Genetic characterization of twelve P2-186 hybrid bacteriophages

Sm, Hocking; Jb, Egan

doi:10.1007/bf00384403

Cited by 14 publications

(8 citation statements)

References 9 publications

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“…As mentioned above, the P2-related phage 186 encodes a gene, B, that is functionally analogous to ogr. The B and Ogr proteins share extensive amino acid similarity (62.5% identity [26]), and B can replace ogr as a positive regulator of P2 late genes in P2-186 hybrid phages (25). The B gene is located just downstream from a 186 late operon similar to the FETUD operon of P2.…”

Section: Methodsmentioning

confidence: 99%

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene

1991

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The bacteriophage P2 ogr gene encodes an 8.3-kDa protein that is a positive effector of P2 late gene transcription. The ogr gene is preceded by a promoter sequence (Pogr) resembling a normal Escherichia coli promoter and is located just downstream of a late transcription unit. We analyzed the kinetics and regulation of ogr gene transcription by using an ogr-specific antisense RNA probe in an Si mapping assay. During a normal P2 infection, ogr gene transcription starts from Pgr an intermediate time between the onset of early and late transcription. At late times after infection the ogr gene is cotranscribed with the late FETUD operon; the ogr gene product thus positively regulates its own synthesis from the P2 late promoter PF. Expression of the P2 late genes also requires P2 DNA replication. Complementation experiments and transcriptional analysis show that a nonreplicating P2 phage expresses the ogr gene from Pg, but is unable to transcribe the late genes.A P2 ogr-defective phage makes an increased level of ogr mRNA, consistent with autogenous control from Pogr. Transcription of the ogr gene in the prophage of a P2 heteroimmune lysogen is stimulated after infection with P2, suggesting that Pogr is under indirect immunity control and is activated by a yet-unidentified P2 early gene product during infection.Bacteriophage P2 belongs to a group of noninducible temperate phages of Escherichia coli that differ from the lambdoid phages in both gene organization and regulation (for reviews, see references 1 and 11). P2 early transcription is confined to the right half of the P2 chromosome (16) and initiates at two divergent promoters, P, and Pe, in the early control region (38). Transcription of the left half of the chromosome, where most of the phage morphogenesis genes are located, is induced later in infection (17). The P2 late genes are clustered into four transcription units based on the polar effects of amber mutations (29,46) (Fig. 1). Transcription of these operons depends on the host RNA polymerase (31), the P2 DNA replication genes A and B (17,28,29), and the P2 ogr gene (3,45). The four late operons are all expressed at the same time during a P2 infection, suggesting independent and coordinated transcription initiation from the late promoters (9,10,28). Late transcription initiates from sites that do not resemble promoters normally recognized by the host RNA polymerase (9, 10).The product of the P2 ogr gene is a 72-amino-acid basic protein (4,12,26,27)

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Section: Methodsmentioning

confidence: 99%

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene

1991

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“…Ogr and P4 6, which are capable of acting at each others promoters (Hocking & Egan, 1982a;Sauer et al, 1982). These proteins exhibit highly homologous primary structures (62% amino acid sequence identity between 186 B and P2 Ogr), including a conserved array of four cysteine residues with a C~S -X~-C~S -X~~-C~S -X~-Cys motif expected to coordinate zinc in a zinc-finger structure ( Table 1).…”

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confidence: 99%

Metal‐ and DNA‐binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc‐finger protein

1997

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Coliphage 186 B is a 72-amino acid protein belonging to the Ogr family of analogous transcription factors present in P2-like phage, which contain a C~S -X~-C~S -X~~-C~S -X , -C~S presumptive zinc-finger motif. The molecular characterization of these proteins has been hampered by their insolubility, a difficulty overcome in the present study by obtaining B as a soluble cadmium-containing derivative (CdB). Atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB. The UV absorption spectrum revealed a shoulder at 250 nm, characteristic of CysS-Cd(I1) ligand-to-metal charge-transfer transitions, and the difference absorption coefficient after acidification 24 mM" cm") indicated the presence of a Cd(Cys-S), center. Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA-binding (KD,app 3-4 pM) and the protein was shown to activate transcription in vitro from a promoter-reporter plasmid construct. The B DNA-binding site was mapped by gel shift and DNAase I cleavage protection experiments to an area between -70 and -43 relative to the transcription start site, coincident with the consensus sequence, G'ITGT-N,-TNANCCA, from -66 to -47 of the 186 and P2 late promoters. Inactive B point mutants were obtained in the putative DNA-binding loop of the N-terminal zinc-finger motif and in a central region thought to interact with the Escherichia coli RNA polymerase a-subunit. A truncated B mutant comprising the first 53 amino acids (Bl-53) exhibited close to wild-type activity, showed a DNA-binding affinity similar to that of the full-length protein, and could be reconstituted with either Cd or Zn. Gel permeation analysis revealed that B 1-53 was a majority dimeric species whereas wild-type B showed larger oligomers. 186 B therefore exhibits a potentially linear organization of functional regions comprising an N-terminal C4 zinc-finger DNA-binding region, a dispensable C-terminal region involved in protein self-association, and a central region that interacts with RNA polymerase.

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“…sizes of their gene products in P2 phage tails, FI is believed to code for the sheath protein and FlI is believed to code for the tube protein (55,90). Hybrids of P2 and the P2-like phage 186 that were selected for P2 host range and 186 immunity lack the P2 E, T, U, and D genes, demonstrating that these genes do not play a role in host specificity (43). Elimination of these genes leads to the deduction that the tail fiber genes are located in the VWJIHG transcription unit.…”

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confidence: 99%

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages

1992

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We have determined the DNA sequence of the bacteriophage P2 tail genes G and H, which code for polypeptides of 175 and 669 residues, respectively. Gene H probably codes for the distal part of the P2 tail fiber, since the deduced sequence of its product contains regions similar to tail fiber proteins from phages Mu, P1, lambda, K3, and T2. The similarities of the carboxy-terminal portions of the P2, Mu, ann P1 tail fiber proteins may explain the observation that these phages in general have the same host range. The P2 H gene product is similar to the products of both lambda open reading frame (ORF) 401 (stf, side tail fiber) and its downstream ORF, ORF 314. If 1 bp is inserted near the end of ORF 401, this reading frame becomes fused with ORF 314, creating an ORF that may represent the complete stf gene that encodes a 774-amino-acid-long side tail fiber protein. Thus, a frameshift mutation seems to be present in the common laboratory strain of lambda. Gene G of P2 probably codes for a protein required for assembly of the tail fibers of the virion. The entire G gene product is very similar to the products of genes U and U' of phage Mu; a region of these proteins is also found in the tail fiber assembly proteins of phages TuIa, TuIb, T4, and lambda. The similarities in the tail fiber genes of phages of different families provide evidence that illegitimate recombination occurs at previously unappreciated levels and that phages are taking advantage of the gene pool available to them to alter their host ranges under selective pressures.

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Genetic characterization of twelve P2-186 hybrid bacteriophages

Cited by 14 publications

References 9 publications

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene

Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene

Metal‐ and DNA‐binding properties and mutational analysis of the transcription activating factor, B, of coliphage 186: A prokaryotic C4 zinc‐finger protein

DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages

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