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Multiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. The role of MprF in commensals remains, however, uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. The L. plantarum ΔmprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on ΔmprF mutant's ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the ΔmprF mutant after infection-induced inflammation in the guts of wild-type flies but not flies lacking AMPs. Additionally, we found that the ΔmprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. Our further analysis suggests that MprF-mediated lipoteichoic acid modifications are involved in host immunomodulation. Overall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important commensal resilience factor. Author summary: Commensal microbial communities residing in the host intestine often experience the exposure to host immune effectors triggered by the pathogens during infection. Despite these perturbations, intestinal commensals stably colonize the host, thus exhibiting resilience to inflammation. Here, we used the fruit fly Drosophila melanogaster and its gut commensal, Lactiplantibacillus plantarum, as a model to investigate the role of a specific bacterial gene, multiple peptide resistance factor (MprF), in commensal-host interactions during infection. We generated an L. plantarum ΔmprF mutant and demonstrated susceptibility of this mutant to AMPs and deficiency in the colonization of flies after infection. Importantly, ΔmprF mutant efficiently colonized flies that lack AMPs, demonstrating that MprF-mediated resistance to host AMPs is crucial for commensal persistence during infection. Additionally, we discovered that the ΔmprF mutant induced due to an increased release of cell-wall fragments enhanced immune response in flies as compared to wild-type bacteria. Hence, MprF confers two highly-relevant functions for commensal-host association: AMP resistance and immune evasion. Finally, we showed a role of MprF in regulating LTA size, implying a broader role of MprF in the bacterial physiology then previously appreciated. Collectively, our work shows that AMP resistance via MprF-mediated lipid and LTA modifications is essential for commensal resilience during inflammation. [ABSTRACT FROM AUTHOR]

Abstract In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light‐dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer‐DNA (T‐DNA) insertion mutant line SALK_008491, previously known as nhd1‐1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non‐photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14‐kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD‐dependent D‐3‐phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow‐up studies employing respective single mutants and complementation with overlapping transformation‐competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion‐carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T‐DNA lines. Although second‐site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T‐DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

Summary: Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host’s transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. Here, we employ Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. We show that microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, we report that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. Our study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes.

The complex, interconnected, and non-contiguous nature of canopy environments present unique cognitive, locomotor, and sensory challenges to their animal inhabitants. Animal movement through forest canopies is constrained; unlike most aquatic or aerial habitats, the three-dimensional space of a forest canopy is not fully realized or available to the animals within it. Determining how the unique constraints of arboreal habitats shape the ecology and evolution of canopy-dwelling animals is key to fully understanding forest ecosystems. With emerging technologies, there is now the opportunity to quantify and map tree connectivity, and to embed the fine-scale horizontal and vertical position of moving animals into these networks of branching pathways. Integrating detailed multi-dimensional habitat structure and animal movement data will enable us to see the world from the perspective of an arboreal animal. This synthesis will shed light on fundamental aspects of arboreal animals’ cognition and ecology, including how they navigate landscapes of risk and reward and weigh energetic trade-offs, as well as how their environment shapes their spatial cognition and their social dynamics.

One of the key limitations of CRISPR-Cas-based genome editing techniques is the PAM dependency. Here, the authors review ongoing efforts towards realizing PAM-free nucleases, address potential consequences of eliminating PAM recognition, and propose an alternative nuclease repertoire covering all possible PAM sequences.

Sleep is fundamental to the health and fitness of all animals. The physiological importance of sleep is underscored by the central role of homeostasis in determining sleep investment – following periods of sleep deprivation, individuals experience longer and more intense sleep bouts. Yet, most sleep research has been conducted in highly controlled settings, removed from evolutionarily relevant contexts that may hinder the maintenance of sleep homeostasis. Using triaxial accelerometry and GPS to track the sleep patterns of a group of wild baboons (Papio anubis), we found that ecological and social pressures indeed interfere with homeostatic sleep regulation. Baboons sacrificed time spent sleeping when in less familiar locations and when sleeping in proximity to more group-mates, regardless of how long they had slept the prior night or how much they had physically exerted themselves the preceding day. Further, they did not appear to compensate for lost sleep via more intense sleep bouts. We found that the collective dynamics characteristic of social animal groups persist into the sleep period, as baboons exhibited synchronized patterns of waking throughout the night, particularly with nearby group-mates. Thus, for animals whose fitness depends critically on avoiding predation and developing social relationships, maintaining sleep homeostasis may be only secondary to remaining vigilant when sleeping in risky habitats and interacting with group-mates during the night. Our results highlight the importance of studying sleep in ecologically relevant contexts, where the adaptive function of sleep patterns directly reflects the complex trade-offs that have guided its evolution.

More refined knowledge of how tropical forests respond to changes in the abiotic environment is necessary to mitigate climate change, maintain biodiversity, and preserve ecosystem services. To evaluate the unique response of diverse Afrotropical forest communities to disturbances in the abiotic environment, we employ country-wide tree species inventories, remotely sensed climate data, and future climate predictions collected from 104 1-ha plots in the central African country of Gabon. We predict a 3–8% decrease in Afrotropical forest species richness by the end of the century, in contrast to the 30–50% loss of plant diversity predicted to occur with equivalent warming in the Neotropics. This work reveals that forecasts of community species composition are not generalizable across regions, and more representative studies are needed in understudied diverse biomes. This study serves as an important counterpoint to work done in the Neotropics by providing contrasting predictions for Afrotropical forests with substantially different ecological, evolutionary, and anthropogenic histories.

By dispersing seeds long distances, large, fruit-eating animals influence plant population spread and community dynamics. After fruit consumption, animal gut passage time and movement determine seed dispersal patterns and distances. These, in turn, are influenced by extrinsic, environmental variables and intrinsic, individual-level variables. We simulated seed dispersal by forest elephants (Loxodonta cyclotis) by integrating gut passage data from wild elephants with movement data from 96 individuals. On average, elephants dispersed seeds 5.3 km, with 89% of seeds dispersed farther than 1 km. The longest simulated seed dispersal distance was 101 km, with an average maximum dispersal distance of 40.1 km. Seed dispersal distances varied among national parks, perhaps due to unmeasured environmental differences such as habitat heterogeneity and configuration, but not with human disturbance or habitat openness. On average, male elephants dispersed seeds farther than females. Elephant behavioral traits strongly influenced dispersal distances, with bold, exploratory elephants dispersing seeds 1.1 km farther than shy, idler elephants. Protection of forest elephants, particularly males and highly mobile, exploratory individuals, is critical to maintaining long distance seed dispersal services that shape plant communities and tropical forest habitat.

Abstract CRISPR‐Cas systems allow bacteria to memorize prior infections as a means to combat the same invader if it attempts another attack in the future. While the underlying mechanisms of this bacterial immunity have been intensely studied over the past decade, little attention has been paid to CRISPR defense at the single‐cell level. In their recent work, Brouns and colleagues (McKenzie et al, 2022) track memory acquisition and defense in individual cells and find a wide range of temporal dynamics that shape how a cell population experiences and combats an active infection.

Much is unknown about the impact of the COVID-19 pandemic on the carceral experience. Firsthand accounts, however, can illuminate how the virus traveled through correctional institutions and how operational changes intended to mitigate virus transmission altered daily life for incarcerated people. Analyzing semi-structured interviews with 53 individuals released from state prisons, county jails, and halfway houses in a northeastern U.S. state between October 2020 and June 2021, this study explores the shifting conditions of confinement in the early months of the pandemic, as well as how incarcerated individuals experienced the pandemic in terms of their mental and physical health, safety, and trust in correctional institutions. Interviewees described the spread of COVID-19 in correctional environments and how it led to several pandemic-related changes, such as stoppages or adjustments to programs, visits, recreation time, and movement around the facility. The data ultimately suggest that pandemic lockdown measures, instituted to prevent virus transmission, led to environments akin to solitary confinement and compromised the ability of incarcerated individuals to connect with loved ones, exacerbated their fears about contracting the virus, and further eroded their trust in correctional institutions. [ABSTRACT FROM AUTHOR]

Understanding how tropical forests respond to abiotic environmental changes is critical for preserving biodiversity, mitigating climate change, and maintaining ecosystem services in the coming century. To evaluate the relative roles of the abiotic environment and human disturbance on Central African tree community composition, we employ tree inventory data, remotely sensed climatic data, and soil nutrient data collected from 30 1‐ha plots distributed across a large‐scale observational experiment in forests that had been differently impacted by logging and hunting in northern Republic of Congo. We show that the composition of Afrotropical plant communities at this scale responds to human disturbance more than to climate, with particular sensitivities to hunting and distance to the nearest village (a proxy for other human activities, including tree‐cutting and gathering). These findings contrast neotropical predictions, highlighting the unique ecological, evolutionary, and anthropogenic history of Afrotropical forests. [ABSTRACT FROM AUTHOR]

CRISPR array generation is difficult due to reoccurring repeat sequences. Here the authors present CRATES—a modular, one-pot assembly method—and demonstrate the creation of arrays for Cas9, Cas12a and Cas13a for cell-free, bacterial, yeast and mammalian systems.

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure-function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer-small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine.

Summary: We present a protocol to rapidly test DNA binding and cleavage activity by CRISPR nucleases using cell-free transcription-translation (TXTL). Nuclease activity is assessed by adding DNA encoding a nuclease, a guide RNA, and a targeted reporter to a TXTL reaction and by measuring the fluorescence for several h. The reactions, performed in a few microliters, allow for parallel testing of many nucleases and guide RNAs. The protocol includes representative results for (d)Cas9 from Streptococcus pyogenes targeting a GFP reporter gene.For complete information on the generation and use of this protocol, please refer to the paper by Marshall et al. (2018).