. Bacteriol. Escherichia coli. J. Biol. Chem. 246:2147-2151. 124:119-126. 24. Wayne, R., and J. B. Neilands. 1975. Evidence for com- 12. Kadner, R. J., and G. L. Liggins. 1973. Transport of mon binding sites for ferrichrome compounds and vitamin B,2 in Escherichia coli: genetic studies. J. bacteriophage 480 in the cell envelope ofEscherichia Bacteriol. 115:514-521. coli. J. Bacteriol. 121:497-503. 13. Konisky, J., B. S. Cowell, and M. J. Gilchrist. 1973.