Evidence for an early regulatory function in phage P22

Summary A temperature sensitive mutant of P22 phage (ts X) was isolated and studied.This mutant seems to have a basic regulatory function: it is defective in an early function like the typical DNA^- mutant ts 12.1; it is unable to direct the phage DNA synthesis and does not lyse infected or induced cells.Unlike ts 12.1, the mutation ts X seems to involve a gene product necessary for the expression of any vegetative function, since no phage protein synthesis, no alteration of host DNA synthesis, and no cell killing can be observed under non-permissive conditions.The possible functional similarity between the N-cistron of the phage and the present X-cistron in P22 is discussed.     

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome

Summary A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a C31 phage vector into a glk mutant that contained a deletion of the entire homolgous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is there-fore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on the the C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the C31 prophage.     

Novel replication mutant of microvirid phage α3 deleted in the complementary strand origin

Summary The bacteriophage 3 origin of complementary strand DNA synthesis (—ori) contains two potential secondary loop structures (I and II), which have been implicated as direct recognition sites for host Escherichia coli DnaG protein. To elucidate to what extent such structures are essential, we introduced a nucleotide deletion within the —ori region, by nuclease digestion of 3 replicative form DNA. A mutant, delB, thus constructed had a 121 nucleotide deletion within the —ori region and was completely lacking in the two putative hairpin loops, I and II. The delB mutant formed smaller plaques on the host E. coli C and had a longer latent period, but the mean burst size at 37° C was almost the same (400 phages) as that of the wild type. In contrast to the parental phage, growth of the mutant depends on host dnaB and dnaC functions. These results indicate that the prototype secondary structures in the 3 origin of complementary strand synthesis are dispensable for delB and that the 3 mutant has an additional replication origin whose function is dependent on DnaB and DnaC proteins, rather than on DnaG protein alone.     

A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaA, a new gene adjacent to the dnA, gene

Summary An Escherichia coli mutant defective in replication of the chromosome has been isolated from temperature-sensitive mutants that cannot support colicin E1 plasmid DNA synthesis in the presence of chloramphenicol. Cellular DNA synthesis of the mutant ceases almost immediately after transfer to the nonpermissive temperature. The defect is due to a single mutation, dna-59, which is located close to the sites of dnaA mutations and a cou ^R mutation conferring DNA gyrase with resistance to coumermycin. The dna-59 mutant is not able to support DNA synthesis of phage at the high temperature. The mutant also restricts growth of X174 phage at the high temperature, but permits formation of supercoiled closedcircular duplex replicative intermediates. T7 phage can grow on the mutant even at the high temperature.A specialized transducing phage imm ²¹[tna dnaA]#2 (Miki et al., 1978) supports growth of dna-59, dnaA46 and dna-167 cells at the high temperature. Some of the EDTA-resistant derivatives of the phage have lost part or all of the dnaA gene, but carry gene function complementing the defect of dna-59 cells, as judged by conversion of the above dna strains to wild type cells by phage infection, and by suppression of the loss of viability of dna-59 cells at the high temperature by phage infection. The gene containing the dna-59 mutation site is thus distinct from the dnaA gene. Since the dna-59 mutation does not affect expression of the cou ^r gene of DNA gyrase, which is another known gene involved in DNA synthesis near the dnaA gene, this mutation is probably in a new gene, dnaN. From analysis of the suppression activities of imm ²¹[tna dnaA]#2 phage and its deletion derivatives against dnaN59 cells, it is suggested that the expression of the dnaN gene function is reduced by deletion in the dnaA region.     

Sequence homology and recombination between the genomes of morphologically dissimilar bacteriophages LP 52 and theta

Summary A restriction fragment map of Bacillus licheniformis temperate phage LP 52 DNA (molecular weight 38.5×10⁶) was established, using restriction endonucleases BamHI (8 target sites), BglI (10 sites), BglII (13 sites) and EcoRI (22 sites). The map is linear, with well-defined ends, without any signs of circular permutation. The DNA of a related phage, LP 51, produced identical restriction fragments. At least 62% DNA of LP 52 has been found homologous to the DNA of the recently discovered, morphologically quite dissimilar, phage , as demonstrated by hybridization of electrophoretically separated restriction fragments of DNA. Under the same conditions, the DNAs of LP 52 and of the morphologically similar Bacillus subtilis phage 105 did not cross-hybridize. The homologous regions in the genomes of phages LP 52 and have been shown to be colinear. Comparison of the cleavage maps of phages LP 52 and has shown that, within the regions of homology, not a single restriction fragment and few restriction sites have been conserved during divergent evolution. Three major regions of heterology were defined; the longest one, covering the right-hand end of the map (73±2.75% up to 100% LP 52 genome length) appeared to contain genes coding for structural proteins of the virions; a shorter region at the left-hand end of the map (coordinates zero to 10.3±3.3% LP 52 genome length) and a very short central region (coordinates 41.8–43.9%) could be identified, the latter apparently containing a regulatory locus responsible for the heteroimmune behavior of the two phages. Recombinants between phages LP 52 and were isolated. Mapping of recombinant genomes has indicated mutual substitution of allelic pieces of LP 52 and DNAs upon strict conservation of overall genome length.     

Physical mapping of Bacillus subtilis phage rho14 cloning vehicles: heteroduplex and restriction enzyme analyses

Summary Four deletion mutants of temperate Bacillus subtilis bacteriophage 14 have been examined utilizing restriction enzyme and DNA heteroduplex methods. This has allowed the orientation and mapping of the deletions on the 14 physical map. A continuous 15% of the genome contains functions not essential for bacteriophage viability. A 7% subsection section of this region contains phage immunity functions. The deletions were found to range in size from about 2.2–3.3 kilobases. In addition, the deletion mutants retain a single Sal I restriction site, which is currently being used as a cloning site for recombinant DNA. We have located the Sal I site to be 400 basepairs from the immunity region. Thus, the clear-and turbid-plaque deletion mutants are all capable of being utilized as molecular cloning vehicles for Bacillus subtilis.     

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd

Summary Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transposon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 to 1.7 , and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In nondefective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducting phage from different defective clones varied from 0.6 to 3.0 and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.This work was presented at the EMBO Workshop on single-stranded DNA viruses, October 1976, Harpert, The Netherlands     

Isolation and characterization of lambda b221poriCasnA, a plaque-forming specialized transducing phage carrying the origin of replication of the Escherichia coli chromosome

Summary A specialized transducing phage, b221poriCasnA has been isolated carrying oriC the origin of chromosomal replication of Escherichia coli. All phage genes required for lytic growth are retained, thus the phage is capable of lytic growth. The presence of the oriC locus confers upon infecting phage DNA the ability to replicate as a plasmid using only host DNA replication functions. The presence of both oriC and asnA markers has allowed the development of a plaque assay for origin function which can be used to identify mutants at these loci. Comparison of restriction endonuclease cleavage sites present on b221poriCasnA DNA to those on tis parent, b221 rex::Tn10 suggests the steps involved in the formation of the transducing phage.     

Kinetics of induction of error-prone repair of bacteriophage lambda by temperature shift in an Escherichia coli dnaB mutant.

Summary Preincubation at 42^o, before infection at permissive temperature by phage , of an Escherichia coli dnaB mutant, provokes a significant increase in survival and mutagenesis of ultraviolet irradiated phage as well as mutagenesis of untreated phage. Similarly to UV irradiation and many chemical mutagens, the inhibition of DNA synthesis by temperature shift of this dnaB mutant induces SOS repair. This work shows that replication blockage in bacterial DNA is not only mutagenic for bacterial DNA itself (Witkin, 1975) but also for normally replicating DNA, probably due to induction of diffusible products.     

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

Summary A set of dilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing dilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO ⁺, while the ilvA538 allele is trans-recessive to ilvA ⁺. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.     

Mutants of phage HK239 defective in excluding phages lambda T4rII, P1, and P2

Summary Escherichia coli cells lysogenic for temperate phage HK239 exclude phages , HK022, P1 vir, P2, and rII mutants of phage T4. After mutagenic treatment, four isolates were obtained for their inability to exclude T4rII. It is shown that this mutation, designated exc, is located in the prophage HK239, and that, it also abolishes the exclusion of phages , HK022, P1 vir, and P2.     

Identification of a PstI polymorphism in the p21Cip1/Waf1 cyclin-dependent kinase inhibitor gene

A PstI polymorphism in the 3 flanking region of the p21^CiP1/Waf1 cyclin-dependent kinase inhibitor gene is described. DNA sequencing analysis identified a CT base substitution in the 3 flanking region of the gene. This substitution leads to the destruction of a PstI site and results in a biallelic DNA polymorphism. This restriction fragment length polymorphism (RFLP) provides the first known genetic marker for this cell cycle regulatory gene.     

DNA relatedness among bacteriophages of the morphological group C3

Six bacteriophages with an elongated head and a short, noncontractile tail were compared by DNA-DNA hybridization, seroneutralization kinetics, mol% G+C and molecular weight of DNA, and host range. Three phage species could be identified. Phage species 1 containedEnterobacter sakazakii phage C2,Erwinia herbicola phages E3 and E16P, andSalmonella newport phage 7–11. These phages had a rather wide host range (4 to 13 bacterial species). DNA relatedness among species 1 phages was above 75% relative binding ratio (S1 nuclease method, 60°C) when labeled DNA from phage C2 was used, and above 41% when labeled DNA from phage E3 was used. Molecular weight of DNA was about 58×10⁶ (C2) to 67 ×10⁶ (E3). The mol% G+C of DNA was 43–45. Anti-C2 serum that neutralizes all phages of species 1 does not neutralize phages of the other two species. Species 2 contains only coliphage Esc-7-11, whose host range was only oneEscherichia coli strain out of 188 strains of Enterobacteriaceae studied; it was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage Esc-7-11 had a base composition of 43 mol% G+C and a molecular weight of about 45×10⁶. Species 3 contains onlyProteus mirabilis phage 13/3a. Its host range was limited to swarmingProteus species. Species 3 was unrelated to the other two species by seroneutralization and DNA hybridization. DNA from phage 13/3a had a base composition of 35 mol% G+C and molecular weight of about 53×10⁶. It is proposed that phage species be defined as phage nucleic acid hybridization groups.     

High efficiency transduction of single strand plasmid DNA into enteric bacteria

Summary This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli.     

Specificity of bacteriophage Mu excision

Summary To study the excision of bacteriophage Mu at the DNA sequence level, the Mu-derived phage placMu3 was transposed to the transcribed but non-translated leader region of a plasmid-borne tetracycline (tet) resistance gene. Revertants (excision products) were then selected by Tet⁺ restoration of Tet⁺ and characterized. Of 21 independent Tet⁺ revertants, 17 contained simple deletions of most or all of placMu3, while the other four contained more complex rearrangements in which one end of placMu3 had been transposed, and most of the prophage had been deleted. The deletion endpoints were found in short direct repeats in each of the complex rearrangements and in 11 of the 17 simple deletion excisants. The results suggest models of slipped mispairing of template and nascent DNA strands facilitated by proteins of the Mu transposition machinery.     

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Summary Three transducing phages have been isolated from pEDR20, an R100:: cointegrate plasmid in which the insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of . Each phage contains IS1b, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA73, contains the entire r-determinant of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.     

Recombinant P4 bacteriophages propagate as viable lytic phages or as autonomous plasmids in Klebsiella pneumoniae

Summary We demonstrate the use of bacteriophage P4 as a molecular cloning vector in Klebsiella pneumoniae. A hybrid P4 phage, constructed in vitro, that contains a K. pneumoniae hisDG DNA fragment can be propagated either as a lytic viable specialized transducing phage or as an autonomous, self-replicating plasmid. Hybrid P4 genomes existing as plasmids can be readily converted into non-defective P4-hybrid phage particles by superinfection with helper phage P2. Infection of a K. pneumoniae hisD non-P2 lysogen with P4-hisD hybrid phage results in approximately 90% of the infected cells becoming stably transduced to HisD⁺. Because P4 interferes with P2 growth, high titre stocks of P4 hybrid phages are relatively free (10^-6) of P2 contamination. The hisG gene product was detected in ultraviolet light irradiated host cells infected by the P4-hisDG hybrid phage. A mutant of P4 (P4sidl) that directs the packaging of P4 DNA into P2 sized capsids should permit the construction of hybrid phages carrying 26 kilobase inserts.