Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Transfection of Escherichia coli Spheroplasts I. General Facilitation of Double-Stranded Deoxyribonucleic Acid Infectivity by Protamine Sulfate

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.     

Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl in Escherichia coli

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.     

Different restriction of bacteriophages T3 and T7 by P1-lysogenic cells and the role of the T3-coded SAMase.

The intracellular growth of the phages T3 and T7 is restricted in the presence of the Escherichia coli prophage P1. Phage T3 has a higher ability to express its genome and to damage the host cell than T7. This partial protection of T3 against P1 restriction is due to the T3-coded SAMase, an enzyme which degrades S-adenosylmethionine, the cofactor of the P1 restriction endonuclease. Since we did not observe DNA cleavage in vivo, we conclude that the in vivo action of the P1 nuclease is limited to a SAM-dependent repressor-like binding to T3 and T7 DNA, while further reactions with the DNA (modification vs cleavage) are blocked.     

Chromosomal relocation of prophage-associated bacterial genes

Taylor, M. W. (Stanford University, Stanford, Calif.), and C. Yanofsky. Chromosomal relocation of prophage-associated bacterial genes. J. Bacteriol. 91:1469-1476. 1966.-Two distinguishable colony types, rough-edged and smooth-edged, were observed when tryptophan auxotrophs of Escherichia coli were transformed to tryptophan independence with DNA from the hybrid nondefective transducing phage i(lambda)h(phi80)T(1) (S)tryp A(+)B(+), and with the helper phage lambdai(434). P1kc transduction experiments with cells of the two types of colonies as genetic donors showed that the i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage was located at different regions of the E. coli chromosome. In cells of rough-edged colonies, the prophage was linked to the tryp-cys region, its normal location, whereas in cells of smooth-edged colonies the prophage was associated with the gal region. When transformation experiments were performed with a T(1) (R)tryp(-) deletion mutant as recipient, and phage lambdai(434) as helper, prophage localization was only detected at the gal region. Localization of (lambda)h(phi80)T(1) (S)tryp A(+)B(+) prophage near gal does not appear to be due to the formation of a recombinant phage carrying tryp A(+)B(+), but is due to some type of interaction between the genomes of i(lambda)h(phi80)T(1) (S)tryp A(+)B(+) and the helper phage. When conditions comparable to those used in transformation studies were employed in transduction experiments, including the use of helper phage, two classes of transductants with either cys or gal linkage were also observed. To examine whether the location of the prophage on the E. coli chromosome had any effect on the ability of the prophage-associated tryp A(+) and tryp B(+) genes to function or respond to different repression conditions, specific activities of the A and B subunits of tryptophan synthetase specified by the phage genome were measured. Similar values were obtained regardless of the location of the prophage-associated tryp genes. Furthermore, the prophage-associated tryp genes, free from their normal operator region, permitted enzyme formation which was unaffected by repression or derepression conditions.     

Genetic mapping of transposon Tn5-induced mutation blocking threonine dehydrogenase in Escherichia coli K-12

The paper deals with a mutant of Escherichia coli K-12 obtained by transposon Tn5 mutagenesis. Insertion of this transposon inactivated the gene for L-threonine dehydrogenase catalysing the first step of L-threonine degradation. The insertion of Tn5 was mapped by using conjugation as well as transduction by T4GT7 and P1. It is located at 81 min of the E. coli genetic map between mtl and pyrE genes.     

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.     

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.     

Cloning and characterization of bacteriophage-like DNA from Haemophilus somnus homologous to phages P2 and HP1.

In an attempt to identify and characterize components of a heme uptake system of Haemophilus somnus, an Escherichia coli cosmid library of H. somnus genomic DNA was screened for the ability to bind hemin (Hmb+). The Hmb+ phenotype was associated with a 7,814-bp HindIII fragment of H. somnus DNA that was subcloned and sequenced. Thirteen open reading frames (orfs) were identified, all transcribed in one direction, and transposon mutagenesis identified orf7 as the gene associated with the Hmb+ phenotype. Orf7 (178 amino acids) has extensive homology with the lysozymes of bacteriophages P-A2, P21, P22, PZA, phi-29, phi-vML3, T4, or HP1. The orf7 gene complemented the lytic function of the K gene of phage P2 and the R gene of phage lambda. A lysozyme assay using supernatants from whole-cell lysates of E. coli cultures harboring plasmid pRAP501 or pGCH2 (both of which express the orf7 gene product) exhibited significant levels of lysozyme activity. The orf6 gene upstream of orf7 has the dual start motif common to the holins encoded by lambdoid S genes, and the orf6 gene product has significant homology to the holins of phages HP1 and P21. When expressed from a tac promoter, the orf6 gene product caused immediate cell death without lysis, while cultures expressing the orf7 gene product grew at normal rates but lysed immediately after the addition of chloroform. Based on this data, we concluded that the Hmb+ phenotype was an artifact resulting from the expression of cloned lysis genes which were detrimental to the E. coli host. The DNA flanking the cloned lysis genes contains orfs that are similar to structural and DNA packaging genes of phage P2. Polyclonal antiserum against Orf2, which is homologous to the major capsid precursor protein (gpN) of phage P2, detected a 40,000-M(r) protein expressed from pRAP401 but did not detect Orf2 in H. somnus, lysates. The phage-like DNA was detected in the serum-susceptible preputial strains HS-124P and HS-127P but was absent from the serum-resistant preputial strains HS-20P and HS-22P. Elucidation of a potential role for this cryptic prophage in the H. somnus life cycle requires more study.     

Mutants defective in the 33K outer membrane protein of Salmonella typhimurium.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.     

Phylogenetically related Argentinean and Australian Escherichia coli O157 isolates are distinguished by virulence clades and alternative Shiga toxin 1 and 2 prophages

Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx(2) (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx(2) is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx(1) prophage. In both Argentinean and Australian LI/II strains, stx(2c) is almost exclusively carried by a prophage inserted at sbcB. However, alternative q(933)- or q(21)-related alleles were identified in the Australian stx(2c) prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.     

Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.     

Characterization of generalized transducing phage phi w39 heteroimmune to phage P1 in Escherichia coli W39

Generalized transducing phage similar to phage P1 in Escherichia coli was isolated from E. coli W39, an antigenic test strain of the O121 group. This phage, designated phi w39, was reciprocally heteroimmune to phages P1 and P7, but nonreciprocally heteroimmune to phage D6. Transduction experiments using various R plasmids with different molecular weights suggested that phage phi w39 could transduce at least 65 megadaltons DNA. As in the case of P1 prophage, phi w39 prophage existed as a plasmid belonging to incompatibility group Y and carried a dnaB-like function. The molecular weight of phi w39 plasmid was nearly the same as that of plasmid, i.e., 58.6 megadaltons. Despite the pronounced structural and functional similarity of phages phi w39 and P1, restriction cleavage patterns of their genomes differed considerably.     

Evidence of Horizontal Transfer of the EcoO109I Restriction-Modification Gene to Escherichia coli Chromosomal DNA

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.     

Isolation and characterization of T-even ghost-tolerant mutants of Escherichia coli.

Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.     

A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis

The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.     

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.     

The cotransduction of pET system plasmids by mutants of T4 and RB43 bacteriophages

The study of the cotransduction of the plasmid pairs pET-3a-pLysE and pET-3a-pLysS by the mutant phage T4alc7 showed that the antibiotic resistance markers of the plasmids were cotransduced with a high frequency. The analysis of the plasmid DNA of cotransductants and cotransformants showed that the mutant phage T4alc7 can be used for obtaining the monomeric and oligomeric forms of plasmids and for the cotransduction of two-plasmid overproduction systems into E. coli strains. The plaque mutants RB43-03 and RB43-13 derived from bacteriophage RB43 were found to be able to cotransduce the antibiotic resistance markers of pET-3a and pLysE plasmids.     

Construction of Escherichia coli strains deleted for the attachment site by transposon-linked generalized transduction

Abstract A strain of Escherichia coli K-12 has been constructed which can be used in combination with P1 or T4GT7-mediated transduction to generate strains deleted for the λ attachment site. This allows the quick and simple construction of well defined deletion strains, with a minimum of transfer of other genes.     

Role of immunoregulatory transcription factors in differential immunomodulatory effects of tocotrienols

Tocotrienols have been shown to possess antioxidant, antitumor, cardioprotective, and antiproliferative effects. This report describes novel immunomodulatory effects of tocotrienols in murine lymphocytes. γ-Tocotrienol (GT) was more effective in suppressing concanavalin A (Con A)-induced T cell proliferation and cytokine production compared to α-tocotrienol (AT) when present continuously in the culture. GT inhibited T cell activation markers and costimulatory molecule. GT modulated intracellular glutathione in lymphocytes, and the suppressive effects of GT could not be abrogated by thiol or nonthiol antioxidants, indicating a poor link between anti-inflammatory properties of tocotrienols and cellular redox status. It was also observed that GT suppressed Con A-induced activation of NF-κB, AP-1, and NF-κB-dependent gene expression. Cellular uptake studies with tocotrienols showed higher accumulation of GT compared to AT. Similar immunosuppressive effects of GT were also observed when administered to mice. In contrast, transient exposure of lymphocytes to GT (4 h) resulted in higher survival and proliferation of lymphocytes in vitro and in vivo in syngeneic and allogeneic hosts. This was attributed to the ability of GT to induce NF-κB, AP-1, and mTOR activation in lymphocytes upon transient exposure. Our results demonstrated that antioxidants such as tocotrienols may exhibit pleiotropic effects by activating multiple mechanisms in cells.