Suppressors of mutations in the bacteriophage T4 gene coding for both RNA ligase and tail fiber attachment activities.

The protein product of T4 gene 63 catalyzes both the attachment of tail fibers to fiberless phage particles and the ligation of single-stranded RNA (Snopek at al., Proc. Natl. Acad. Sci. U.S.A. 74:3355-3359, 1977). To investigate whether the gene 63 product has a role in nucleotide metabolism, we isolated false revertants of amM69 in gene 63. We screened for revertants that could grow at 30 degrees C but not at 43 degrees C on Escherichia coli OK305 when nucleotides were limiting. These false revertants contained the original mutation in gene 63 and new suppressor mutations. Some of these suppressor mutations caused temperature sensitivity by themselves, allowing single mutants carrying the suppressor to be recognized and isolated. The results of mapping and complementation studies indicated that most of these ts suppressors were in the t gene (lysis), one was in gene 5 (baseplate), and one was in gene 18 (sheath). The mutation in gene 18, tsDH638, suppressed three different amber mutations in gene 63 but did not suppress amber mutations in several other genes. None of the suppressors that were characterized were in genes with known functions in nucleotide metabolism. However, an intriguing property of these false revertants was that they were very sensitive to hydroxyurea, an inhibitor of nucleotide metabolism.     

Assembly of the tail of bacteriophage T4

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.     

Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.     

Effect of mutation to streptomycin resistance on amber suppressor genes

Three classes of nonidentical streptomycin-resistant mutations were distinguished in Escherichia coli by their effect on the efficiency of suppression by an amber suppressor gene, sup E. The first class of mutation caused a strong restriction in efficiency of suppression of an amber codon in various cistrons of phage lambda and in an alkaline phosphatase structural gene of E. coli. The second class caused weak restriction, and the third class caused no restriction. The restrictive effect of the streptomycin resistance mutation of the first class on the sup E gene was reduced by addition of streptomycin. This mutation had little effect on efficiencies of suppression by amber suppressor genes sup D and sup F. Analyses on the alkaline phosphatase formed in the suppressor strain indicated that mutation to restrictive streptomycin resistance causes a reduction in translation of the amber codon in the alkaline phosphatase structural gene.     

Molecular reorganization in the hexagon to star transition of the baseplate of bacteriophage T4

The baseplate of bacteriophage T4 is a complex structure containing at least 14 different structural proteins. It undergoes a transition from a hexagonal to a star-shaped configuration during infection of the host bacterial cell. We have used a combination of genetics and image processing of electron micrographs to analyse both the wild-type structure and a series of mutant structures lacking specific gene products. Besides describing the basic anatomy of the hexagon and star configurations, we have been able to locate the products of genes 9, 11 and 12.Gene 9 product occupies a peripheral position in hexagons and stars consistent with its providing a binding site for the long tail fibres. Gene 11 product in the hexagon forms the distal part of the tail pin, which folds out to form the point of the hexagram in the star configuration. Gene 12 product is visualized as an extended 350 Å fibre in stars and broken baseplates but appears to have a more compact configuration in hexagons and intact phage.We demonstrate the structural relationship between the hexagonal and starshaped configurations and show how the positions of the specific gene products alter as a result of the structural transition. We suggest a speculative model for the role of gene 9 and gene 12 products in triggering the rearrangement of the baseplate and tail contraction.     

Attachment of tail fibers in bacteriophage T4 assembly. Identification of the baseplate protein to which tail fibers attach

A phage-neutralizing rabbit antiserum collected after immunization with tail-fiberless bacteriophage T4 particles was adsorbed with complete T4 phage. The resulting adsorbed serum inhibited tail fiber attachment in vitro. To identify the antigens against which this inhibitory activity was directed, blocking experiments were carried out with the adsorbed serum. Isolated complete baseplates and mutant-infected-cell extracts lacking known baseplate gene products but containing gene 9 product showed similar high levels of blocking activity. By contrast, both tail-fiberless particles lacking gene 9 product and infected-cell extracts made with gene 9 mutants showed 30-fold to 100-fold lower blocking activity. These results strongly support the conclusion that gene 9 product is the baseplate protein to which tail fibers attach.     

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB.

We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid. Mutations at this locus do not affect the ability of F plasmid-containing bacteria to undergo conjugation or be infected by the F plasmid-specific RNA phage f2. The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria. All fii mutants become tolerant to colicins E1, E2, and E3. Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage. Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe. The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins. The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome. Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus. Studies in maxicells show that the product of fii appears to be a 24-kilodalton protein which copurifies with the cell envelope. The product of tolA has been identified tentatively as a 51-kilodalton protein. Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E. coli map.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA

Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment. However, whether these sequences belong to genes which are functional as tRNA is generally not known. In this article, we demonstrate that the attachment site of temperate phage 16-3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41. A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase. We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R. meliloti 41 and in Agrobacterium tumefaciens GV2260. Upon lysogenization of R. meliloti by phage 16-3, the proline tRNA gene retained its structural and functional integrity. Aspects of the co-evolution of a temperate phage and its bacterium host is discussed. The side product of this work, i.e. construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study.     

Analysis of phage resistance in Staphylococcus aureus SA003 reveals different binding mechanisms for the closely related Twort-like phages ɸSA012 and ɸSA039

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage ɸSA012 through a long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed eight genes that acquired mutations: six point mutations (five missense mutations and one nonsense mutation) and two deletions. Complementation of the phage-resistant strains by the wild-type alleles showed that five genes were linked to phage adsorption of ɸSA012, and two mutated host genes were linked to the inhibition of post-adsorption. Unlike ɸSA012, infection by ɸSA039, a close relative of ɸSA012, onto early coevolved phage-resistant SA003 (SA003R2) was impaired drastically. Here, we identified that ɸSA012 and ɸSA039 adsorb to the cell surface S. aureus SA003 through a different mechanism. ɸSA012 requires the backbone of wall teichoic acids (WTA), while ɸSA039 requires both backbone and the β-GlcNAc residue. In silico analysis of the ɸSA039 genome revealed that several proteins in the tail and baseplate region were different from ɸSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ɸSA012 and ɸSA039.

     

Suppression of Amber and Ochre Mutants in Salmonella typhimurium by a Mutant F′-1-gal Factor Carrying an Ochre Suppressor Gene

A Salmonella typhimurium strain was given the amber mutation hisC527 by transduction, made galactose-negative by mutation, then infected with the F'-1-gal factor. Of 107 spontaneous and mutagen-induced histidine-independent mutants tested, 3 proved to result from suppressor mutations within the F' factor. The mutant F' factors, when transferred to S. typhimurium and E. coli auxotrophs, suppressed amber and ochre but not UGA or missense mutants, and are inferred to carry ochre suppressor genes. Attempts to isolate an F' amber suppressor mutant were unsuccessful. A suppressor F' factor was transferred to 14 rough mutants which had been isolated from LT2 hisC527 (amber) by selection for resistance to phage P22.c2. One rough mutant was partly suppressed, as shown by its acquisition of O agglutinability and by alterations in its phage resistance pattern. Phage P22h grown on the suppressed mutant contransduced its rf. gene with cysE(+) and with pyrE(+), and the affected locus is inferred to be rfaL. Both the original and the mutant F' factors conferred resistance to the rough-specific phage Br60, which is therefore "female-specific."     

Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Effect of mutations in Escherichia coli genes PolAm RecA, RecB and RecC on the frequency of genetic recombination in amber-mutants involving the early genes of bacteriophage T4]

Effects of mutations in genes PolA, RecA, RecB and RecC of Escherichia coli on the recombination frequencies between rII markers of T4 have been studied in conditions of partial inhibition of some early functions. It was found that the presence of the mutations in genes PolA or RecA decreased significantly the recombination frequency of phage amber mutant in the gene 43 (DNA polymerase), increased it in the case of amber mutation in the gene 46 (exonuclease) and had no effect on the recombination of amber mutants in genes 30, 32, 33, 41, 42, 45, 44, 52. None of the amber mutants studied changed recombination frequencies in the presence of the mutations in genes RecB or RecC. Possible mechanisms of some of the effects observed are discussed.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Assembly of bacteriophage T4 tail fibers: Identification and characterization of the nonstructural protein gp57

Summary Formation of both the tail fiber and the baseplate of bacteriophage T4 depends on the product of T4 gene 57. A single amber mutation in that gene causes loss of two T4-specific proteins. Their molecular weights are 18,000 and about 6,000, respectively, based on their electrophoretic mobilities in SDS-polyacrylamide gels. E. coli carrying a cloned T4 DNA fragment of about 700 basepairs, which directs the synthesis of the smaller protein only, specifically supports the growth of gene 57 amber mutants. We conclude that the small protein is a functional product of gene 57.Abbreviations Am ampicillin - Cm chloramphenicol - Tet tetracycline - SDS sodium dodecyl sulfate - bp basepairs - wt wildtype - Su suppressor - Km kanamycin - ds double stranded - ss single stranded - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme

Summary The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants. The 15K lysozyme was also found to be expressed in E. coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25. These observations indicate that the 15K lysozyme is the gene 25 product.