Members of the generaHydrogenovibrio,Thiomicrospira, andThiomicrorhabdusfix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking inThiomicrorhabdussp. strain Milos-T2 andThiomicrorhabdus arctica, and the inability ofThiomicrorhabdussp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed inEscherichia coli. The expression of these genes in the carbonic anhydrase-deficientE. colistrain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded byTcr_0853andTcr_0854and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCEAutotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophicBacteriaandArchaeaare likely to be diverse but have been well characterized only for “Cyanobacteria.” Based on genome sequences, members of the generaHydrogenovibrio,Thiomicrospira, andThiomicrorhabdushave a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed inE. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.
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
Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria (“Candidatus Endoriftia persephone”) dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the “Ca. Endoriftia persephone” genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis. IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the “Ca. Endoriftia persephone” genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ. However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.
Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and Calvin-Benson-Bassham cycle (CBB) in “ Candidatus Endoriftia persephonae”, the autotrophic sulfur-oxidizing bacterial endosymbiont from giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than the CBB. R. pachyptila were incubated under in situ conditions in high-pressure aquaria under low (28-40 μmol hr −1 ) or high (180 - 276 μmol hr −1 ) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under either condition were capable of similar rates of CO 2 fixation. Activities of rCAC enzyme ATP-dependent citrate lyase and CBB enzyme RubisCO did not differ between the two conditions, though transcript abundances for ATP-dependent citrate lyase were 4 to 5-fold higher under low sulfide conditions. δ 13 C values of internal dissolved inorganic carbon pools were variable, and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ 13 C values of lipids fell between those collected for organisms using either the rCAC or CBB cycles exclusively. These observations are consistent with co-occurring activities of rCAC and CBB cycles in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from –omics studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila were maintained and monitored in high-pressure aquaria prior to measuring their CO 2 -fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO 2 -fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.
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