Differential Spo0A-mediated effects on transcription and replication of the related Bacillus subtilis phages Nf and ϕ29 explain their different behaviours in vivo

Members of groups 1 (e.g. ϕ29) and 2 (e.g. Nf) of the ϕ29 family of phages infect the spore forming bacterium Bacillus subtilis. Although classified as lytic phages, the lytic cycle of ϕ29 can be suppressed and its genome can become entrapped into the B. subtilis spore. This constitutes an alternative infection strategy that depends on the presence of binding sites for the host-encoded protein Spo0A in the ϕ29 genome. Binding of Spo0A to these sites represses ϕ29 transcription and prevents initiation of DNA replication. Although the Nf genome can also become trapped into B. subtilis spores, in vivo studies showed that its lytic cycle is less susceptible to spo0A-mediated suppression than that of ϕ29. Here we have analysed the molecular mechanism underlying this difference showing that Spo0A differently affects transcription and replication initiation of the genomes of these phages. Thus, whereas Spo0A represses all three main early promoters of ϕ29, it only represses one out of the three equivalent early promoters of Nf. In addition, contrary to ϕ29, Spo0A does not prevent the in vitro initiation of Nf DNA replication. Altogether, the differences in Spo0A-mediated regulation of transcription and replication between ϕ29 and Nf explain their different behaviours in vivo

Antagonistic interactions between phage and host factors control arbitrium lysis–lysogeny decision

Phages can use a small-molecule communication arbitrium system to coordinate lysis–lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin–antitoxin system MazE–MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis–lysogeny by domesticating and fine-tuning a phage-defence mechanism

Novel regulatory mechanism of establishment genes of conjugative plasmids

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg. Reg also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg. Each operator is bound with extreme cooperativity by two Regdimers. The X-ray structure revealed that Reg has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition

Performance and calibration of the CHORUS scintillating fiber tracker and opto-electronics readout system

An essential component of the CERN WA95/CHORUS experiment is a scintillating fiber tracker system for precise track reconstruction of particles. The tracker design, its opto-electronics readout and calibration system are discussed. Performances of the detector are presented

The CHORUS neutrino oscillation search experiment

The CHORUS experiment has successfully finished run I (320~000 recorded \numu\ CC in 94/95) and performed half of run II (225~000 \numu\ CC in 96). The analysis chain was exercised on a small data sample for the muonic \tdecay\ search using for the first time fully automatic emulsion scanning. This pilot analysis, resulting in a limit \sintth \leq 3 \cdot 10^{-2}, confirms that the CHORUS proposal sensitivity (\sintth \leq 3 \cdot 10^{-4}) is within reach in two years

Relevance of UP elements for three strong Bacillus subtilis phage φ29 promoters

Various Escherichia coli promoters contain, in addition to the classical –35 and –10 hexamers, a third recognition element, named the UP element. Located upstream of the –35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase α subunit (αCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early σ(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage φ29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the –35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed

Different responses to Spo0A-mediated suppression of the related Bacillus subtilis phages Nf and φ29

The φ29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis. Previous studies showed that group 1 phage φ29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of φ29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of φ29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than φ29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host.This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.Peer reviewe

kinC/D-mediated heterogeneous expression of spo0A during logarithmical growth in Bacillus subtilis is responsible for partial suppression of ø29 development

The host of the lytic bacteriophage φ29 is the spore-forming bacterium Bacillus subtilis. When infection occurs during early stages of sporulation, however, φ29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. Recently, we have shown that Spo0A, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic φ29 cycle in cells having initiated sporulation. Surprisingly, we found that φ29 development is suppressed in a subpopulation of logarithmically growing culture and that spo0A is heterogeneously expressed during this growth stage. Furthermore, we showed that kinC and, to a minor extent, kinD, are responsible for heterogeneous expression levels of spo0A during logarithmical growth that are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of φ29. Whereas spo0A was known to be heterogeneously expressed during the early stages of sporulation, our findings show that this also occurs during logarithmical growth. These insights are likely to have important consequences, not only for the life cycle of φ29, but also for B. subtilis developmental processes.This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.Peer reviewe

Different responses to Spo0A-mediated suppression of the related Bacillus subtilis phages Nf and φ29

The φ29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis. Previous studies showed that group 1 phage φ29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of φ29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of φ29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than φ29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host.This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.Peer reviewe