Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli

A virulent double-stranded DNA bacteriophage, Phi K1-5, has been isolated and found to be capable of infecting Escherichia coli strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the Podoviridae family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of Phi K5, which allows this phage to specifically infect K5 E. coli strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the N-acetylneuraminidase (endosialidase) protein of Phi K1E, which allows this phage to specifically infect K1 strains of E. coli. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of Phi K5, Phi K1E, and Phi K1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.     

Escherichia coli K Bacteriophages I. Isolation and Introductory Characterization of Five Escherichia coli K Bacteriophages

A set of five Escherichia coli K phages has been isolated. These phages are adsorbed to and lyse the capsular forms of the host bacteria, whereas their spontaneous, acapsular mutants are not affected. All host strains are heavily encapsulated test strains for E. coli K antigens of the thermostable A type and they readily segregate acapsular mutants. In four of the phage-host systems, all secondary growth obtained was found to be acapsular. When tested for host-range mutants on 38 strains of E. coli and Klebsiella, less than one mutant per 10(5) plaque-forming units was found. No cross-reacting neutralizing antibodies were obtained when rabbits were immunized with the K phages. The latent periods (between 16 and 30 min) and average burst sizes (between 145 and 580) were determined by one-step growth experiments.     

Two different Escherichia coli capsular polysaccharide depolymerases each associated with one of the coliphage phi K5 and phi K20

The Escherichia coli capsular polysaccharides (K antigens) K5 and K20 are known as primary receptors for the coliphage phi K5 and phi K20, respectively. A host range study of the phage revealed that E. coli K5 strains were not only lysed by phi K5 but also by phi K20, and furthermore that the E. coli K95 test strain was attacked by phi K5 in addition to K5 strains. In order to find out whether the phage can degrade the K antigens, the interaction of the phage with isolated polysaccharides was studied. It could be demonstrated that phi K5 was able to depolymerize the K5 and K95 polysaccharides and that phi K20 showed degrading activity towards the antigens K20 and K5. Obviously, each of the phages was associated with two different enzyme systems which enabled them to recognize and depolymerize chemically unrelated polysaccharides.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.     

The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages

Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 10(5) phage organisms. It could be prevented by doses as low as 10(2), by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K-. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.     

The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli

Bacteriophage K1F specifically infects Escherichia coli strains that produce the K1 polysaccharide capsule. Like several other K1 capsule-specific phages, K1F encodes an endo-neuraminidase (endosialidase) that is part of the tail structure which allows the phage to recognize and degrade the polysaccharide capsule. The complete nucleotide sequence of the K1F genome reveals that it is closely related to bacteriophage T7 in both genome organization and sequence similarity. The most striking difference between the two phages is that K1F encodes the endosialidase in the analogous position to the T7 tail fiber gene. This is in contrast with bacteriophage K1-5, another K1-specific phage, which encodes a very similar endosialidase which is part of a tail gene "module" at the end of the phage genome. It appears that diverse phages have acquired endosialidase genes by horizontal gene transfer and that these genes or gene products have adapted to different genome and virion architectures.     

CMP-KDO-synthetase activity in Escherichia coli expressing capsular polysaccharides

The temperature-regulated expression of capsular group II polysaccharides of Escherichia coli (B. Jann and K. Jann, (1990) Curr. Top. Microbiol. Immunol. 150: 19-42) depends on an elevated concentration of CMP-KDO, as evidenced by an increased activity of CMP-KDO synthetase. The increase in activity of CMP-KDO synthetase is observed only in cytoplasmic fractions of bacteria which had been grown at 37 degrees C but not after growth at 18 degrees C. The activity of CMP-KDO synthetase thus parallels the activity of the (membrane-associated) system synthesizing capsules of group II in E. coli. No such dependence of capsule expression on CMP-KDO was observed with E. coli with capsules of group I. A number of E. coli strains with capsular polysaccharides, which on the basis of genetic determination and chemical characteristics are considered as group II capsules, show no temperature regulation of their capsules and do not depend on an elevated CMP-KDO concentration for capsule expression. The capsular polysaccharides of these E. coli strains, which possibly represent a new group of E. coli capsules are tentatively classified as group I/II.     

Accelerated Adsorption of Bacteriophage T5 to Escherichia coli F, Resulting from Reversible Tail Fiber-Lipopolysaccharide Binding

A dual specificity for phage T5 adsorption to Escherichia coli cells is shown. The tail fiber-containing phages T5(+) and mutant hd-3 adsorbed rapidly to E. coli F (1.2 x 10(-9) ml min(-1)), whereas the adsorption rate of the tail fiber-less mutants hd-1, hd-2, and hd-4 was low (7 x 10(-11) ml min(-1)). The differences in adsorption rates were due to the particular lipopolysaccharide structure of E. coli F. Phage T4-resistant mutants of E. coli F with an altered lipopolysaccharide structure exhibited similar low adsorption for all phage strains with and without tail fibers. The same held true for E. coli K-12 and B which also differ from E. coli F in their lipopolysaccharide structures. Only the tail fiber-containing phages reversibly bound to isolated lipopolysaccharides of E. coli F. Infection by all phage strains strictly depended on the tonA-coded protein in the outer membrane of E. coli. We assume that the reversible preadsorption by the tail fibers to lipopolysaccharide accelerates infection which occurs via the highly specific irreversible binding of the phage tail to the tonA-coded protein receptor. The difference between rapid and slow adsorption was also revealed by the competition between ferrichrome and T5 for binding to their common tonA-coded receptor in tonB strains of E. coli. Whereas binding of T5(+) to E. coli K-12 and of the tail-fiber-less mutant hd-2 to E. coli F and K-12 was inhibited 50% by about 0.01 muM ferrichrome, adsorption of T5 to E. coli F was inhibited only 40% by even 1,000-fold higher ferrichrome concentrations.     

Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT-. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli.

With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide.     

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.     

Structural investigation of the capsular polysaccharide from Escherichia coli O9:K37 (A 84a)

The structure of the capsular polysaccharide from E. coli O9:K37 (A 84a) has been studied, using methylation analysis, Smith degradation, and graded acid hydrolysis. The configurations at the anomeric centres were assigned by 1H-n.m.r. spectroscopy of the polysaccharide and its derivatives and oligosaccharide fragments. The polysaccharide has the following trisaccharide repeating-unit which is unique in the E. coli series of capsular polysaccharides in possessing a 1-carboxyethylidene group as the sole acidic function. (Formula: see text) E. coli capsular polysaccharides have been classified into seventy-four serotypes. The structures of about twenty of these polysaccharides have been elucidated, one of which, K29, has been reported to contain a 1-carboxyethylidene group. In continuation of a programme aimed at establishing the structural basis for the serology and immunochemistry of the E. coli capsular antigens, we now report on the structure of the capsular polysaccharide from E. coli O9:K37.     

Isolation and characteristics of new phages from a serine-producing Escherichia coli K-12

Two lytic phages, designated S1 and S2, were isolated from culture lysates of a genetically modified serine-producing Escherichia coli K-12. Both phages were highly species-specific for E. coli. S1 was specific for strains of K-12, while S2 attacked strains B and C in addition to K-12 strains. S1 had an icosahedral head 75 nm in diameter and a contractile tail 150 nm long. S2 had an icosahedral head 60 nm in diameter and a noncontractile tail 160 nm long. They were serologically unrelated. Their serotypes were different from those of the other E. coli phages. The latent period and burst size were 28 min and 450, respectively, for S1, and 15 min and 100, respectively, for S2. The phages contained double-stranded DNA with four normal bases. The G+C contents were about 31% for S1 DNA and about 37% for S2 DNA. Restriction patterns of their DNAs were different from each other. The genome sizes were 52 kbp for S1 and 49 kbp for S2. No homology was observed between their genomes. Furthermore, the structural proteins of the two phages also differend. W conclude that phages S1 and S2, differing from each other, could be new phages for E. coli.     

Degrees of relatedness of T-even type E. coli phages using different or the same receptors and topology of serologically cross-reacting sites

The relatedness of a series of T-even like phages which use the Escherichia coli outer membrane protein OmpA as a receptor, and the classical phages T2, T4 and T6 has been investigated. Immunoelectron microscopy and the pattern of phage resistance in bacterial mutants revealed that: (i) phages of this morphology do not necessarily cross-react serologically; (ii) phages using different receptors may bind heterologous IgG everywhere except to the tip (comprising approximately 10% of one fiber polypeptide) of the long tail fibers; (iii) cross-reacting OmpA-specific phages may bind heterologous IgG only to the tip of these fibers: (iv) OmpA-specific phages not cross-reacting at the tip of the tail fibers use different receptor sites on the protein. Absence of cross-reactivity appears to reflect high degrees of dissimilarity. A DNA probe consisting of genes encoding the two most distal tail fiber proteins of T4 detected homologies only in DNA from phages serologically cross-reacting at this fiber. Even under conditions of low stringency, allowing the formation of stable hybrids with almost 30% base mismatch, no such homologies could be found in serologically unrelated phages. Thus, in the collection of phages examined, there are sets of very similar and very dissimilar tail fiber genes and even of such gene segments.     

Partial characterization of coliphage WPK and a comparison with coliphage T3.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.