Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.     

Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12

Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.     

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.     

The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2

Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.     

Integration of specialized transducing bacteriophage lambda cI857 St68 h80 dgnd his by an unusual pathway promotes formation of deletions and generates a new translocatable element.

Molecular and genetic studies have revealed that several illegitimate recombinational events are associated with integration of the specialized transducing bacteriophage lambda cI57 St68 h80 dgnd his into either the Escherichia coli chromosome or into a plasmid. Most Gnd+ His+ transductants did not carry the prophage at att phi-80, and 10% were not immune to lambda, i.e., "nonlysogenic." Integration of the phage was independent of the phage Int and Red gene products and of the host's general recombination (Rec) system. In further studies, bacterial strains were selected which carried the phage integrated into an R-factor, pSC50. Restriction endonuclease analysis of plasmid deoxyribonucleic acid (DNA) purified from these strains showed that formation of the hybrid plasmids resulted from recombination between a single region of pSC50 and one of several sites within the lambda-phi 80 portion of the phage. Furthermore the his-gnd region of the phage, present in the chromosome of one nonlysogenic transductant, was shown to be able to translocate to pSC50. Concomitant deletion of phage DNA sequences or pSC50 DNA was frequently observed in conjunction with these integration or translocation events. In supplemental studies, a 22- to 24-megadalton segment of the his-gnd region of the chromosome of a prototrophic recA E. coli strain was shown to translocate to pSC50. One terminus of this translocatable segment was near gnd and was the same as a terminus of the his-gnd segment of the phage which translocated from the chromosome of the nonlysogenic transductant. These data suggest that integration of lambda cI857 St 68 h80 dgnd his may be directed by a recombinationally active sequence on another replicon and that the resulting cointegrate structure is subject to the formation of deletions which extend from the recombinationally active sequence. Translocation of the his-gnd portion of the phage probably requires prior replicon fusion, whereas the his-gnd region of the normal E. coli chromosome may comprise a discrete, transposable element.     

Sequence analysis of Stx2-converting phage VT2-Sa shows a great divergence in early regulation and replication regions.

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.     

Evidence that the outer membrane protein gene nmpC of Escherichia coli K-12 lies within the defective qsr'' prophage.

Recombinants between phage lambda and the defective qsr' prophage of Escherichia coli K-12 were made in an nmpC (p+) mutant strain and in the nmpC+ parent. The outer membrane of strains lysogenic for recombinant qsr' phage derived from the nmpC (p+) strain contained a new protein identical in electrophoretic mobility to the NmpC porin and to the Lc porin encoded by phage PA-2. Lysogens of qsr' recombinants from the nmpC+ strain and lysogens of lambda p4, which carries the qsr' region, did not produce this protein. When observed by electron microscopy, the DNA acquired from the qsr' prophage showed homology with the region of the DNA molecule of phage PA-2 which contains the lc gene. Relative to that of the recombinant from the nmpC (p+) mutant, the DNA molecule of the recombinant from the nmpC+ parent contained an insertion near the lc gene. These results were supported by blot hybridization analysis of the E. coli chromosome with probes derived from the lc gene of phage PA-2. A sequence homologous to the lc gene was found at the nmpC locus, and the parental strains contained an insertion, tentatively identified as IS5B, located near the 3' end of the porin coding sequence. We conclude that the structural gene for the NmpC porin protein is located within the defective qsr' prophage at 12.5 min on the E. coli K-12 map and that this gene can be activated by loss of an insertion element.     

Inactivation of prophage in ultraviolet-irradiated Escherichia coli: dependence on recA gene activity.

The fate of the prophage part of the lysogenic chromosome was followed in the course of post-ultraviolet incubation. For this purpose, lambda cI857 ind prophage, which can be induced by heat but not by ultraviolet light, was used. The prophage, intially more resistant than its repair-proficient host cell, was rapidly inactivated. This inactivation was not caused by the impaired capacity of irradiated cells to support growth of the phage. Over the entire dose range tested, little, if any, sensitivity difference between the host and the prophage was found at the end of cell division delay. Rapid inactivation of the prophage was also observed in uvr cells after small doses of ultraviolet light. The same small doses did not cause inactivation in lysogens carrying a mutation in the gene recA. This suggests that the functional gene recA is required for inactivation of the prophage part of the lysogenic chromosome.     

Effects of DNA Heterologies on Bacteriophage λ Packaging

We have examined the impact of DNA heterologies on the packaging of λ DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI(+) phage and DNA from phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsidate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus. The size of the connector portal could be the basis for this limit.     

Evolutionary coorigin of the RNA polymerase sigma factor and the lambda repressor inferred from gene nucleotide sequence homologies

The lambda phage cI gene and E. coli rpoD gene encoding the lambda repressor and sigma factor, respectively, were aligned with each other based on the internal homologies found in the rpoD gene. Statistical evaluations for these intragenic and intergenic base sequence homologies in the corresponding alignments have conclusively demonstrated that the rpoD gene must have evolved by repeated gene duplications from a primitive gene closely similar to and co-ancestral to the cI gene.     

Yet another way that phage λ manipulates its Escherichia coli host: λrexB is involved in the lysogenic–lytic switch

The life cycle of phage λ has been studied extensively. Of particular interest has been the process leading to the decision of the phage to switch from lysogenic to lytic cycle. The principal participant in this process is the λcI repressor, which is cleaved under conditions of DNA damage. Cleaved λcI no longer acts as a repressor, allowing phage λ to switch from its lysogenic to lytic cycle. The well‐known mechanism responsible for λcI cleavage is the SOS response. We have recently reported that the Escherichia coli toxin‐antitoxin mazEF pathway inhibits the SOS response; in fact, the SOS response is permitted only in E. coli strains deficient in the expression of the mazEF pathway. Moreover, in strains lysogenic for prophage λ, the SOS response is enabled by the presence of λrexB. λRexB had previously been found to inhibit the degradation of the antitoxin MazE, thereby preventing the toxic action of MazF. Thus, phage λ rexB gene not only safeguards the prophage state by preventing death of its E. coli host but is also indirectly involved in the lysogenic–lytic switch.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector

A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation.     

DNA sequence at the end of the cI gene in bacteriophage lambda.

The nucleotide sequence of 57 base pairs near the end of the cI gene in phage lambda is presented. This sequence was determined by direct sequencing techniques and includes the codons for 11 carboxyterminal aminoacids of the cI product, the lambda repressor. The sequence reveals that the cI gene, which has recently been shown to have a unique initiation region, is terminated by a UGA codon. A GUG triplet, which could act as a translation start signal for the rex gene occurs 8 base pairs beyond the cI termination codon. This GUG triplet is preceded by a sequence that could serve as a strong ribosome binding site for the rex message.     

Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light

In targeted mutagenesis of lambda phage by ultraviolet light, the mutations are caused by radiation-induced lesions in the phage DNA. Of 62 mutations in the lambda cI gene that were sequenced, 41 (63%) of the targeted mutations were transitions, with similar numbers of C X G to T X A and T X A to C X G base changes. The remaining 21 mutations were about equally divided among eight transversions, seven frameshifts (5 additions and 2 deletions), and six double events with either two nearby base changes or a base change and a nearby frameshift. Of the 62 mutations, 60 could be associated with -Pyr-Pyr- sequences in the DNA, sites of likely photoproducts. For more information on this point, lambda phage were irradiated with 313 nm light in the presence of acetophenone, for which the major photoproduct is reported to be the thymine-thymine cyclobutyl dimer, with no measurable Pyr(6-4)Pyo photoproducts. Of 22 mutations sequenced, 19 were transversions and only one was a transition, permitting the conclusion that thymine-thymine cyclobutyl dimers are not the primary cause of ultraviolet light-induced transitions. A consideration of all the data strongly suggests that Pyr(6-4)Pyo photoproducts are mutagenic lesions.     

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.     

A model for the recA-dependent repair of excision gaps in UV-irradiated Escherichia coli

DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coli. Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C. Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation. At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.     

PY54, a linear plasmid prophage of Yersinia enterocolitica with covalently closed ends

PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.