Coliphage HK243: Biological and Physicochemical Characteristics

Coliphage HK243 can form plaques on Escherichia coli C and K-12, but not B. The plaques are 1–2 mm in diameter and are opaque areas which clear upon exposure to chloroform vapor. During one-step growth, the eclipse and the latent periods are 20 and 30 min, respectively. Phage-infected cells continue to produce cell-free plaque-forming units for as long as 80 min after the end of the latent period, although at high multiplicities of infection (MOI) most cells lyse. No lysogenic bacteria have been found among survivors, so HK243 is considered a virulent phage. Some of the cells surviving a high MOI challenge are maltose negative and resistant to both HK243 and coliphage lambda. This fact has made possible the isolation of lambda-resistant mutants of lambda-lysogens. However, no serological cross-reaction between the phages lambda and HK243 has been detected. Genetic data involving three essential loci and a locus controlling plaque morphology suggest a circular linkage map. The virions are tadpole-shaped with an icosahedral head 68 nm long which is attached to a flexible tail 131 nm long. The phage has a linear, duplex DNA genome of molecular weight approximately 44 × 10⁶ and a base composition of 33% adenine, 31% thymine, 16% guanine, and 20% cytosine.     

Lambdoid Coliphage HK139 Integrates Between his and supD

Phage HK139 is UV inducible and lambda homoimmune and has the host range of phi80. It can recombine with lambda as well as with phi80, and in the prophage form it is found integrated between the loci his and supD.     

Position and direction of strand exchange in bacteriophage HK022 integration

The positions of the endonucleolytic cleavages promoted by the integrase protein (Int) of coliphage HK022 within its attB site were determined. The protein catalyses a staggered cut, which defines an overlap sequence of 7 by within the core site. The overlap region is at the center of symmetry of a palindromic sequence which appears in all four putative att core binding sites for Int. We confirm that the order of strand exchange is similar to that in phage .     

Specificity determinants in the attachment sites of bacteriophages HK022 and lambda.

The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.     

Solution structure and stability of the full-length excisionase from bacteriophage HK022.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the lambda-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28-->Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined alpha-helices (residues 5-11 and 18-27) and five beta-strands (residues 2-4, 30-31, 35-36, 41-44 and 48-49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage lambda excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical 'winged-helix' DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51-72, which were not resolved in the lambda Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Nitrate Nonutilizing Mutants and Vegetative Compatibility Groups in Fusarium poae

Nitrate nonutilizing (nit) mutants were recovered from 24 isolates of Fusarium poae and used to force heterokaryons between these isolates and to determine vegetative compatibility. Between 30 and 90% of the mycelial blocks, cultured on medium containing chlorate, produced nit mutants. The amount of chlorate in the medium altered the frequency and spectrum of nit mutants recovered. Most of the mutants (63%) had lesions at a nitrate reductase structural locus (nit1). Another 30% were mutants at one or more loci that control the production of a molybdenum-containing cofactor necessary for nitrate reductase activity (NitM). A few (6%) of the mutations occurred in a regulatory gene specific for the nitrate reduction pathway (nit3). Pairings between nit1 and NitM mutants were made on minimal medium containing nitrate as the sole nitrogen source. A mutant grows thinly unless it forms a complementary heterokaryon upon contact with another mutant. Heterokaryon formation was indicated by dense growth where the two mutant colonies touched. The 24 isolates could be divided into 13 nonoverlapping vegetative compatibility groups, suggesting that asexual exchange of genetic information within F. poae is subject to significant limitations.     

Temperate coliphage HK253: attachment site and restricted transduction of proAB mutants of Escherichia coli K-12.

Temperate coliphage HK253 integrates near the proAB locus on the Escherichia coli K-12 chromosome. It can bring about specialized transduction of proAB and phoE mutants of E. coli, but it is incapable of general transduction. One of the proline-transducing particles was found to be nondefective.     

Vegetative Replication and Transfer Replication of Deoxyribonucleic Acid in Temperature-Sensitive Mutants of Escherichia coli K-12

Crosses were carried out at 34 C and 42 C between eight pairs of isogenic strains of Escherichia coli K-12. The donor and recipient of each pair carried the same mutation for temperature-sensitive deoxyribonucleic acid (DNA) synthesis; they differed only in the presence of F-lac in the donor and a spectinomycin-resistance marker in the recipient. A different temperature-sensitive mutation was present in each of the eight pairs, the eight temperature-sensitive mutations being located in at least two different genes. In all eight pairs, the transfer of F-lac occurred at high and equal rates at 34 C and 42 C, although vegetative DNA replication at 42 C was approximately 10⁻⁴ of that at 34 C. The transfer of F-lac at 42 C was accompanied in seven of the eight crosses by an equivalent amount of DNA synthesis in excess of that observed in the unmated controls. The DNA synthesized during transfer at 42 C was characterized by equilibrium centrifugation in cesium chloride and by its sedimentation velocity in sucrose gradients. It was found to have a density and a molecular weight characteristic of F-lac DNA. A small proportion of the material labeled during transfer was recovered in the form of covalently closed DNA. It is concluded that vegetative replication of the chromosome and transfer replication of F are separate processes, the former requiring at least two gene products which are nonessential for the latter.     

Isolation and Preliminary Characterization of Temperature-Sensitive Mutants of Rhizobiophage C

By using mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine, 150 temperature-sensitive mutants of rhizobiophage c were isolated. All were able to form plaques at 14 C but not at 29 C. They were classified into 21 complementation groups. Representative temperature-sensitive mutants from each complementation group were analyzed with regard to gene function. The approximate time of expression of the genes defined by the mutants was measured by temperature-shift experiments. Most genes began to be expressed at 4.0 to 7.5 h after infection at 14 C. Four genes were found which were expressed 2.5 to 3.5 h after infection. Some mutants showed no DNA synthesis at 29 C; some showed a delay in lysis. Some produced apparently normal particles after infection and lysis at 29 C; others produced various types of defective particles. Some mutants showing no DNA synthesis at 29 C nevertheless caused lysis at the normal time together with the production of phage structural components. Representative mutants from each complementation group were mapped by using two-factor crosses. A preliminary genetic map of phage c was constructed from the data.     

Development of Coliphage T5: Ultrastructural and Biochemical Studies

Electron microscopic studies of Escherichia coli infected with bacteriophage T5(+) have revealed that host nuclear material disappeared before 9 min after infection. This disappearance seemed to correspond to the breakdown of host deoxyribonucleic acid (DNA) into acid-soluble fragments. Little or no host DNA thymidine was reincorporated into phage DNA, except in the presence of 5-fluorodeoxyuridine (FUdR). Progeny virus particles were observed in the cytoplasm 20 min postinfection. Most of these particles were in the form of hexagonal-shaped heads or capsids, which were filled with electron-dense material (presumably T5 DNA). A small percentage (3 to 4%) of the phage heads appeared empty. On rare occasions, crystalline arrays of empty heads were observed. Nalidixic acid, hydroxyurea, and FUdR substantially inhibited replication of T5 DNA. However, these agents did not prevent virus-induced degradation of E. coli DNA. Most of the phage-specified structures seen in T5(+)-infected cells treated with FUdR or with nalidixic were in the form of empty capsids. Infected cells treated with hydroxyurea did not contain empty capsids. When E. coli F was infected with the DO mutant T5 amH18a (restrictive conditions), there was a small amount of DNA synthesis. Such cells contained only empty capsids, but their numbers were few in comparison to those in cells infected under permissive conditions or infected with T5(+). The cells also failed to lyse. These results confirm other reports which suggest that DNA replication is not required for the synthesis of late proteins. The data also indicate that DNA replication influences the quantity of viral structures being produced.     

Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.