Co-existence of CBB cycle and rTCA carbon fixation pathway in thermophilic Actinomycetota Carbonactinospora thermoautotrophica StC and correlation with the CO2-concentrating microcompartment

Seven carbon autotrophic fixation pathways were described so far. However, it is not common to find the co-existence of more than one cycle in a single cell. Here, we describe a thermophilic bacterium Carbonactinospora thermoautotrophica StC with a unique and versatile carbon metabolism. StC was isolated from a consortium found in a burning organic pile that exhibits an optimal growth temperature between 55° and 65° C. The genome analyses suggested that the strain StC potentially performs two-carbon fixation pathways, Calvin-Benson-Bassham (CBB) cycle and the Reductive citrate cycle (rTCA) and preserve a microcompartment related with CO2 concentration. To better understand the carbon fixation in StC strain, the expression of the genes of bacterial cells grown autotrophically and heterotrophically were analyzed. For our surprise the data showed the co-existing of the both carbon fixation pathways - CBB and rTCA cycles - in a cultivable thermophilic chemoautotrophic bacterium Carbonactinospora thermoautotrophica strain StC, based on integrated omics of genomics, transcriptomics, and proteomics. These two cycles working together may help microorganisms to improve the CO2 fixation. The knowledge about the co-occurrence of carbon cycle in a single cell leads open a question ‘why microorganisms use multiple pathways to fix carbon and what the advantage for this strategy?’. Advancing on this is a key to better understand the biological carbon fixation mechanism in thermophiles and prospecting the repurposing of enzymes in synthetic biology for biotechnological applications. The cells of Carbonactinospora thermoautotrophica strain StC were grown for four days at 60 °C under three different conditions: i) autotrophically in a N-FIX solid culture medium with a gas atmosphere (CO/CO2/N2/O2); ii) heterotrophically in R2A culture medium (glucose and peptone as carbon and nitrogen source respectively) solidified with Noble Ágar (Sigma-Aldrich) and iii) N-FIX solid culture medium with a gas atmosphere (CO/CO2/) + NH4Cl2. The experiment was conducted in quadruplicate, and RNA was extracted using NucleoSpin TriPrep kit (Macherey-Nagel) with some modifications, as follows: 480 µL of 0.5M EDTA and 120 µL of lysozyme (100 mg/mL) were used to lyse the bacterium cells by incubating at 37 °C for 30 minutes, followed by FastPrep grind and homogenization (MP Biomedicals). Total RNA was then purified with the Ambion’s Dnase Treatment Kit (Thermo Fisher Scientific) and quantified by spectrophotometry, using NanoDrop 2000 (Thermo Fisher Scientific) and Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific).