Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

Structure and inherent properties of the bacteriophage lambda head shell. V. Amber mutants in gene E

A total of 940 amber mutants in gene E of bacteriophage lambda was isolated to study the structure-function relationship of the gene product, the major capsid protein. The mutants were mapped to 43 mutation sites, most of which have been located, albeit tentatively, at exact points in the known base sequence, by deletion mapping and by the specificity of mutagenesis and the patterns of suppression. The patterns of suppression were interpreted in terms of both the efficiency of insertion of amino acid residues by suppressors and the exchangeability of amino acid residues. The exchangeability seems to be related to the hydrophilicity of the residues themselves and their environment, as well as to the functional similarity between the replaced and the inserted amino acid residues. Suppression of two of the mutations resulted in the production of characteristic aberrant head-related structures, each showing a defect in a different functional site in the protein. This, together with the approximate positions of some specific missense mutations as determined in this study, revealed the distribution of the functional sites along the polypeptide chain of the gene E product.     

Attachment site mutants of bacteriophage lambda

Insertion and excision of the chromosome of phage λ occurs by recombination at special regions of the phage and bacterial chromosomes known as attachment sites (alt's). We have isolated att mutants which display reduced recombination frequencies. The mutations are cis-dominant, trans-recessive, and can be crossed into a phage, bacterial or prophage att. These results suggest that the att's, although different over-all, include the same DNA sequence as part of their structure, and that the mutations reside in these sequences. Crosses between mutant and wild-type att's occasionally yield heterozygotes. This result suggests that recombination of the att's generates complementary single-strands via staggered nicks in these common sequences. Recombinant att's are then formed by the interannealing of single-strands of different att's followed by ligation.     

Studies on partially virulent mutants of lambda bacteriophage

Summary Genetic studies coupled with functional analysis of gene action have demonstrated that there are two classes of partially virulent CP mutants which differ in the mechanism by which they overcome the immunity repressor. Class I contains a mutation within the cI region which causes the modified cI product to negatively complement the active repressor present in the immune cells. Class II achieve their virulence by a mutation which renders the x-y-cII-O operon insensitive to repression.     

Structure and inherent properties of the bacteriophage lambda head shell. II Isolation and initial characterization of prophage mutants defective in gene E

Prophage mutants defective in gene E of bacteriophage lambda were isolated and characterized to analyze the physiological functions of the major head protein (the gene E product). Seventy-one mutants were classified into the following five groups according to their phenotype under the electron microscope: two producing polyheads, two producing icosahedral structures and one producing no head-related structures detectable by electron microscopy. The former four phenotypes probably arose from defects in specific functional sites of the major head protein. Deletion mapping showed that some of the mutants belonging to the same phenotype mapped in distantly separated regions in gene E. Such regions may code for adjacent parts of the three-dimensional structure after folding of the polypeptide chain.     

Recombination of bacteriophage lambda in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.     

Characterization of bacteriophage lambda excisionase mutants defective in DNA binding

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.     

Role of the cro gene in bacteriophage lambda development

Previous experiments have shown that the product of the cro gene of baeteriophage λ can exert an anti-repression activity, defined by the capacity of certain “cro-constitutive” defective lysogens to channel a superinfecting λ phage toward lytic development. We have used a combination of biological and biochemical assays to draw two main conclusions concerning this anti-repression activity: (1) after infection of a cro-constitutive cell, the superinfecting phage is unable to establish repression because it is unable to commence synthesis of cI protein (λ repressor) at a substantial rate; (2) the cause of this diminished synthesis of cI protein is the capacity of cro product to repress synthesis of the cII and cIII proteins, which normally activate the cI gene to establish repression in an infected cell. From our experiments and those of others, we suggest that cro product possesses a repression activity which is similar to that of the cI protein itself, but normally exerts a very different physiological role: the turnoff of synthesis of replication, recombination and regulation proteins as the virus enters the late stage of lytic development.     

DNA-replication of spi - mutants of bacteriophage lambda in recombination-deficient bacteria

Summary Phage imm ²¹ c spi ^– infecting recA ^– cells gives a burst of 6 progeny phages compared to 120 in rec ⁺ cells. Parental spi ^– DNA is not degraded in recA ^– cells. The synthesis of early replication products is enhanced by a factor of 2 yielding 30 closed circular progeny DNA molecules per cell compared to 15 in the control. These DNA supercoils include 9% of dimer molecules under red ^– recA ^– and red ^– rec ⁺ conditions. On the other hand, the formation of linear phage DNA molecules in recA ^– cells is reduced by a factor of 5 to 6, if compared to spi ^– DNA in rec ⁺ and spi ⁺ DNA in recA ^– cells, respectively. The specific biological activity of these linear molecules in the helper phage assay system is unimpaired. Intermediates of late spi ^– replication under recA ^– conditions are supposed to be the unprotected targets of the action of the recB ⁺ recC ⁺ nuclease.     

The cIII gene and protein of bacteriophage lambda

The cIII and cII gene products of bacteriophage λ control the lysogenic response through positive regulation of the viral repressor and integration genes and negative regulation of lytic functions. Although many aspects of cII action have been defined biochemically, little is known about cIII. As a first step in defining the molecular role of cIII in the regulation of lysogeny, we have determined the precise location and DNA sequence of the cIII gene. In addition, we have identified the cIII gene product as a polypeptide with a molecular weight of approximately 6000.     

Explanations accounting for transduction by bacteriophage lambda in maltose negative bacteriophage lambda resistant mutants of Escherichia coli K-12.

Summary Entry of DNA from phages particles into rMal^- mutants of Escherichia coli K-12 is shown to be due to two distinguishable processes. One, residual transduction, results from a low level expression of lamB. The other one, background transduction, is independent of gene lamB.Interpretations are presented for these results. It is proposed that residual transduction is due to a weak promoter pB3 located within or near the distal part of the gene preceeding lamB in the same operon. It is proposed that background transduction is due to a secondary receptor structure for phage . Finally a tentative hypothesis relating pB3 to insertion sequences is presented.