Bacteriophage P1 gene 10 encodes a trans-activating factor required for late gene expression

Abstract

Amber mutants of bacteriophage P1 were used to identify functions involved in late regulation of the P1 lytic growth cycle. A single function has been genetically identified to be involved in activation of the phage-specific late promoter sequence P. In vivo, P1 gene 10 amber mutants fail to trans activate a lacZ operon fusion under the transcriptional control of promoter Ps. Several P1 segments, mapping around position 95 on the P1 chromosome, were cloned into multicopy plasmid vectors. Some of the cloned DNA segments had a deleterious effect on host cells unless they were propagated in a P1 lysogenic background. By deletion and sequence analysis, the harmful effect could be delimited to a 869-bp P1 fragment, containing a 453-bp open reading frame. This open reading frame was shown to be gene 10 by sequencing the amber mutation amlO.1 and by marker rescue experiments with a number of other gene 10 amber mutants. Gene 10 codes for an 18.1-kDa protein showing an unusually high density of charged amino acid residues. No significant homology to sequences present in the EMBL/GenBank data base was found, and the protein contained none of the currently known DNA-binding motifs. An in vivo trans activation assay system, consisting of gene 10 under the transcriptional control of an inducible promoter and a gene SllacZ fusion transcribed from Ps, was used to show that gene 10 is the only phage-encoded function required for late promoter activation.The temperate bacteriophage P1 propagates on several enterobacterial species, including Escherichia coli (64). The linear DNA molecule carried in a phage particle is about 100 kb in size and has a terminal redundancy of 8 to 12% (32, 65). As a prophage, P1 is a low-copy-number plasmid of about 90 kb (3, 33).The genome of P1 is organized in relatively short transcriptional units and in this respect resembles the virulent bacteriophage T4 (43) rather than the temperate lambdoid phages (57). For example, P1 genes for both immunity control and morphogenesis are widely dispersed over the genome (50,62,63). The presence of at least 14 operator sites for the P1 cI repressor protein (8,15,16,28,29,59) and the tripartite immunity system of the phage (64) indicate the degree of complexity which P1 faces in regulating and expressing its genetic information.While the lysogenic condition of P1 has been studied intensively (for a recent review, see reference 64), relatively little is known about the regulation of the lytic growth cycle. A few functions which are under transcriptional control of the P1 cI repressor have been identified (8,10). Among those is the repL function, which is essential for the lytic replication of the phage genome (9,25,52).Recently, a new class of P1 promoters, controlling the late genes encoded by the tail fiber and the dar operons, has been identified (21,22

Genome of Bacteriophage P1

“…Genes of the lytic pathway have been divided into early and late. Transcription of the late genes requires, in addition to bacterial RNA polymerase, a P1-encoded activator protein, Lpa (188), and an E. coli RNA polymeraseassociated stringent starvation protein, SspA (111).…”

The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III θ subunit, is expressed during both lysogenic and lytic growth stages

“…Previously, this gene was indicated to be a late phage gene [14,15], a possibility not readily reconciled with a replication function for the Hot protein. Our present results clearly indicate that hot is not a typical late gene like, as exemplified, for example, by gene 16 in this study.…”

“…The previous studies classifying hot as a late gene were based on its promoter structure, containing −22 and −10 elements, rather than −35 and −10 elements, which serve as recognition elements for the late-gene specific P1 transcription factor gp10 [6,14,15]. The hot gene is unusual among this group of genes in that the spacing between the −10 and −22 regions is nine instead of the canonical four [6].…”

“…Of interest in this respect is the previous description of hot as a "late" viral gene [6,14,15]. This classification as a late gene is based on its unusual promoter structure (containing a −10 and −22 consensus sequence rather than the normal −10 and −35 sequence), which has been associated with expression of late phage genes [14,15]. This implies that Hot might not be expressed until the phage packaging stage when DNA replication has largely ceased, a somewhat unusual expression profile for a presumed replication protein.…”

Three functions of bacteriophage P1 involved in cell lysis

“…In the cycle of vegetative reproduction of the temperate bacteriophage P1, a number of late functions become activated by the product of gene 10 (16,18). Two phage-specific late promoter sequences, P S and LPdar, from which the tail-fiber operon RSU and the dar operon (defense against restriction), respectively, are expressed were identified (10,11,17).…”