Identification of a segment of the Escherichia coli Tsx protein that functions as a bacteriophage receptor area.

The Escherichia coli outer membrane protein Tsx functions as a nucleoside-specific channel and serves as the receptor for colicin K and a number of T-even-type bacteriophages, including phage T6. To identify those segments of the Tsx protein that are important for its phage receptor function, we devised a selection and screening procedure which allowed us to isolate phage-resistant strains synthesizing normal amounts of Tsx. Three different Tsx-specific phages (T6, Ox1, and H3) were employed for the selection of phage-resistant derivatives of a strain expressing a tsx(+)-lacZ+ operon fusion, and 28 tsx mutants with impaired phage receptor function were characterized. Regardless of the Tsx-specific phage used for the initial mutant selection, cross-resistance against a set of six different Tsx phages invariably occurred. With one exception, these mutant Tsx proteins could still serve as a colicin K receptor. DNA sequence analysis of 10 mutant tsx genes revealed the presence of four distinct tsx alleles: two point mutations, an 18-bp deletion, and a 27-bp tandem duplication. In three isolates, Asn-249 was replaced by a Lys residue (tsx-504), and in four others, residue Asn-254 was replaced by Lys (tsx-505). The deletion (tsx-506; one isolate) removed six amino acids (residue 239 to residue 244) from the 272-residue Tsx polypeptide chain, and the DNA duplication (tsx-507; two isolates) resulted in the addition of nine extra amino acids (residue 229 to residue 237) to the Tsx protein. In contrast to the wild-type Tsx protein and the other mutant Tsx proteins the Tsx-507 protein was cleaved by trypsin when intact cells were treated with this protease. The Tsx proteins encoded by the four tsx alleles still functioned in deoxyadenosine uptake in vivo, demonstrating that their nucleoside-specific channel activity was not affected by the alterations that caused the loss of their phage receptor function. HTe changes in the Tsx polypeptide that confer resistance against the Tsx-specific phages are clustered in a small region near the carboxy terminus of Tsx. Our results are discussed in terms of a model for the topological organization of the carboxy-terminal end of the Tsx protein within the outer membrane.     

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     

DNA sequence heterogeneity in the genes of T-even type Escherichia coli phages encoding the receptor recognizing protein of the long tail fibers

Summary Genes (g) 36 and 37 code for the proteins of the distal half of the long tail fibers of phage T4, gene product (gp) 35 links the distal half to the proximal half of this fiber. The receptor, lipopolysaccharide, most likely is recognized by gp37. Using as probe a restriction fragment consisting of most of g36 and g37 of phage T4 the genes corresponding to g35, g36, and g37 of phages T2 and K3 (using the E. coli outer membrane proteins OmpF and OmpA, respectively, as receptors) have been cloned into plasmid pUC8. Partial DNA sequences of g37 of phage K3 have been determined. One area, corresponding to residues 157 to 210 of the 1026 residue gp37 of phage T4, codes for an identical sequence in phage K3. Another area corresponds to residues 767 to 832 of the phage T4 sequence. Amino acid residues 767 to 832 of the phage T4 sequence are almost identical in both phage proteins while the remainder is rather different. DNAs of T2, T4, T6, another T-even type phage using protein Tsx as a receptor, and 10 different T-even type phages using the OmpA protein as a receptor have been hybridized with restriction fragments covering various parts of the g37 area of phage K3. With probably only one exception all of the 13 phages tested possess unique genes 37 and within the majority of these, sequences highly homologous to parts of g37 of K3 are present in a mosaic type fashion. Other regions of these genes 37 did not show any homology with the K3 probes; in case of the OmpA specific phage M1 absence of homology was evident in most of its g37 even including the area that should serve for recognition of the cellular receptor. In sharp contrast to this situation it was found that a major part of the gene (g23) coding for the major capsid protein is rather highly conserved in all phages studied. The extreme variability in sequences existing in genes 37 might be a consequence of phages during evolution being able to more or less drastically change their receptor specifities.     

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)     

Outer membrane proteins of Escherichia coli K-12: Isolation of a common receptor protein for bacteriophage T6 and colicin K

Summary tsx mutants, resistant to T6-like bacteriophages and colicin K, of Escherichia coli K-12 lack an outer membrane protein of 26,000 molecular weight. This protein is shown to have receptor activity for both bacteriophage T6 and colicin K. The protein has been purified and its amino acid composition determined. Some tsx mutants appear to have an altered receptor protein, as indicated by their ability to plate extended host-range mutants of bacteriophage T6. These mutants are also cotransducible with proC and can be arranged in an order of increasing resistance to the host range phages, which appear to have differing degrees of ability to propagate on the tsx mutants.The tsx protein was shown to be catabolite repressible both by use of varying growth conditions and cya and crp mutants.     

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.     

IgA response of BALB/c mice to orally administered Salmonella typhimurium flagellin‐displaying T2 bacteriophages

Salmonella typhimurium antigens were displayed on the capsid of a T2 bacteriophage to explore the potential of phage display for an oral vaccine. Segments of the flagellin proteins FliC (H1 antigen) and FljB (H2) were fused to the N‐terminal of T2 phage SOC to give two recombinant phages, T2FliCm and T2FljBm. Over 14 days, 19 BALB/c mice were orally administered twice, either with purified recombinant FliCm and FljBm protein, or T2FliCm and T2FljBm with or without host Escherichia coli. Feces were sampled over 10 weeks and examined for phage by plaque assay and for the presence of mucosal IgA by ELISA. Relatively few phages were detected relative to the amount administered (up to 8.21 × 10³ PFU/g faeces) and none were detected five days after initial administration. The administration of a large number of phages appeared to cause no clinical symptoms. IgA concentration in feces peaked around four weeks after the second administration and subsided after eight weeks. The highest relative titers were observed in the protein group (0.37% for anti‐FliCm and 0.22% for anti‐FljBm) and the mouse group which received no E. coli (0.33% and 0.35%) despite the theoretical amount of protein contained in a phage dose being at least 80–465 times lower than the protein dose administered. The possibility that the immuno‐stimulatory properties of the phage create an adjuvant effect to enhance the immunogenic properties of the displayed proteins is discussed. We conclude that phage may be valuable as a vector for oral vaccines. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system

Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication. Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E. coli host-cells. All stable spontaneous Albr mutants of E. coli simultaneously became resistant to phage T6. The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations. Albicidin does not closely resemble a nucleoside in structure. However, Albs E. coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations. An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance. Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E. coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells. We conclude that albicidin is effective at very low concentrations against E. coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake. Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.     

T-even type phages can change their host range by recombination with gene 34 (tail fibre) or gene 23 (head)

Summary T-even type phages recognize their cellular receptors with the tip of their long tail fibres. The gene products involved in receptor recognition are proteins 37 and 38. While screening libraries of phage K3 with a probe of gene38 from phage T2, a class of weakly hybridizing clones was found in addition to the expected clones of gene38 of K3. One of these clones was identified as being from gene23 of the phage which codes for the major head subunit; another clone originated from gene34, which codes for the proximal half of the long tail fibres. Neither gene product 23 nor 34 is involved in receptor recognition. Phages can recombine with the DNA of the gene23 and gene34 clones and change the host range. This paper is dedicated to Prof. Dr. Wolfgang Beermann, Tübingen, on the occation of his 65^th birthday     

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

Summary We have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.     

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. Constitutively activated mutants of a bone morphogenetic protein (BMP) receptor, ALK2, have been identified in patients with FOP. Recently, a novel ALK2 mutation, L196P, was found in the most benign case of FOP reported thus far. In the present study, we examined the biological activities of ALK2(L196P) in vitro. Over-expression of ALK2(L196P) induced BMP-specific activities, including the suppression of myogenesis, the induction of alkaline phosphatase activity, increased BMP-specific luciferase reporter activity, and increased phosphorylation of Smad1/5 but not Erk1/2 or p38. The activities of ALK2(L196P) were higher than those of ALK2(G356D), another mutant ALK2 allele found in patients with FOP and were equivalent to those of ALK2(R206H), a typical mutation found in patients with FOP. ALK2(L196P) was equally or more resistant to inhibitors in comparison to ALK2(R206H). These findings suggest that ALK2(L196P) is an activated BMP receptor equivalent to ALK2(R206H) and that ALK2(L196P) activity may be suppressed in vivo by a novel molecular mechanism in patients with this mutation.     

T-even-type bacteriophages use an adhesin for recognition of cellular receptors

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.     

Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site

The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro. When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro. This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine. Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively. We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.