Preparation of plasmids from lambdoid phages and studies on their incompatibilities.

Various lambdoid phages, including those carrying the immunity region of 434, 21, and φ80, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm21dv, and φ80dv, were similar to the already known plasmid λdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids λdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.     

Differences in host controlled reactivation of lambdoid and T phages

Summary Phages 434, T4, T5 and T7 are studied with regard to host controlled reactivation of damage produced by UV or photodynamic action sensitized by thiopyronine. Repair of 434 phages proceeds under control of both hcr and rec genes. UV irradiated T5 and T7 phages are reactivated under control of the host's hcr genes only. If these phages are inactivated by photodynamic action they are reactivated not at all. T4 phages inactivated by both treatments are also refractory to host controlled reactivation. These differences might reflect different degrees of autarchy and different abilities of phage DNAs to serve as substrate for recombination enzymes of the host.These results were presented in an abstracted version at the V. International UV colloquium Grundlagen der UV-Wirkung, Kühlungsborn, DDR, in October 1969. The experiments with T5 were done by Mrs. E. Marx.     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.     

Recombination in the genesis and evolution of hepatitis B virus genotypes

Hepatitis B virus (HBV) infection is widely distributed in both human and ape populations throughout the world and is a major cause of human morbidity and mortality. HBV variants are currently classified into the human genotypes A to H and species-associated chimpanzee and gibbon/orangutan groups. To examine the role of recombination in the evolution of HBV, large-scale data retrieval and automated phylogenetic analysis (TreeOrder scanning) were carried out on all available published complete genome sequences of HBV. We detected a total of 24 phylogenetically independent potential recombinants (different genotype combinations or distinct breakpoints), eight of which were previously undescribed. Instances of intergenotype recombination were observed in all human and ape HBV variants, including evidence for a novel gibbon/genotype C recombinant among HBV variants from Vietnam. By recording sequence positions in trees generated from sequential fragments across the genome, violations of phylogeny between trees also provided evidence for frequent intragenotype recombination between members of genotypes A, D, F/H, and gibbon variants but not in B, C, or the Asian B/C recombinant group. In many cases, favored positions for both inter- and intragenotype recombination matched positions of phylogenetic reorganization between the human and ape genotypes, such as the end of the surface gene and the core gene, where sequence relationships between genotypes changed in the TreeOrder scan. These findings provide evidence for the occurrence of past, extensive recombination events in the evolutionary history of the currently classified genotypes of HBV and potentially in changes in its global epidemiology and associations with human disease.     

The integration-excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages

Excision of Tn1545 and related conjugative transposons of Gram-positive bacteria occurs by reciprocal site-specific recombination between non-homologous regions of the transposon-target junctions. Excisive recombination requires two transposon-encoded proteins designated Xis-Tn and Int-Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon-target junctions. Using a trans-complementation assay, we have demonstrated that Int-Tn is able to catalyse in vivo integration of a circular intermediate of Tn1545 defective for integration and excision. comparison of integration sites suggests that limited sequence homology at the vicinity of the recombining sites is required for integration of the element. These data support the hypothesis that the integration/excision systems of conjugative transposons from Gram-positive cocci and of lambdoid phages from Gram-negative bacilli have evolved from a common ancestor.     

Evidence for the exchange of segments between genomes during the evolution of lambdoid bacteriophages

Heteroduplexes between the DNA molecules of 12 lambdoid phages were analysed by electron microscopy. The positions of the regions of base sequence homology between the DNA molecules divide them into 35 segments, most of which have a number of alternative forms (alleles), which in general must be functionally homologous but which differ in base sequence and length. The positions of the boundaries between segments in phage lambda show that each segment is probably a gene or a group of genes, and that each phage genome is a different combination of the alleles of the segments. The frequency of the occurrence of the different alleles indicates that the total number in the natural population may be small. The different combinations of alleles of separate segments, found among the phages, indicate the exchange of segments between the phages during their evolution.     

Three new temperate phages of Bacillus subtilis

Three temperate bacteriophages of Bacillus subtilis were isolated from soil samples and analysed, together with all the other known temperate phages of this organism, with respect to their host range, immunity, serology and DNA restriction pattern, and by other tests. The results show that the newly isolated phages are new members of the immunity sub-group I of the group III of B. subtilis temperate bacteriophages. We named these new phages IG1, IG3 and IG4.     

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.