Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4.

DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control.

Identification of a phage-coded DNA-binding protein that regulates transcription from late promoters in bacteriophage P4.

The genetic element P4 can propagate as a temperate phage or as a multicopy plasmid in its host Escherichia coli. Late in the lytic cycle and in the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P(LL) and P(sid), which control the transcription of the left and right operons, respectively. Both P4 late promoters are positively regulated by the product of the P4 delta gene, which is transcribed from P(sid). We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control. vis is the first gene downstream of P(LL) and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif. Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P(LL). By Northern hybridization analysis we showed that vis negatively regulates transcription from P(LL) and enhances transcription from P(sid). Thus, vis auto-regulates its expression by repressing its own promoter and enhancing transcription of delta, which is required for P(LL) activation. The vis gene was fused with the glutathione S-transferase gene and the GST-Vis fusion protein was partially purified. By gel retardation assays and DNA footprinting we demonstrated that GST-Vis binds to a 32 bp long region immediately downstream of P(LL). We also showed, by gel retardation, that GST-Vis binds to the P sid region. A sequence present in both P(LL) and P(sid) regions may represent the Vis binding consensus sequence. The dual role of Vis on the control of P4 late transcription may be required for a regulated expression of the replication functions when P4 propagates in the plasmid state.

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.

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.

Organisation of the tmb catabolic operons of Pseudomonas putida TMB and evolutionary relationship with the xyl operons of the TOL plasmid pWW0.

In Pseudomonas putida (Pp) TMB the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pWW0, but are chromosomally encoded. We have analysed by cloning. Southern blotting and sequencing of selected regions the organisation of the tmb cluster. This analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high degree of homology and are similarly organised in operons. However the operons are differently arranged on the Pp TMB chromosome and on the pWW0 plasmid. Comparison of the two systems suggests that the operon arrangement found in pWW0 may have originated from that found in Pp TMB via cointegration mediated by replicative transposition or by intermolecular recombination between two copies of the insertion element IS1246.

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

Expression of phage P4 integrase is regulated negatively by both Int and Vis.

Phage P4 int gene encodes the integrase responsible for phage integration into and excision from the Escherichia coli chromosome. Here, the data showing that P4 int expression is regulated in a complex manner at different levels are presented. First of all, the Pint promoter is regulated negatively by both Int and Vis, the P4 excisionase. The N-terminal portion of Int appears to be sufficient for such a negative autoregulation, suggesting that the Int N terminus is implicated in DNA binding. Second, full-length transcripts covering the entire int gene could be detected only upon P4 infection, whereas in P4 lysogens only short 5'-end covering transcripts were detectable. On the other hand, transcripts covering the 5'-end of int were also very abundant upon infection. It thus appears that premature transcription termination and/or mRNA degradation play a role in Int-negative regulation both on the basal prophage transcription and upon infection. Finally, comparison between Pint-lacZ transcriptional and translational fusions suggests that Vis regulates Int expression post-transcriptionally. The findings that Vis is also an RNA-binding protein and that Int may be translated from two different start codons have implications on possible regulation models of Int expression.

Circular genetic map of satellite bacteriophage P4.

A genetic map of satellite bacteriophage P4 has been constructed by means of standard multifactor crosses. The genetic map appears to be a circular permutation of the mature DNA physical map. In addition, a set of markers appear to be linked both to the left and to the right of the same gene alpha. These facts suggest that the P4 genetic map is circular. Since terminal redundancy and/or cyclic permutation are not known to be present in P4 mature DNA, the circularity of P4 genetic map may reflect the physical circularity of the molecules involved in the recombination process. The low frequency of recombination and the strong negative interference observed are in agreement with the above hypothesis.

Bacteriophage P2: recombination in the superinfection preprophage state and under replication control by phage P4.

Genetic crosses (mixed infection, lytic cycle) with bacteriophage P2 are known to give extremely low recombination frequencies, and these are unaffected by the recA status of the host bacterium. We now show the following: (1) the satellite bacteriophage P4, which interacts with P2 in a number of ways, but is quite different from it in terms of DNA replication and its control, is clearly dependent on the host recA+ function for recombination; (2) a chimeric phage (Lindqvist's P2/P4 Hy19), in which P2 replication early genes have been replaced by those of P4, recombines in a recA+-dependent manner; (3) immunity-sensitive P2 phages, in mixed infections of P2-immune bacteria, and hence blocked in their replication, recombine in a recA+-dependent manner; (4) an analysis of the distribution of exchanges based on a simple model confirms that in mixed infections of sensitive cells (where P2 is actively multiplying) recombinational exchanges tend to be statistically clustered in a segment of the chromosome containing the origin of replication, and also shows that, under conditions in which P2 DNA replication is blocked, the distribution of exchanges correlates well with the physical distances between markers on the P2 DNA.

Antisense RNA-dependent transcription termination sites that modulate lysogenic development of satellite phage P4.

In the lysogenic state, bacteriophage P4 prevents the expression of its own replication genes, which are encoded in the left operon, through premature transcription termination. The phage factor responsible for efficient termination is a small, untranslated RNA (CI RNA), which acts as an antisense RNA and controls transcription termination by pairing with two complementary sequences (seqA and seqC) located within the leader region of the left operon. A Rho-dependent termination site, timm, was previously shown to be involved in the control of P4 replication gene expression. In the present study, by making use of phage PhiR73 as a cloning vector and of suppressor tRNAGly as a reporter gene, we characterized two additional terminators, t1 and t4. Although transcription termination at neither site requires the Rho factor, only t1 has the typical structure of a Rho-independent terminator. t1 is located between the PLE promoter and the cI gene, whereas t4 is located between cI and timm. Efficient termination at t1 requires the CI RNA and the seqA target sequence; in vitro, the CI RNA enhanced termination at t1 in the absence of any bacterial factor. A P4 mutant, in which the t1 terminator has been deleted, can still lysogenize both Rho+ and Rho- strains and exhibits increased expression of CI RNA. These data indicate that t1 and the Rho-dependent timm terminators are not essential for lysogeny. t1 is involved in CI RNA autoregulation, whereas t4 appears to be the main terminator necessary to prevent expression of the lytic genes in the lysogenic state.

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.

Mechanisms of genome propagation and helper exploitation by satellite phage P4.

Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

Determination of capsid size by satellite bacteriophage P4.

Satellite bacteriophage P4 requires all morphogenic gene products provided by a helper phage, such as coliphage P2, to assemble its own capsid, which is one-third the volume of the larger helper capsid. We have isolated a satellite phage P4 sid (size determination) mutant that is unable to direct the assembly of the small wild-type-size P4 capsid. Instead, this mutant produces P4 plaque-forming units with large P2-size capsids which contain two or three copies of the P4 sid1 genome. P4 sid1 is evidently mutated in a protein that is specifically responsible for determining the precise size and symmetry of the structure into which the helper P2 gene products will assemble. In addition, we have found that the physical size of the genome does not appear to play an essential role in the proper assembly of the icosahedral capsid, since the majority of the P4 sid1 plaque-forming units do not contain a complete capsidful of DNA.

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.

Characterization of the oriI and oriII origins of replication in phage-plasmid P4.

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.

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.