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Utilization and degradation of laminarin-based substrates by marine yeasts suggests their niche-specific role in microbial loop dynamics. | 10.1101/2024.09.13.612705 | Reich, M.; Arslan-Gatz, B.; Schultz-Johansen, M.; Hollwedel, T.-N.; Niggemeier, S.; Nimzyk, R.; Wichels, A.; Gerdts, G.; Hehemann, J.-H.; Harder, T. | In the oceans, the diversity of phytoplankton primary products supports a wide range of microbial heterotrophs, including bacteria and fungi. The organic substrate dynamics within pelagic microbial communities are strongly controlled by microorganismal interactions, resulting in a dense interactome. While the role of bacteria in the microbial loop is well documented, the degradation capacity and substrate specificity of marine fungi, as well as their role and function in metabolic guilds with bacteria, is comparatively less understood. We chose the polysaccharide laminarin, a major product of marine primary production, as well as oligomeric laminarin subunits and monomeric glucose, to study the degradation capacity of eleven marine yeast isolates from the pelagic microbial community of Helgoland Roads. Our aim was to measure yeast growth and correlate degradation yields and putative intermediate degradation products with the size of laminarin-based organic precursor substrates. We developed a reproducible, temporally resolved, high-throughput growth protocol to measure resource-specific yeast growth. Measurement of temporally fine-scaled growth kinetic models of isolates were accompanied with qualitative and quantitative chemical analyses of substrates and degradation intermediates. Our data showed that yeast growth was negatively correlated with oligomer length. Fluorophore-assisted carbohydrate electrophoresis suggested the lack of enzymatic endo-activity for laminarin in yeasts under investigation, suggesting they may occupy a niche in the microbial loop, benefitting from extracellular hydrolysis of carbohydrates by other microorganisms. In terrestrial environments, namely forest soil ecosystems, yeasts have been assigned a similar niche, supporting a prominent role of yeasts in microbial interactomes. | 2024-09-14 | |
Surface-mediated Bacteriophage Defense Incurs Fitness Tradeoffs for Interbacterial Antagonism | 10.1101/2024.09.13.612980 | Tsai, C.-E.; Wang, F.-Q.; Yang, C.-W.; Yang, L.-L.; Chen, Y.-C.; Chen, P.-Y.; Hwang, I.-S.; Ting, S.-Y. | Bacteria in polymicrobial habitats are constantly exposed to biotic threats from bacteriophages, antagonistic bacteria, and predatory eukaryotes. These antagonistic interactions play crucial roles in shaping the evolution and physiology of bacteria. To survive, bacteria have evolved mechanisms to protect themselves from such attacks, but the fitness costs of resisting one threat and rendering bacteria susceptible to others remain unappreciated. Here, we examined the fitness consequences of bacteriophage resistance in Salmonella enterica, revealing that phage-resistant variants exhibited significant fitness loss upon co-culture with competitor bacteria. These phage-resistant strains display varying degrees of lipopolysaccharide (LPS) deficiency and increased susceptibility to contact-dependent interbacterial antagonism, such as the type VI secretion system (T6SS). Utilizing mutational analyses and atomic force microscopy, we show that the long-modal length O-antigen of LPS serves as a protective barrier against T6SS-mediated intoxication. Notably, this competitive disadvantage can also be triggered independently by phages possessing LPS-targeting endoglycosidase in their tail spike proteins, which actively cleave the O-antigen upon infection. Our findings reveal two distinct mechanisms of phage-mediated LPS modifications that modulate interbacterial competition, shedding light on the dynamic microbial interplay within mixed populations. | 2024-09-14 | |
tRNA lysidinylation is essential for the minimal translation system found in the apicoplast of Plasmodium falciparum | 10.1101/2024.09.13.612944 | Elahi, R.; Prigge, S. T. | For decades, researchers have sought to define minimal genomes to elucidate the fundamental principles of life and advance biotechnology. tRNAs, essential components of this machinery, decode mRNA codons into amino acids. The apicoplast of malaria parasites encodes 25 tRNA isotypes in its organellar genome - the lowest number found in known translation systems. Efficient translation in such minimal systems depends heavily on post-transcriptional tRNA modifications, especially at the wobble anticodon position. Lysidine modification at the wobble position (C34) of tRNACAU distinguishes between methionine (AUG) and isoleucine (AUA) codons, altering the amino acid delivered by this tRNA and ensuring accurate protein synthesis. Lysidine is formed by the enzyme tRNA isoleucine lysidine synthetase (TilS) and is nearly ubiquitous in bacteria and essential for cellular viability. We identified a TilS ortholog (PfTilS) located in the apicoplast of Plasmodium falciparum parasites. By complementing PfTilS with a bacterial ortholog, we demonstrated that the lysidinylation activity of PfTilS is critical for parasite survival and apicoplast maintenance, likely due to its impact on apicoplast protein translation. Our findings represent the first characterization of TilS in an endosymbiotic organelle, advancing eukaryotic organelle research and our understanding of minimal translational machinery. Due to the absence of lysidine modifications in humans, this research also exposes a potential vulnerability in malaria parasites that could be targeted by antimalarial strategies. | 2024-09-14 | |
In silico analysis reveals differential targeting of enterovirus species by commonly used PCR assays | 10.1101/2024.09.13.612945 | Trinh, V. N.; Mulakken, N. J.; Nelson, K. L.; Be, N. A.; Kantor, R. S. | Quantitative polymerase chain reaction (qPCR) assays are sensitive molecular tools commonly used to quantify pathogens in environmental samples. These assays have become a staple of wastewater-based surveillance and often form the basis of quantitative microbial risk assessments. However, PCR assays may fail to capture all of their intended targets due to signature erosion over time. Here, we performed an in silico analysis of four human enterovirus PCR assays to assess signature erosion against the NCBI virus database. The predicted number of genomes hit by each assay rose alongside total genomes in the database through 2010 but the proportion of predicted hits began to level off with the emergence of the clinically important enterovirus D-68. We found that although all assays captured a majority of enterovirus species, only one recently developed assay adequately captured enterovirus D species. Some assays also appeared more likely to capture non-human enteroviruses than others, an important consideration for data interpretation. We conclude that broad-spectrum virus assays applied to environmental samples should be regularly checked against expanding sequence databases and provide methods to do so. | 2024-09-14 | |
Translational profiling of stress-induced small proteins uncovers an unexpected connection among distinct signaling systems | 10.1101/2024.09.13.612970 | Vellappan, S.; Sun, J.; Favate, J.; Jagadeesan, P.; Cerda, D.; Shah, P.; Yadavalli, S. S. | Signaling networks in bacteria enable environmental sensing and adaptation to challenging conditions by activating specific genes that help counteract stressors. Small proteins ([≥]50 amino acids long) are a rising class of bacterial stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack known phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we investigate small proteins induced in response to stress using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, a majority of which are transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, which underscore the physiological significance of their expression in low magnesium stress. Most remarkably, our study reveals that a small membrane protein YoaI is an unusual connector of the major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli, advancing our understanding of small protein regulators of cellular signaling. | 2024-09-14 | |
Genomic Reconstruction and Dietary Response Assessment of Three Acutalibacteraceae Bacteria Isolated from Fecal Samples of Singapore Subjects. | 10.1101/2024.09.13.612987 | Park, M. A.; Almunawar, S. N. A.; Lim, R. R. X.; Haldar, S.; Henry, C. J.; Moskvin, O. V. | Clostridium leptum, a key player in gut butyrate production, has a profound impact on various facets of intestinal health. A recent clinical trial highlighted a significant increase in the relative abundance of this species in response to dietary interventions using beneficial oils. We isolated microbial strains corresponding to \'Clostridium leptum\' (at the 16S rRNA gene similarity level) and sequenced their genomes. All three genomes were successfully reconstructed, maintaining the chromosome as a single contig. Subsequent genome-wide analysis unveiled the phylogenetic diversity of the isolates, including the discovery of a new species - Gallacutalibacter singaporense. Based on the reconstructed metabolic model, we predicted growth condition patterns of this new species and confirmed the predictions in vitro. Leveraging the assembled genomes, we dissected the components of the strong dietary intervention response signal previously ascribed to \'C.leptum\' and revealed distinct individual dynamics of all three bacteria in the clinical trial context. The transitional behavior of the novel species, in particular, revealed intriguing patterns, blazing the path to uncovering previously unrecognized interactions along the diet - gut microbiome - human health axis. | 2024-09-14 | |
Dynamic network regulating phosphatidic acid homeostasis revealed using membrane editing coupled to proximity labeling | 10.1101/2024.09.14.612979 | Tei, R.; Baskin, J. M. | Cellular lipid metabolism is subject to strong homeostatic regulation, but players involved in and mechanisms underlying these pathways remain mostly uncharacterized. Here, we develop and exploit a \'\'Feeding-Fishing\'\' approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics via proximity labeling (fishing) to elucidate molecular players and pathways involved in homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. By performing proximity biotinylation using a membrane-tethered TurboID alongside membrane editing to selectively deliver phosphatidic acid to the same membrane, we identified numerous PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Subsequent mechanistic analysis established that PA homeostasis in the cytosolic leaflets of the plasma membrane and of lysosomes is governed by a select subset of PA metabolic pathways and, via divergent molecular mechanisms, several members of the lipid transfer protein superfamily capable of mediating interorganelle lipid transport. More broadly, the interfacing of membrane editing with organelle membrane proteomics using proximity labeling represents a powerful and generalizable strategy for revealing mechanisms governing lipid homeostasis. | 2024-09-14 | |
Effects of Nf1 on sleep behavior are mediated through starvation caused by deficits in SARM1 dependent NAD+ metabolism. | 10.1101/2024.09.14.612058 | Sofela, F. A.; Lopez Valencia, M.; Jongens, T. A.; Sehgal, A. | Neurofibromatosis 1 (NF1) is a relatively common autosomal dominant disease which predisposes to the formation of tumors, and is also associated with behavioral phenotypes, including sleep disturbances. As loss of the NF1 protein has been recently associated with metabolic dysfunction, we explored the relationship between metabolic and behavioral phenotypes through metabolomic analysis of Drosophila Nf1-null mutants. Nf1-null mutants exhibit a metabolic signature indicative of starvation, with diminished metabolites related to glucose, glycogen, and fatty acid processing and increased mRNA of Akh, a hormone that promotes foraging during starvation. Reduced sleep in Nf1-null mutants was rescued by genetic manipulation of the AKH pathway and by a high-sucrose diet, which also partially corrected hypolipidemia, suggesting that sleep loss is due to starvation-induced foraging. Interestingly, behavioral phenotypes can be recapitulated by loss of NF1 only in the periphery and trace to mitochondrial defects that include elevated levels of the NADase SARM1. Indeed, inhibition of SARM1 activity rescues sleep behavior in Nf1-null flies. These findings suggest a novel connection between loss of NF1 and mitochondrial dysfunction caused by SARM1 hyperactivation, setting the scene for new pharmacological and dietary approaches that could provide relief to NF1 patients. | 2024-09-14 |