Anthropogenic activities affect the diverse autotrophic communities of coastal sediments.

Coastal sediments are a critical domain for carbon sequestration and are profoundly impacted by human activities. Therefore, it is essential to understand the structure and components of benthic autotrophs that play a crucial role in carbon sequestration processes, as well as the influence of anthropogenic activities on their communities. This study utilized an urban estuary, an industrial sea bay, a maricultural sea region, and two mangrove coastlines within the coastal areas of Guangdong Province, China. The micro-benthos in these environments, including prokaryotes and eukaryotes, were identified through high-throughput sequencing of 16S rRNA and 18S rRNA genes. The findings show that the autotrophic composition was altered by the interactions of anthropogenic heavy metals (Cd and Zn) and micro-eukaryotes (protazoa, metazoa, and parasitic organisms). Industrial pollution reduced the abundance of both prokaryotic and eukaryotic autotrophs. Mangroves induced a substantial transformation in the sediment eukaryotic and prokaryotic composition, increasing the proportion of autotrophs, notably sulfur-oxidizing and iron-oxidizing bacteria and microalgae. This alteration suggests an increase in specific sulfur and iron cycling with simultaneous carbon sequestration within mangrove sediments. These results indicate that anthropogenic activities affect sediment carbon sequestration by altering autotrophic assemblages along coastlines, thereby inducing consequential shifts in overall elemental cycling processes.

A new type of carboxysomal carbonic anhydrase in sulfur chemolithoautotrophs from alkaline environments.

Autotrophic bacteria are able to fix CO2 in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO2 fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO2-concentrating mechanisms (CCMs). CCMs consist of CO2 and HCO3- transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophic Bacteria that use the Calvin-Benson-Basham cycle. Members of the genus Thiomicrospira lack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO2 fixation by carboxysomes from members of Thiomicrospira and potentially other Bacteria. Carboxysome enrichments from Thiomicrospira pelophila and Thiomicrospira aerophila were found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted in T. pelophila, cells could no longer grow under low-CO2 conditions, and CA activity was no longer detectable in their carboxysomes. When T. pelophila ιCA was expressed in a strain of Escherichia coli lacking native CA activity, this strain recovered an ability to grow under low CO2 conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCE: Here, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO2 fixation by some organisms with CO2-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions.

Polyhydroxyalkanoate Copolymer Production by Recombinant Ralstonia eutropha Strain 1F2 from Fructose or Carbon Dioxide as Sole Carbon Source.

Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB-4, cannot produce PHA. Strain 1F2, derived from strain PHB-4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO2. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 106 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA.

Corrigendum: Atypical carboxysome loci: JEEPs or junk?

[This corrects the article DOI: 10.3389/fmicb.2022.872708.].

Prevalence and Preferred Niche of Small Eukaryotes with Mixotrophic Potentials in the Global Ocean.

Unicellular eukaryotes that are capable of phago-mixotrophy in the ocean compete for inorganic nutrients and light with autotrophs, and for bacterial prey with heterotrophs. In this study, we ask what the overall prevalence of eukaryotic mixotrophs in the vast open ocean is, and how the availability of inorganic nutrients, light, and prey affects their relative success. We utilized the Tara Oceans eukaryotic 18S rRNA gene and environmental context variables dataset to conduct a large-scale field analysis. We also performed isolate-based culture experiments to verify growth and nutritional resource relationships for representative mixotrophic taxa. The field analysis suggested that the overall prevalence of mixotrophs were negatively correlated with nutrient concentrations and positively associated with light availability. Concentrations of heterotrophic bacteria as a single variable also presented a positive correlation with mixotrophic prevalence, but to a lesser extent. On the other hand, the culture experiments demonstrated a taxa-specific relationship between mixotrophic growth and nutrition resources, i.e., the growth of one group was significantly dependent on light availability, while the other group was less affected by light when they received sufficient prey. Both groups were capable of growing efficiently with low inorganic nutrients when receiving sufficient prey and light. Therefore, our field analysis and culture experiments both suggest that phago-mixotrophy for ocean eukaryotes is seemingly an efficient strategy to compensate for nutrient deficiency but unnecessary to compensate for light scarcity. This study collectively revealed a close relationship between abiotic and biotic nutritional resources and the prevalence of trophic strategies, shedding light on the importance of light and nutrients for determining the competitive success of mixotrophs versus autotrophic and heterotrophic eukaryotes in the ocean.

Widespread dissolved inorganic carbon-modifying toolkits in genomes of autotrophic Bacteria and Archaea and how they are likely to bridge supply from the environment to demand by autotrophic pathways.

Using dissolved inorganic carbon (DIC) as a major carbon source, as autotrophs do, is complicated by the bedeviling nature of this substance. Autotrophs using the Calvin-Benson-Bassham cycle (CBB) are known to make use of a toolkit comprised of DIC transporters and carbonic anhydrase enzymes (CA) to facilitate DIC fixation. This minireview provides a brief overview of the current understanding of how toolkit function facilitates DIC fixation in Cyanobacteria and some Proteobacteria using the CBB and continues with a survey of the DIC toolkit gene presence in organisms using different versions of the CBB and other autotrophic pathways (reductive citric acid cycle, Wood-Ljungdahl pathway, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate cycle, and dicarboxylate-hydroxybutyrate cycle). The potential function of toolkit gene products in these organisms is discussed in terms of CO2 and HCO3- supply from the environment and demand by the autotrophic pathway. The presence of DIC toolkit genes in autotrophic organisms beyond those using the CBB suggests the relevance of DIC metabolism to these organisms and provides a basis for better engineering of these organisms for industrial and agricultural purposes.

Autotrophic Production of the Sesquiterpene α-Humulene with Cupriavidus necator in a Controlled Bioreactor.

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L-1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

Denitrification shifted autotroph-heterotroph interactions in Microcystis aggregates.

Denitrification is the most important process for nitrogen removal in eutrophic lakes and was mostly investigated in lake sediment. Denitrification could also be mediated by cyanobacterial aggregates, yet how this process impacts nitrogen (N) availability and the associated autotroph-heterotroph relationships within cyanobacterial aggregates has not been investigated. In this study, incubation experiments with nitrate amendment were conducted with Microcystis aggregates (MAs). Measurement of nitrogen contents, 16S rRNA-based microbial community profiling and metatranscriptomic sequencing were used to jointly assess nitrogen turnover dynamics, as well as changes in microbial composition and gene expression. Strong denitrification potential was revealed, and maximal N removal was achieved within two days, after which the communities entered a state of severe N limitation. Changes of active microbial communities were further promoted both with regard to taxonomic composition and transcriptive activities. Expression of transportation-related genes confirmed competition for N sources by Microcystis and phycospheric communities. Strong stress response to reactive oxygen species by Microcystis was revealed. Notably, interspecific relationships among Microcystis and phycospheric communities exhibited a shift toward antagonistic interactions, particularly evidenced by overall increased expression of genes related to cell lysis and utilization of cellular materials. Patterns of fatty acid and starch metabolism also suggested changes in carbon metabolism and cross-feeding patterns within MAs. Taken together, this study demonstrated substantial denitrification potential of MAs, which, importantly, further induced changes in both metabolic activities and autotroph-heterotroph interactions. These findings also highlight the key role of nutrient condition in shaping autotroph-heterotroph relationships.

"Candidatus Hydrogenisulfobacillus filiaventi" strain R50 gen. nov. sp. nov., a highly efficient producer of extracellular organic compounds from H2 and CO2.

Production of organic molecules is largely depending on fossil fuels. A sustainable alternative would be the synthesis of these compounds from CO2 and a cheap energy source, such as H2, CH4, NH3, CO, sulfur compounds or iron(II). Volcanic and geothermal areas are rich in CO2 and reduced inorganic gasses and therefore habitats where novel chemolithoautotrophic microorganisms for the synthesis of organic compounds could be discovered. Here we describe "Candidatus Hydrogenisulfobacillus filiaventi" R50 gen. nov., sp. nov., a thermoacidophilic, autotrophic H2-oxidizing microorganism, that fixed CO2 and excreted no less than 0.54 mol organic carbon per mole fixed CO2. Extensive metabolomics and NMR analyses revealed that Val, Ala and Ile are the most dominant form of excreted organic carbon while the aromatic amino acids Tyr and Phe, and Glu and Lys were present at much lower concentrations. In addition to these proteinogenic amino acids, the excreted carbon consisted of homoserine lactone, homoserine and an unidentified amino acid. The biological role of the excretion remains uncertain. In the laboratory, we noticed the production under high growth rates (0.034 h-1, doubling time of 20 h) in combination with O2-limitation, which will most likely not occur in the natural habitat of this strain. Nevertheless, this large production of extracellular organic molecules from CO2 may open possibilities to use chemolithoautotrophic microorganisms for the sustainable production of important biomolecules.

Heterotrophic Growth Dominates in the Most Extremotolerant Extremophile Cultures.

Due to their ability to withstand "extreme" conditions, Earth's extremophilic organisms can constrain habitability windows for other planetary systems. However, there are many other considerations to microbial growth requirements beyond environmental extremes, such as nutrient availability. Here, we conduct a literature review of the most extremotolerant extremophiles in culture, since working with cultured organisms allows environmental and nutrient variables to be constrained with a high level of specificity. We generated a database that includes the isolation environment, carbon source(s) used, and growth preferences across temperature, pressure, salinity, and pH extremes. We found that the "most extreme" conditions were primarily sustained by heterotrophs, except for hyperthermophiles. These results highlight the importance of considering organic carbon availability when using extremophiles for habitability constraints. We also interrogated polyextreme potential across temperature, pressure, salinity, and pH conditions. Our findings suggest that the investigation of growth tolerance rather than growth optimum may reveal wider habitability parameters. Overall, these results highlight the potential polyextremes, environments, nutrient requirements, and additional analyses that could improve the application of cultured investigations to astrobiology questions.

Continuous Supply of Non-Combustible Gas Mixture for Safe Autotrophic Culture to Produce Polyhydroxyalkanoate by Hydrogen-Oxidizing Bacteria.

Polyhydroxyalkanoates (PHAs) are eco-friendly plastics that are thermoplastic and biodegradable in nature. The hydrogen-oxidizing bacterium Ralstonia eutropha can biosynthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], the most common PHA, from carbon dioxide using hydrogen and oxygen as energy sources. In conventional autotrophic cultivation using R. eutropha, a gas mixture containing 75−80 vol% hydrogen is supplied; however, a gas mixture with such a high hydrogen content has a risk of explosion due to gas leakage. In this study, we aimed to develop an efficient cell culture system with a continuous supply of a non-combustible gas mixture (H2: O2: CO2: N2 = 3.8: 7.3: 13.0: 75.9) for safe autotrophic culture to produce P(3HB) by hydrogen-oxidizing bacteria, with a controlled hydrogen concentration under a lower explosive limit concentration. When the gas mixture was continuously supplied to the jar fermentor, the cell growth of R. eutropha H16 significantly improved compared to that in previous studies using flask cultures. Furthermore, an increased gas flow rate and agitation speed enhanced both cell growth and P(3HB) production. Nitrogen source deficiency promoted P(3HB) production, achieving up to 2.94 g/L P(3HB) and 89 wt% P(3HB) content in the cells after 144 h cultivation. R. eutropha NCIMB 11599, recombinant R. eutropha PHB-4, and Azohydromonas lata grew in a low-hydrogen-content gas mixture. R. eutropha H16 and recombinant R. eutropha PHB-4 expressing PHA synthase from Bacillus cereus YB-4 synthesized P(3HB) with a high weight-average molecular weight of 13.5−16.9 × 105. Thus, this autotrophic culture system is highly beneficial for PHA production from carbon dioxide using hydrogen-oxidizing bacteria as the risk of explosion is eliminated.

Marine Autotroph-Herbivore Synergies: Unravelling the Roles of Macroalgae in Marine Ecosystem Dynamics.

Species invasion is a leading threat to marine ecosystems worldwide, being deemed as one of the ultimate jeopardies for biodiversity along with climate change. Tackling the emerging biodiversity threat to maintain the ecological balance of the largest biome in the world has now become a pivotal part of the Sustainable Development Goals (SDGs). Marine herbivores are often considered as biological agents that control the spread of invasive species, and their effectiveness depends largely on factors that influence their feeding preferences, including the specific attributes of their food-the autotrophs. While the marine autotroph-herbivore interactions have been substantially discussed globally, many studies have reported contradictory findings on the effects of nutritional attributes and novelty of autotrophs on herbivore feeding behaviour. In view of the scattered literature on the mechanistic basis of autotroph-herbivore interactions, we generate a comprehensive review to furnish insights into critical knowledge gaps about the synergies based largely on the characteristics of macroalgae; an important group of photosynthetic organisms in the marine biome that interact strongly with generalist herbivores. We also discuss the key defence strategies of these macroalgae against the herbivores, highlighting their unique attributes and plausible roles in keeping the marine ecosystems intact. Overall, the feeding behaviour of herbivores can be affected by the nutritional attributes, morphology, and novelty of the autotrophs. We recommend that future research should carefully consider different factors that can potentially affect the dynamics of the marine autotroph-herbivore interactions to resolve the inconsistent results of specific attributes and novelty of the organisms involved.

Atypical Carboxysome Loci: JEEPs or Junk?

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria, primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3 - in the cytoplasm via active transport, HCO3 - enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3 - to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and ß-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts.

Hydrogenophilus thiooxidans sp. nov., a moderately thermophilic chemotrophic bacterium unable to grow on hydrogen gas, isolated from hot spring microbial mats.

A novel thermophilic chemotrophic bacterium, strain SS56T, was isolated from Nakabusa Hot Spring, Japan. The isolate was a rod-shaped (1.5-2.1×0.6-0.8 µm), Gram-stain-negative bacterium. The cells of this strain grew chemoheterotrophically under aerobic and anaerobic conditions. Autotrophic growth was observed with thiosulphate and elemental sulphur under aerobic conditions but not with H2 as the electron donor. Heterotrophic growth in the presence of O2 occurred on yeast extract, tryptone, polypeptone and organic acids. Strain SS56T used nitrite as an alternative electron acceptor under anaerobic chemoheterotrophic conditions. The isolate grew between 35 and 65 °C, with the optimum at 55 °C. The pH range for growth was pH 6.0-9.0; optimal growth occurred at pH 7.0-8.0. The 16S rRNA gene sequence of strain SS56T was 98.9% identical to that of Hydrogenophilus thermoluteolus TH-1T. The draft genome sequence of 2401804 bp for strain SS56T gave values of 53.7% for digital DNA-DNA hybridization, 92.9% for average nucleotide identity and 93.6% for average amino acid identity compared with the genome sequence of 2223143 bp for H. thermoluteolus TH-1T. Based on the information described above, strain SS56T (=DSM 111892T=JCM 34254T) is proposed as the type strain of a novel species, Hydrogenophilus thiooxidans sp. nov.

Comparative Genomics on Cultivated and Uncultivated Freshwater and Marine "Candidatus Manganitrophaceae" Species Implies Their Worldwide Reach in Manganese Chemolithoautotrophy.

Chemolithoautotrophic manganese oxidation has long been theorized but only recently demonstrated in a bacterial coculture. The majority member of the coculture, "Candidatus Manganitrophus noduliformans," is a distinct but not yet isolated lineage in the phylum Nitrospirota (Nitrospirae). Here, we established two additional MnCO3-oxidizing cultures using inocula from Santa Barbara (California) and Boetsap (South Africa). Both cultures were dominated by strains of a new species, designated "Candidatus Manganitrophus morganii." The next most abundant members differed in the available cultures, suggesting that while "Ca. Manganitrophus" species have not been isolated in pure culture, they may not require a specific syntrophic relationship with another species. Phylogeny of cultivated "Ca. Manganitrophus" and related metagenome-assembled genomes revealed a coherent taxonomic family, "Candidatus Manganitrophaceae," from both freshwater and marine environments and distributed globally. Comparative genomic analyses support this family being Mn(II)-oxidizing chemolithoautotrophs. Among the 895 shared genes were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse complex 1 containing 2 additional pumping subunits was also shared by the family, as were genes for the reverse tricarboxylic acid carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the family lacked genes for nitrification found in Nitrospira species. The results suggest that "Ca. Manganitrophaceae" share a core set of candidate genes for the newly discovered manganese-dependent chemolithoautotrophic lifestyle and likely have a broad, global distribution. IMPORTANCE Manganese (Mn) is an abundant redox-active metal that cycles in many of Earth's biomes. While diverse bacteria and archaea have been demonstrated to respire Mn(III/IV), only recently have bacteria been implicated in Mn(II) oxidation-dependent growth. Here, two new Mn(II)-oxidizing enrichment cultures originating from two continents and hemispheres were examined. By comparing the community composition of the enrichments and performing phylogenomic analysis on the abundant Nitrospirota therein, new insights are gleaned on cell interactions, taxonomy, and machineries that may underlie Mn(II)-based lithotrophy and autotrophy.

Desulfovulcanus ferrireducens gen. nov., sp. nov., a thermophilic autotrophic iron and sulfate-reducing bacterium from subseafloor basalt that grows on akaganéite and lepidocrocite minerals.

A deep-sea thermophilic bacterium, strain Ax17T, was isolated from 25 °C hydrothermal fluid at Axial Seamount. It was obligately anaerobic and autotrophic, oxidized molecular hydrogen and formate, and reduced synthetic nanophase Fe(III) (oxyhydr)oxide minerals, sulfate, sulfite, thiosulfate, and elemental sulfur for growth. It produced up to 20 mM Fe2+ when grown on ferrihydrite but < 5 mM Fe2+ when grown on akaganéite, lepidocrocite, hematite, and goethite. It was a straight to curved rod that grew at temperatures ranging from 35 to 70 °C (optimum 65 °C) and a minimum doubling time of 7.1 h, in the presence of 1.5-6% NaCl (optimum 3%) and pH 5-9 (optimum 8.0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was 90-92% identical to other genera of the family Desulfonauticaceae in the phylum Pseudomonadota. The genome of Ax17T was sequenced, which yielded 2,585,834 bp and contained 2407 protein-coding sequences. Based on overall genome relatedness index analyses and its unique phenotypic characteristics, strain Ax17T is suggested to represent a novel genus and species, for which the name Desulfovulcanus ferrireducens is proposed. The type strain is Ax17T (= DSM 111878T = ATCC TSD-233T).

Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments.

In nature, concentrations of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO2 fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations, were chosen for study. A high-CO2-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) ΔyadF ΔcynT to grow under low-CO2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO2, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with various subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO2.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

Ubiquity and functional uniformity in CO2 concentrating mechanisms in multiple phyla of Bacteria is suggested by a diversity and prevalence of genes encoding candidate dissolved inorganic carbon transporters.

Autotrophic microorganisms catalyze the entry of dissolved inorganic carbon (DIC; = CO2 + HCO3- + CO32-) into the biological component of the global carbon cycle, despite dramatic differences in DIC abundance and composition in their sometimes extreme environments. "Cyanobacteria" are known to have CO2 concentrating mechanisms (CCMs) to facilitate growth under low CO2 conditions. These CCMs consist of carboxysomes, containing enzymes ribulose 1,5-bisphosphate oxygenase and carbonic anhydrase, partnered to DIC transporters. CCMs and their DIC transporters have been studied in a handful of other prokaryotes, but it was not known how common CCMs were beyond "Cyanobacteria". Since it had previously been noted that genes encoding potential transporters were found neighboring carboxysome loci, α-carboxysome loci were gathered from bacterial genomes, and potential transporter genes neighboring these loci are described here. Members of transporter families whose members all transport DIC (CHC, MDT and Sbt) were common in these neighborhoods, as were members of the SulP transporter family, many of which transport DIC. 109 of 115 taxa with carboxysome loci have some form of DIC transporter encoded in their genomes, suggesting that CCMs consisting of carboxysomes and DIC transporters are widespread not only among "Cyanobacteria", but also among members of "Proteobacteria" and "Actinobacteria".