Alternative promoters in the development of bacteriophage plasmid P4 (original) (raw)
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Genetic analysis of the immunity region of phage-plasmid P4
Molecular Microbiology, 1992
In the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418-8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity-defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.The immunity region covers PLE and the cl locus. We were unable to obtain evidence of translation of the region, thus we suggest that P4 immunity is not elicited by a protein but by a transcript (or transcripts) encoded by the region downstream of the promoter PLE, The promoter PLE appears to be necessary for the expression of P4 immunity: fragments in which the PLE region is deleted did not complement P4 cl405 for lysogenization, although they still interfered with P4 growth. Two complementary sequences downstream of PLE(seqA and seqB) at the 5’and 3’ends of the immunity region play an essential role in the control of P4 immunity.
Multiple regulatory mechanisms controlling phage-plasmid P4 propagation
Fems Microbiology Reviews, 1995
Abstract: Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic / plasmid vs. the lysogenic condition is the regulation of P4 α operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE. PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.
Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli
Journal of Molecular Biology, 1981
We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 300/b of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gpll , before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 D?r'A have @loi, and mini cells have lTO of the gpll levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gpl0 and gpll formed from the cloned genes after T4 infection.
Bacteriophage P4 immunity controlled by small RNAs via transcription termination
Molecular Microbiology, 1992
Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post-infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity. In order to understand how P4 immunity is elicited, we have characterized the transcription pattern during the establishment and the maintenance of the satellite phage P4 lysogenic condition. We found that prophage transcription starting at PLE ends prematurely and the transcripts do not extend beyond 300-400 nucleotides downstream of P|.E-Thus P4 immunity acts by causing premature transcription termination rather than by repressing transcription initiation. The P4 immunity region is transcribed in the prophage, but it does not seem to be translated; this region contains two elements {seqA and seqB) of a palindromic sequence. In addition to transcripts about 300 nucleotides long, P4 prophage produces a family of shorter transcripts, about 80 nucleotides long, containing seqA or seqB. Evidence is presented suggesting that SeqB RNA is the frans-acting immunity factor, and that interaction of SeqB RNA with the complementary nascent RNA containing seqA may be involved in bringing about premature transcription termination.
Genetic analysis of bacteriophage P4 using P4-plasmid ColE1 hybrids
MGG Molecular & General Genetics, 1980
A set of plasmids that contain fragments of the bacteriophage P4 genome has been constructed by deleting portions of a P4-ColE1 hybrid. A P4 genetic map has been established and related to the physical map by examining the ability of these plasmids to rescue various P4 mutations. The P4 virl mutation and P4 genes involved in DNA replication (~), activation of P2 helper genes (3 and e), polarity suppression (psu) and head size determination (sid) have been mapped, as has the region responsible for synthesis of a nonessential P4 protein. One of the deleted plasmids contains only 5900 base pairs (52%) of P4 but will form plaques if additional DNA is added to increase its total size to near that of P4. This plasmid is also unique in that it will not form stable associations with P2 lysogens of E. coli which are recA +. P4 c~ mutants can be suppressed as a result of replication under control of the ColE1 part of the hybrid.
Immunity Determinant of Phage-plasmid P4 is a Short Processed RNA
Journal of Molecular Biology, 1995
In the phage-plasmid P4, both lysogenic and lytic functions are coded by the Dipartimento di Gentica e di Biologia dei Microrganismi same operon. Early after infection the whole operon is transcribed from the constitutive promoter P LE . In the lysogenic condition transcription from P LE Università di Milano terminates prematurely and only the immunity functions, which are Via Celoria 26, 20133 Milano proximal to the promoter, are thus expressed. Fragments of the P4 immunity Italy region were cloned in an expression vector. A DNA fragment as short as 91 bp was sufficient, when transcribed, to express P4 immunity and to complement P4 immunity deficient mutants. This fragment, like prophage P4, produced a 69 nt long RNA (CI RNA). A shorter P4 fragment neither expressed immunity nor synthesized the CI RNA. Thus the CI RNA is the P4 trans-acting immunity factor. The 5' end of the CI RNA, mapped by primer extension, does not correspond to the transcription initiation point, thus suggesting that the CI RNA is produced by processing of the primary transcript.
Deletion analysis of a bacteriophage P2 late promoter
Gene, 1990
We have fused the promoter (PF) for the P2 late FETUD oceron to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, 6. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpod 109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of Pv were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides-69 and-64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.
Prophage P4 immunity is elicited by a short, 69-nucleotide RNA (CI RNA) coded for within the untranslated leader region of the same operon it controls. CI RNA causes termination of transcription that starts at the promoter P LE and prevents the expression of the distal part of the operon that codes for P4 replication functions (␣ operon). In this work, we identify two sequences in the untranslated leader region of the ␣ operon, seqA and seqC, that are the targets of the P4 immunity factor. seqA and seqC exhibit complementarity to a sequence internal to the CI RNA (seqB). Mutations in either seqA or seqC that alter its complementarity to seqB abolished or reduced P4 lysogenization proficiency and delayed the shutoff of the long transcripts originating from P LE that cover the entire operon. Both seqA and seqC single mutants were still sensitive to P4 prophage immunity, whereas P4 seqA seqC double mutants showed a virulent phenotype. Thus, both functional sites are necessary to establish immunity upon infection, whereas a single site appears to be sufficient to prevent lytic gene expression when immunity is established. A mutation in seqB that restored complementarity to both seqA and seqC mutations also restored premature termination of P LE transcripts, thus suggesting an important role for RNA-RNA interactions between seqB and seqA or seqC in P4 immunity.
Cloning of the immunity repressor determinant of bacteriophage P2 in the pBR322 plasmid
MGG Molecular & General Genetics, 1980
Through in vitro recombination of DNA restriction fragments, we have constructed a plasmid, which expresses in vivo the immunity repressor gene (C) of bacteriophage P2. A bacterial strain carrying such a plasmid showed a high level of P2 specific immunity. It was lysogenized normally by an infecting P2, but the frequency of spontaneous phage production was reduced about 10 4 fold as compared to a normal P2 lysogen. Satellite phage P4, known to derepress P2 lysogens, was unable to derepress the plasmid-carrying lysogenic strain so to allow growth of coinfecting P2. Phage P4 multiplied on the plasmidcarrying, P2-1ysogenic strain, but due to a prolonged latent period failed to form plaques on this strain.