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.

Characterization of a unique repression system present in arbitrium phages of the SPbeta family.

Arbitrium-coding phages use peptides to communicate and coordinate the decision between lysis and lysogeny. However, the mechanism by which these phages establish lysogeny remains unknown. Here, focusing on the SPbeta phage family's model phages phi3T and SPß, we report that a six-gene operon called the "SPbeta phages repressor operon" (sro) expresses not one but two master repressors, SroE and SroF, the latter of which folds like a classical phage integrase. To promote lysogeny, these repressors bind to multiple sites in the phage genome. SroD serves as an auxiliary repressor that, with SroEF, forms the repression module necessary for lysogeny establishment and maintenance. Additionally, the proteins SroABC within the operon are proposed to constitute the transducer module, connecting the arbitrium communication system to the activity of the repression module. Overall, this research sheds light on the intricate and specialized repression system employed by arbitrium SPß-like phages in making lysis-lysogeny decisions.

Rationally designed antifungal protein chimeras reveal new insights into structure-activity relationship.

Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.

Insights into the mechanism of action of the arbitrium communication system in SPbeta phages.

The arbitrium system is employed by phages of the SPbeta family to communicate with their progeny during infection to decide either to follow the lytic or the lysogenic cycle. The system is controlled by a peptide, AimP, that binds to the regulator AimR, inhibiting its DNA-binding activity and expression of aimX. Although the structure of AimR has been elucidated for phages SPß and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPß. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPß receptor. The crystal structures of this AimR (apo, AimP-bound and DNA-bound) together with in vitro and in vivo analyses confirm a mechanism of action by AimP-induced conformational restriction, shedding light on peptide specificity and cross regulation with relevant biological implications.

The arbitrium system controls prophage induction.

Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPß prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPß lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPß-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPß phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.

A regulatory cascade controls Staphylococcus aureus pathogenicity island activation.

Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.

Molecular Basis of Lysis-Lysogeny Decisions in Gram-Positive Phages.

Temperate bacteriophages (phages) are viruses of bacteria. Upon infection of a susceptible host, a temperate phage can establish either a lytic cycle that kills the host or a lysogenic cycle as a stable prophage. The life cycle pursued by an infecting temperate phage can have a significant impact not only on the individual host bacterium at the cellular level but also on bacterial communities and evolution in the ecosystem. Thus, understanding the decision processes of temperate phages is crucial. This review delves into the molecular mechanisms behind lysis-lysogeny decision-making in Gram-positive phages. We discuss a variety of molecular mechanisms and the genetic organization of these well-understood systems. By elucidating the strategies used by phages to make lysis-lysogeny decisions, we can improve our understanding of phage-host interactions, which is crucial for a variety of studies including bacterial evolution, community and ecosystem diversification, and phage therapeutics.

Deciphering the Molecular Mechanism Underpinning Phage Arbitrium Communication Systems.

Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system.

Structural basis of Rap phosphatase inhibition by Phr peptides.

Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change.

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain.

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.

Energetics of 5-bromo-4-chloro-3-indolyl-alpha-D-mannose binding to the Parkia platycephala seed lectin and its use for MAD phasing.

Parkia platycephala belongs to the most primitive group of Leguminosae plants. Its seed lectin is made up of three homologous beta-prism repeats and exhibits binding specificity for mannose/glucose. The properties of the association between the lectin from P. platycephala seeds and monosaccharide ligands were analysed by isothermal titration calorimetry and surface plasmon resonance. The results are consistent with the lectin bearing three thermodynamically identical binding sites for mannose/glucose per monomer with dissociation constants in the millimolar range. Binding of each ligand by the lectin is enthalpically driven. Crystals have been obtained of the lectin in complex with a brominated derivative of mannose (5-bromo-4-chloro-3-indolyl-alpha-D-mannose), which were suitable for deriving an electron-density map by MAD phasing. In agreement with the thermodynamic data, six Br atoms were found in the asymmetric unit of the monoclinic P2(1) crystals, which contained two P. platycephala lectin molecules. The availability of other Br derivatives of monosaccharides (glucose, galactose, fucose) may make this strategy widely useful for structure elucidation of novel lectins or when (as in the case of the P. platycephala lectin) molecular-replacement methods fail.

Crystallization and preliminary X-ray diffraction analysis of HML, a lectin from the red marine alga Hypnea musciformis.

HML, a lectin from the red marine alga Hypnea musciformis, defines a novel lectin family. Orthorhombic crystals of HML belonging to space group P2(1)2(1)2(1) grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. A complete data set was collected at 2.4 A resolution. HML is the first marine alga lectin to be crystallized.

Crystallization and preliminary X-ray diffraction analysis of the seed lectin from Parkia platycephala.

The crystallization and preliminary X-ray diffraction analysis of the seed lectin of Parkia platycephala, a Mimosoideae, regarded as the most primitive group of the Leguminosae plants, are reported. Its amino-acid sequence consists of three tandemly arranged jacalin-related beta-prism domains, which is a novel fold for a leguminous lectin. Furthermore, no other lectin structure with this arrangement of domains has been described. P2(1)2(1)2(1) crystals (unit-cell parameters a = 63.6, b = 68.5, c = 208.5 A), which diffract to a maximum resolution of 2.2 A, were obtained in hanging drops at pH 8 and 293 K by the vapor-diffusion method using 10% 2-propanol and 20% polyethylene glycol 4000 as precipitants. The asymmetric unit contains two lectin molecules and has a solvent content of 46%. Only a single beta-prism domain could be located by molecular replacement using the structure of the Helianthus tuberosus lectin (PDB code 1c3k) as the search model. Isomorphous heavy-atom derivatives are currently being produced to solve the complete structure of the P. platycephala seed lectin.