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Abstract Reproduction, embryological development, and settlement of corals are critical for survival of coral reefs through larval propagation. Yet, for many species of corals, a basic understanding of the early life‐history stages is lacking. In this study, we report our observations forex situreproduction in the massive reef‐building coralPoritescf.P. lobataacross 2 years. Spawning occurred in April and May, on the first day after the full moon with at least 2 h of darkness between sunset and moonrise, on a rising tide. Only a small proportion of corals observed had mature gametes or spawned (14–35%). Eggs were 185–311 μm in diameter, spherical, homogenous, and provisioned with 95–155 algal cells (family Symbiodiniaceae). Males spawned before females, andex situfertilization rates were high for the first 2 h after egg release. Larvae were elliptical, ~300 μm long, and symbiotic. Just 2 days after fertilization, many larvae swam near the bottom of culture dishes and were competent to settle. Settlers began calcification 2 days after metamorphosis, and tentacles were developed 10 days after attachment. Our observations contrast with previous studies by suggesting an abbreviated pelagic larval period inPoritescf.P. lobata, which could lead to the isolation of some populations. The high thermal tolerance and broad geographic range ofPoritescf.P. lobatasuggest that this species could locally adapt to a wide range of environmental conditions, especially if larvae are locally retained. The results of this study can inform future work on reproduction, larval biology, dispersal, and recruitment inPoritescf.P. lobata, which could have an ecological advantage over less resilient coral species under future climate change. 

ABSTRACT As ocean warming threatens reefs worldwide, identifying corals with adaptations to higher temperatures is critical for conservation. Genetically distinct but morphologically similar (i.e. cryptic) coral populations can be specialized to extreme habitats and thrive under stressful conditions. These corals often associate with locally beneficial microbiota (Symbiodiniaceae photobionts and bacteria), obscuring the main drivers of thermal tolerance. Here, we leverage a holobiont (massivePorites) with high fidelity for C15 photobionts to investigate adaptive variation across classic (“typical” conditions) and extreme reefs characterized by higher temperatures and light attenuation. We uncovered three cryptic lineages that exhibit limited micro‐morphological variation; one lineage dominated classic reefs (L1), one had more even distributions (L2), and a third was restricted to extreme reefs (L3). L1 and L2 were more closely related to populations ~4300 km away, suggesting that some lineages are widespread. All corals harboredCladocopiumC15 photobionts; L1 and L2 shared a photobiont pool that differed in composition between reef types, yet L3 mostly harbored unique photobiont strains not found in the other lineages. Assemblages of bacterial partners differed among reef types in lineage‐specific ways, suggesting that lineages employ distinct microbiome regulation strategies. Analysis of light‐harvesting capacity and thermal tolerance revealed adaptive variation underpinning survival in distinct habitats: L1 had the highest light absorption efficiency and lowest thermal tolerance, suggesting that it is a classic reef specialist. L3 had the lowest light absorption efficiency and the highest thermal tolerance, showing that it is an extreme reef specialist. L2 had intermediate light absorption efficiency and thermal tolerance, suggesting that is a generalist lineage. These findings reveal diverging holobiont strategies to cope with extreme conditions. Resolving coral lineages is key to understanding variation in thermal tolerance among coral populations, can strengthen our understanding of coral evolution and symbiosis, and support global conservation and restoration efforts. 

Abstract A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized forE. colifunction across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks. 

Reef-building crustose coralline algae (CCA) are known to facilitate the settlement and metamorphosis of scleractinian coral larvae. In recent decades, CCA coverage has fallen globally and degrading environmental conditions continue to reduce coral survivorship, spurring new restoration interventions to rebuild coral reef health. In this study, naturally produced chemical compounds (metabolites) were collected from two pantropical CCA genera to isolate and classify those that induce coral settlement. In experiments using four ecologically important Caribbean coral species, we demonstrate the applicability of extracted, CCA-derived metabolites to improve larval settlement success in coral breeding and restoration efforts. Tissue-associated CCA metabolites induced settlement of one coral species,Orbicella faveolata, while metabolites exuded by CCA (exometabolites) induced settlement of three species:Acropora palmata,Colpophyllia natansandOrbicella faveolata. In a follow-up experiment, CCA exometabolites fractionated and preserved using two different extraction resins induced the same level of larval settlement as the unfractionated positive control exometabolites. The fractionated CCA exometabolite pools were characterized using liquid chromatography tandem mass spectrometry, yielding 145 distinct molecular subnetworks that were statistically defined as CCA-derived and could be classified into 10 broad chemical classes. Identifying these compounds can reveal their natural prevalence in coral reef habitats and facilitate the development of new applications to enhance larval settlement and the survival of coral juveniles. 

Our work targets automated analysis to quantify the growth dynamics of a population of bacilliform bacteria. We propose an innovative approach to frame-sequence tracking of deformable-cell motion by the automated minimization of a new, specific cost functional. This minimization is implemented by dedicated Boltzmann machines (stochastic recurrent neural networks). Automated detection of cell divisions is handled similarly by successive minimizations of two cost functions, alternating the identification of children pairs and parent identification. We validate the proposed automatic cell tracking algorithm using (i) recordings of simulated cell colonies that closely mimic the growth dynamics of E. coli in microfluidic traps and (ii) real data. On a batch of 1100 simulated image frames, cell registration accuracies per frame ranged from 94.5% to 100%, with a high average. Our initial tests using experimental image sequences (i.e., real data) of E. coli colonies also yield convincing results, with a registration accuracy ranging from 90% to 100%. 

The increased complexity of synthetic microbial biocircuits highlights the need for distributed cell functionality due to concomitant increases in metabolic and regulatory burdens imposed on single-strain topologies. Distributed systems, however, introduce additional challenges since consortium composition and spatiotemporal dynamics of constituent strains must be robustly controlled to achieve desired circuit behaviors. Here, we address these challenges with a modeling-based investigation of emergent spatiotemporal population dynamics using cell-length control in monolayer, two-strain bacterial consortia. We demonstrate that with dynamic control of a strain’s division length, nematic cell alignment in close-packed monolayers can be destabilized. We find that this destabilization confers an emergent, competitive advantage to smaller-length strains—but by mechanisms that differ depending on the spatial patterns of the population. We used complementary modeling approaches to elucidate underlying mechanisms: an agent-based model to simulate detailed mechanical and signaling interactions between the competing strains, and a reductive, stochastic lattice model to represent cell-cell interactions with a single rotational parameter. Our modeling suggests that spatial strain-fraction oscillations can be generated when cell-length control is coupled to quorum-sensing signaling in negative feedback topologies. Our research employs novel methods of population control and points the way to programming strain fraction dynamics in consortial synthetic biology. 

Abstract Pyrrolysine (Pyl, O) exists in nature as the 22^ndproteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes inEscherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins inE. coli.