Genetic studies of coliphage P1. III. Extended genetic map.

Using semiquantitative spot tests, 107 independently isolated amber mutants of P1 were shown to be rescued by a nonpermissive strain of Escherichia coli lysogenic for P7 (previously called phiamp), indicating extensive genetic relatedness between P1 and P7. The amount of rescue observed varied with mutants from different genetic linkage clusters of P1. Although these rescue tests cannot distinguish between recombination, complementation, transactivation, or combinations thereof, a major role is indicated for recombination.     

Genetic studies of coliphage P1. I. Mapping by use of prophage deletions.

One hundred and ten amber mutants of coliphage P1 were isolated and localized into groups with respect to the existing genetic map by use of nonpermissive Escherichia coli K-12 strains lysogenic for P1 with deletions. These lysogens contain one of three types of deletion prophages: P1cry and its derivatives, P1dlacs, and P1dpros. Fourteen such lysogens were tested for their ability to rescue the amber mutants which were then assigned to one of nine deletion segments of the P1 genome defined by the termini of the various prophage deletions. The relationship of the nine deletion segments with the published P1 map is described, two new segments having been added. The deletions of the 14 prophages overlapped sufficiently to indicate that the P1 genetic prophage map should be represented in circular form, which is consistent with the fact that P1 is normally a circular plasmid in the prophage state. The distribution of mutants into deletion segments is nonrandom for at least one segment. In addition, the deletion termini of the 14 defective prophages coincided in five out of nine regions separating the nine deletion segments. Various possible explanations are discussed for the nonrandom recurrence of these deletion termini, including the evidence of hot spots of recombination.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites

We have studied P1 site-specific recombination by cloning a 6·5 × 10³ base EcoRI fragment (fragment 7) of P1 DNA into a λ vector and then asking whether that fragment can promote efficient recombination for λ markers that flank the fragment. Our results indicate that fragment 7 can reassort these markers very efficiently, and that this recombination can occur in the absence of the bacterial recA and recBC functions. The fragment 7 recombination system has been dissected by an analysis of deletion mutations into two components, a site (called loxP) that must be present in both partners in the recombination in order for recombination to occur, and a P1 gene (called cre), whose product is necessary for recombination. The location of the loxP site at the end of the P1 genetic map suggests that this site-specific recombination system is responsible for the lack of linkage between terminal P1 markers and therefore for the linearity of that map.     

Stimulation of IS1 excision by bacteriophage P1 ref function.

Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation. The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1. The responsible P1 gene, ref, has been cloned and sequenced. ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre. Expression of ref was repressed by P1 c+. The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo. Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein. Ref stimulated precise excision in recB or himA cells, but not in recA mutants. Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction. We have constructed a P1c1.100 ref::Tn10 mutant. The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens. Thus, the role of ref in the physiology of P1 remains to be determined.     

Deletions in bacteriophage P2. Circularity of the genetic map and its orientation relative to the DNA denaturation map

Several types of viable chromosomal deletions of bacteriophage P2 were isolated. One type gives the immunity insensitive phenotype and may extend to the genes for the immunity repressor (C) and for integrative recombination (int). Two other types delete genes (old and fun) known to be active in the lysogenic state. For such deletion mutants the relationship between particle density and DNA length was established. The deletions were located in respect to previously mapped genes and the results were compared with electron microscopical studies (by Inman and collaborators) of the P2 chromosome. It is concluded that the best representation of the genetic map of P2 is circular. The cohesive ends of the linear P2 DNA molecule are most likely formed between genes old and Q. Except for the neighborhood of gene old, the previously published, linear genetic map of P2 (Lindahl) is colinear with the melting map of the P2 chromosome (Inman). Preliminary evidence for some specific recombination event often accompanying integrative recombination between phage chromosomes is presented.     

P+HA- charge recombination reaction rate constant in Rhodobacter sphaeroides reaction centers is independent of the P/P+ midpoint potential.

The kinetics of the P+HA- (oxidized donor, reduced bacteriopheophytin acceptor) recombination reaction was measured in a series of reaction center mutants of Rhodobacter sphaeroides with altered P/P+ midpoint potentials between 410 and 765 mV. The time constant for P+HA- recombination was found to range between 14 and 26 ns and was essentially independent of P/P+ midpoint potential. Previous work has shown that the time constant for initial electron transfer in these mutants at room temperature is also only weakly dependent on the P/P+ midpoint potential, ranging from about 2.5 ps to about 50 ps. These results, taken together, imply that heterogeneity in the P/P+ midpoint potential within the reaction center population is not likely the dominant cause of the substantial kinetic complexity observed in the decay of the excited singlet state of P on the picosecond to nanosecond time scale. In addition, the pathway of P+HA- decay appears to be direct or via P+BA- rather than proceeding back through P, even in the highest-potential mutant, as is evident from the fact that the rate of P+HA- recombination is unaltered by pushing P+HA- much closer to P in energy. Finally, the midpoint potential independence of the P+HA- recombination rate constant suggests that the slow rate of P+HA- recombination arises from an inherent limitation in the maximum rate of this process rather than because it occurs in the inverted region of a classical Marcus rate vs free energy curve.     

Requirement for protein synthesis for survival of unmodified bacteriophage T1 in a restricting host.

At high multiplication of infection, a substantial fraction of restricting cells (P1 lysogens) could be productively infected by unmodified coliphage T1 (T1.0) provided that protein synthesis was uninhibited during the first 5 min of infection. Successful infection under restricting conditions was accompanied by more genetic recombination than was seen under nonrestricting host, the recombination frequency declined for markers on T1.0 genomes; no effect was seen on recombination between markers on modified (T1.P) genomes. This suggested that recombination between unmodified genomes may be essential for their survival under conditions of host restriction. In a restricting host, genetic markers on T1.0 could recombine with T1.P even when the rescuing phage was added 6 min after T1.0 infection. However, even marker rescue recombination was diminished when protein synthesis was inhibited during early infection. Since DNA restriction is an early event, protein synthesis may be required soon after infection of a restricting host by T1.0 in order to preserve restriction-damaged DNA in a form that can participate in recombination. Experiments are also described that rule out some possibilities for the role of such a protein(s).     

A dnaB analog function specified by bacteriophage P7 and its comparison to the similar function specified by bacteriophage P1

Summary Evidence is presented that bacteriophage P7 specifies an analog of the E. coli DNA replication protein, dnaB. As in the related bacteriophage P1 (D'Ari et al., 1975; Ogawa, 1975), in lysogens of P7, the production of the analog protein is repressed and constitutive mutants could be isolated. Such constitutive of several dnaB(ts) mutations and also rescue a strain carrying a dnaB amber mutation. While neither P7 nor the mutant P1bacban (defective in the structural gene ban) could suppress dnaB(ts) mutations efficiently, recombinants between these two phages could do so, indicating the presence of a functional dnaB analog gene (called sdb) on P7. In a dnaB amber strain suppressed by the presence of the constitutive mutant P7csb, bacteriophage failed to replicate which is a further similarity between P7 and P1. P7csb mutants or P7-P1bacban recombinants were found to be less thermoresistant than P1bac1 suggesting that the P7-specified dnaB analog protein or its production is relatively less tolerant of temperatures above 37°C.     

Fine-structure mapping of herpes simplex virus type 1 temperature-sensitive mutations within the short repeat region of the genome.

Cloned herpes simplex virus type 1 (HSV-1) DNA fragments were used to fine-structure map the temperature-sensitive (ts) lesions from four mutants, ts T, D, c75, and K, by marker rescue. These mutants all overproduced immediate-early viral polypeptides at the nonpermissive temperature. Although one of these viruses, ts K, gave a more restricted infected-cell polypeptide profile under these conditions than the other three, no complementation was detected between pairwise crosses of these mutants in the yield test. Recombination, however, was obtained between all mutant pairs except ts T and D. In physical mapping experiments, ts+ virus was recovered from cells coinfected with DNA of ts T, D, or c75 and BamHI fragment k from wild-type strain 17 HSV-1 DNA cloned in pAT153, whereas ts K was rescued by cloned HSV-1 BamHI-y. Both of these cloned DNA fragments contained sequences from the short repeat region of the HSV-1 genome. The ts mutations were more precisely mapped by marker rescue, using restriction enzyme fragments within BamHI-k and -y from cloned DNA. The smallest fragment able to rescue a mutant was 320 base pairs long. The order of the four mutations derived from these studies was consistent with the assignment by genetic recombination. All four lesions mapped within the coding sequences of the immediate-early polypeptide Vmw IE 175 (ICP4) which lie outside the "a" sequence. The results showed that mutations in different regions of the gene encoding Vmw IE 175 could produce similar phenotype effects at the nonpermissive temperature.     

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.     

Recombination properties of P1 dlac.

The P1 dlac prophage plasmid of Escherichia coli K-12 has been utilized as the recipient DNA substrate in experiments with lambda plac5 transduction and with Hfr and F' conjugation. The P1 dlac plasmid does not recombine with lambda plac5 at the elevated levels seen for the F42lac plasmid. Recombination between lambda plac5 and P1 dlac is essentially indistinguishable from recombination between lambda plac5 and a chromosomal lac gene in tems of both level of recombination and recombination pathway (RecBC, RecE, and RecF) dependence. The initiation of recombination between P1 dlac and lac genes from an Hfr or F' donor is severalfold more efficient than it is for a recipient chromosomal lac gene.     

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.     

Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG.

ruvC mutants of Escherichia coli appear to lack an activity that resolves Holliday intermediates into recombinant products. Yet, these strains produce close to normal numbers of recombinants in genetic crosses. This recombination proficiency was found to be a function of recG. A "mini-kan" insertion in recG was introduced into ruvA, ruvB, and ruvC strains. Conjugational recombination was reduced by more than 100-fold in recG ruvA::Tn10, recG ruvB, and recG ruvC strains and by about 30-fold in a recG ruvA strain carrying a ruvA mutation that is not polar on ruvB. The double mutants also proved very deficient in P1 transduction and are much more sensitive to UV light than ruv single mutants. Since mutation of recG alone has very modest effects on recombination and sensitivity to UV, it is concluded that there is a functional overlap between the RecG and Ruv proteins. However, this overlap does not extend to circular plasmid recombination. The possibility that RecG provides a second resolvase that can substitute for Ruv is discussed in light of these findings.     

Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay.

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.     

Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.     

Genetic analysis of temperature-sensitive mutants which define the genes for the major herpes simplex virus type 2 DNA-binding protein and a new late function.

Eleven temperature-sensitive mutants of herpes simplex virus type 2 (HSV-2) exhibit overlapping patterns of complementation that define four functional groups. Recombination tests confirmed the assignment of mutants to complementation groups 1 through 4 and permitted the four groups to be ordered in an unambiguous linear array. Combined recombination and marker rescue tests (A. E. Spang, P. J. Godowski, and D. M. Knipe, J. Virol. 45:332-342, 1983) indicate that the mutations lie in a tight cluster near the center of UL to the left of the gene for DNA polymerase in the order 4-3-2-1-polymerase. The seven mutants that make up groups 1 and 2 fail to complement each other and mutants in HSV-1 complementation group 1-1, the group thought to define the structural gene for the major HSV-1 DNA-binding protein with a molecular weight of 130,000. At 38 degrees C, mutants in groups 1 and 2 synthesize little or no viral DNA, and unlike cells infected with the wild-type virus, mutant-infected cells exhibit no detectable nuclear antigen reactive with monoclonal or polypeptide-specific antibody to the major HSV-2 DNA-binding protein. The four mutants that make up groups 3 and 4 do not complement each other, nor do they complement mutants in group 2. They do, however, complement mutants in group 1 as well as representative mutants of HSV-1 complementation group 1-1. At 38 degrees C, mutants in groups 3 and 4 are phenotypically DNA+, and nuclei of mutant-infected cells contain the HSV-2 DNA-binding protein. Thus, the four functional groups appear to define two closely linked genes, one encoding an early viral function affecting both viral DNA synthesis and expression of the DNA-binding protein with a molecular weight of 130,000 (groups 1 and 2), and the other encoding a previously unidentified late viral function (groups 3 and 4). The former gene presumably represents the structural gene for the major HSV-2 DNA-binding protein.     

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.     

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.