Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

Outer membrane of Escherichia coli K-12: Tsx mutants (resistant to bacteriophage T6 and colicin K) lack an outer membrane protein

$\text{Tsx}$ mutants of $\text{Escherichia coli}$ are fully resistant to a set of T6-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. $\text{Tsx}$ mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations.We suggest that the tsx-protein is the receptor for both the bacteriophage and colicin.     

Evidence for the Direct Action of Colicin K on Aerobic 32Pi Uptake in Escherichia coli In Vivo and In Vitro

Pentachlorophenol (PCP)-sensitive incorporation of (32)P-labeled orthophosphate ((32)P(i)) into nucleotides and nucleic acids by disrupted spheroplasts of Escherichia coli was inhibited by addition of colicin K. Incorporation by intact cells was also inhibited by a similar concentration of colicin K. Various colicin K-resistant mutants were isolated, and their ability to incorporate (32)P(i) was tested. When T6(r)-colK(r) mutants (T6 phage-resistant) and tol I mutants (T6-sensitive, colicin E-sensitive) were converted to disrupted spheroplasts, their (32)P(i)-incorporation became sensitive to colicin K. On the contrary, incorporation by disrupted spheroplasts from tol II mutants (T6-sensitive, colicin E-resistant) was fairly resistant to colicin K like that of intact cells. A modification of the cell surface of T6(r)-colK(r) mutants, caused by mutation to novobiocin-permeable, T4 phage-resistant cells, restored the sensitivity of the cells to colicin K. The modified T6(r)-colK(r) cells did not adsorb T6 phage or colicin K, indicating that the receptors for T6 phage or colicin K are not reactivated by this modification. Similar treatment of tol I mutants did not have this effect. These observations strongly suggest that colicin K can act on its target on the cell membrane if it can penetrate the cell surface to reach this target. The receptor for colicin K on the cell surface, which may be part of the T6 phage-receptor, may have some unknown function in relation to the action of colicin K in normal cells, but tends to become dispensable if the cells become permeable to colicin K.     

Analysis of a mutated phage T6 receptor protein of Escherichia coli K 12

Summary The tsx-206 allele encodes an altered Tsx protein, Tsx-206, that can no longer function as the T6 receptor. We show here that this allele also confers resistance to the Tsx-specific phages III, H3, H8, K9, K18 and Oxl but not to colicin K. The Tsx-206 protein still mediates the efficient permeation of deoxyadenosine across the outer membrane at low substrate concentration. A host-range mutant of phage T6, T6h3.1, was isolated which can use both the Tsx-206 and the Tsx wild-type protein as its receptor. Cloning and DNA sequence analysis of the tsx-206 allele showed that the phage resistant phenotype was associated with an Asn to Tyr substitution at position 254 of the 272-residue Tsx protein.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.     

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

Mutation analysis of the receptor for colicins E1–E7. A pilot study

Thirty eight mutant clones of the colicin indicator strainEscherichia coli K 12 ROW, selected by their insensitivity to any of the colicins El–E7, were isolated. Comparison of their sensitivity-resistance patterns to colicins El–E7 enabled us to draw a rough preliminary map of the receptor for E colicins. In this receptor, the highly specific binding site for colicin El partially overlaps with the domain shared by all colicins E2 through E7. A specific binding site of this domain appears to be common for colicins E3 and E6; a part of the E3 and E6 binding site is also common for colicins E4 and E5 and a small, least specific, part also for colicins E2 and E7. Using colicin assay experiments, the binding capacity of coliein E receptor mutants could be estimated. A decreased, but not completely lost ability of certain mutants to bind colicins E, correlated to their lowered sensitivity to them, was found. Thus the phenomenon of partial colicin resistance was established, showing that colicin sensitivity—resistance is not a qualitative but a quantitative marker.     

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.     

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids

Summary The DNA polymerase induced by bacteriophage T7 is composed of a phage-specified subunit, the gene 5 protein, and a host-specified subunit, the 12,000 dalton thioredoxin of Escherichia coli. tsnC mutants of E. coli B (Chamberlin, 1974) have no detectable thioredoxin, and thus cannot support the growth of phage T7, although they are killed by phage infection. A mutant of E. coli K12 affecting thioredoxin has been isolated by a modification of the procedure used by Chamberlin (1974) to isolate tsnC mutants of E. coli B. The gene affecting thioredoxin has been designated trxA. This mutant, E. coli JM109, shows the TsnC phenotype in that it is killed by, but cannot support the growth of, bacteriophage T7. T7 DNA replication does not occur in mutantinfected cells. These phenotypic expressions of the tsnC mutation have enabled us to screen recombinants for the trxA allele in HfrxF^- crosses and F-ductants in episome transfer experiments. Extracts of transductants in generalized transduction by P1 phage were screened for their ability to complement partially purified phage T7 gene 5 protein to form T7 DNA polymerase. The trxA gene is located at 84 min on the E. coli linkage map, between uvrE and metE; trxA is 34% co-transducible with metE.     

Isolation of conjugation-constitutive mutants of colicin factor Ib

Summary Colicin factor ColIb-P9 is known to act as a sex factor in E. coli or Salmonella. Although ColIb-P9 confers mating ability on its host bacteria, this ability appears to be repressed since only a small proportion of cells in a culture of a colicinogenic strain are able to pair with, and transmit the factor to recipient bacteria. We have isolated mutants of ColIb-P9 which confer constitutive donor ability on their host. De-repression in these mutants is probably due to failure to produce repressor, rather than to insensitivity to repressor. As the colicin production by the mutants is still repressed, colicin synthesis and conjugation ability are subject to independent systems of regulation.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

A common receptor protein for phage T5 and colicin M in the outer membrane of Escherichia coli B

The receptor protein for phage T5 was isolated from the outer membrane of Escherichia coli B and found to be also a receptor for colicin M. The receptor protein from a phage-resistant mutant inactivates neither the phage nor the colicin. Binding of colicin M to the receptor prevents binding of phage T5. It is concluded that phage T5 and colicin M bind to the same active area of this receptor protein. The receptor protein seems to consist of one polypeptide chain with a molecular weight of 85000.     

The immunity and lysis genes of ColN plasmid pCHAP4

Summary Nucleotide sequencing of part of the plasmid pCHAP4, which encodes the ca. 42000 Da putative poreforming colicin N, confirmed previous results indicating that the colicin N immunity gene (cni) and the colicin release or lysis gene (cnl) are located immediately downstream from the colicin N structural gene (cna) in the order cna-cni-cnl. The cni gene is transcribed in the opposite direction to cna and probably encodes an M_r 15239 Da protein. The putative immunity protein was detected among the [³⁵S]methionine-labelled proteins produced by minicells carrying cni cloned under lac promoter control, and when the gene was subcloned into expression vectors under the control of a bacteriophage T7 promoter. Deletion of the region immediately upstream from cni completely abolished colicin N immunity, presumably because the natural promoter had been deleted. cnl is in the same operon as cna, and encodes a typical Col plasmid pro-lysis protein comprising a signal peptide and a 34 residue mature polypeptide with high homology to all but one of the other known Col lysis proteins, including the fatty acylated amino-terminal cysteine residue which was specifically labelled with ³H-palmitate. Cell fractionation studies indicated that the cnl gene product was located predominantly in the outer membrane.     

Colicin M is only bactericidal when provided from outside the cell

Summary The colicin M structural gene, cma, was subcloned in a vector which allowed temperature-inducible control of its expression. Induction of expression of cma in colicin M uptake proficient strains was lethal for the host cell when the colicin M immunity protein was not present. In liquid culture cells lysed, and no colonies were formed on solid media. These effects were not observed in mutants defective in the colicin receptor (FhuA) or uptake functions (TonB, TolM), nor in wild-type cells treated with trypsin prior to induction of cma expression. It was concluded that cytoplasmic colicin M is not toxic for the producing cell. To exert a lethal effect the colicin has to enter the cell from outside. Cells expressing cma released small amounts of colicin M.     

Daring to be different: colicin N finds another way

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. Johnson et al. show that the receptor‐binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor‐binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.     

Isolation of metabolically active Petite mutants of Kluyveromyces lactis, a petite-negative yeast

Summary Conditions under which complete cultures of the petite-negative yeast Kluyveromyces lactis can be converted to metabolically active petite mutants have been found. These mutants, which lack mitochondrial protein synthesis have been shown to be metabolically active by their ability to exclude the dye trypan blue. They appear to possess a functional protein synthesising system, which is sensitive to the inhibitor trichodermin. However, on transfer to solid nutrient medium, these mutants fail to grow normally, and give rise to microcolonies composed of up to a thousand cells. These colonies autolyse after several days.     

On the receptor for bacteriophage T4 inEscherichia coli K12

Purified lipopolysaccharide (LPS) from a mutant strain ofEscherichia coli K12 altered in its LPS has been shown to serve as a receptor for bacteriophage T4, which contrasts with LPS from a wild-type strain. Studies of extragenic suppression of a mutation in the gene specifying protein 1b revealed that the galactose residue in the LPS normally masks the LPS receptor and that in the absence of this residue protein 1b is not a necessary component of the T4 receptor.