A mutation of the transactivation gene of satellite bacteriophage P4 that suppresses the rpoA109 mutation of Escherichia coli

Abstract

Satellite bacteriophage P4 requires the products of the late genes of a helper such as P2 in order to grow lytically. The Escherichia coli rpoA109 mutation, which alters the a subunit of RNA polymerase, prevents transcription of the late genes of bacteriophage P2. Suppressor mutations that define the P2 ogr gene overcome this block. We found that P4 lytic growth using a P2 ogr+ prophage helper was prevented by the rpoA109 mutation but that this block was overcome when the P2 helper carried the suppressor mutation in the ogr gene. Furthermore, we isolated and characterized four independent mutations in P4, called org, that suppress the E. coli rpoAl09 mutation by allowing P4 lytic growth using a P2 ogr+ helper. DNA sequence analysis revealed that the four independent org mutations are identical and that they occur in the P4 8 gene, which codes for a factor that positively regulates the transcription of the P2 and P4 late genes. 8 is predicted to Fig. 1. In the lytic pathway, P4 must obtain the products of the P2 late genes in order to make the phage particle and lyse the bacterial host, Escherichia coli (51, 52). Transcription of the P2 late genes in the absence of P4 requires P2 DNA replication and the product of the P2 ogr gene (8,9,18,37). P4 facilitates its lytic growth in two ways: (i) by derepressing a P2 prophage, which leads to P2 DNA replication and subsequent expression of the P2 late genes; and (ii) by directly activating the transcription of the P2 late genes in a process termed transactivation. P4 is also able to transactivate the late genes of a replication-deficient P2 helper (51,53,55). Transactivation is controlled by the P4 8 gene, which codes for a positive transcription factor that acts at the late promoters of the helper phage (8,9,48,55). When P4 uses a replicationdeficient helper, a functional 8 gene is essential for lytic multiplication (24, 55).Transcription of the late genes of bacteriophage P2 is dependent on the bacterial RNA polymerase (36). P2 late gene transcription is blocked by the E. coli rpoA109 mutation, which substitutes a histidine for a leucine residue in the a subunit of RNA polymerase (16,56). Dominant mutations of P2 were isolated that suppressed rpoA109 by restoring transcription of the P2 late genes, and these mutations defined the P2 ogr gene (6,10,56 during coinfection with a wild-type P2 helper (48). We report here the isolation and characterization of four independent P4 mutants that overcome the rpoA109-imposed block. MATERIALS AND METHODSMedia and buffers. L broth, which was used for growth of bacteria, contained 1% tryptone, 0.5% yeast extract, and 0.5 or 1% NaCl. LGCM, which was used for growing phage, contained L broth supplemented to 0.2% glucose, 1 mM CaCl2, and 2 mM MgCl2. LC agar, which was used for plating P2 and P4 phage, contained L broth supplemented to 2.5 mM CaCl2 and 1% agar. Antibiotics were added to media when necessary to a final concentration of 200 ,ug/ml for streptomycin, 50 ,ug/ml for ampicillin, and 15 ,ug/ml for tetracycline. P2 phage was...

Association of a retroelement with a P4-like cryptic prophage (retronphage phi R73) integrated into the selenocystyl tRNA gene of Escherichia coli

“…It appears to be composed of two homologous domains, which show sequence similarity to the 72-residue P2 org gene product (17). It will be interesting to test whether the pro-retronphage 8 gene product functions as a transcriptional activator like the P2 Ogr and the P4 8 gene product.…”

Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

“…The rpoA341 mutation interferes with expression of the cysA locus, araBAD operon, and mel operon (14). The rpoA109 mutation inhibits the expression of late genes of P2 (10) and P4 (11) phages and is suppressed by mutation in the P2 ogr gene (10) or in the P4 8 gene (33), both of which encode transcriptional activators of the late genes. The rpoA341 and rpoA109 mutations cause a single amino acid substitution at residue 271 (35) and at residue 289 or 290 (34), respectively, both substitution sites being within the C-terminal region of the a subunit.…”

Mutations in rpoA affect expression of anaerobically regulated genes in Salmonella typhimurium

“…(i) The E. coli mutation rpoA109 that interferes with late gene expression in phage P2 (42) and its satellite P4 (15) changes to His (7,10). This mutation appears to block the action of the positive transcriptional activators of late gene promoters encoded by the ogr (P2) and 8 (P4) genes (14,15).…”

“…(i) The E. coli mutation rpoA109 that interferes with late gene expression in phage P2 (42) and its satellite P4 (15) changes to His (7,10). This mutation appears to block the action of the positive transcriptional activators of late gene promoters encoded by the ogr (P2) and 8 (P4) genes (14,15). (ii) The rpoA341 (phs) mutation of E. coli causes a pleiotropic growth defect including a requirement for cysteine or methionine, an inability to grow on melibiose, and a substantially reduced rate of growth on arabinose (13,34).…”