Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans

Abstract

This study was motivated by surprising gaps in the current knowledge of microbial inorganic carbon (Ci) uptake and assimilation at acidic pH values (pH < 3). Particularly striking is the limited understanding of the differences between Ci uptake mechanisms in acidic versus circumneutral environments where the Ci predominantly occurs either as a dissolved gas (CO2) or as bicarbonate (HCO3-), respectively. In order to gain initial traction on the problem, the relative abundance of transcripts encoding proteins involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using ferrous iron as an energy source, although they are able to grow at pH 5 when using sulfur as an energy source. The relative abundance of transcripts of five operons (cbb1 -5) and one gene cluster (can-sulP) was monitored by RT-qPCR and, in selected cases, at the protein level by Western blotting, when cells were grown under different regimens of CO2 concentration in elemental sulfur. Of particular note was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP, previously annotated as a sulfate transporter, is a novel type of bicarbonate transporter. A conceptual model of CO2 fixation was constructed from combined bioinformatic and experimental approaches that suggests strategies for providing ecological flexibility under changing concentrations of CO2 and provides a portal to elucidating Ci uptake and regulation in acidic conditions. The results could advance the understanding of industrial bioleaching processes to recover metals such as copper at acidic pH. In addition, they may also shed light on how chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally occurring low pH environments.

This study was motivated by surprising gaps in the current knowledge of microbial inorganic carbon (Ci) uptake and assimilation at acidic pH values (pH < 3). Particularly striking is the limited understanding of the differences between Ci uptake mechanisms in acidic versus circumneutral environments where the Ci predominantly occurs either as a dissolved gas (CO2) or as bicarbonate (HCO3-), respectively. In order to gain initial traction on the problem, the relative abundance of transcripts encoding proteins involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using ferrous iron as an energy source, although they are able to grow at pH 5 when using sulfur as an energy source. The relative abundance of transcripts of five operons (cbb1 -5) and one gene cluster (can-sulP) was monitored by RT-qPCR and, in selected cases, at the protein level by Western blotting, when cells were grown under different regimens of CO2 concentration in elemental sulfur. Of particular note was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP, previously annotated as a sulfate transporter, is a novel type of bicarbonate transporter. A conceptual model of CO2 fixation was constructed from combined bioinformatic and experimental approaches that suggests strategies for providing ecological flexibility under changing concentrations of CO2 and provides a portal to elucidating Ci uptake and regulation in acidic conditions. The results could advance the understanding of industrial bioleaching processes to recover metals such as copper at acidic pH. In addition, they may also shed light on how chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally occurring low pH environments.