Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.     

Virulent Mutants of Bacteriophage P22 I. Isolation and Genetic Analysis

Mutants of phage P22 which form plaques on a P22 lysogen have been isolated. These virulent mutants have been classified into three groups. (i) VirA mutants arise spontaneously in wild-type stocks and form very small turbid plaques on a P22 lysogen. The single mutation responsible for VirA virulence maps near the mnt locus, one of the immunity regions of phage P22. (ii) VirB mutants do not arise spontaneously and have been isolated only from mutagenized P22 stocks. VirB mutants form small, clear plaques on a P22 lysogen. One of the VirB mutants, virB-3, was analyzed in detail. The virB-3 mutant is comprised of two mutations: K5, which maps within the c(2) gene, and Vx, which maps in the region between the c(2) and c(3) genes. Phages carrying either the K5 or Vx mutation are not virulent in themselves but mutate to VirB virulence at a frequency of 10(-5) to 10(-6). It is concluded that K5 and Vx are mutations at specific sites which together confer the ability to undergo phage development in the presence of repressor. (iii) VirC mutants are defined by a large clear plaque morphology when plated on a P22 lysogen. VirC mutants are comprised of the determinants of both VirA and VirB virulence.     

Regulation of Bacteriophage P22 DNA synthesis and repressor levels in P22cly infections.

A rifampin-resistant mutant of Salmonella typhimurium carries an altered RNA polymerase. Wild-type (c+) phage P22 displays clear plaques and a reduced lysogenization frequency on this mutant host. The cly mutants of P22 were isolated on the basis of their ability to lysogenize such mutant hosts. Two classes of regulatory events, both of which are dependent on P22 gene c1 activity, are necessary for the establishment of lysogeny in P22. The positive events culminate in repressor synthesis; the negative events cause a retardation in phage DNA synthesis. Neither the positive nor the negative events are observed in P22c+ infections of the mutant host. Both effects are found in P22cly infections of the mutant host. Observable results of both the negative and the positive events are exaggerated in P22cly infections of wild-type hosts as compared to P22c+ infections. The cly mutation apparently increases the positive and negative regulatory events so that they are detectable in the mutant host and exaggerated in wild-type hosts. Possible mechanisms that result in the high frequency of lysogenization that characterizes the cly mutation and the nature of the cly mutation are discussed.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

Oligopeptidase A is required for normal phage P22 development.

The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carrying opdA mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations. Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host. This decrease resulted from a reduced efficiency of plating of particles from an opdA infection. In the absence of a functional opdA gene, most of the P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated. Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment. The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins. The opdA-independent mutations lead to mutant forms of gp7 which function without processing.     

Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su^- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.     

Repressor synthesis in regulatory mutants of bacteriophage P22

Summary SDS-polyacrylamide gel analysis of P22-infected Salmonella has allowed identification of the c₂ repressor (MW 31,000) and study of repressor synthesis in regulatory mutants of P22. Repressor is synthesized in reduced amounts or is absent in infections with P22, c1#7, P22, c2 am08, P22 c3 am03, and P22 c3 am012, but is synthesized in markedly increased amounts in the virulent mutant, P22 virB3, and its component mutants, vx and k5. Higher levels of repressor are also found in the P22 cly 17 mutant.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Packaging of an oversize transducing genome by Salmonella phage P22

The DNA in specialized transducing particles of the Salmonella phage P22 was examined by electron microscopy. The transducing particles of P22Tc-10 (which transduce tetracycline-resistance) are shown to contain DNA molecules that are incomplete permuted fragments of an oversize genome, as predicted by the genetic results of Chan et al. (1972). The oversize transducing genome differs from the P22 wild-type genome by a large (mol. wt 2.5 × 10⁶) insertion of foreign DNA. The insertion, as seen in heteroduplexes, has an unusual lariat-like structure, which suggests that the insertion contains a non-tandem reverse duplication.By comparing wild-type P22 with P22Tc-10 and deletion revertants of P22Tc-10, we show by direct physical means, that the amount of terminal repetition in P22 phage DNA is a direct function of the genome size, as predicted from the model for circular permutation and terminal repetition suggested by Streisinger et al. (1967).     

Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22

The bipartite immunity and repression system of the temperate Salmonella bacteriophage P22 has been analyzed by genetic means. Both parts of the immunity system, immI and immC, are necessary to confer upon lysogens immunity to superinfection with P22. The product of the c2 gene (which lies in immC) is a repressor which apparently regulates directly the expression of phage genes in a manner analogous (if not identical) with that found for coliphage λ.The immI region contains three genetic elements. One of these (mnt; Gough, 1968) appears to specify another repressor whose specific activity is continuously required for the maintenance of lysogeny. We have identified two new regulatory elements in immI through the isolation of mutants. Virulent mutations (virA) in the Vy element confer the ability to grow in immune P22 lysogens by destroying or inactivating the repression functions of the lysogen (possibly the c2-repressor itself). The third element in immI is a structural gene (ant) for a protein (antirepressor) which is regulated by mnt (repressor) and Vy (promoter/operator).We have shown that the ability of P22 to grow on immI-deletion lysogens, the dominant virulence of virA virulents, and the requirement for mnt for the maintenance of lysogeny, all depend on an intact ant⁺ gene. It is proposed that P22 antirepressor represents a new type of regulatory protein which acts by controlling other regulatory proteins.     

New Deoxyribonuclease Activity After Bacteriophage P22 Infection

Extracts from P22-infected and uninfected cultures of Salmonella typhimurium were subjected to deoxyribonucleic acid (DNA)-cellulose and diethylaminoethyl-cellulose chromatography. Comparison of the elution patterns revealed that in infected cells there is a decrease in the amount of nuclease activity specific for denatured DNA and an increase in the amount of nuclease activity specific for native DNA. The latter activity was shown to differ from a similar host enzyme in Mg²⁺, Mn²⁺, and pH optima. This new activity is not found after infection of a lysogen with a nonvirulent phage or after infection under nonpermissive conditions with P22ts25.1 (a mutant in gene 25 that carries out no known functions other than adsorption and injection) and thus appears to be specified by the phage genome.     

Kinetics of P22 early gene expression suggests a cro-like regulatory function

Summary Analysis of phage-specified protein synthesis after phage infection of UV-irradiated cells shows a turn-off of early gene expression, a regulatory event that is independent of the known P22 regulatory functions. This supports the suggestion of a cro-like function in P22. We have identified the products of genes 18 and int as contributing to the complex 40,000 dalton band in our SDS-polyacrylamide gels. Both gene products appear to be subject to regulation by the cro-like function of P22. Proteins of 33,000, 29,000, 27,000, 25,000, and 24,000 MW, specified by as yet unidentified P22 genes of the early leftward operon, are regulated by the same function. Our data suggest that the cro-like function is expressed from the early rightward operon.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

Structure and Functions of the Bacteriophage P22 Tail Protein

The product of gene 9 (gp9) of Salmonella typhimurium bacteriophage P22 is a multifunctional structural protein. This protein is both a specific glycosidase which imparts the adsorption characteristics of the phage for its host and a protein which participates in a specific assembly reaction during phage morphogenesis. We have begun a detailed biochemical and genetic analysis of this gene product. A relatively straightforward purification of this protein has been devised, and various physical parameters of the protein have been determined. The protein has an s_20,w of 9.3S, a D_20,w of 4.3 × 10⁻⁷ cm²/s, and a molecular weight, as determined by sedimentation equilibrium, of 173,000. The purified protein appears as a prolate ellipsoid upon electron microscopic examination, with an axial ratio of 4:1, which is similar to the observed shape when it is attached to the phage particle. The molecular weight is consistent with the tail protein being a dimer of gp9 and each phage containing six of these dimers. An altered form of the tail protein has been purified from supF cells infected with a phage strain carrying an amber mutation in gene 9. Phage “tailed” with this altered form of gp9 adsorb to susceptible cells but form infectious centers with a severely reduced efficiency (ca. 1%). Biochemical analysis of the purified wild-type and genetically altered tail proteins suggests that loss of infectivity correlates with a loss in the glycosidase activity of the protein (2.5% residual activity). From these results we propose that the glycosidic activity of the P22 tail protein is not essential for phage assembly or adsorption of the phage to its host but is required for subsequent steps in the process of infection.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation

The functions of ten known late genes are required for the intracellular assembly of infectious particles of the temperate Salmonella phage P22. The defective phenotypes of mutants in these genes have been characterized with respect to DNA metabolism and the appearance of phage-related structures in lysates of infected cells. In addition, proteins specified by eight of the ten late genes were identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis; all but two are found in the mature phage particle. We do not find cleavage of these proteins during morphogenesis.The mutants fall into two classes with respect to DNA maturation; cells infected with mutants of genes 5, 8, 1, 2 and 3 accumulate DNA as a rapidly sedimenting complex containing strands longer than mature phage length. 5^? and 8^? lysates contain few phage-related structures. Gene 5 specifies the major head structural protein; gene 8 specifies the major protein found in infected lysates but not in mature particles. 1^?, 2^? and 3^? lysates accumulate a single distinctive class of particle (“proheads”), which are spherical and not full of DNA, but which contain some internal material. Gene 1 protein is in the mature particle, gene 2 protein is not.Cells infected with mutants of the remaining five genes (10, 26, 16, 20 and 9) accumulate mature length DNA. 10^? and 26^? lysates accumulate empty phage heads, but examination of freshly lysed cells shows that many were initially full heads. These heads can be converted to viable phage by in vitro complementation in concentrated extracts. 16^? and 20^? lysates accumulate phage particles that appear normal but are non-infectious, and which cannot be rescued in vitro.From the mutant phenotypes we conclude that an intact prohead structure is required to mature the virus DNA (i.e. to cut the overlength DNA concatemer to the mature length). Apparently this cutting occurs as part of the encapsulation event.     

Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI

The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo, mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A.Proteins made in cells infected with the fin mutants were labelled with [³⁵S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique.gpA overproducers can also be isolated by selecting for λAam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called Aop and maps in the Nu1-A region. Aop is also a fin mutation, since its presence in λFI^? enables it to plate on non-permissive hosts.Therefore, it seems that one condition sufficient for normal growth of FI^? phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.     

The Salmonella typbimurium RecJ function permits growth of P22 abc phage on recBCD+ hosts

Summary We describe recJ mutants of Salmonella typhimurium. The recJ gene maps between sufD and serA (min 62) and is transcribed counterclockwise. Unlike recJ mutants of Escherichia coli, recJ strains of S. typhimurium are sensitive to irradiation with UV light. This sensitivity is equivalent to or greater that that displayed by recBCD mutant strains. The residual ability of phage P22 abc (anti-recBCD) mutants to form plaques on recBCD ⁺ strains is eliminated in recJ hosts. Thus host RecJ function appears to substitute for the anti-RecBCD functions of phage P22 and may serve to limit RecBCD activity.