Deletion of late genes of the prophage lambda

The deletions in tandem prophage lambda appear with high frequency (to 10%) in rec A- strain of Escherichia coli. The deletions were shown by marker rescue and hybridization of fragments of DNA on nitrocellulose filters with nick-translated phage lambda DNA localized only in prophage area. Right and left att sites are not involved. The majority of defective lysogens had all regulatory regions and deletions of late structural genes. These strains may be used for construction of the host-vector systems with the strongest promoter p'R of phage lambda.     

Isolation and characterization of plaque-forming lambdadnaZ+ transducing bacteriophages.

The Escherichia coli dnaZ gene, a deoxyribonucleic acid (DNA) polymerization gene, is located 1.2 min counterclockwise from purE, at approximately min 10.5 on the E. coli map. From a lysogen with lamdacI857 integrated at a secondary attachment site near purE, transducing phages (lambdadnaS+) that transduced a dnaZts (lambda+) recipient to temperature insensitivity (TS+) were discovered. Three different plaque-forming transducing phages were isolated from seven primary heterogenotes. Genetic tests and heteroduplex mapping were used to determine the length and position of E. coli DNA within the lambda DNA. Complementation tests demonstrated that the deletions in all three strains removed both att P and the int gene, i,e., DNA from both prophage ends. Heteroduplex mapping confirmed this result by demonstrating that all three strains had deletions of lambda DNA that covered the b2 to red region, thereby removing both prophage ends. Specifically, the deletions removed lambda DNA between the points 39.3 to 66.5% of lambda length (measured in percent length from the left and of lambda phage DNA) in all three strains. The three strains are distinct, however, because they had differing lengths of host DNA insertions. These phages must have been formed by an anomalous procedure, because standard lambda transducing phages are deleted for one prophage end only. In lambdagal and lambdabio strains, the deletions of lambda DNA begin at the union of prophage ends (i.e., position 57.3% of lambda length) and extend leftward or rightward, respectively (Davidson and Szybalski, in A, D. Hershey [ed.], The Bacteriophage Lambda, p. 45-82, 1971). Models for formation of the lambdadnaZ+ phages are discussed.     

Bacteriophage mu-induced deletions in a plasmid containing the nif (N2 fixation) genes of Klebsiella pneumoniae.

Five plasmids with insertions of a heat-inducible Mu prophage in a Mu-sensitive and P1-sensitive derivative of plasmid pRD1, a recombinant R factor containing the his-nif region of Klebsiella pneumoniae, were isolated and characterized. In one plasmid containing the Mu prophage integrated at the his-distal end of nif, selection for heat resistance resulted in the generation of deletions extending from the Mu prophage into the nif region. Thirty of these deltions were used to map 26 point mutations in nif.     

Deletions Induced by Heat Treatment of E. COLI K12 Lysogenic for λ Prophages

We have investigated the production of prophage deletions in heat-induced λ lysogens of E. coli K12. Our results are indicative of a direct action of the heat-induced prophage in producing deletions. The temperature of 40° used for the experiments may be critical to prove this effect. The phage function involved in deletion formation is not known.     

Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.     

Deletion mapping of the polA-metB region of the Escherichia coli chromosome.

A lambdacI857 prophage inserted into one of the genes of the rha locus was used to select deletions unambiguously ordering the markers polA-glnA-rha-pfkA-tpi-metBJF. Transduction with phage P1 indicates at least 70% linkage between glnA and polA. The order of the pfk and tpi markers is reversed from that previously published. Despite the relatively large distance separating the glnA and rha loci, deletions removing this entire region have no obvious phenotype. The isolation of Tn10 transposons integrated at different sites between rha and glnA greatly facilitated this work.     

Regional Specificity of Illegitimate Recombination by the Translocatable Amplicillin-Resistance Element Tn1 in the Genome of Phage P22

Insertions of the translocatable ampicillin-resistance element Tn1 were selected in the genome of the temperate Salmonella phage P22 by growing the phage on hosts carrying the resistance plasmid RP4. Insertions of Tn1 into phage P22 are rare (10(-10) per phage) and nonrandomly distributed in the P22 genome. They are found mainly in the vicinity of the P22 ant gene. Insertions within the ant gene are found at many (at least 15) genetically separable sites, are found equally frequently in both orientations and cause irreversible loss of gene function. Some insertions in ant appear to be associated with an adjecent deletion. Prophage deletions were derived from P22::Tn1 phages by two methods. Low multiplicity transductants have nonrandomly distributed endpoints. One end is at or very near the site of the Tn1 insertion, and the other is in the vicinity of gene 12; however, there are many genetically distinguishable endpoints within gene 12. Prophage deletions selected as survivors of induction of a P22Ap mnt-ts lysogen have similarly nonrandom endpoints, with the Tn1-distal end frequently near the ant gene, as well as gene 12. Physical analysis of several prophage deletions suggests that the Tn1 is intact to the resolution of DNA electron microscopy and that the deletions begin at the end of the Tn1 insertion. These results suggest that illegitimate recombination associated with Tn1 shows regional specificity (i.e., preference for some large areas of the P22 genome over other areas), but that within these regions is quite nonspecific.     

Fine Structure Genetic and Physical Map of the Phage P22 Tail Protein Gene

Bacteriophage P22 which are incapable of making functional tail protein can be propagated by the addition of purified mature tail protein trimers to either liquid or solidified medium. This unique in vitro complementation condition has allowed us to isolate 74 absolute lethal tail protein mutants of P22 after hydroxylamine mutagenesis. These phage mutants have an absolute requirement for purified P22 tail protein to be present in a soft agar overlay in order to form plaques and do not grow on any nonsense suppressing strains of Salmonella typhimurium. In order to genetically map and physically locate these mutations we have constructed two complementary sets of fine structure deletion mapping strains using a collection of Tn1 insertions in gene 9, the structural gene for the tail protein. Fourteen bacteriophage P22 strains carrying unique Tn1 transposon insertions (Ap phage) in gene 9 have been crossed with Ap phage carrying Tn1 insertions in gene 20. Phage carrying deletions that arose from homologous recombination between the Tn1 elements were isolated as P22 lysogens. The deletion prophage were shown to be missing all genetic information bracketed by the parental Tn1 elements and thus form a set of deletions into gene 9 from the 5' end of the gene. From the frequency of production of these deletion phage the orientation of the Tn1 insertions in gene 9 could be deduced. The genetic end points of the deletions in gene 9 and thus the order of Tn1 insertions were determined by marker rescue experiments using the original Ap phage. The genetic end points of the deletions in gene 20 were determined in similar experiments using nonsense mutations in gene 20. To locate the physical end points of these deletions in gene 9, DNA containing the Tn1 element has been cloned from each of the original Ap phage into plasmids. The precise point of insertion of Tn1 into gene 9 was determined by restriction enzyme mapping and DNA sequencing of the relevant portions of each of these plasmids. In vitro deletion of different 3' gene 9 sequences in the plasmid clones was accomplished through the use of unique restriction endonuclease sites in Tn1. The resulting plasmids form a set of deletions extending into the 3' end of the gene which are complementary compared to the deletion lysogens.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Variability of the immunoglobulin heavy chain constant region locus: a population study

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.     

Mapping of insertion mutations in gnd of Escherichia coli with deletions defining the ends of the gene.

A genetic map was prepared for gnd, the gene of Escherichia coli which encodes the metabolically regulated 6-phosphogluconate dehydrogenase. Direct selection methods were used for the isolation of mutants with deletions that define the respective ends of gnd. These selections depended on the availability of a defective lysogen in which gnd was present on a lambda h80 dgnd his prophage located at the att phi 80 region of the chromosome. Mutants with deletions entering gnd from the his-distal end were selected as Gnd- TonB- mutants. Mutants with his-proximal gnd deletions were selected as Gnd-, temperature-resistant mutants of a specially prepared stable lysogen. Gnd- mutants were also isolated after mutagenesis with bacteriophage Mu cts61, and genetic tests were used to determine which mutants carry a Mu cts61 prophage in gnd. The deletion mutations were mapped against each other and against the insertion mutations through the use of F' merodiploid strains. The insertion mutations mapped at seven distinct sites in gnd; three mapped under the deletions defining the his-proximal portion of the gene and three mapped with the his-distal deletions.     

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.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.     

Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene.

Deletion of the Escherichia coli K-12 chromosome associated with P2 mediated education extend through the structural gene for uridine kinase, udk, and the dcd gene encoding 2'-deoxycytidine 5'-triphosphate deaminase. The lack of uridine kinase makes a positive selection possible for these strains. Due to the dcd mutation, P2 eductants show large alterations in their deoxyribonucleoside triphosphate pools.     

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.     

Studies with a Gluconate-6-Phosphate Dehydrogenase Mutant in Escherichia coli Strain C

A map location of the gluconate-6-phosphate dehydrogenase (gnd) marker was estimated in Escherichia coli C at approximately 46 min by P1 transduction. The gnd locus appears to lie between the co-transducible histidine and prophage P2 location I markers.     

Characterization of serracin P,a phage-tail-like bacteriocin,and its activity against Erwinia amylovora,the fire blight pathogen

Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens. This activity increased significantly upon induction with mitomycin C. A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S. plymithicum J7. It was found to be the only compound involved in the antibacterial activity against sensitive strains. The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the phiCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis. This strongly suggests a common ancestry for serracin P and these bacteriophages.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

Prophage type of recombinants produced by cell fusion between various combinations of lysogenic or non-lysogenic substrains from two Staphylococcus aureus L-forms

Streptomycin (SM)- or erythromycin (EM)-resistant lysogenic and non-lysogenic substrains were produced from two Staphylococcus aureus L-form strains lysogenic for different prophages, namely, EMT-L (prophage alpha) and 209P (prophage beta). Cells of these L-form substrains were fused in various combinations using polyethylene glycol (PEG), and the frequency of recombinants selected as double resistance to both SM and EM and the prophage types of these recombinants were examined. In all the combinations, the frequency of recombinants was greater when the cells were treated with PEG than when they were not, and the difference was statistically significant (p less than 0.01) in 13 combinations. Combination between the lysogenic SM-resistant EMT-L substrain [EMT(Smr-alpha)] and lysogenic EM-resistant 209P-L substrain [209P(Emr-beta)] and the reverse combination, between 209P(Smr-beta) and EMT(Emr-alpha), resulted in a majority of recombinants harboring prophage beta. The former combination yielded recombinants that all held both prophage alpha and beta.