Thermophilic Dehalococcoidia with unusual traits shed light on an unexpected past.

Although the phylum Chloroflexota is ubiquitous, its biology and evolution are poorly understood due to limited cultivability. Here, we isolated two motile, thermophilic bacteria from hot spring sediments belonging to the genus Tepidiforma and class Dehalococcoidia within the phylum Chloroflexota. A combination of cryo-electron tomography, exometabolomics, and cultivation experiments using stable isotopes of carbon revealed three unusual traits: flagellar motility, a peptidoglycan-containing cell envelope, and heterotrophic activity on aromatics and plant-associated compounds. Outside of this genus, flagellar motility has not been observed in Chloroflexota, and peptidoglycan-containing cell envelopes have not been described in Dehalococcoidia. Although these traits are unusual among cultivated Chloroflexota and Dehalococcoidia, ancestral character state reconstructions showed flagellar motility and peptidoglycan-containing cell envelopes were ancestral within the Dehalococcoidia, and subsequently lost prior to a major adaptive radiation of Dehalococcoidia into marine environments. However, despite the predominantly vertical evolutionary histories of flagellar motility and peptidoglycan biosynthesis, the evolution of enzymes for degradation of aromatics and plant-associated compounds was predominantly horizontal and complex. Together, the presence of these unusual traits in Dehalococcoidia and their evolutionary histories raise new questions about the timing and selective forces driving their successful niche expansion into global oceans.

Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota.

Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 µm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

Incomplete denitrification phenotypes in diverse Thermus species from diverse geothermal spring sediments and adjacent soils in southwest China.

A few members of the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite (NO2-) or nitrous oxide (N2O). However, the denitrification abilities of the genus as a whole remain poorly characterized. Here, we describe diverse denitrification phenotypes and genotypes of a collection of 24 strains representing ten species, all isolated from a variety of geothermal systems in China. Confirmed terminal products of nitrate reduction were nitrite or N2O, while nitric oxide (NO) was inferred as the terminal product in some strains. Most strains produced N2O; complete denitrification was not observed. Denitrification phenotypes were largely consistent with the presence of denitrification genes, and strains of the same species often had the same denitrification phenotypes and largely syntenous denitrification gene clusters. Genes for nirS and nirK coexisted in three Thermus brockianus and three Thermus oshimai genomes, which is a unique hallmark of some denitrifying Thermus strains and may be ecologically important. These results show that incomplete denitrification phenotypes are prominent, but variable, within and between Thermus species. The incomplete denitrification phenotypes described here suggest Thermus species may play important roles in consortial denitrification in high-temperature terrestrial biotopes where sufficient supply of oxidized inorganic nitrogen exists.

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.

Diversity of Root Nodule-Associated Bacteria of Diverse Legumes Along an Elevation Gradient in the Kunlun Mountains, China.

Bacteria in root nodules of legumes play important roles in promoting plant growth. In this study, we investigated root nodule-associated bacteria isolated from leguminous plants along an elevation gradient on the northern slope of the Kunlun Mountains, China, using a cultivation approach. In total, 300 isolates were obtained from seven legume species within six ecological zones. Isolates were identified based on 16S rRNA gene phylogenetic analysis and potential rhizobia were further identified using a recA gene phylogeny. Among the isolates, Bacillales (particularly Bacillus) were the dominant isolates from all host legumes and all elevations (63.5%), followed by Rhizobiales (13%) and Pseudomonadales (11.7%). Less than 3% of the isolates belonged to Burkholderiales, Paenibacillales, Enterobacteriales, Actinomycetales, Sphingomonadales, Xanthomonadales, Chitinophagales, Brevibacillales, Staphylococcales, or Mycobacteriales. A few elevation-specific patterns emerged within the Bacillales and Pseudomonadales. For example, isolates related to the psychrotroph Bacillus psychrosaccharolyticus were only isolated from the highest elevation sites (>3,500 m) whereas those related to the mesophile Bacillus endophyticus were only isolated from lowest elevation sites (1,350 m), suggestive of a role of soil temperature in their distribution. Similarly, isolates related to Pseudomonas brassicacearum were the dominant Pseudomonadales isolates, but they were only isolated from middle and low elevations (<3,200 m). A total of 39 isolates belonged to the Rhizobiales, 36 of which were confirmed to the genus level using the recA gene. In all, Rhizobiales isolates were obtained from five different host legumes spanning the entire elevation gradient. Those from the low-elevation Qira Desert-Oasis Transition Zone (1,350-1,960 m) suggested some patterns of host preference. For example, most isolates from Albizia julibrissin formed a monophyletic group related to Rhizobium lemnae and most from Alhagi sparsifolia were closely related to Ensifer kummerowiae. In general, this study shows that most bacteria associated with root nodules of legumes are widely distributed in distinct ecological zones within a single geographic region but suggests that both climate and host interactions may influence their distributions.

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.

Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA.