Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Defective prophages of bacteriophage Mu

Summary A method is described for the isolation of thermoinducible defective Mu lysogens. Four of these defective lysogens were studied more extensively. By marker-rescue experiments it was shown that the strain harbouring the smallest defective prophage contains the immunity gene cts and the genes A and B; the strain with the largest defective prophage still contains all the known essential genes of Mu, A to S (see Fig. 1).After induction at 43° C all the defective lysogens are killed, whereas no lysis occurs.Although in all the thermoinducible defective lysogens the A and B gene products could be demonstrated by complementation, these gene products are not responsible for the killing of the host, suggesting the presence of another unknown early gene product of Mu. The level of complementation of a mutation in gene A is reduced by the presence in the cell of another defective Mu prophage containing the G part of Mu. This effect on A gene complementation is markedly enhanced when the defective prophage, containing the G part, is located on an episome instead of on the chromosome. Complementation of late genes by a defective prophage located on the chromosome, is extremely low or undetectable. A stimulation of complementation by a factor of 10 to 40 was found when the same defective prophage was situated on a F factor. A possible explanation for this episome effect will be discussed.     

Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli.

EcoRI-cleaved deoxyribonucleic acid segments carrying two genes from Bacillus subtilis, pyr and leu, have been cloned in Escherichia coli by insertion into a derivative of the E. coli bacteriophage lambda. Lysogenization of pyrimidine- and leucine-requiring auxotrophs of E. coli by the hybrid phages exhibited prototrophic phenotypes, suggesting the expression of B. subtilis genes in E. coli. Upon induction, these lysogens produced lysates capable of transducing E. coli pyr and leu auxotrophs to prototrophy with high frequency. Isolated DNAs of these bacteriophages have the ability to transform B. subtilis auxotrophs to pyr and leu independence and contain EcoRI-cleaved segments which hybridize to corresponding segments of B. subtilis.     

Isolation and characterization of nondefective transducing lambda bacteriophages carrying fla genes of Escherichia coli K-12.

In Escherichia coli K-12, 11 fla genes and a hag gene are located between his and uvrC, making two clusters at map positions 42.5 and 43.0 min. Nondefective transducing lambda phages for these genes were isolated. Low-frequency-transducing donors were constructed starting from lysogens of lambda cI857 in which the prophage is integrated at a secondary attachment site at 44 min on the E. coli map. Two strategies were used to delete the region between the prophage and the fla genes. Deletion mutants of the supD locus between fla and the prophage were isolated by selecting for loss of Su1+, an allele of supD. A strain with a deletion starting within the prophage and ending at a position close to the fla genes was isolated from heat-resistant derivatives of the lysogen. A lysogen of lambda b2 was then constructed in which the prophage had integrated at the site of the defective prophage by means of recombination with residual lambda deoxyribonucleic acid. From low-frequency-transducing lysate of the donor strains thus constructed, either directly or in combination with a procedure that extends the loci transduced, various lambda pfla's were isolated. lambda pflaL1 carries all nine fla genes at 43 min, and lambda pflaH14 carries hag and two fla genes at 42.5 min.     

sub, a host mutation that specifically allows growth of replication-deficient gene B mutants of coliphage P2

Summary Bacteriophage P2 normally requires the products of its early genes A and B for lytic growth in its host, Escherichia coli C. A host mutation, sub-1, which allows P2 to grow without a functional B gene product is described. The sub-1 mutation is recessive and maps at approximately 10 min on the E. coli genetic map.     

Physical characterisation of the "Rac prophage" in E. coli K12.

Summary We confirm the hypothesis of Low (1973) that many E. coli K 12 strains contain a prophage (the Rac prophage) located a few minutes clockwise of the trp operon on the genetic map. We have used restriction endonucleases and ³²P-labelled probes to construct a physical map of this prophage. Some E. coli K 12 strains, including AB1157, have lost the entire prophage, apparently by a specific deletion. This is consistent with prophage excision by site-specific recombination. reverse (rev) phages (Zissler et al., 1971) are recombination proficient derivatives of phage in which the phage recombination functions have been replaced by analogous functions (RecE) derived from the host chromosome (Gottesman et al., 1974; Gillen et al., 1977). Our data support the origin of rev phages by recombination between and the Rac prophage following excision of the Rac prophage from the E. coli chromosome.Important experimental data are included in the Figure legends.     

Structure of cryptic lambda prophages

When Escherichia coli cells lysogenic for bacteriophage lambda are induced with ultraviolet light, cells carrying cryptic lambda prophages are occasionally found among the apparently cured survivors. The lambda variant crypticogen (lambda crg) carries an insertion of the transposable element IS2, which increases the frequency of cryptic lysogens to about 50% of cured cells: 43 of these cryptic prophages have been characterized. They all contain substitutions that replace the early segment of the prophage genome (from the IS2 to near the cos site) with a duplicate copy of a large segment of the host chromosome. The right end of the substitution always results from recombination between the nin-QSR-cos region of the prophage and the homologous incomplete lambdoid prophage Qsr' at 12.5 minutes in the E. coli chromosome. The left end of the substitution is usually a crossover that recombines the IS2 element in the prophage with an E. coli IS2 at 8.5 minutes, near the lac gene, or with a second IS2 located counterclockwise from leu at 2 minutes, generating duplications of at least 200,000 bases. Five cryptic lysogens derived from cells lysogenic for a reference strain of lambda (which lacks the IS2 present in lambda crg) have been characterized. They contain substitutions whose right termini are generated by a crossover with the Qsr' prophage. The left termini of these substitutions are formed either by a crossover between the lambda exo gene and a short exo-homologous segment of Qsr' (2/5), or by a crossover between sequences to the left of attL and an unmapped distant region of the host chromosome (3/5). The large duplications carried by these cryptic lysogens are stable, unlike tandem duplications, and so may significantly influence the cell's evolutionary potential.     

Gross map location of Escherichia coli transfer RNA genes.

Chromosomal locations of Escherichia coli genes specifying more than 20 different transfer RNA species were determined by utilizing two different methods. One was based upon gene dosage effects caused by F′ factors. In 15 different F′ strains and their corresponding F^? strains, relative contents of individual tRNAs were measured after separating the tRNAs by two-dimensional polyacrylamide gel electrophoresis. Approximate doubling of the content of particular tRNA was found in individual F′ strains, as showing gross map location of the tRNA gene. The other method was based on the amplified synthesis of tRNAs occurring after prophage induction of λ lysogens. Synthesis of individual tRNAs was measured after the induction of λ phages integrated at five different bacterial sites. Characteristic overproduction of different tRNAs was observed in individual prophage strains. This finding also gave approximate map locations of tRNA genes close to the prophage sites. The mapping data obtained by the two methods were consistent with each other and also with the reported positions in the cases where previously mapped. On the basis of map location of the tRNA_f1^Met gene newly determined, the λ-transducing phage carrying the tRNA_f1^Met gene was found.     

Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4

Temperate phages can integrate their genomes into a specific region of a host chromosome to produce lysogens (prophage). During genome insertion, prophages may interrupt the gene coding sequence. In Bacillus subtilis, the sigma factor gene sigK is interrupted by a 48 kb prophage‐like element. sigK is a composite coding sequence from two partial genes during sporulation. For over two decades, however, no further examples of DNA element‐mediated gene reconstitution other than sigK have been identified in spore formers. Here we report that the gene for dipicolinic acid (DPA) synthetase β subunit spoVFB in B. weihenstephanensis KBAB4 is interrupted by a prophage‐like element named vfbin. DPA is synthesized in the mother cell and required for maintaining spore dormancy. We found that spoVFB was a composite coding sequence generated in the mother cell via chromosomal rearrangement that excised vfbin. Furthermore, vfbin caused excision after phage‐inducer treatment, but vfbin appeared to be defective as a prophage. We also found various spore‐forming bacteria in which sporulation‐related genes were disrupted by prophage‐like DNA elements. These results demonstrate the first example of a similar mechanism that affects a sporulation gene other than sigK and suggest that this prophage‐mediated DNA rearrangement is a common phenomenon in spore‐forming bacteria.     

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.     

Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field <Emphasis Type="Italic">E. coli</Emphasis> O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.     

Cloning the trpR gene.

Summary In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage . From one such secondary site lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB ⁺ and trpR ⁺ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for bamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment.A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the trpR gene.In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR ⁺ pseudolysogens and strains harboring pBR322 trpR ⁺ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyltryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.Journal Paper No. 7426 of the Purdue University Agricultural Experiment Station     

Function,expression, specificity,diversity and incompatibility of actinobacteriophage parABS systems

More than 180 individual phages infecting hosts in the phylum Actinobacteria have been sequenced and grouped into Cluster A because of their similar overall nucleotide sequences and genome architectures. These Cluster A phages are either temperate or derivatives of temperate parents, and most have an integration cassette near the centre of the genome containing an integrase gene and attP. However, about 20% of the phages lack an integration cassette, which is replaced by a 1.4 kbp segment with predicted partitioning functions, including plasmid‐like parA and parB genes. Phage RedRock forms stable lysogens in Mycobacterium smegmatis in which the prophage replicates at 2.4 copies/chromosome and the partitioning system confers prophage maintenance. The parAB genes are expressed upon RedRock infection of M. smegmatis, but are downregulated once lysogeny is established by binding of RedRock ParB to parS‐L, one of two centromere‐like sites flanking the parAB genes. The RedRock parS‐L and parS‐R sites are composed of eight directly repeated copies of an 8 bp motif that is recognized by ParB. The actinobacteriophage parABS cassettes span considerable sequence diversity and specificity, providing a suite of tools for use in mycobacterial genetics.     

Induction of Phage Production in the Lysogenic Escherichia coli by Hydroxyurea

1) Hydroxyurea, a reversible DNA synthesis inhibitor, was used to study the mechanism of prophage λ induction in Escherichia coli K12. Induction of prophage was judged on two criteria: increase of phage-producing cells and loss of colony-forming ability of the cells. 2) Hydroxyurea induced an increase of phage-producing cells only in lysogenic strains known to be inducible with ultraviolet irradiation for prophage development and not in strains such as E. coli K12 (λind^–) or E. coli K12 recA (λ⁺). 3) When protein synthesis was inhibited, hydroxyurea did not increase phage-producing cells of lysogenic strains; it showed a bacteriocidal effect on lysogenic recA⁺ strains, but not on nonlysogenic strains. 4) The sensitivity of E. coli K12 recA to hydroxyurea was independent of whether or not the cells were lysogenic. 5) From the results it is suggested that certain steps leading to loss of colony-forming ability (i.e. prophage induction) do not require de novo protein synthesis but require the presence of the host recA⁺ gene.     

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

Background  

The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Prophage map of converting corynebacteriophage beta.

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.     

Coping with inadvertent lysis of Escherichia coli cultures: Strains resistant to lysogeny and infection by the stealthy lysogenic phage Φ80

Phage Φ80 can infect Escherichia coli in a stealthy manner and persist by forming lysogens. Such Φ80 lysogens are fairly common and often go undetected unless the host is grown at temperatures below 37°C. Since low growth temperatures are required for growing temperature-sensitive mutants and often preferred for large-scale applications such as protein production, Φ80-resistant strains would be useful. We report the construction of E. coli strains that cannot be efficiently lysogenized or infected by bacteriophage Φ80. These strains contain combinations of deletions or mutations in the bacterial attachment site for Φ80 integration and/or deletions in the genes required for phage absorption to the host outer membrane. These strains should help contain and prevent Φ80 infection of E. coli cultures in a laboratory or industrial setting.