Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Microbiome Shifts Associated With the Introduction of Wild Atlantic Horseshoe Crabs (Limulus polyphemus) Into a Touch-Tank Exhibit.

The Atlantic horseshoe crab (Limulus polyphemus) is a common marine aquarium species and model organism for research. There is potential monetary and conservation value in developing a stable captive population of horseshoe crabs, however, one major impediment to achieving captivity is a lack of knowledge regarding captive diseases. We utilized 16S rRNA gene amplicon sequencing to track changes in the microbiomes of four body locations in three wild-caught (tracked over 14 months in captivity) and three tank-acclimated (>2 years in captivity) adult L. polyphemus in a touch tank at Shark Reef Aquarium at Mandalay Bay in Las Vegas, NV. The wild population hosted diverse and distinct microbiomes on the carapace (260 ± 96 amplicon sequence variants or ASVs), cloaca (345 ± 77 ASVs), gills (309 ± 36 ASVs), and oral cavity (359 ± 37 ASVs), which were dominated by classes Gammaproteobacteria, Bacteroidia, and Alphaproteobacteria. A rapid decline in richness across all body locations was observed within 1 month of captivity, with tank-acclimated (>2 years) animals having <5% of the initial microbiome richness and a nearly completely restructured microbial community. Tank-acclimated horseshoe crabs possessed distinct microbiomes that were highly uneven and low in species richness on the carapace (31 ± 7 ASVs), cloaca (53 ± 19 ASVs), gills (17 ± 2 ASVs), and oral cavity (31 ± 13 ASVs). The carapace, oral cavity, and gills of the tank-acclimated animals hosted abundant populations of Aeromonas (>60%) and Pseudomonas (>20%), both of which are known opportunistic pathogens of aquatic animals and can express chitinases, providing a plausible mechanism for the development of the carapace lesion pathology observed in this and other studies. The cloaca of the tank-acclimated animals was slightly more diverse than the other body locations with Aeromonas, Enterococcus, Shewanella, and Vagococcus dominating the community. These results provide an important baseline on the microbiomes of both wild and tank-acclimated horseshoe crabs and underscore the need to continue to investigate how native microbial populations may protect animals from pathogens.

Position-Specific Metabolic Probing and Metagenomics of Microbial Communities Reveal Conserved Central Carbon Metabolic Network Activities at High Temperatures.

Temperature is a primary driver of microbial community composition and taxonomic diversity; however, it is unclear to what extent temperature affects characteristics of central carbon metabolic pathways (CCMPs) at the community level. In this study, 16S rRNA gene amplicon and metagenome sequencing were combined with 13C-labeled metabolite probing of the CCMPs to assess community carbon metabolism along a temperature gradient (60-95°C) in Great Boiling Spring, NV. 16S rRNA gene amplicon diversity was inversely proportional to temperature, and Archaea were dominant at higher temperatures. KO richness and diversity were also inversely proportional to temperature, yet CCMP genes were similarly represented across the temperature gradient and many individual metagenome-assembled genomes had complete pathways. In contrast, genes encoding cellulosomes and many genes involved in plant matter degradation and photosynthesis were absent at higher temperatures. In situ 13C-CO2 production from labeled isotopomer pairs of glucose, pyruvate, and acetate suggested lower relative oxidative pentose phosphate pathway activity and/or fermentation at 60°C, and a stable or decreased maintenance energy demand at higher temperatures. Catabolism of 13C-labeled citrate, succinate, L-alanine, L-serine, and L-cysteine was observed at 85°C, demonstrating broad heterotrophic activity and confirming functioning of the TCA cycle. Together, these results suggest that temperature-driven losses in biodiversity and gene content in geothermal systems may not alter CCMP function or maintenance energy demands at a community level.