Is abortive infection by bacteriophage BF23 of Escherichia coli harboring ColIb plasmids due to cell killing by internally liberated colicin Ib?

Infection of Escherichia coli harboring ColIb+ plasmids with bacteriophage BF23+ is abortive and resulted in changes of membrane permeability as measured by efflux of nucleotides and K+. A single pre-early gene product of BF23+ was necessary and sufficient to elicit the abortive response. Appropriate mutations in this pre-early gene allowed a productive infection in ColIb+ cells. Appropriate mutations in the ColIb plasmid also allowed a productive infection with BF23+. A comparison of changes occurring during abortive infection and during killing of sensitive cells by external colicin Ib or Ia, together with certain genetic data, has led to the conclusion that membrane changes accompanying the two phenomena are the result of a common mechanism, namely, the interaction of free colicin with the cytoplasmic membrane.     

Arrangement on the Chromosome of the Known Pre-Early Genes of Bacteriophages T5 and BF23

Genetic crosses between mutants defective in the known pre-early genes of T5 and BF23 and the detection of putative N-terminal fragments have allowed the determination of the order of genes along the initially transferred 8% section of DNA of these phages.     

Identification of a second tetracycline-inducible polypeptide encoded by Tn10.

Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10. One of these polypeptides was the previously identified 36,000-dalton TET polypeptide. The other two had approximate sizes of 25,000 and 13,000 daltons. The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts. Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts. However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts. An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides. The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E. coli strains carrying the amber suppressors supD, supE, and supF. In general, strains containing these nonsense suppressors were less resistant to tetracycline.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

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

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

Induction of pre-early nuclear antigen(s) in HEL cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV)-specific nuclear antigen could be detected within 1 hr after infection in human embryo lung cells by the anticomplement immunofluorescence (ACIF) test. This antigen has been named the pre-early nuclear antigen (PENA) in this paper. Serum absorption tests suggested that PENA is immunologically different from the early antigen and the major nuclear inclusion antigens detected by the indirect immunofluorescence test before and after viral DNA replication, respectively. PENA-forming ability of the virus corresponded to its plaque forming ability. PENA formation was not affected by phosphonoacetate but was inhibited by the addition of inhibitors of RNA and protein syntheses or by UV-irradiation of infecting virus, suggesting that the formation of PENA depends on the expression of infecting virus gene functions. Virus-specific proteins were isolated by indirect immunoprecipitation from HCMV-infected cells exposed to 35S-methionine. SDS-polyacrylamide gel electrophoresis of the immunoprecipitate showed that at least two species of virus-specific polypeptides with molecular weights o.f 70,000 and 30,000 were synthesized de novo within 3 hr after infection.     

Effect of Infection with Ribonucleic Acid Bacteriophage R23 on the Inducible Synthesis of β-Galactosidase in Escherichia coli

Infection by ribonucleic acid (RNA) bacteriophage R23 inhibited the synthesis of beta-galactosidase in Escherichia coli. The inhibition, although not complete, was apparent shortly after infection and was maximal after the first 20 min of infection. R23 diminished the beta-galactosidase-synthesizing capacity when inducer was added after phage infection, but not when infection followed inducer removal. These findings suggested that the primary effect of R23 on enzyme-forming capacity was limitation of synthesis of enzyme-specific messenger RNA. Studies with ultraviolet irradiated phage and amber mutants of R23 indicated that the inhibitory process could be separated into two phases. Early inhibition did not require the expression of the viral genome, whereas late inhibition required the expression of the viral RNA synthetase cistron.     

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 30% of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gp11, before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 DNA have 0.1% and mini cells have 1% of the gp11 levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gp10 and gp11 formed from the cloned genes after T4 infection.     

Abortive infection by bacteriophage BF23 due to the colicin Ib factor. II. Involvement of pre-early proteins

The mechanism by which the growth of phage BF23 is arrested in cells carrying colicinogenic factor Ib involves certain phage-specific pre-early proteins. BF23 induces the extensive formation of proteins lc and ld, but very little formation of protein le, whereas BF23h^? (a mutant that is not arrested in cells carrying colicinogenic factor Ib) induces very small amounts of proteins lc and ld, but extensive amounts of protein le. Proteins lc and ld may be oligomers of protein le, and it is the presence of these putative oligomers that is necessary for the arrest of phage growth.     

Terminally redundant deletion mutants of bacteriophage BF23.

Deletion mutants of bacteriophage BF23 were isolated and the positions of the deletions were determined. Two different deletable regions were detected: one in the same region as previously reported for bacteriophage T5, which is closely related to BF23; and the other within both terminal repetitions. The former deletable region lay between positions 0.31 and 0.36, which represented the fractional lengths of the BF23 ( + ) DNA as measured from its left end. The latter deletion was evenly divided between the two terminal repetitions. The deletion in the left terminal repetition lay between positions 0.044 and 0.078 and was repeated in the corresponding region of the right terminal repetition between positions 0.966 and 1.0. The size of the DNA transferred to host cells during the first step of DNA transfer by BF23 carrying deletions in the terminal repetitions of its DNA was less than the size of DNA transferred during the first step by wild-type BF23 by an amount equal to the size of the deletion in each terminal repetition. This finding suggests the existence of a specific mechanism for delineating the position at which the first step of DNA transfer is stopped.     

Identification of bacteriophage T4D gene 29 product, a baseplate hub component, as a folylpolyglutamate synthetase

An assay for folylpolyglutamate synthetase activity in extracts of uninfected and bacteriophage T4D-infected Escherichia coli B has been developed. T4D infection induced the formation of a new synthetase raising the total synthetase activity three-fold. Extracts obtained after infection with T4 gene 51, 27 or 28 amber mutants showed increased synthetase activities while extracts obtained from cells infected with a T4D gene 29 amber mutant did not show any increase in synthetase activity. The phage-induced synthetase was found to copurify with the gene 29 product and a 100-fold purified synthetase of molecular size of 74,000 daltons has been obtained. The purified synthetase has a folate substrate specificity different from the host synthetase since it added glutamate residues to dihydrofolate as well as to the usual tetrahydrofolate substrate.     

Novel segregation patterns of infecting-mutant genotypes in plate complementation tests among amber mutants of bacteriophage BF23

Amber mutants of bacteriophage BF23 were classified into two functional groups, types I and II, by the yields of the infecting-mutant genotypes in plate complementation tests. Type I mutants produced their genotypes at levels more than 20% of the total progeny phages, and type II mutants did so at levels of less than 5%. Comparison of the results of plate complementation tests with those of extract complementation tests revealed that all the type I mutants were defective in the tail formation, while most type II mutants were defective in the formation of either mature heads (type IIa) or both mature heads and tails (type IIb). Since in extract complementation tests the activated phages are always of genotypes corresponding to mutations defective in only the tail formation, the plate complementation test is comparable with the extract complementation test when judged on the basis of the yield of the mutant genotypes. Of 29 complementation groups, 8 type I, 14 type IIa, and 5 type IIb mutants were identified. Previously, amber mutations of BF23 were mapped on four genetic segments. These segments were ordered in one linkage map by crosses between deletion and amber mutants.     

Translation of RNAs Synthesized In Vivo and In Vitro from Bacteriophage SP82 DNA

The synthesis of 69 phage-specific polypeptides during the infection of Bacillus subtilis with bacteriophage SP82 was detected by pulse-labeling, one-dimensional electrophoresis, and autoradiography. SP82 virions were found to contain approximately 22 polypeptides, most of which were synthesized late in infection; evidence was obtained for the processing of the major virion protein. RNAs extracted at different times during infection were translated by using an Escherichia coli cell-free extract. Only smaller-molecular-weight peptides were produced efficiently in vitro; in the 9,000- to 60,000-molecular-weight range, 50 to 60% of the peptides synthesized in vivo were produced by translation of RNAs extracted from infected cells. Eight of the virion peptides were produced by in vitro translation of RNAs extracted from infected cells. RNAs were synthesized under defined conditions by RNA polymerase extracted from uninfected B. subtilis and by polymerases isolated from cells 8 and 20 min after infection with SP82. Translation of these RNAs yielded characteristic and different patterns of polypeptides. Nine of the 12 polypeptides produced by translation of RNAs synthesized by the host polymerase corresponded in mobility to peptides appearing in vivo in the 0 to 3 and 3 to 6 min intervals of pulse-labeling after infection; 12 of the 25 peptides synthesized from RNAs produced by polymerase extracted 8 min after infection corresponded in mobility to peptides detected in vivo 8 min after infection, and 15 of the 22 peptides directed by RNAs made by the polymerase isolated 20 min after infection corresponded to peptides present in vivo late in infection. Five of the peptides produced in vitro from the latter RNA corresponded to virion peptides.     

Fibrous projections from the core of a bacteriophage T7 procapsid

A cylindrical core previously demonstrated in a bacteriophage T7 procapsid (capsid I) has been further examined by electron microscopy. Fibrous extensions of the core have been observed; these fibers appear to connect the core to the capsid I envelope. After infection of a nonpermissive host with bacteriophage T7 amber mutant in any gene coding for a core protein, the resulting lysates contained more noncapsid assemblies of capsid envelope protien than did wild-type lysates; these assemblies had a mass two to at least 500 times greater than the mass of capsid I. This suggests that the internal core and fibers assist the assembly of subunits in the envelope of capsid I.     

Nucleotide sequence of a DNA fragment that contains the abi gene of the ColIb plasmid

A 1989-bp PstI DNA fragment from the ColIb plasmid, which contains the abi gene that is necessary for the abortive response to infections by bacteriophage BF23 or T5, was sequenced. A candidate open reading frame for the abi gene has been suggested on the basis of a Shine-Dalgarno sequence appropriately placed ahead of its ATG initiation codon, a promoter upstream from the Shine-Dalgarno sequence, and a location compatible with deletion mapping. The polypeptide that would be coded by this open reading frame is 89 amino acids long and strongly hydrophobic. A promoter that could serve this open reading frame was detected by exonuclease III "footprinting" using RNA polymerase from uninfected Escherichia coli as the DNA-binding protein.     

Early development of bacteriophages SP01 and SP82G in minicells of Bacillus subtilis.

SP01- and SP82G-infected Bacillus subtilis CU403 divIVBI minicells synthesize 13 easily detectable early RNA species with molecular weights ranging from 60 × 10³ to 430 × 10³. Comparison of in vivo and in vitro translation of early messenger RNA indicates that five early mRNAs of SP01 are synthesized but not translated unless protein synthesis has been permitted in the infected minicell, providing evidence for a translation control mechanism. A sequential appearance of 48 polypeptides has been determined in SP01-infected minicells. The polypeptides have been grouped into two classes of early polypeptides, i.e. those encoded by early mRNA and three subsequent classes as demonstrated by the analysis of polypeptides synthesized in minicells infected with the SP01 mutants, susF21, susF4 and susF14. Phage capsid proteins are not synthesized in minicells. RNA synthesized in infected minicells is subject to turnover. The individual mRNA species have differing functional stabilities ranging from a loss of only 50% functional activity, in 20 minutes at 37 °C, to loss of over 99% activity.Infection of anucleate minicells has been shown to be a very simple method for comparison of closely related phages (slight differences are detected between SP01- and SP82G-encoded mRNA and polypeptides), detection of polypeptides affected by amber mutations and the analysis of early events in phage development in the absence of host syntheses.