Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus Ferrooxidans
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fmicb-10-00603 April 2, 2019 Time: 17:28 # 1 ORIGINAL RESEARCH published: 04 April 2019 doi: 10.3389/fmicb.2019.00603 Effect of CO2 Concentration on Uptake and Assimilation of Inorganic Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans Mario Esparza1, Eugenia Jedlicki2, Carolina González2, Mark Dopson3 and David S. Holmes2,4* 1 Laboratorio de Biominería, Departamento de Biotecnología, Facultad de Ciencias del Mar y Recursos Biológicos, Universidad de Antofagasta, Antofagasta, Chile, 2 Center for Bioinformatics and Genome Biology, Fundación Ciencia & Vida, Santiago, Chile, 3 Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Linnaeus University, Kalmar, Sweden, 4 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile 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 Edited by: in acidic versus circumneutral environments where the Ci predominantly occurs either − Gloria Paz Levicán, as a dissolved gas (CO2) or as bicarbonate (HCO3 ), respectively. In order to gain Universidad de Santiago de Chile, initial traction on the problem, the relative abundance of transcripts encoding proteins Chile involved in Ci uptake and assimilation was studied in the autotrophic, polyextreme Reviewed by: Sabrina Hedrich, acidophile Acidithiobacillus ferrooxidans whose optimum pH for growth is 2.5 using Federal Institute for Geosciences ferrous iron as an energy source, although they are able to grow at pH 5 when and Natural Resources, Germany Kathleen Scott, using sulfur as an energy source. The relative abundance of transcripts of five University of South Florida, operons (cbb1-5) and one gene cluster (can-sulP) was monitored by RT-qPCR and, United States in selected cases, at the protein level by Western blotting, when cells were grown *Correspondence: under different regimens of CO concentration in elemental sulfur. Of particular note David S. Holmes 2 [email protected] was the absence of a classical bicarbonate uptake system in A. ferrooxidans. However, bioinformatic approaches predict that sulP, previously annotated as a sulfate transporter, Specialty section: is a novel type of bicarbonate transporter. A conceptual model of CO fixation was This article was submitted to 2 Extreme Microbiology, constructed from combined bioinformatic and experimental approaches that suggests a section of the journal strategies for providing ecological flexibility under changing concentrations of CO2 and Frontiers in Microbiology provides a portal to elucidating Ci uptake and regulation in acidic conditions. The Received: 02 November 2018 Accepted: 11 March 2019 results could advance the understanding of industrial bioleaching processes to recover Published: 04 April 2019 metals such as copper at acidic pH. In addition, they may also shed light on how Citation: chemolithoautotrophic acidophiles influence the nutrient and energy balance in naturally Esparza M, Jedlicki E, González C, occurring low pH environments. Dopson M and Holmes DS (2019) Effect of CO2 Concentration on Keywords: CO2 fixation, CCM, carbon concentration mechanism, Acidithiobacillus ferrooxidans, acidic Uptake and Assimilation of Inorganic environment, low pH environment, bicarbonate uptake, RubisCO Carbon in the Extreme Acidophile Acidithiobacillus ferrooxidans. Front. Microbiol. 10:603. Abbreviations: AMD, acid mine drainage; CBB, Calvin–Benson–Bassham; CCM, carbon concentration mechanism; Ci, doi: 10.3389/fmicb.2019.00603 inorganic carbon. Frontiers in Microbiology| www.frontiersin.org 1 April 2019| Volume 10| Article 603 fmicb-10-00603 April 2, 2019 Time: 17:28 # 2 Esparza et al. CO2 Fixation at Extremely Low pH INTRODUCTION In contrast, less is known about Ci uptake and assimilation in extremely acidic environments where the dominant source Acidithiobacillus ferrooxidans is a polyextremophile inhabiting of Ci is the dissolved gas CO2 (Carroll and Mather, 1992; very acidic (pH < 3) and often metal laden environments Cardenas et al., 2010; Valdés et al., 2010; Mangan et al., 2016; that belongs to the Acidithiobacillia class within the WikiVividly, 2018). A. ferrooxidans fixes carbon by the CBB Proteobacteria (Williams and Kelly, 2013). It is an obligate cycle (Esparza et al., 2010). Bioinformatic analyses, EMSA chemolithoautotrophic, mesophilic microorganism that gains assays, and complementation of mutants in the surrogate energy and reducing power by the aerobic oxidation of host Cupriavidus necator (formerly Ralstonia eutropha) have hydrogen, inorganic sulfur compounds, and ferrous iron demonstrated the presence of four operons (cbb1-4) of CBB (Bonnefoy and Holmes, 2012; Dopson and Johnson, 2012) cycle genes in A. ferrooxidans that are involved in Ci uptake and anaerobically via sulfur or formate oxidation coupled to and assimilation. Operons cbb1-3 were shown experimentally reduction of ferric iron (Pronk et al., 1991; Hedrich and Johnson, to be regulated by CbbR, a LysR-family transcription regulator 2013; Osorio et al., 2013). (Esparza et al., 2009, 2010, 2015). In the present study, RNA A. ferrooxidans is one of the most abundant microorganisms transcript and protein abundance profiles were determined for found at ambient temperatures in industrial bioleaching heaps genes present in A. ferrooxidans operons cbb1-4 under different used for the recovery of, e.g., copper (Soto et al., 2013; Vera et al., CO2 concentrations. In addition, a fifth cbb operon (cbb5) 2013; Zhang et al., 2016). It also forms an integral part of natural and a gene cluster predicted to encode a bicarbonate uptake occurring acidic ecosystems such as the Rio Tinto and deep transporter and a carbonic anhydrase were detected and were subsurface in the Iberian pyrite belt (Amils et al., 2014), acidic also evaluated for expression under different CO2 concentration springs, cave systems plus volcanic soils (reviewed in Johnson, regimes. Acquiring this knowledge is important considering 2012; Hedrich and Schippers, 2016), and acid mine drainage the central roles that the CCM and CBB cycle genes play in (AMD) (Chen et al., 2015; Teng et al., 2017). A. ferrooxidans the determination of CO2 fixation and biomass formation in is considered a model species for understanding genetic and extremely acidic environments. metabolic functions reviewed in Cardenas et al., 2016) and survival mechanisms at extremely low pH (Chao et al., 2008) and reviewed in Slonczewski et al.(2009). It has also provided MATERIALS AND METHODS useful information for understanding how microorganisms can contribute to the nutrient and energy balance in bioleaching Bacterial Strains and Culture Conditions heaps (Valdes et al., 2008; Valdés et al., 2010). A. ferrooxidans ATCC 23270 was cultured in 9K medium The dominant source of available inorganic carbon (Ci) (Quatrini et al., 2007) adjusted to pH 3.5 with H2SO4 and ◦ in circumneutral and slightly alkaline environments such as containing 5 g/L elemental sulfur at 30 C under aerobic − terrestrial fresh water and oceans is bicarbonate (HCO 3) with conditions (0.036% CO2). Increased concentrations of CO2 were lower concentrations of dissolved CO2 (Mangan et al., 2016). The obtained by sparging with a mixture of CO2 and air by changing majority of models for prokaryotic Ci uptake and assimilation the ratio of CO2 in the gas mixture. A. ferrooxidans cultures were have been elucidated for organisms, such as cyanobacteria, grown to mid-log phase (Guacucano et al., 2000) as measured by that inhabit these environments (Burnap et al., 2015; Klanchui cell counts using a Neubauer chamber. Cells were rapidly cooled ◦ et al., 2017). Cyanobacteria fix carbon via the Calvin-Benson- on ice and then centrifuged at 800 × g for 5 min at 4 C to remove Bassham (CBB) cycle and use a variety of carbon concentration solid sulfur particles followed by cell capture by centrifugation at × ◦ mechanisms (CCMs) to take up CO2 or bicarbonate and provide 8,000 g for 10 min at 4 C. The cell pellet was re-suspended CO2 to the carbon fixation enzyme, ribulose bisphosphate in ice-cold 9K salt solution for further washing. Total RNA was carboxylase-oxygenase (RubisCO). Five Ci uptake systems have prepared immediately after cell harvesting. been reported including three bicarbonate transporters: BCT1, SbtA, and BicA that vary in affinity and flux for bicarbonate Isolation of RNA and Real-Time and two intracellular CO2 “uptake” systems, that convert CO2, Quantitative PCR (RT-qPCR) Assays passively diffusing into the cell, into bicarbonate (Burnap et al., Total RNA was isolated from A. ferrooxidans cells as described 2015; Klanchui et al., 2017). The transporters vary in affinity previously (Guacucano et al., 2000). The RNA preparations and flux for bicarbonate providing a selective advantage to were treated with DNase I (Fermentas) before proceeding with organisms in environments with a wide dynamic range of the cDNA synthesis step. One microgram of total cellular − HCO3 availability. For example, freshwater b-cyanobacteria RNA was used for each reaction. Real-time quantitative RT- that live at about pH 7 not only use the high affinity SbtA PCR (RT-qPCR) was performed using RevertAid H Minus transporter and the low affinity, high flux BicA transporter Reverse Transcriptase (Fermentas). The sequences of the but also the medium affinity BCT1, an inducible bicarbonate qPCR primers for genes