Escherichia coli K-12 tolZ mutants tolerant to colicins E2, E3, D, Ia, and Ib: defect in generation of the electrochemical proton gradient

Spontaneous Escherichia coli K-12 mutants tolerant to colicin E3 were isolated, and on the basis of their tolerance patterns to 19 kinds of colicins, a new phenotypic class of tolZ mutants was found. The tolZ gene was located between min 77 and 78 on the E. coli K-12 genetic map. The tolZ mutants were tolerant to colicins E2, E3, D, Ia, and Ib, and showed an increased sensitivity to ampicillin, neomycin, and EDTA, but not to deoxycholate; they were able to grow on glucose minimal medium, but not on nonfermentable carbon sources (succinate, acetate, pyruvate, lactate, malate, etc.). The pleiotropic phenotype of the tolZ mutant was due to a single mutation. Both respiration and membrane ATPase activity of the tolZ mutant were normal. The tolZ mutant had a defect in the uptake of proline, glutamine, thiomethyl-beta-D-galactoside, and triphenylmethylphosphonium ion; these uptake systems are driven by an electrochemical proton gradient (delta-mu H+) or a membrane potential (delta psi). In contrast, the uptake of methionine and alpha-methyl-D-glucoside, which is not dependent on delta-mu H+ and delta psi, was normal in the tolZ mutant. Glucose 6-phosphate uptake at pH 5.5, which is driven by a transmembrane pH gradient, in the tolZ mutant was similar to the parent level. These results indicate that the tolZ mutant has a defect in the generation of delta-mu H+ and delta psi.     

Cross-resistance between bacteriophages and colicins in Escherichia coli K-12.

Cross-resistance between bacteriophages and colicins was studied using collections of bacteriophage- and colicin-resistant mutants of Escherichia coli K-12. No new examples were found of highly specific one-to-one cross-resistance of the type suggestive of common receptors. However, several groups of mutants showed tolerance to colicins and resistance to bacteriophages. Mutants known to be very defective in lipopolysaccharides composition were found to commonly show tolerance to certain colicins in addition to their bacteriophage resistance. Another group of mutants showed varying patterns of resistance to colicins E2, E3, K, L, A, S4, N, and X and bacteriophages E4, K2, K20, K21, K29, and H+. However, many bacteriophage-resistant mutants were fully colicin sensitive, and most colicin-resistant mutants were fully sensitive to bacteriophages.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

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.     

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.     

The structurally related exbB and tolQ genes are interchangeable in conferring tonB-dependent colicin, bacteriophage, and albomycin sensitivity.

Double exbB tolQ mutants of Escherichia coli were completely resistant to bacteriophages T1 and phi 80, in contrast to strains with exbB or tolQ mutations, which were sensitive. Cells carrying mutations in exbB were partially tolerant to colicins B, D, and M and became fully tolerant by the introduction of tolQ mutations. This suggested involvement of both exbB and tolQ in tonB-dependent uptake.     

Effects of colicins K and E1 on the glucose phosphotransferase system.

1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of alpha-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of alpha-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake. 2. Colicins K and E1 decreased the apparent Km for alpha-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins. 3. In the presence of colicins, alpha-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated alpha-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound. 4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.     

Effect of mutation, electric membrane potential, and metabolic inhibitors on the accessibility of nucleic acids to ethidium bromide in Escherichia coli cells

The uptake of ethidium bromide by Escherichia coli K 12 cells has been studied by using 14C-labeled ethidium and spectrofluorometry on three E. coli strains: the first one (AB1157) has an ethidium-resistant phenotype; the second one derives from the first one after a single mutation (at 10 min on the E. coli genetic map) and has an ethidium-sensitive (Ebs) phenotype; the third one is the acrA strain which appeared to have the same phenotype as the Ebs strain. When the cells are in exponential growth, no ethidium enters wild-type cells, and a very limited amount of ethidium enters Ebs and acrA cells. Massive quantities of ethidium enter AB1157, Ebs, and acrA cells treated by uncouplers and respiring Ebs cells treated by the membrane ATPase-inhibitor dicyclohexylcarbodiimide. A small amount of ethidium enters cells treated in M9 succinate medium by metabolic inhibitors such as KCN or cells starved with oxygen in the same M9 medium. The amount of ethidium and ethidium dimer retained at equilibrium by either type of cell, and by cells infected by T5 phage, as well as the kinetics of influx and efflux, has been measured under a variety of situations (membrane energized or not, and/or membrane ATPase inhibited or not). Furthermore, it was shown that ethidium binds to both RNA and DNA when it enters CCCP-treated wild-type E. coli cells, whereas it binds mainly to DNA when it enters Ebs and acrA cells in exponential growth. As it will be discussed, it is difficult to account for the EthBr uptake by invoking only membrane functions and active transport. Therefore, it is proposed that the variations of the nucleic acid accessibility in E. coli cells might play a role in the control of this uptake. Accordingly, in ethidium-sensitive cells, the mutation would have caused a significant part of the chromosomal DNA (10-20%) to become accessible to ethidium. Hansen [Hansen M. T. (1982) Mutat. Res. 106, 209-216], after a study of the photobinding of psoralen to nucleic acids in the acrA mutant, also suggested that DNA environment was modified in acrA cells.     

Uptake of the lipophilic cation dibenzyldimethylammonium into Saccharomyces cerevisiae. Interaction with the thiamine transport system

The distribution ratio of the lipophilic cation dibenzyldimethylammonium between the cells of Saccharomyces cerevisiae and the medium appears to reflect changes in the membrane potential in a way that is qualitatively correct: the addition of a proton conductor or of an agent which blocks metabolism causes an apparent depolarization of the cell membrane; monovalent cations cause also a lowering of the equilibrium distribution, whereas the addition of divalent cations results in an increase of the partition ratio.However, uptake of dibenzyldimethylammonium and probably also of other liophilic cations proceeds via the thiamine transport system of the yeast. Dibenzyldimethylammonium transport is inducible, like thiamine transport. A kinetic analysis of the mutual interaction between thiamine and dibenzyldimethylammonium uptake shows that these compounds share a common transport system; moreover, dibenzyldimethylammonium uptake is inhibited completely by thiamine disulfide, a competitive inhibitor of thiamine transport and dibenzyldimethylammonium uptake in a thiamine-transport mutant is reduced considerably.It is concluded that one should be cautious when using lipophilic cations to measure the membrane potential of cells of S. cerevisiae.     

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.     

The TolA protein interacts with colicin E1 differently than with other group A colicins.

The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli. Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins. TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity. The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively. TolA and TolA deletion mutants were expressed from a plasmid in an E. coli strain producing no chromosomally encoded TolA. Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability. Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca. 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B. In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable. In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells. It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N. Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.     

Effect of acriflavine on the plasma membrane of Escherichia coli K12

Plasma membranes of acriflavine-sensitive mutant (acrA) and acriflavine-resistant (acrA+, wild-type and true revertant) Escherichia coli K12 strains treated with acriflavine were observed under the electron microscope by means of the freeze-fracture technique. The plasma membrane of the acrA mutant exhibited a complex lamellar structure at the end of the cell when treated with 20 micrograms acriflavine ml-1. However, the membrane of the acrA+ cells also gave the lamellar complex when treated with a very high concentration of acriflavine (100 micrograms ml-1). The size of the intramembranous particles was not affected by the acriflavine treatments.     

Evidence for a negative membrane potential and for movement of C1- against its electrochemical gradient in the ascomycete Neocosmospora vasinfecta.

The iodides of three lipid-soluble cations (dibenzyldimethylammonium; tribenzylmethylammonium, TBMA+; ethyldimethylbenzylammonium) were synthesized by the reaction of 14C-labeled methyl or 14C-labeled ethyl iodide with the appropriate secondary of tertiary amine and used in an attempt to measure the transmembrane electrical potential difference in Neocosmospora. Only mycelium containing high levels of Na+ accumulated measureable amounts of these cations and only above pH 6. Uptake was reduced in the presence of exogenous K+, Na+, Mg2+, or tris(hydroxymethyl)aminomethane. The velocity of TBMA+ uptake was proportional to its concentration between 46 and 427 muM. Neither the rate nor the extent of TBMB+ uptake was greatly affected by the presence of a fivefold excess of either dibenzyldimethylammonium or ethyldimethylbenzylammonium, even though these cations were themselves accumulated. The uncoupler m-chlorophenylhydrazone induced loss of previously accumulated TBMA+ from the mycelium. Anaerobiosis and cold (5 degrees C) temperature both inhibited TBMA+ uptake but did not induce the loss of previously accumulated TBMA+. The uptake of lipophilic cations by Na+-rich mycelium indicated a minimum transmembrane electrical potential of -60 to -70 mV (inside negative). Net uptake of these cations appeared to be strongly influenced by the availability of endogenous exchangeable cations and by the presence of other exogenous cations, as well as by the membrane potential. Despite these limitations, transport of C1- by Na+-rich mycelium appeared to take place against the electrochemical gradient for C1-.     

Requirement for membrane potential in active transport of glutamine by Escherichia coli.

The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells.     

Genetics and physiology of colicin-tolerant mutants of Escherichia coli

A series of colicin-tolerant (tol) mutants of Escherichia coli K-12, which adsorbed colicins but were not killed by them, were isolated and studied genetically and physiologically. Three major classes of mutants were found: tol II, tolerant to colicins A, E1, E2, E3, and K; tol III, tolerant to A, E2, E3, and K; and tol VIII, tolerant to E1 only. The sites of tol II and tol III mutations mapped near the gal region (gene order: tol-gal-bio) and were cotransduced with gal by P1. In heterozygous diploids, tol(+) was dominant over tol; tol II and tol III gave full complementation. All the tol mutations that mapped near gal rendered the bacteria more fragile during growth and hypersensitive to deoxycholate and to ethylenediaminetetraacetic acid. The tol VIII mutation mapped between str and his. These mutants were extremely sensitive to deoxycholate and were also hypersensitive to methylene blue, acridines, and various other compounds. The sensitivity is attributed to increased uptake due to selective alteration of the permeability barrier. The colicin-tolerant mutations are interpreted as affecting some components of the cytoplasmic membrane which mediate between the adsorbed colicin molecules and the target sites of their biochemical effects in the bacterial cell.     

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.     

Acriflavine-binding capacity controlled by the acrA gene of Escherichia coli

The acrA mutation in Escherichia coli led to a substantial increase of the acriflavine-binding capacity of the cell, whereas the related mutations acrB (gyrB) and arcC did not. Metal ions such as Na+, K+, Mg2+, Ca2+ and Al3+ effectively released the bound acriflavine, in proportion to their ionic strengths. The presence of cations, in fact, increased the survival fraction of the cells in the acriflavine-containing medium. Polymyxin B, an antibiotic which binds to membrane phospholipid, competed with acriflavine for binding sites. Cell wall digestion by treatment with lysozyme and EDTA slightly decreased the acriflavine-binding capacity. Almost no difference was observed in acriflavine-binding capacity between intact cells and cells from which lipopolysaccharide has been extracted (46.9% removed from the acrA cells and 47.4% from the acrA+ cells). Acriflavine bound to the cells was most effectively extracted by ethanol containing 1% HCl or by 2% (w/v) SDS. The difference in the acriflavine-binding capacity between the acrA and acrA+ cells was also observed in the spheroplasts. These facts indicate a relationship between the acrA gene product and the acriflavine-binding capacity of the cells.     

Fuctional organization of the outer membrane of escherichia coli: Phage and colicin receptors as components of iron uptake systems

The functional interaction of outer memberane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 anf ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all colicins. The interaction of the ton A, Ton B, and feu functions apparently permits quite different “substrates” to overcome the permeablility barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of ³H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferriferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source waw used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: (1) ton A; (2) ton B mutants; (3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; (4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; (5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000–83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene. The nature of these proteins and their possible role in iron transport is disussed.     

Isolation and characterization of T-even ghost-tolerant mutants of Escherichia coli.

Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.     

Functional stability of the bfe and tonB gene products in Escherichia coli.

The expression of several functional properties of the products of the bfe and tonB genes in Escherichia coli was measured after the specific termination of the synthesis of the products of these genes. This was accomplished by the use of a temperature-sensitive amber suppressor mutation, which allowed control, by manipulation of the growth temperature, of the level of product formed from suppressible mutant alleles of the bfe or tonB gene. The bfe product is an outer membrane receptor protein for vitamin B12, the E-colicins, and bacteriophage BF23. The identity of the tonB product is unknown, but it is necessary for a subsequent step of uptake of vitamin B12, iron chelates, all of the group B colicins, and bacteriophages T1 and phi 80. Results from a different experimental system had shown that the termination of expression of the bfe locus was rapidly followed by loss of sensitivity to colicins E2 and E3 and, subsequently, to bacteriophage BF23. This was confirmed with this experimental system. Receptors that were no longer functional for colicin or phage uptake remained fully effective for B12 uptake, showing that receptors are stable on the cell surface. This supports previous contentions for the presence of different functional states for colicin receptors. The functional properties of the tonB product, measured by B12 uptake or sensitivity to the group B colicin D, were unstable, declining extensively after cessation of its synthesis.