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.

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.

Phage-inducible chromosomal islands promote genetic variability by blocking phage reproduction and protecting transductants from phage lysis.

Phage-inducible chromosomal islands (PICIs) are a widespread family of highly mobile genetic elements that disseminate virulence and toxin genes among bacterial populations. Since their life cycle involves induction by helper phages, they are important players in phage evolution and ecology. PICIs can interfere with the lifecycle of their helper phages at different stages resulting frequently in reduced phage production after infection of a PICI-containing strain. Since phage defense systems have been recently shown to be beneficial for the acquisition of exogenous DNA via horizontal gene transfer, we hypothesized that PICIs could provide a similar benefit to their hosts and tested the impact of PICIs in recipient strains on host cell viability, phage propagation and transfer of genetic material. Here we report an important role for PICIs in bacterial evolution by promoting the survival of phage-mediated transductants of chromosomal or plasmid DNA. The presence of PICIs generates favorable conditions for population diversification and the inheritance of genetic material being transferred, such as antibiotic resistance and virulence genes. Our results show that by interfering with phage reproduction, PICIs can protect the bacterial population from phage attack, increasing the overall survival of the bacterial population as well as the transduced cells. Moreover, our results also demonstrate that PICIs reduce the frequency of lysogenization after temperate phage infection, creating a more genetically diverse bacterial population with increased bet-hedging opportunities to adapt to new niches. In summary, our results identify a new role for the PICIs and highlight them as important drivers of bacterial evolution.

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.

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.

Lunar Phase Modulates Circadian Gene Expression Cycles in the Broadcast Spawning Coral Acropora millepora.

Many broadcast spawning corals in multiple reef regions release their gametes with incredible temporal precision just once per year, using the lunar cycle to set the night of spawning. Moonlight, rather than tides or other lunar-regulated processes, is thought to be the proximate factor responsible for linking the night of spawning to the phase of the Moon. We compared patterns of gene expression among colonies of the broadcast spawning coral Acropora millepora at different phases of the lunar cycle, and when they were maintained under one of three experimentally simulated lunar lighting treatments: i) lunar lighting conditions matching those on the reef, or lunar patterns mimicking either ii) constant full Moon conditions, or iii) constant new Moon conditions. Normal lunar illumination was found to shift both the level and timing of clock gene transcription cycles between new and full moons, with the peak hour of expression for a number of genes occurring earlier in the evening under a new Moon when compared to a full Moon. When the normal lunar cycle is replaced with nighttime patterns equivalent to either a full Moon or a new Moon every evening, the normal monthlong changes in the level of expression are destroyed for most genes. In combination, these results indicate that daily changes in moonlight that occur over the lunar cycle are essential for maintaining normal lunar periodicity of clock gene transcription, and this may play a role in regulating spawn timing. These data also show that low levels of light pollution may have an impact on coral biological clocks.

Photoreception and signal transduction in corals: proteomic and behavioral evidence for cytoplasmic calcium as a mediator of light responsivity.

Little is known about how corals sense and respond to light. In this report the proteome of coral is explored using 2D protein electrophoresis in two species, Montastraea cavernosa and Acropora millepora. Multiple protein species have major shifts in abundance in both species when sampled in daylight compared to corals sampled late in the night. These changes were observed both in larvae lacking zooxanthellae and in adult tissue containing zooxanthellae, including both Pacific and Caribbean corals. When larvae kept in the dark were treated with either thapsigargin or ionomycin, compounds that raise the level of cytoplasmic calcium, the night pattern of proteins shifted to the day pattern. This implies that photoreceptors responding to light elevate calcium levels and that calcium acts as the second messenger relaying light responses in corals. Corals spawn at night, and spawning can be delayed by exposure to light or pushed forward by early artificial sunsets. In a series of behavioral experiments, treatment of corals with ionomycin or thapsigargin was found to delay broadcast spawning in M. franksi, demonstrating that pharmacologically altering cytoplasmic calcium levels generates the same response as light exposure. Together these results show that the photo-responsive cells of corals detect and respond to light by altering cytoplasmic calcium levels, similarly to the transduction pathways in complex invertebrate eyes. The primacy of cytoplasmic calcium levels in light responsivity has broad implications for coral reproduction, including predicting how different species spawn at different times after sunset and how reproductive isolation is achieved during coral speciation.

Circadian cycles of gene expression in the coral, Acropora millepora.

BACKGROUND: Circadian rhythms regulate many physiological, behavioral and reproductive processes. These rhythms are often controlled by light, and daily cycles of solar illumination entrain many clock regulated processes. In scleractinian corals a number of different processes and behaviors are associated with specific periods of solar illumination or non-illumination--for example, skeletal deposition, feeding and both brooding and broadcast spawning. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken an analysis of diurnal expression of the whole transcriptome and more focused studies on a number of candidate circadian genes in the coral Acropora millepora using deep RNA sequencing and quantitative PCR. Many examples of diurnal cycles of RNA abundance were identified, some of which are light responsive and damped quickly under constant darkness, for example, cryptochrome 1 and timeless, but others that continue to cycle in a robust manner when kept in constant darkness, for example, clock, cryptochrome 2, cycle and eyes absent, indicating that their transcription is regulated by an endogenous clock entrained to the light-dark cycle. Many other biological processes that varied between day and night were also identified by a clustering analysis of gene ontology annotations. CONCLUSIONS/SIGNIFICANCE: Corals exhibit diurnal patterns of gene expression that may participate in the regulation of circadian biological processes. Rhythmic cycles of gene expression occur under constant darkness in both populations of coral larvae that lack zooxanthellae and in individual adult tissue containing zooxanthellae, indicating that transcription is under the control of a biological clock. In addition to genes potentially involved in regulating circadian processes, many other pathways were found to display diel cycles of transcription.