rII Mutants Of T4 Research Articles

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins.

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49. In the present paper, we tested this hypothesis in vivo. We used an experimental model system that combines site-specific double-strand breaks with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. We compared recombination of homoallelic HR and LR markers in the S17 and S17 E727 background (amber mutations in the uvsX and in the uvsX and 49 genes, respectively). In S17-crosses, the HR and LR markers retain their respective high-recombination and low-recombination behavior. In S17 E727-crosses, however, the HR and LR markers show no difference in the recombination frequency and both behave as LR markers. We conclude that endo VII is the enzyme that recognizes mismatches in recombinational heteroduplexes and performs their incision. This role for endo VII was suggested previously from biochemical studies, but this is its first in vivo demonstration.

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.

Polarity withinpMandpEpromoted phage lambdacI-rexA-rexBtranscription and its suppression

The cI-rexA-rexB operon of bacteriophage lambda confers 2 phenotypes, Imm and Rex, to lysogenic cells. Immunity to homoimmune infecting lambda phage depends upon the CI repressor. Rex exclusion of T4rII mutants requires RexA and RexB proteins. Both Imm and Rex share temperature-sensitive conditional phenotypes when expressed from cI[Ts]857 but not from cI+ lambda prophage. Plasmids were made in which cI-rexA-rexB was transcribed from a non-lambda promoter, pTet. The cI857-rexA-rexB plasmid exhibited Ts conditional Rex and CI phenotypes; the cI+-rexA-rexB plasmid did not. Polarity was observed within cI-rexA-rexB transcription at sites in cI and rexA when CI was nonfunctional. Renaturation of the Ts CI857 repressor, allowing it to regain functionality, suppressed the polar effect on downstream transcription from the site in cI. The second strong polar effect near the distal end of rexA was observed for transcription initiated from pE. The introduction of a rho Ts mutation into the host genome suppressed both polar effects, as measured by its suppression of the conditional Rex phenotype. Strong suppression of the conditional Rex[Ts] phenotype was imparted by ssrA and clpP (polar for clpX) null mutations, suggesting that RexA or RexB proteins made under conditions of polarity are subject to 10Sa RNA tagging and ClpXP degradation.

The roles of the bacteriophage T4 r genes in lysis inhibition and fine-structure genetics: a new perspective.

Seldom has the study of a set of genes contributed more to our understanding of molecular genetics than has the characterization of the rapid-lysis genes of bacteriophage T4. For example, T4 rII mutants were used to define gene structure and mutagen effects at the molecular level and to help unravel the genetic code. The large-plaque morphology of these mutants reflects a block in expressing lysis inhibition (LIN), the ability to delay lysis for several hours in response to sensing external related phages attacking the cell, which is a unique and highly adaptive attribute of the T4 family of phages. However, surprisingly little is known about the mechanism of LIN, or how the various r genes affect its expression. Here, we review the extensive old literature about the r genes and the lysis process and try to sort out the major players affecting lysis inhibition. We confirm that superinfection can induce lysis inhibition even while infected cells are lysing, suggesting that the signal response is virtually instantaneous and thus probably the result of post-translational regulation. We identify the rI gene as ORF tk.-2, based on sequence analysis of canonical rI mutants. The rI gene encodes a peptide of 97 amino acids (Mr = 11.1 kD; pI = 4.8) that probably is secreted into the periplasmic space. This gene is widely conserved among T-even phage. We then present a model for LIN, postulating that rI is largely responsible for regulating the gpt holin protein in response to superinfection. The evidence suggests that the rIIA and B genes are not directly involved in lysis inhibition; rather, when they are absent, an alternate pathway for lysis develops which depends on the presence of genes from any of several possible prophages and is not sensitive to lysis inhibition.

The Rex phenotype of altruistic cell death following infection of a λ lysogen by T4 rII mutants is suppressed by plasmids expressing OOP RNA

The coordinate expression from induced λ prophages of p Lit-rexB-t Imm (late immunity transcription, LIT RNA) and p O-oop-t O (OOP RNA) has remained unexplained. The initial assigned sequence for p Lit bore no relationship to p O . We have identified two promoter sites for independent rexB transcription, denoted here p Lit2 and p Lit1 , which are separated by about 330 bp. The upstream p Lit1 site shares with p O a common 9 bp sequence between the −10 and −35 regions, with strong homology to aspects of the SOS box or LexA operator site. This sequence is also found within OOP RNA, suggesting that OOP RNA, or another regulatory factor recognizing the common sequence, was involved in the regulation of rexB expression and hence Rex exclusion. We measured the influence of OOP synthesis from plasmids on the Rex phenotype, finding that plasmids producing OOP can suppress Rex exclusion by a λ prophage. The possibility was suggested that low level constitutive rexB transcription occurs from p Lit2 . Potential binding sites were identified for DnaA, for the LexA, CI and Cro repressors and for λ O protein in the 80 nt DNA interval upstream from and including p Lit1 , suggesting a complex regulatory pattern for rexB expression from this promoter. © 1997 Elsevier Science B.V. All rights reserved.

The Rex system of bacteriophage lambda: tolerance and altruistic cell death.

The rexA and rexB genes of bacteriophage lambda encode a two-component system that aborts lytic growth of bacterial viruses. Rex exclusion is characterized by termination of macromolecular synthesis, loss of active transport, the hydrolysis of ATP, and cell death. By analogy to colicins E1 and K, these results can be explained by depolarization of the cytoplasmic membrane. We have fractionated cells to determine the intracellular location of the RexB protein and made RexB-alkaline phosphatase fusions to analyze its membrane topology. The RexB protein appears to be a polytopic transmembrane protein. We suggest that RexB proteins form ion channels that, in response to lytic growth of bacteriophages, depolarize the cytoplasmic membrane. The Rex system requires a mechanism to prevent lambda itself from being excluded during lytic growth. We have determined that overexpression of RexB in lambda lysogens prevents the exclusion of both T4 rII mutants and lambda ren mutants. We suspect that overexpression of RexB is the basis for preventing self-exclusion following the induction of a lambda lysogen and that RexB overexpression is accomplished through transcriptional regulation.

Sequences and studies of bacteriophage T4 rII mutants

We have sequenced more than 80 mutants of the bacteriophage T4 rIIA and rIIB genes. These include deletions about whose origin we have speculated, mutations affecting the rIIB promoters, various pseudo-revertants of the rII − phenotype, including mutations that bring about the reinitiation of translation following termination, mutations that affect regulation of rIIB translation by regA, the toxic minute plaquing mutants FC237 and FC238 and their detoxifiers, and many more of the classic frameshifts from the Cambridge collection. These mutants have been sequenced using dideoxy-mediated chain termination by either Escherichia coli DNA polymerase using single-stranded DNA as a template or by avian retroviral reverse transcriptase using mRNA or DNA as the template molecule. We list the sequence changes of the mutants with pertinent historic and phenotypic data. The mutants that facilitate translation reinitiation are discussed, and we discuss a model that could account for the generation of many of the mutations.

Membrane ATPase activation on infection of E. coli K (lambda) cells with phage T4rII mutants.

Membrane ATPase activation on infection of E. coli K (lambda) cells with phage T4rII mutants.

The isolation and characterization of TabR bacteria: Hosts that restrict bacteriophage T4 rII mutants

We have isolated mutants of Escherichia coli B (called TabR) that restrict the growth of bacteriophage T4 rII mutants at high temperature. TabR strains lysed very rapidly after infection with rII mutants, and no progeny phage were produced. T4+-infected TabR cells also lysed quickly, but the cells remained intact long enough to give a small burst. We have selected pseudorevertants of rII deletion mutants that grow on TabR at high temperature; tk (thymidine kinase) is a component of one class of these pseudorevertants.

Reversion of bacteriophage T4rII mutants by high levels of pyrimidine deoxyribonucleosides.

High levels of pyrimidine deoxyribonucleosides, but not purine deoxyribonucleosides, increase the reversion rate of bacteriophage T4rII mutants to r+. This increased reversion rate is not observed when a thymidine kinase mutation is introduced into the bacteriophage, but the high reversion rate persists when the host, E. coli, harbors a thymidine kinase mutation.

Suppressors of mutations in the rII gene of bacteriophage T4 affect promoter utilization.

Homyk, Rodriguez and Weil (1976) have described T4 mutants, called sip, that partially suppress the inability of T4rII mutants to grow in lambda lysogens. We have found that mutants sip1 and sip2 are resistant to folate analogs and overproduce FH2 reductase. The results of recombination and complementation studies indicate that sip mutations are in the mot gene. Like other mot mutations (Mattson, Richardson and Goodin 1974; Chace and Hall 1975; Sauerbier, Hercules and Hall 1976), the sip2 mutation affects the expression of many genes and appears to affect promoter utilization. The mot gene function is not required for T4 growth on most hosts, but we have found that it is required for good growth on E. coli CTr5X. Homyk, Rodriguez and Weil (1976) also described L mutations that reverse the effects of sip mutations. L2 decreases the folate analog resistant and the inability of sip2 to grow on CTr5X. L2 itself is partially resistant to a folate analog, and appears to reverse the effects of sip2 on gene expression. These results suggest that L2 affects another regulatory gene related to the mot gene.

Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.

Superinfection exclusion by P22 prophage in lysogens of Salmonella typhimurium: IV. Genetics and physiology of sieB exclusion

The sieB exclusion system of phage P22 interferes with superinfection by several Salmonella phages, but not P22 itself. Vegetative development of the Salmonella phage L proceeds normally in P22 sieB + lysogens until midway through the latent period, when there is a rapid and complete cessation of DNA, RNA, and protein synthesis, resulting in failure to produce any progeny phage. It appears that expression of vegetative functions by the excluded superinfecting phage contributes to the premature arrest of macromolecular synthesis, since superinfecting L phage can establish lysogeny normally in P22 sieB + lysogens. Superinfecting P22 phage escape sieB exclusion by expressing an early phage function ( esc + P22), lacking in L phage. Escape through expression of esc + P22 is dominant in mixed infections. The esc + P22 determinant maps near the P22 c 3 gene and is separable from the P22 immunity regions. Recombination resulting in acquisition of the esc + P22 determinant from the prophage limits the efficiency of exclusion of L phage by P22 sieB + lysogens. The sieB gene of phage P22 is located between erf and gene 24 on the P22 genetic map. This fact poses an interesting problem, since the prophage sieB gene is expressed during superinfection while flanking genes in this region remain repressed. These results indicate that the P22 sieB exclusion mechanism may have elements in common with several other phage exclusion mechanisms, such as those responsible for the exclusion of T4 rII mutants by coliphage λ lysogens and for the exclusion of λ by coliphage P2 lysogens.

Mutants of phage HK239 defective in excluding phages lambda T4rII, P1, and P2.

Escherichia coli cells lysogenic for temperate phage HK239 exclude phages λ, HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages λ, HK022, P1 vir, and P2.

HK239: a P2 related temperate phage which excludes rII mutants of T4.

Biological properties of a temperate phage, HK239, isolated from cow dung were studied. This phage produces clear plaques on E. coli B and most strains of E. coli K-12, but produces turbid centered plaques on K-12 strain HfrC and E. coli C strain, CIa. Bacterial growth in turbid centered plaques on CIa and HfrC is due to lysogenic, immune bacteria. Delayed bacterial growth in clear plaques on K-12 strains is mostly that of phage resistant clones and, infrequently, of lysogenic, immune cells. HK239 shows an interesting combination of properties of two well known coliphages. It bears a close resemblance to phage P2 in its antigenic properties and in interfering with the growth of λ. On the other hand, K-12 strains lysogenic for HK239 exclude r ll mutants of phage T4 and in this respect, HK239 resembles λ.

Anomalously revertible r II mutants of phage T 4 .

SUMMARYA class of rII mutants revertible by both base analogues and acridines is described. The members of this anomalous class are base-substitution mutants suppressed by a phase-shift mutation in a phage gene mapping outside the rII region.

A mutation in Escherichia coli enhancing the UV-mutability of phage lambda but not of its infectious DNA in a spheroplast assay.

A mutant (called mul) has been isolated from E. coli AB1157 which increases up to 13 fold the rate of clear plaque mutations of extracellularly UV-irradiated phage λ. The mul-mutation does not affect the UV-mutability of T4rII mutants or various bacterial markers and therefore seems to act specifically on UV-irradiated phage λ. However, when spheroplasts prepared from mul cells were infected with either irradiated or unirradiated λ-DNA equal frequencies of clear plaques were produced. As there is indirect evidence (host cell reactivation) that the spheroplasts do not significantly exclude irradiated phage DNA it seems that the mul phenotype can be expressed only in complete cells.

Phenotypic reversion by hydroxylamine. A new group of suppressible phage T4 rII mutants.

Phenotypic reversion by hydroxylamine. A new group of suppressible phage T4 rII mutants.

DNA replication by phage T4 rII mutants without polynucleotide ligase (gene 30)

In phage T4-infected E. coli , the polynucleotide ligase coded for by T4 gene 30 is not required for DNA replication and phage production when the infecting phage is an rII mutant. The wild-type T 4 rII gene product may be a nuclease or control a nuclease activity.