Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-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 protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore 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 of enterobactin (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 of 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 of the siderophores tested protect cells against the killing action of colicin E1 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 Ia 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.     

Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB.

The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb. Like tonB mutants, exb mutants were resistant to colicin M and albomycin but, in contrast to tonB mutants, showed only reduced sensitivity to colicins B and D. Overexpressed tonB on the multicopy vector pACYC177 largely restored the sensitivity of exb mutants to colicins B, D, and M but only marginally increased sensitivity to albomycin. Suppression of the btuB451 mutation in the structural gene for the vitamin B12 outer membrane receptor protein by a mutation in tonB occurred only in an exb+ strain. Degradation of the unstable overproduced TonB protein was prevented by overproduced ExbB protein. The ExbB protein also stabilized the ExbD protein. Pulse-chase experiments with radiolabeled ferrichrome revealed release of ferrichrome from exbB, tonB, and fhuC mutants, showing that ferrichrome had not crossed the cytoplasmic membrane. It is concluded that the ExbB and ExbD proteins contribute to the activity of TonB and, like TonB, are involved in receptor-dependent transport processes across the outer membrane.     

Internal deletions in the FhuA receptor of Escherichia coli K-12 define domains of ligand interactions.

The ferrichrome-iron receptor encoded by the fhuA gene of Escherichia coli K-12 is a multifunctional outer membrane receptor required for the binding and uptake of ferrichrome and bacteriophages T5, T1, phi 80, and UC-1 as well as colicin M. To identify domains of the protein which are important for FhuA activities, a library of 31 overlapping deletion mutants in the fhuA gene was generated. Export of FhuA deletion proteins to the outer membrane and receptor functions of the deletion proteins were analyzed. All but three of the deletion mutant FhuA proteins cofractionated with the outer membrane; no FhuA proteins were detected in outer membrane preparations or in cell extracts when the deletions spanned amino acids 418 to 440. Most deletion proteins were susceptible to cleavage by endogenous proteolytic activity; some degradation products were detected on Coomassie blue-stained gels and on Western blots (immunoblots). Receptor functions were measured with the mutated genes present on multicopy plasmids. Two deletion mutants, FhuA delta 060-069 and FhuA delta 129-168, conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating of bacteriophages as that of wild-type FhuA; killing by colicin M was also unaffected. For FhuA delta 021-128 and FhuA delta 406-417, reduced sensitivity to colicin M was detected; wild-type phenotypes were observed for all other FhuA functions. Deletions from amino acids 169 to 195 slightly reduced sensitivities to bacteriophages and to colicin M; ferrichrome growth promotion was unaffected. When deletions extended into the region of amino acids 196 to 405, all FhuA functions were either reduced or abolished. The results indicate that selected regions of the FhuA protein have receptor activities and demonstrate the presence of both shared and unique ligand-responsive domains.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Import of biopolymers into Escherichia coli: nucleotide sequences of the exbB and exbD genes are homologous to those of the tolQ and tolR genes, respectively.

Escherichia coli with mutations in the exb region are impaired in outer membrane receptor-dependent uptake processes. They are resistant to the antibiotic albomycin and exhibit reduced sensitivity to group B colicins. A 2.2-kilobase-pair DNA fragment of the exb locus was sequenced. It contained two open reading frames, designated exbB and exbD, which encoded polypeptides of 244 and 141 amino acids, respectively. Both proteins were found in the cytoplasmic membrane. They showed strong homologies to the TolQ and TolR proteins, respectively, which are involved in uptake of group A colicins and infection by filamentous bacteriophages. exbB and exbD were required to complement exb mutations. Osmotic shock treatment rendered exb mutants sensitive to colicin M, which was taken as evidence that the ExbB and ExbD proteins are involved in transport processes across the outer membrane. It is concluded that the exb- and tol-dependent systems originate from a common uptake system for biopolymers.     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), 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 apparently only required for the irreversible adsorption of the phage T1, 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 T1 and T1h, 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 T1h (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. Ferrichrome 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 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional 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.     

Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.     

Characterization of group B colicin-resistant mutants of Escherichia coli K-12: colicin resistance and the role of enterochelin.

Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors.

Colicin B formed by Escherichia coli kills sensitive bacteria by dissipating the membrane potential through channel formation. The nucleotide sequence of the structural gene (cba) which encodes colicin B and of the upstream region was determined. A polypeptide consisting of 511 amino acids was deduced from the open reading frame. The active colicin had a molecular weight of 54,742. The carboxy-terminal amino acid sequence showed striking homology to the corresponding channel-forming region of colicin A. Of 216 amino acids, 57% were identical and an additional 19% were homologous. In this part 66% of the nucleotides were identical in the colicin A and B genes. This region contained a sequence of 48 hydrophobic amino acids. Sequence homology to the other channel-forming colicins, E1 and I, was less pronounced. A homologous pentapeptide was detected in colicins B, M, and I whose uptake required TonB protein function. The same consensus sequence was found in all outer membrane proteins involved in the TonB-dependent uptake of iron siderophores and of vitamin B12. Upstream of cba a sequence comprising 294 nucleotides was identical to the sequence upstream of the structural gene of colicin E1, with the exception of 43 single-nucleotide replacements, additions, or deletions. Apparently, the region upstream of colicins B and E1 and the channel-forming sequences of colicins A and B have a common origin.     

Protein import into Escherichia coli: colicins A and E1 interact with a component of their translocation system.

Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. Their mode of action involves three steps: binding to specific receptors located in the outer membrane, translocation across this membrane, and action on their targets. A specific colicin domain can be assigned to each of these steps. Colicins have been subdivided into two groups (A and B) depending on the proteins required for them to cross the external membrane. Plasmids were constructed which led to an overproduction of the Tol proteins involved in the import of group A colicins. In vitro binding of overexpressed Tol proteins to either Tol-dependent (group A) or TonB-dependent (group B) colicins was analyzed. The Tol dependent colicins A and E1 were able to interact with TolA but the TonB dependent colicin B was not. The C-terminal region of TolA, which is necessary for colicin uptake, was also found to be necessary for colicin A and E1 binding to occur. Furthermore, only the isolated N-terminal domain of colicin A, which is involved in the translocation step, was found to bind to TolA. These results demonstrate the existence of a correlation between the ability of group A colicins to translocate and their in vitro binding to TolA protein, suggesting that these interactions might be part of the colicin import process.     

Outer Membrane-Dependent Transport Systems in Escherichia coli: Effect of Repression or Cessation of Colicin Receptor Synthesis on Colicin Receptor Activities

Proteins in the outer membrane of gram-negative bacteria serve as general porins or as receptors for specific nutrient transport systems. Many of these proteins are also used as receptors initiating the processes of colicin or phage binding and uptake. The functional activities of several outer membrane proteins in Escherichia coli K-12 were followed after cessation or repression of their synthesis. Cessation of receptor synthesis was accomplished with a thermolabile suppressor activity acting on amber mutations in btuB (encoding the receptor for vitamin B(12), the E colicins, and phage BF23) and in fepA (encoding the receptor for ferric enterochelin and colicins B and D). After cessation of receptor synthesis, cells rapidly became insensitive to the colicins using that receptor. Treatment with spectinomycin or rifampin blocked appearance of insensitive cells and even increased susceptibility to colicin E1. Insensitivity to phage BF23 appeared only after a lag of about one division time, and the receptors remained functional for B(12) uptake throughout. Therefore, possession of receptor is insufficient for colicin sensitivity, and some interaction of receptor with subsequent uptake components is indicated. Another example of physiological alteration of colicin sensitivity is the protection against many of the tonB-dependent colicins afforded by provision of iron-supplying siderophores. The rate of acquisition of this nonspecific protection was found to be consistent with the repression of receptor synthesis, rather than through direct and immediate effects on the tonB product or other components of colicin uptake or action.     

Domains of colicin M involved in uptake and activity

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the Tong, ExbB and ExbD proteins. These colicins contain a sequence, called the Tong box, which has been implicated in transport via Tong. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with Tong may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This Colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193–197 and 223–252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the Tong box at the N-terminal end of colicin M must be involved in colicin uptake via Tong across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.     

Activity domains of the TonB protein

Escherichia coli and related Gram-negative bacteria contain an energy-coupied transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E. coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C-terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the M-terminal region of TonB within or close to the cytoplasmic membrane since an N-terminal 44-residue fragment of TonB was stabilized by ExbB and interfered with wild-type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine-7 to histidine and alanine-22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB pöint mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid-encoded tonB mutants of this region showed negative complementation of chromosomal wild-type tonB, and certain tonB mutants suppressed colicin B TonB-box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin la, a domain closer to the C-terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)×(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the praline-rich sequence since its removal resulted in a normal mobility.     

Fragmentation of colicins A and E1 by cell surface proteases.

Interaction of either colicin A or E1 with the surface of Escherichia coli cells resulted in extensive cleavage of the colicins into many peptide fragments in the molecular weight range of 10,000 to 30,000 released into the supernatants of colicin-cell mixtures. The protease inhibitor P-aminobenzamidine inhibited the cleavage of colicin A and enhanced colicin killing activity, suggesting that proteolysis is not required for the killing action of colicin. Fragments derived from the supernatants of the mixtures were inactive against sensitive cells. Proteolytic activity against both colicins was localized primarily in the outer membrane fraction of the cell envelope. At least two distinct protease activities appear to be present. Examination of the patterns of cleavage and inactivation of the colicins by a series of resistant mutants indicates that specific colicin receptors play no essential role in colicin proteolysis. In addition, evidence is presented that adsorption of colicin to specific receptors is a reversible process.     

Primary structure of colicin M, an inhibitor of murein biosynthesis.

The DNA sequence of the colicin M activity gene cma was determined. A polypeptide consisting of 271 amino acids was deduced from the nucleotide sequence. The amino acid sequence agreed with the peptide sequences determined from the isolated colicin. The molecular weight of active colicin M was 29,453. The primary translation product was not processed. In the domain required for uptake into cells, colicin M contained the pentapeptide Glu-Thr-Leu-Thr-Val. A similar sequence was found in all colicins which are taken up by a TonB-dependent mechanism and in outer membrane receptor proteins which are constituents of TonB-dependent transport systems. The structure of colicin M in the carboxy-terminal activity domain had no resemblance to the pore-forming colicins or colicins with endonuclease activity. Instead, the activity domain contained a sequence which exhibited homology to the sequence around the serine residue in the active site of penicillin-binding proteins of Escherichia coli. The colicin M activity gene was regulated from an SOS box upstream of the adjacent colicin B activity gene on the natural plasmid pColBM-Cl139.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function.

Vitamin B12 (CN-Cbl) and iron-siderophore complexes are transported into Escherichia coli in two energy-dependent steps. The first step is mediated by substrate-specific outer membrane transport proteins and the energy-coupling TonB protein complex, and the second step uses separate periplasmic permeases for transport across the cytoplasmic membrane. Genetic and biochemical evidence suggests that the TonB-dependent outer membrane transporters contact TonB directly, and thus they might compete for limiting amounts of functional TonB. The transport of iron-siderophore complexes, such as ferrichrome, causes a partial decrease in the rate of CN-Cbl transport. Although CN-Cbl uptake does not inhibit ferrichrome uptake in wild-type cells, in which the amount of the outer membrane ferrichrome transporter FhuA far exceeds that of the cobalamin transporter BtuB, CN-Cbl does inhibit ferrichrome uptake when BtuB is overexpressed from a multicopy plasmid. This inhibition by CN-Cbl is increased when the expression of FhuA and TonB is repressed by growth with excess iron and is eliminated when BtuB synthesis is repressed by CN-Cbl. The mutual inhibition of CN-Cbl and ferrichrome uptake is overcome by increased expression of TonB. Additional evidence for interaction of the Cbl and iron transport systems is provided by the strong stimulation of the BtuB- and TonB-dependent transport of CN-Cbl into a nonexchangeable, presumably cytoplasmic pool by preincubation of cells with the iron(II) chelator 2,2'-dipyridyl. Other metal ion chelators inhibited CN-Cbl uptake across the outer membrane. Although the effects of chelators are multiple and complex, they indicate competition or interaction among TonB-dependent transport systems.     

Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000. It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M. The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region. A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites. All mutations inserted a hexamer that encoded a unique SacI site. A large deletion in fhuA was also isolated by TAB linker mutagenesis. Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type. Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody. Receptor functions were measured with the mutated genes present in a single copy on the chromosome. Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. TAB linker insertions after amino acids 69 and 128 abolished all receptor functions. Phage T5 id not bind to these mutant FhuA proteins in detergent extracts. The deletion mutant was also defective in all FhuA functions. Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude. Insertion at other selected sites decreased some or all receptor functions only slightly. An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion. Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype. These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands.