Early Abortive Lysis by Phage BF23 in Escherichia coli K-12 Carrying the Colicin Ib Factor

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.     

Regulation of Bacteriophage T5 Development by ColI Factors

The I-type colicinogenic factor ColIb transforms Escherichia coli from a permissive to a nonpermissive host for bacteriophage T5 reproduction by preventing complete expression of the phage genome. T5-infected ColIb(+) cells synthesize only class I (early) phage protein and ribonucleic acid (RNA). Neither phage-specific class II proteins [associated with viral deoxyribonucleic acid (DNA) replication] nor class III proteins (phage structural components) are formed due to the failure of the infected ColIb(+) cells to synthesize class II or class III phage-specific messenger RNA. Comparable studies with T5-infected cells colicinogenic for the related ColIa factor revealed no decrease in the yield of progeny phage although the presence of the ColIa factor leads to a significant reduction in the amount of phage-directed class III protein synthesis.     

Membrane damage in abortive infections of colicin Ib-containing Escherichia coli by bacteriophage T5.

Strains of Escherichia coli K-12 containing the colicin Ib (Col Ib) factor did not produce progeny phage when infected by T5 bacteriophage. The cells were killed but did not lyse. If sodium dodecyl sulfate (SDS) was added to T5-infected E. coli (Col Ib), lysis occurred prematurely, but no phage were produced. SDS had no effect on infected cells that did not contain the Col Ib factor or on uninfected cells with or without the Col Ib factor. Cells that contained a mutant Col Ib factor that allowed phage production were not prematurely lysed after infection in the presence of SDS. When the Col Ib-containing cells were infected, protein and RNA synthesis stopped at about 10 min postinfection, and the cells released abnormal amounts of 32P-containing material, ATP, and beta-galactosidase into the medium. They also became inhibited in their ability to accumulate thiomethyl-beta-D-galactopyranoside and to utilize glycerol. Two alternative hypotheses are presented to explain these results.     

Genes affecting coliphage BF23 and E colicin sensitivity in Salmonella typhimurium.

Rough strains of Salmonella typhimurium were sensitive to coliphage BF23. Spontaneous mutants resistant to BF23 (bfe) were isolated, and the trait was mapped using phage P1. The bfe gene in S. typhimurium was located between argF (66% co-transducible) and rif (61% co-transducible). The BF23-sensitive S. typhimurium strains were not sensitive to the E colicins. Cells of these rough strains absorbed colicin, as measured by loss of E2 or E3 killing units from colicin solutions and by specific adsorption of 125I-colicin E2 to bfe+ cells. Sensitivity to colicins E1, E2, and E3 was observed in a S. typhimurium strain carrying the F'8 gal+ episome. This episome complemented the tolB mutation of Escherichia coli. We conclude that the bfe+ protein satisfies requirements for adsorption of both phage BF23 and the E colicins. In addition, expression of a gene from E. coli, possibly tolB, is necessary for efficient E colicin killing of S. typhimurium.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.     

Induction of immunity and specific unresponsiveness in vitro by cell-bound antigen: I. Symmetry in enhancement of both responses

Pulse treatment of lymphoid cells from rabbits with solubilized antigens from T2 phage results in the firm binding of small but highly active amounts of antigen. Binding of phage antigens to viable, nonviable, or disrupted cells enhances their ability to evoke antibody formation or specific unresponsiveness in the primary in vitro response of rabbit spleen cells. Transfer of sonicate containing the equivalent of 10₂ to 10₃ antigen-pulsed cells carrying 10^?8 to 10^?7 μg phage protein nitrogen into spleen cell cultures regularly evokes antibody formation, while introduction to such cultures of 10^?3 μg phage protein nitrogen in cell-bound form evokes unresponsiveness. These findings indicate a 10- to 100-fold amplification of tolerogenic and immunogenic activities of cell-bound over soluble T2 antigen.     

Major proteins of the outer cell envelope membrane of Escherichia coli K12: multiple species of protein I differ in primary structure.

Summary Protein I, one of the major outer membrane proteins of E. coli in most K12 strains is represented by two very similar polypeptides Ia and Ib. Sequential mutations (involving selections for phage resistance) can lead to loss of proteins Ia and Ib. Among revertants of such Ia^- Ib^- mutants clones exist that instead of Ia or Ib produce a third species of protein I, polypeptide Ic.Ichihara and Mizushima [J. Biochem. 83, 1095–1100 (1978)] have shown that proteins Ia and Ib exhibit differences in primary structure. Here evidence is presented indicating that protein Ic also is not identical in primary structure with Ia or Ib. Thus, 3 very similar structural genes appear to exist for the protein I species known to date, and that for Ic normally is silent. Introduction of a functional Ic locus into a Ia⁺ Ib⁺ strain caused expression of all three proteins with a reduced rate of synthesis of protein Ia.     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

Escherichia coli K317, formerly used to define colicin group E2, produces colicin E7, is immune to colicin E2, and carries a bacteriophage-restricting conjugative plasmid.

Escherichia coli strain CL137, a K-12 derivative made E colicinogenic by contact with Fredericq's strain K317, was unaffected by colicin E2-P9, but K-12 carrying ColE2-P9 was sensitive to the E colicin made by strains CL137 and K317. This colicin we named E7-K317 because by the test of colicinogenic immunity it differed from colicins E1-K30, E2-P9, and E3-CA38 and from recently recognized colicins termed E4Horak, E5, and E6. Strain K317 as conjugational donor transmitted E7 colicinogeny; about half the E7-colicinogenic transconjugants were immune to colicin E2-P9. A spontaneous variant of CL137 retained E7 colicinogeny but was sensitive to E2 colicins. We attribute the E2 immunity of strain CL137 and some E7-coliconogeic transconjugants to a "colicin-immunity plasmid," ColE2imm-K317, from strain K317. Tra+ E7-colicinogenic transconjugants restricted phage BF23 in the same way as strains carrying ColIb-P9. We attribute Tra+ and restricting ability to a plasmid, pRES-K317, acquired from strain K317, and related to the ColI plasmids.     

Maturation of the head of bacteriophage T4. II. Head-related, aberrant tau-particles

Three somewhat different types of particle accumulate in cells infected with a phage carrying a mutation in gene 21 (in addition to the tubular variant (polyhead) of the head). The major type is the so-called τ-particle. These particles are very fragile, associated with the cell membrane, and have a sedimentation coefficient of about 420 S. They possess no DNA if isolated, and contain predominantly the precursor proteins P23, P24, P22 and the internal protein IP III, in addition to protein P20 and several proteins of unknown genetic origin.The remainder of the particles are partially or completely filled with DNA. The ratio of τ-particles to these partially or completely filled particles depends upon the particular mutant (in gene 21) phage used. In cells infected with a phage carrying the amber mutation (N90) in gene 21, about 10% of the precursor head protein P23 is cleaved to P231, and correspondingly about 10% of the particles are partially or completely filled with DNA. In cells infected with the temperature-sensitive mutant (N8) in gene 21, about 1% of the particles are fully or partially filled, and correspondingly about 1% of the P23 is cleaved to P231. In either case, the DNA-associated particles contain predominantly the cleaved proteins P231 and IP III1, and have none of the P22 and IP III found in τ-particles. This observation, and the correlation of the amount of partially or completely filled particles with the extent of the cleavage of P23 in the lysates, strongly suggest that cleavage of the head proteins is required for DNA packaging to occur.The τ-particles have properties similar to the so-called prohead I particles which we have isolated as intermediates in wild-type head assembly (preceding paper). However, temperature shift-down experiments, using several different phage carrying temperature-sensitive mutations in gene 21, indicate that the bulk of the τ-particles cannot be used for normal phage production.     

Interaction of Colicins with Bacterial Cells IV. Immunity Breakdown Studied with Colicins Ia and Ib

Colicinogenic cells are immune to the lethal effect of the colicin which they produce. In the presence of very high concentrations of colicin, however, colicinogenic cells are no longer immune to the homologous colicin. This phenomenon, immunity breakdown, was studied with colicins Ia and Ib. The biochemical effects of colicin Ib on Escherichia coli were studied with a standard noncolicinogenic strain. At multiplicities of about 10 or higher, colicin Ib inhibited incorporation of leucine into protein and incorporation of (32)P-inorganic phosphate into deoxyribonucleic acid and ribonucleic acid by more than 95%. Under the same conditions, (32)P incorporation into phospholipid and nucleotide fractions was inhibited only partially (about 80 and 60%, respectively). Inhibition of (32)P incorporation into the terminal phosphorus of adenosine triphosphate was also considerably less than that of macromolecular synthesis (50 to 60%). (32)P incorporation into the nonnucleotide organic phosphate fraction was not inhibited. Respiration was not affected. Colicin Ia showed the same biochemical effects as colicin Ib. A mutant of an Ib-colicinogenic E. coli strain selected for resistance to low concentrations of colicin Ia was shown to be resistant to high concentrations of homologous colicin Ib, whereas the parent Ib-colicinogenic strain is sensitive to high concentrations of colicin Ib. This mutant lost its specific receptors for colicin Ib. Moreover, the biochemical effects of high concentrations of colicin Ib on Ib-colicinogenic cells during immunity breakdown were similar to the effects found in sensitive cells exposed to low concentrations of the same colicin. It is concluded that the killing of colicinogenic cells in the presence of high concentrations of homologous colicin is indeed caused by the homologous colicin molecules.     

Major proteins of the outer cell envelope membrane of Escherichia coli K-12: multiple species of protein I.

Summary Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia⁺ Ib^-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II^*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.     

Irreversible binding to the receptor of bacteriophages T5 and BF23 does not occur with the tip of the tail.

Treatment of purified tails of bacteriophage T5 with 0.05% sodium dodecyl sulfate specifically removed pb2, a protein of 108,000 molecular weight (108K), from the tail. Although these tails were devoid of the single straight tail fiber, they still inhibited adsorption of T5 to Escherichia coli cells. Reconstitution of these tails with pb2 increased the efficiency of inhibition of T5 adsorption. Treatment of tails with 0.1% sodium dodecyl sulfate removed, in addition to pb2, a protein of 67K from phage T5 and one of 60K from phage BF23. These tails failed to inhibit phage adsorption, and no reconstitution was achieved. Reconstitution of T5 tails with pb2 from BF23, and of BF23 tails with pb2 from T5, did not alter the host receptor specificity of the tails. Binding of untreated T5 tails to small FhuA receptor particles revealed that binding occurred with the conical part of the tail and that pb2 was most likely released from the tail upon binding. From these results and from recent observations with T5-BF23 hybrid phages (K.J. Heller, Virology 139:11-21, 1984), we conclude that the receptor-binding proteins of T5 and BF23 are the 67K and 60K proteins, respectively, and that they are not located at the tip of the tail but rather at or near the site where the straight tail fiber is attached to the conical part of the tail.     

Membrane protein biosynthesis in bacteriophage BF23-infected Escherichia coli.

When Escherichia coli is infected with bacteriophage BF23, two new proteins with molecular weights greater than 10,000, as indicated by polyacrylamide gel electrophoresis, are found associated with the cells' membranes. One of these, found associated with both the inner and outer membrane, has a molecular weight of about 55,000 and is regulated by the A1 gene of this phage, a gene found on the spontaneously injected 8% piece of BF23 DNA, DNA that codes for the synthesis of proteins necessary for the injection of the whole phage genome. The other protein, often undetected in whole membrane preparations, is found exclusively associated with the inner membrane. Evidence indicates that this protein is also regulated by the initially injected 8% piece of the DNA.     

Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid

A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.     

Major outer membrane proteins of E. coli K12 serve as receptors for the phages T2 (protein Ia) and 434 (protein Ib).

Summary Mutants of E. coli resistant to bacteriophage T2 have lowered amounts of protein Ia in their outer membrane. Bacteriophage T2 was inactivated by a mixture of protein Ia-lipopolysaccharide. Protein Ia or lipopolysaccharide alone had no neutralizing activity. However, only protein Ia was required to inactivate a T2 host range mutant. In the presence of polymyxin B T2 receptor activity of protein Ia — lipopolysaccharide mixtures could not be restored. E. coli strains missing protein Ib were resistant against the lambdoid phage 434. Purified protein Ib inactivated 434 and virh ⁴³⁴. Addition of lipopolysaccharide did not enhance the neutralizing activity of protein Ib, indicating that lipopolysaccharide may not be necessary for the inactivation of the phage.     

Characteristics of hybrid plasmids carrying the genes of colicinogenic plasmid Collb-P9 responsible for colicin Ib synthesis and inhibition of phage T5 development

The EcoRI and HindII restriction endonucleases and pBR325 vector plasmid were used to obtain a set of hybrid plasmids containing ColIb-P9 fragments carrying the characters for colicin Ib synthesis and immunity and the ability to inhibit T5 phage growth. The genes responsible for colicin synthesis and immunity are closely linked and localized in the EcoRI fragment with a molecular weight of 1.85 MD (pIV41) or in the HindII fragment of 2.4 MD (pIV1). The clones containing these plasmids show an increased level of both spontaneous and mitomycin C-induced colicin synthesis and an increased level of immunity due to a larger dosage of the genes. The genes controlling T5 growth inhibition are localized in other restriction fragments of ColIb DNA: the EcoRI fragment of 1.45 MD (pIV7) and the HindII fragment of 4.3 MD (pIV5). We have demonstrated by means of hybrid plasmids that T5 growth inhibition is not connected with the colicin Ib synthesized in infected cells and is controlled by other specific product(s) of the ColIb plasmid genes. T5 phage growth was as efficient in clones containing plasmids with cloned colicin Ib genes as in a strain without plasmids. An investigation of the expression of the genes inhibiting T5 phage growth in an in vitro protein synthesis system has revealed a protein with a molecular weight of 36 000 which seems to take part in the process.     

Ion fluxes during T5 bacteriophage infection of Escherichia coli

When T5 bacteriophage infects Escherichia coli B, ⁴²K⁺ is immediately released from cells that have been preloaded with this ion. The rate of ion release and the total amount released are dependent on the multiplicity of infection and are not diminished by the use of mutants which can only inject 8% of their DNA. Normally, the ion release stops at about 6 min postinfection. If the host cells contain the colicinogenic factor, Col Ib, so that the infection is abortive, K⁺ release continues. Evidence is presented to show that this continued ion release cannot be explained by a “damage and repair” hypothesis. The results are, however, consistent with the interpretation of membrane depolarization due to ion pore formation as the cause of the abortive infection.     

Biosynthesis of the outer membrane receptor for vitamin B12, E colicins, and bacteriophage BF23 by Escherichia coli: kinetics of phenotypic expression after the introduction of bfe+ and bfe alleles.

The bfe locus codes for the cell surface receptor for vitamin B12, the E colicins, and bacteriophage BF23 in the Escherichia coli outer membrane. When the bfe+ allele, which is closely linked to the argH locus, was introduced into an argH bfe recipient by conjugation, arg+ recombinant cells rapidly and simultaneously acquired sensitivity to colicin E3 and phage BF23. In the reciprocal experiment introducing bfe into an argH bfe+ recipient, it was found that colicin E3-resistant, arg+ cells began to appear shortly after the arg+ recombinant population began to divide. This was far earlier than would have been predicted on the basis of 220 receptors per haploid cell. Moreover, there was a lag between the appearance of colicin resistance and the appearance of resistance to killing by phage BF23, and hence a period of time during which some arg+ recombinant cells were sensitive to the phage but resistant to the colicin. Colicin E3 added to cells during this period of time protected against phage killing, indicating that the colicin-resistant cells still had receptors capable of binding colicin on their surface. The modification of the phenotypic expression of colicin and phage resistance by inhibitors of deoxyribonucleic acid, ribonucleic acid, and protein synthesis was also investigated. The results obtained indicate that the receptor protein coded for by the bfe locus can exist on the cell surface in several different functional states.