Proteomic and Mutant Analysis of the CO ₂ Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

Abstract

Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO 2 ϩ HCO 3 Ϫ ϩ CO 3 2Ϫ ) with CO 2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low-and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea. In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota. Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.KEYWORDS autotroph, bicarbonate transporter, carbon concentrating mechanism, carbon fixation, chemolithoautotroph, hydrothermal vent

Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography

“…Previous studies suggested that in some proteobacterial species, cytosolic form IAq RuBisCOs exhibit a higher affinity and specificity for CO 2 even in the presence of O 2 [53]. During periods of high dissolved inorganic carbon (DIC) and NH 3 -limited conditions, cells favor upregulated production of the cytosolic form RuBisCO because of its higher sensitivity to fluctuating inorganic carbon concentrations compared to the carboxysomal counterpart [54].…”

Diversity in CO 2 -Concentrating Mechanisms among Chemolithoautotrophs from the Genera Hydrogenovibrio , Thiomicrorhabdus , and Thiomicrospira , Ubiquitous in Sulfidic Habitats Worldwide

“…Under low-DIC conditions, all genes assayed here encoding members of two distinct clusters within the SbtA family, and two clusters of homologs of the Tcr_0853-and Tcr_0854-encoded transporter from H. crunogenus (17), were upregulated, whether adjacent to carboxysome loci or not (Table 2). When genes encoding members of the Chr and SulP families were adjacent to carboxysome loci, they were also upregulated under low-DIC conditions (Table 2).…”

“…To determine whether carboxysome-associated transporter gene transcripts were more abundant when cells were grown under low-DIC conditions, taxa that were amenable to cultivation in chemostats were grown under two conditions: DIC limitation (low-DIC condition) and NH 3 limitation (high-DIC condition) (14). Cells were harvested via centrifugation (10,000 ϫ g, 5 min, 4°C), flash-frozen with liquid nitrogen, and stored at -80°C for subsequent RNA extraction using the Ambion RiboPure-Bacteria kit (17). Primers were designed to target genes encoding carboxysome components (csoS2; csoS3; positive control for CCM induction), citrate synthase (calibrator for the 2 -ΔΔCT method) (47), and transporters (Table 4), and qRT-PCR assays were implemented in an Applied Biosystems Step One real-time PCR system, using QuantiTect SYBR green RT-PCR (Qiagen, Inc.) as described in reference 17.…”

“…Instead of heating to 95°C, which can cause membrane proteins to aggregate (50), samples were incubated at 37°C for 1 h to facilitate dissolution before they were subjected to SDS-PAGE (51). Coomassie-stained gel fragments were excised from the molecular weight region corresponding to those predicted from the amino acid sequence of the target protein and were processed as described previously (17).…”

“…Random and site-directed mutagenesis of the gene loci Tcr_0853 and Tcr_0854, which are located downstream of the carboxysome locus in this organism, result in a high-CO 2 -requiring phenotype and loss of the ability to generate high intracellular DIC concentrations, suggesting that these genes encode a two-component DIC transporter (17). Homologs of these genes are common among autotrophic Bacteria, and one of them (Tcr_0854) is from a protein family without prior biochemical characterization (Pfam10070) (17).…”