Vegetative DNA of temperate coliphage P2

Summary In order to study P2 DNA synthesis by incorporation of tritium-labeled thymine, mitomycin C has been used to selectively suppress host DNA synthesis. Several conditional lethal mutants of P2 (temperature-sensitive as well as amber) have been tested for their ability to synthesize DNA under non-permissive conditions. Mutants representing the two early genes were found unable to synthesize DNA normally.P2 DNA with the characteristics of covalently closed circular DNA (closed-circular DNA) has been detected during infection with P2. The synthesis of these and of open-circular DNA molecules (=circular DNA duplexes containing one or more single-strand breaks) has been studied. It appears that both closed and open-circular DNA serve as precursors for linear P2 DNA.     

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.     

The old exonuclease of bacteriophage P2.

The Old protein of bacteriophage P2 is responsible for interference with the growth of phage lambda and for killing of recBC mutant Escherichia coli. We have purified Old fused to the maltose-binding protein to 95% purity and characterized its enzymatic properties. The Old protein fused to maltose-binding protein has exonuclease activity on double-stranded DNA as well as nuclease activity on single-stranded DNA and RNA. The direction of digestion of double-stranded DNA is from 5' to 3', and digestion initiates at either the 5'-phosphoryl or 5'-hydroxyl terminus. The nuclease is active on nicked circular DNA, degrades DNA in a processive manner, and releases 5'-phosphoryl mononucleotides.     

Control of gene expression in the P2-related temperate coliphage 186. VI. Sequence analysis of the early lytic region

We have completed the sequence of the 186 early lytic region and established that this region encodes the four genes CP75, CP76, CP77 and CP78, with CP79 the first gene of the next region. Functions have been assigned to the four early genes.     

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

Summary P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12 ^- and P22 23 ^- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24 ^- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L can be turned on by superinfecting P22 24 ^- 12 ^- or P22 24 ^- 23 ^- mutants (and also not by P22 12 ^- and P22 23 ^-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12 ^- or L virB 23 ^- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.     

Characterisation of coliphage K30, a bacteriophage specific for Escherichia coli capsular serotype K30

Abstract Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.