A thermophilic chemolithoautotrophic bacterial consortium suggests a mutual relationship between bacteria in extreme oligotrophic environments

Abstract

A thermophilic, chemolithoautotrophic, and aerobic microbial consortium (termed carbonitroflex) growing in a nutrient-poor medium and an atmosphere containing N₂, O₂, CO₂, and CO is investigated as a model to expand our understanding of extreme biological systems. Here we show that the consortium is dominated by Carbonactinospora thermoautotrophica (strain StC), followed by Sphaerobacter thermophilus, Chelatococcus spp., and Geobacillus spp. Metagenomic analysis of the consortium reveals a mutual relationship among bacteria, with C. thermoautotrophica StC exhibiting carboxydotrophy and carbon-dioxide storage capacity. C. thermoautotrophica StC, Chelatococcus spp., and S. thermophilus harbor genes encoding CO dehydrogenase and formate oxidase. No pure cultures were obtained under the original growth conditions, indicating that a tightly regulated interactive metabolism might be required for group survival and growth in this extreme oligotrophic system. The breadwinner hypothesis is proposed to explain the metabolic flux model and highlight the vital role of C. thermoautotrophica StC (the sole keystone species and primary carbon producer) in the survival of all consortium members. Our data may contribute to the investigation of complex interactions in extreme environments, exemplifying the interconnections and dependency within microbial communities.

Fig. 1. Morphological overview of the chemolithoautotrophic consortium (carbonitroflex).

Initial growth from soil samples. a Colonies growing in mineral medium supplemented with NH₄Cl; b growth on N-FIX medium supplemented with clinoptilolite; c growth on N-FIX medium filled with synthetic air, CO, and CO₂; d confocal electron photomicrograph of carbonitroflex grown in N-FIX medium without nitrogen-fixation sources (magnification 11,210×) showing non-filamentous bacteria (yellow arrow) deposited on actinobacterial filamentous growth (red arrow); e scanning electron photomicrograph depicting the details of the spore-like structure (red arrows) (magnification 51,000×); and f transmission electron photomicrograph (magnification 71,000×) showing the vacuoles (blue arrows), well-delimited membrane, and cell wall (red arrow).

Fig. 2. Transmission electron photomicrographs (magnification 71,000×) showing the highly symmetrical bacterial microcompartments (BMCs)/carboxysome-like structures.

a Red arrows indicate carboxysome-like structures in the cellular cytoplasm of Carbonactinospora thermoautotrophica StC; b a close view of a carboxysome- like structure; and c The Fast Fourier Transform (FFT) image of the carboxysome-like structure, which is a mathematical representation of the spatial frequencies of the image, showing that the StC carboxysome-like structure has a periodic organization with a well-ordered and highly symmetrical pattern.

Fig. 3. Carbonitroflex metagenome composition.

a Taxonomic composition and b molecular phylogenetic analysis of the carbonitroflex consortium based on the gene rrs (16 S); the maximum-likelihood method and Tamura–Nei distance model were used to infer the evolutionary history. The tree with the maximum log probability is shown. The percentage of bootstrap trees in which the associated taxa clustered in this manner is shown adjacent to the branches. The rrs copies filtered from MAGs are highlighted with a diamond shape. The initial trees for the heuristic search were automatically obtained by applying the neighbor-joining and BioNJ algorithms to a matrix of estimated pairwise distances, using the maximum composite likelihood approach, and selecting the topology with the maximum logarithmic likelihood value. The tree is drawn to scale, with branch lengths proportional to the Tamura–Nei distance. The analysis included 32 nucleotide sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were performed using MEGA7.

Fig. 4. Circular representation of the comparison between carbonitroflex MAGs and their closest reference strains.

a Actinomycete isolated from the carbonitroflex consortium (Carbonactinospora thermoautotrophica St_consortium) and strains UBT1 and H1; b Chelatococcus spp. consortium strain and the reference strain DMS18167; c Sphaerobacter thermophilus from the consortium and the reference strain DSM20745; and d Geobacillus spp. strain LEMMY01 and the reference strain DSM13240. More-intense colors represent higher BLASTN identities; GC content is indicated in the inner circles.

Fig. 5. Overall metabolic functions across the MAGs and Geobacillus spp. genome comprising the carbonitroflex consortium.

Metabolic markers of carbon, nitrogen, sulfur, oxygen, and hydrogen cycles were identified via HMM search using the metabolisHMM tool. Metabolic markers with * represent genes with high homology. (See also Supplementary Data 2).

Fig. 6. Proposed metabolic pathways that drive the carbonitroflex consortium interactions.

Main energy and carbon capture are provided by Carbonactinospora thermoautotrophica StC (the breadwinner and sole member capable of carbon fixation), whereas the fixed carbon integrated into the consortium is used by Sphaerobacter thermophilus, Chelatococcus spp., and Geobacillus spp.