Alicia Bravo

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid.

SummaryWe provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two protiens whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.

Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1

SummaryThe proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1. It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression. This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein. These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein. The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed.

Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis.

Promoter-probe and terminator-probe plasmid vectors make possible to rapidly examine whether particular sequences function as promoter or terminator signals in various genetic backgrounds and under diverse environmental stimuli. At present, such plasmid-based genetic tools are very scarce in the Gram-positive pathogenic bacteria Streptococcus pneumoniae and Enterococcus faecalis. Hence, we developed novel promoter-probe and terminator-probe vectors based on the Streptococcus agalactiae pMV158 plasmid, which replicates autonomously in numerous Gram-positive bacteria. As reporter gene, a gfp allele encoding a variant of the green fluorescent protein was used. These genetic tools were shown to be suitable to assess the activity of promoters and terminators (both homologous and heterologous) in S. pneumoniae and E. faecalis. In addition, the promoter-probe vector was shown to be a valuable tool for the analysis of regulated promoters in vivo, such as the promoter of the pneumococcal fuculose kinase gene. These new plasmid vectors will be very useful for the experimental verification of predicted promoter and terminator sequences, as well as for the construction of new inducible-expression vectors. Given the promiscuity exhibited by the pMV158 replicon, these vectors could be used in a variety of Gram-positive bacteria.

Polymerization of bacteriophage ø29 replication protein p1 into protofilament sheets

Protein p1 (85 amino acids) of the Bacillus subtilis phage ø29 is a membrane‐associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose‐binding protein (MalE)‐p1 and MalE‐p1ΔN33. By using both sedimentation assays and negative‐stain electron microscopy analysis, we demonstrated that MalE‐p1 molecules self‐associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE‐p1ΔN33 fusion protein, however, did not self‐interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1ΔN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two‐dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo ø29 DNA synthesis in a temperature‐dependent manner. We propose that protein p1 is a component of a viral‐encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.

Phage Ø29 protein p6: A viral histone-like protein

Abstract Phage O29 protein p6 is one of the most abundant viral proteins in O29-infected B subtilis cells, constituting about 4% of the total cellular proteins (about 3 × 106 copies/cell) at late infection. Electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along O29 DNA, suggesting that protein p6 may have a role in genome packaging and organization. The low stability of the protein p6-O29 DNA complexes observed in vitro could reflect the dynamic nature of these complexes, to allow replication, transcription, and encapsidation of the genome. The protein p6-DNA complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. The DNA in this complex is strongly distorted and compacted. Protein p6 recognition signals have been mapped near the ends of the linear O29 DNA and act as nucleation sites for complex formation. Protein p6 does not recognize a specific sequence, but sequences with specific bendable properties that would favor the formation of the complex. Protein p6 represses transcription from the O29 C2 early promoter, and activates initiation of O29 DNA replication that occurs from both DNA ends. The formation of nucleoprotein complexes at the origins of replication, as well as the specific positioning of protein p6 with respect to the DNA ends are required for the activation of replication. This suggests that the proteins involved in the initiation step of O29 DNA replication, either directly interact with protein p6, or recognize a conformational change at a specific location in the DNA. The mechanism of activation could be the local and transient unpairing of DNA at specific sites, facilitated by the strong distortion of DNA conformation in the nucleoprotein complex.

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ø29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su−) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

Protein p56 from the Bacillus subtilis phage ϕ29 inhibits DNA-binding ability of uracil-DNA glycosylase

Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.

A Uracil-DNA Glycosylase Inhibitor Encoded by a Non-uracil Containing Viral DNA

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage ϕ29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5′-end. Replication of ϕ29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from ϕ29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by ϕ29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.

Compartmentalization of phage φ29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1

The bacteriophage φ29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis‐infected cells. The C‐terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross‐linking experiments, we demonstrate here that p1ΔC43, a C‐terminally truncated p1 protein that neither associates with membranes in vivo nor self‐interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid‐encoded protein p1ΔC43 reduces the rate of φ29 DNA replication in vivo in a dosage‐dependent manner. We also show that truncated p1 proteins that retain the N‐terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP·dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP–φ29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or φ29 DNA polymerase. We propose a model for initiation of in vivo φ29 DNA replication in which the viral replisome attaches to a membrane‐associated p1‐based structure.

Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage φ29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of φ29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the φ29 DNA amplification system, we found that φ29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from φ29-infected cells revealed that uracil residues arise in φ29 DNA during replication, probably as a result of misincorporation of dUMP by the φ29 DNA polymerase. On the other hand, the action of UDG on uracil-containing φ29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing φ29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the φ29 genome.