What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function.

Recent studies have generated interest in the use of the homologous recombination system of bacteriophage lambda for genetic engineering. The system, called Red, consists primarily of three proteins: lambda exonuclease, which processively digests the 5'-ended strand of a dsDNA end; beta protein, which binds to ssDNA and promotes strand annealing; and gamma protein, which binds to the bacterial RecBCD enzyme and inhibits its activities. These proteins induce a 'hyper-rec' state in Escherichia coli and other bacteria, in which recombination events between DNA species with as little as 40 bp of shared sequence occur at high frequency. Red-mediated recombination in the hyper-rec bacterium proceeds via a number of different pathways, and with the involvement of different sets of bacterial proteins, depending in part on the nature of the recombining DNA species. The role of high-frequency double-strand break repair/recombination in the life cycle of the lambdoid phages is discussed.

PCR-mediated gene replacement in Escherichia coli.

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli. Three of these replacements were performed without prior cloning of the genes.

Genetic requirements of phage lambda red-mediated gene replacement in Escherichia coli K-12.

Recombination between short linear double-stranded DNA molecules and Escherichia coli chromosomes bearing the red genes of bacteriophage lambda in place of recBCD was tested in strains bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell; formation of Lac(-) chloramphenicol-resistant bacterial progeny was measured. Recombinant formation was found to be reduced in ruvAB and recQ strains. In this genetic background, mutations in recF, recO, and recR had large effects on both cell viability and on recombination. In these cases, deletion of the sulA gene improved viability and strain stability, without improving recombination ability. Expression of a gene(s) from the nin region of phage lambda partially complemented both the viability and recombination defects of the recF, recO, and recR mutants and the recombination defect of ruvC but not of ruvAB or recQ mutants.

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.

Roles of RuvC and RecG in phage lambda red-mediated recombination.

The recombination properties of Escherichia coli strains expressing the red genes of bacteriophage lambda and lacking recBCD function either by mutation or by expression of lambda gam were examined. The substrates for recombination were nonreplicating lambda chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in ruvC+ recG+, ruvC recG+, ruvC+ recG, and ruvC recG hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome's restriction site marker. In this case, recombinant formation was found to be partially dependent upon ruvC function, especially in a recG mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the cat gene, with flanking lac sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher in recG than in recG+. These observations favor models in which RecG tends to push invading 3'-ended strands back out of recombination intermediates.

Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.

Model-free approaches (random mutagenesis, DNA shuffling) in combination with more "rational," three-dimensional information-guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage lambda, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque-forming ability. Compensating mutations in W138P T4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid lambda phage with sufficient lysozyme activity to produce normal-size plaques, even at elevated temperature (42 degrees C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target-randomized variants.

Alteration of T4 lysozyme structure by second-site reversion of deleterious mutations.

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.

Annealing vs. invasion in phage lambda recombination.

Genetic recombination catalyzed by lambda's Red pathway was studied in rec+ and recA mutant bacteria by examining both intracellular lambda DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec+ cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of lambda catalyzes recombination primarily by annealing.

Second-site reversion of a structural defect in bacteriophage T4 lysozyme.

A plasmid-borne gene encoding bacteriophage T4 lysozyme with a structural mutation, Tyr161-Ala, was mutagenized by by the use of polymerase chain reaction. The mutagenized gene was inserted into a specialized bacteriophage lambda cloning vector that must acquire a functional lysozyme gene in order to form plaques. Functional variants of the mutant lysozyme were selected. Three compensatory second-site revertants were obtained: Thr152-Met, Lys43-Ile, and Thr151-Ala. The effects of these mutations are interpreted in light of previous structural and genetic studies of T4 lysozyme.

Bacterial expression of Scapharca dimeric hemoglobin: a simple model system for investigating protein cooperatively.

Recombinant Scapharca homodimeric hemoglobin has been expressed at high levels from a synthetic gene in Escherichia coli. Addition of the heme precursor delta-aminolevulinic acid to the expression culture results in a considerable increase in the yield of soluble hemoglobin. The recombinant hemoglobin exhibits cooperative oxygen binding properties indistinguishable from native protein. Crystals of the recombinant protein, like those of native hemoglobin, diffract to high resolution which will allow functional studies of site-directed mutants to be correlated with detailed structural analyses.

Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda.

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped with phage deletion mutants. The DNA sequence in the region was reexamined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies with the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes, the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start site of sieB was identified by the use of an RNase protection assay. The sieB promoter thus identified was inactivated by a 2-base substitution in its -10 hexamer. The sieB gene of coliphage lambda was also identified. The promoter for lambda sieB was identified by homology to that of P22 sieB.

A switch in translation mediated by an antisense RNA.

Antisense RNAs regulate expression of target genes in a variety of ways--transcription termination, translation initiation, and mRNA stability. We describe a case in which the target gene encodes two polypeptides, and antisense RNA causes a switch in its translation by selectively inhibiting synthesis of one of the polypeptides. Bacteriophage P22 is a temperate Salmonella phage; in the prophage state it expresses only a handful of its genes. One of these genes, sieB, aborts the lytic development of some phages. P22 itself is insensitive to the lethal effect of SieB because it harbors a determinant called esc. We show that the sieB gene encodes two polypeptides--SieB, which is the exclusion protein, and Esc, which is a truncated version of SieB that inhibits its action. Superinfecting P22 synthesizes an antisense RNA, sas, that inhibits synthesis of SieB but allows continued synthesis of Esc, thus allowing P22 to bypass SieB-mediated exclusion. This translational switch induced by sas RNA is essential to vegetatively developing P22; a mutation that prevents this switch causes P22 to commit SieB-mediated suicide. Finally, we show that P22's Esc allows it to circumvent the SieB-mediated exclusion system of bacteriophage lambda.

Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22.

To examine bacteriophage recombination in vivo, independent of such other processes as replication and packaging, substituted lambda phages bearing restriction site polymorphisms were employed in a direct physical assay. Bacteria were infected with two phage variants; DNA was extracted from the infected cells and cut with a restriction endonuclease. The production of a unique recombinant fragment was measured by Southern blotting and hybridization with a substitution sequence-specific probe. High frequency recombination was observed under the following conditions: the substituted lambda phages infected a wild-type host cell bearing a lambda repressor-expressing plasmid designed to shut down phage transcription and inhibit phage DNA replication as well. The same plasmid expressed the lambda red and gam genes. In addition, the host cell bore a second plasmid which expressed the EcoRI restriction-modification system. Both phage chromosomes possessed a single EcoRI site in the middle of the marked substitution sequence; however, as the site was modified in one of the parent phages, only the other partner was cut. Recombination was found to be dependent upon (1) red, (2) recA, (3) inactivation of the host recBCD function, either by Gam protein or by mutation and (4) double-strand breaks. The homologous recombination system of phage P22 could substitute for that of lambda.

Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme.

The sensitivity of bacteriophage T4 lysozyme function to amino acid substitutions at defined positions in and around the longitudinal, hydrophobic strips of 9 alpha-helices was assessed after systematic replacement of each residue in the protein with a series of 13 amino acids. The hydrophobic strips were defined by identifying the longitudinal sectors in the helices with the highest mean residue hydrophobicities. Sensitivity to mutation (the percentage of replacements leading to loss of function) was calculated for each residue in the following positions: whole protein, helices, hydrophobic strips, other positions within the helices, and various positions within the hydrophobic strips as well as their extensions beyond the helices. Substitutions at positions in the hydrophobic strips led more frequently to loss of function than substitutions in the protein as a whole. One subset, the COOH-terminal hydrophobic strip residues, is apparently critical; substitutions of these residues (but not of their NH2-terminal counterparts) led at least as frequently to loss of function as substitutions of solvent-inaccessible residues, and nearly as frequently as substitutions of the most highly conserved residues.

Functional significance of conserved amino acid residues.

A systemic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues. Substitutions of amino acid residues that are conserved among five bacteriophage-encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues. Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions.

Systematic mutation of bacteriophage T4 lysozyme.

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme.

Replacement of Asp20 in T4 lysozyme by Cys produces a variant with (1) nearly wild-type specific activity, (2) a newly acquired sensitivity to thiol-modifying reagents, and (3) a pH-activity profile that is very similar to that of the wild-type enzyme. These results indicate that the residue at position 20 has a significant nucleophilic function rather than merely an electrostatic role. The intermediate in catalysis by lysozyme is probably a covalent glycosyl-enzyme instead of the ion pair originally proposed.

Bacteriophage P22 accessory recombination function.

The accessory recombination function (arf) gene of bacteriophage P22 is located immediately upstream of the essential recombination function (erf) gene. Three mutant alleles of arf were constructed and installed in P22 in place of the wild-type allele: an out-of-frame internal deletion, an in-frame internal deletion, and an amber mutation. The deletion mutant phages are partially defective in homologous recombination and plaque formation in wild-type and recA hosts; their defects are more severe in recB and recA recB hosts. The amber mutant phage exhibits the same growth phenotypes in nonsuppressing hosts, but not in an amber-suppressor host. Plasmids that express arf complement the growth defect of arf- phages. These plasmids stimulate erf-mediated recombination; they were also found to cause a small stimulation of recA-recBCD-mediated homologous recombination of phage lambda.