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S Zangrossi

Publications and source records attributed to S Zangrossi.

13 recordsLinked to original sources
PubMed2000

Transcriptional and post-transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli.

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock-induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures. We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10-fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift-like pattern in a PNPase-independent manner. The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs. The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase. In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho-dependent termination site within pnp. In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.

Acclimatization↗
PubMed1996

A Rho-dependent transcription termination site regulated by bacteriophage P4 RNA immunity factor.

The genes required for replication of the temperate bacteriophage P4, which are coded by the phage left operon, are expressed from a constitutive promoter (PLE). In the lysogenic state, repression of the P4 replication genes is achieved by premature transcription termination. The leader region of the left operon encodes all the genetic determinants required for prophage immunity, namely: (i) the P4 immunity factor, a short, stable RNA (CI RNA) that is generated by processing of the leader transcript; (ii) two specific target sequences that exhibit complementarity with the CI RNA. RNA-RNA interactions between the CI RNA and the target sites on the mRNA leader region are essential for transcription termination. To understand how transcription termination is elicited by the P4 immunity mechanism, it is relevant to identify the transcription termination site. This, however, could not be directly inferred from the 3'-end of the transcription products because of the extensive and complex processing and degradation of the leader RNA. In this work, by making use of a tRNA gene as a reporter, we identify the termination site of the immunity transcripts (timm). This is a Rho-dependent terminator located within the first translated gene of the left operon and is regulated by P4 immunity. Analysis of the P4 transcription pattern in Escherichia coli rho mutants suggests that termination at timm may also be important for the efficient processing of the CI RNA.

Base Sequence↗
PubMed1996

Immunity specificity determinants in the P4-like retronphage phi R73.

Retronphage phi R73 exhibits extensive sequence homology to the satellite bacteriophage P4. Bacteriophage P4 superinfection immunity is elicited by a small RNA (CI RNA) that causes premature transcription termination within the operon coding for the P4 replication functions. This control is exerted via interaction of the CI RNA with two complementary target sites on the untranslated leader RNA of the replication operon. We found that phi R73 is endowed with a similar immunity system but is heteroimmune to P4. The heteroimmunity is due to six base differences in the CI RNA and to compensatory base substitutions in the target sequences. The sequence differences in the CI RNA are all located in single-stranded regions, which appear to play a predominant role in the interaction with the target sites. Analysis of phage carrying a hybrid immunity system indicates that, although two target sequences are required for the establishment of lysogeny, a single site is sufficient to make a phage sensitive to the prophage immunity.

Bacteriophages↗
PubMed1995

Immunity determinant of phage-plasmid P4 is a short processed RNA.

In the phage-plasmid P4, both lysogenic and lytic functions are coded by the same operon. Early after infection the whole operon is transcribed from the constitutive promoter PLE. In the lysogenic condition transcription from PLE terminates prematurely and only the immunity functions, which are proximal to the promoter, are thus expressed. Fragments of the P4 immunity 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. In an RNase P mutant host the processing of the 5' end and the production of a functional CI RNA were impaired. The requirement of RNase P for the correct processing of CI appears to be related to the predicted secondary structure of the precursor CI RNA. A region (seqB) within the CI RNA shows complementarity with two cis-acting sequences (seqA and seqC) required for P4 immunity, suggesting that transcription termination may be caused by pairing of the CI RNA with the complementary target sequences on the nascent transcript.

Base Sequence↗
PubMed1995

Multiple regulatory mechanisms controlling phage-plasmid P4 propagation.

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 alpha 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.

Bacteriophage P1↗
PubMed1992

Genetic analysis of the immunity region of phage-plasmid P4.

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.

Base Sequence↗
PubMed1992

Bacteriophage P4 immunity controlled by small RNAs via transcription termination.

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 PLE. 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 trans-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.

Coliphages↗
PubMed1988

Alternative promoters in the development of bacteriophage plasmid P4.

Infection of Escherichia coli with the satellite virus P4 without its helper bacteriophage P2 leads either to the immune integrated state or to the nonimmune multicopy plasmid condition. We analyzed the transcription pattern of the phage plasmid P4 early and late after infection and during the stable plasmid or lysogenic condition. The early postinfection phase is characterized by the leftward transcription of an operon including the genes cI (P4 immunity) and alpha (replication). This early transcript starts from the promoter PLE, which shows a good homology with the E. coli sigma 70 promoter. At later times, the transcription of this operon starts from a different promoter, PLL, located 400 base pairs upstream of PLE, and sharing little homology with the canonical E. coli promoter sequence; a longer transcript encoding an additional open reading frame is thus produced. PLL shares two boxes of homology with the P4 late promoter PSID, positively regulated by the P4 delta gene product, and depends on delta function for its full activation. In the multicopy plasmid state, the transcription pattern is similar to that observed at late times after infection. Since in the plasmid state not only is P4 immunity not expressed but its establishment is prevented, even though the P4 cI gene is transcribed, the P4 cI function may be regulated at the posttranscriptional level. In the immune state, transcription starts from PLE but does not continue to cover the P4 alpha gene. This suggests that P4 immunity acts by prematurely terminating transcription initiated at PLE.

Base Sequence↗
PubMed1986

Regulation of the plasmid state of the genetic element P4.

After infection of sensitive cells in the absence of a helper phage, the satellite bacteriophage P4 enters a temporary phase of uncommitted replication followed by commitment to either the repressed-integrated condition or the derepressed-high copy number mode of replication. The transient phase and the stable plasmid condition differ from each other in the pattern of protein synthesis, in the rate of P4 DNA replication and in the expression of some gene functions. The regulatory condition characteristic of the P4 plasmid state affects a superinfecting genome, preventing the establishment of the P4 immune condition.

Coliphages↗
PubMed1984

Plasmid mode of propagation of the genetic element P4.

The satellite bacteriophage P4, in the presence of a helper phage, can enter either the lytic or the lysogenic cycle. In the absence of the helper, P4 can integrate in the bacterial chromosome. In addition, the partially immunity-insensitive mutant P4 vir1 can be maintained as a plasmid. We have found that in the absence of the helper, P4 wt also can be maintained as a plasmid, and that both P4 wt and P4 vir1 have two options for their intracellular propagation: a repressed-integrated or a derepressed-high copy number plasmid mode of maintenance. In the repressed mode, the P4 wt genome is only found integrated into the bacterial chromosome, while the P4 vir1 is found also as a low copy number plasmid coexisting with the integrated P4 vir1 genome. The clones carrying P4 in the derepressed-high copy number plasmid state are obtained at low frequency (0.3%) upon infection with P4 wt, while the vir1 mutation increases this frequency about 300-fold. Such clones can be distinguished easily because of their typical colony morphology (rosettes), due to the presence of filamentous cells. Filamentation of the bacterial host suggests that the presence of derepressed P4 genomes in high copy number interferes with the normal cell division mechanism. The derepressed clones are rather stable, but may revert spontaneously to the repressed state. Spontaneous transition from the repressed to the derepressed state was not observed; however, it can be induced by P2 or P4 vir1 superinfection of P4 wt and P4 vir1 lysogens or by growing the P4 vir1 lysogens up to the late exponential phase. The ability of P4 to choose either of two stable states and the potential to shift between these two modes of propagation indicate that the synthesis of the immunity repressor is regulated.

Cloning, Molecular↗
PubMed1983

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.

Coliphages↗
PubMed1979

X-ray sensitivity of Escherichia coli lysogenic for bacteriophage P2.

Strains of Escherichia coli C or K lysogenic for the non-inducible phage P2 show a lower survival following X-ray irradiation as compared to nonlysogenic strains. This difference in X-ray sensitivity is not accompanied by a significant difference in X-ray induced mutability. The capacity of X-irradiated P2 lysogens to multiply any of a number of unirradiated infecting phages is severely impaired. These effects of X-ray treatment can be most simply explained as a consequence of the fact that protein and RNA syntheses are strongly inhibited in P2 lysogens after X-irradiation. All the above events specifically occurring in X-rayed P2 lysogens are dependent on the P2 gene old.

Bacterial Proteins↗