Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH3SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H2S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 μM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments.

The enzyme 4-hydroxy-tetrahydrodipicolinate synthase (DapA) is involved in the production of lysine and precursor molecules for peptidoglycan synthesis. In a multistep reaction, DapA converts pyruvate and L-aspartate-4-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. In many organisms, lysine binds allosterically to DapA, causing negative feedback, thus making the enzyme an important regulatory component of the pathway. Here, the 2.1 Å resolution crystal structure of DapA from the thermoacidophilic methanotroph Methyl­acidiphilum fumariolicum SolV is reported. The enzyme crystallized as a contaminant of a protein preparation from native biomass. Genome analysis reveals that M. fumariolicum SolV utilizes the recently discovered aminotransferase pathway for lysine biosynthesis. Phylogenetic analyses of the genes involved in this pathway shed new light on the distribution of this pathway across the three domains of life.

Emissions of the strong greenhouse gas methane (CH4) to the atmosphere are mitigated by methanotrophic microorganisms. Methanotrophs found in extremely acidic geothermal systems belong to the phylum Verrucomicrobia. Thermophilic verrucomicrobial methanotrophs from the genus Methylacidiphilum can grow autotrophically on hydrogen gas (H2), but it is unknown whether this also holds for their mesophilic counterparts from the genus Methylacidimicrobium. To determine this, we examined H2 consumption and CO2 fixation by the mesophilic verrucomicrobial methanotroph Methylacidimicrobium tartarophylax 4AC. We found that strain 4AC grows autotrophically on H2 with a maximum growth rate of 0.0048 h–1 and a yield of 2.1 g dry weight⋅mol H2–1, which is about 12 and 41% compared to the growth rate and yield on methane, respectively. The genome of strain 4AC only encodes for an oxygen-sensitive group 1b [NiFe] hydrogenase and H2 is respired only when oxygen concentrations are below 40 μM. Phylogenetic analysis and genomic comparison of methanotrophs revealed diverse [NiFe] hydrogenases, presumably with varying oxygen sensitivity and affinity for H2, which could drive niche differentiation. Our results show that both thermophilic and mesophilic verrucomicrobial methanotrophs can grow as autotrophs on H2 as a sole energy source. Our results suggest that verrucomicrobial methanotrophs are particularly well-equipped to thrive in hostile volcanic ecosystems, since they can consume H2 as additional energy source.

Soda lakes are saline alkaline lakes characterized by high concentrations of sodium carbonate/bicarbonate which lead to a stable elevated pH (>9), and moderate to extremely high salinity. Despite this combination of extreme conditions, biodiversity in soda lakes is high, and the presence of diverse microbial communities provides a driving force for highly active biogeochemical cycles. The sulfur cycle is one of the most important of these and bacterial sulfur oxidation is dominated by members of the obligately chemolithoautotrophic genus Thioalkalivibrio. Currently, 10 species have been described in this genus, but over one hundred isolates have been obtained from soda lake samples. The genomes of 75 strains were sequenced and annotated previously, and used in this study to provide a comprehensive picture of the diversity and distribution of genes related to dissimilatory sulfur metabolism in Thioalkalivibrio. Initially, all annotated genes in 75 Thioalkalivibrio genomes were placed in ortholog groups and filtered by bi-directional best BLAST analysis. Investigation of the ortholog groups containing genes related to sulfur oxidation showed that flavocytochrome c (fcc), the truncated sox system, and sulfite:quinone oxidoreductase (soe) are present in all strains, whereas dissimilatory sulfite reductase (dsr; which catalyzes the oxidation of elemental sulfur) was found in only six strains. The heterodisulfide reductase system (hdr), which is proposed to oxidize sulfur to sulfite in strains lacking both dsr and soxCD, was detected in 73 genomes. Hierarchical clustering of strains based on sulfur gene repertoire correlated closely with previous phylogenomic analysis. The phylogenetic analysis of several sulfur oxidation genes showed a complex evolutionary history. All in all, this study presents a comprehensive investigation of sulfur metabolism-related genes in cultivated Thioalkalivibrio strains and provides several avenues for future research.

Thiocyanate is a C1 compound containing carbon, nitrogen, and sulfur. It is a (by)product in a number of natural and industrial processes. Because thiocyanate is toxic to many organisms, including humans, its removal from industrial waste streams is an important problem. Although a number of bacteria can use thiocyanate as a nitrogen source, only a few can use it as an electron donor. There are two distinct pathways to use thiocyanate: (i) the "carbonyl sulfide pathway," which has been extensively studied, and (ii) the "cyanate pathway," whose key enzyme, thiocyanate dehydrogenase, was recently purified and studied. Three species of Thioalkalivibrio, a group of haloalkaliphilic sulfur-oxidizing bacteria isolated from soda lakes, have been described as thiocyanate oxidizers: (i) Thioalkalivibrio paradoxus ("cyanate pathway"), (ii) Thioalkalivibrio thiocyanoxidans ("cyanate pathway") and (iii) Thioalkalivibrio thiocyanodenitrificans ("carbonyl sulfide pathway"). In this study we provide a comparative genome analysis of these described thiocyanate oxidizers, with genomes ranging in size from 2.5 to 3.8 million base pairs. While focusing on thiocyanate degradation, we also analyzed the differences in sulfur, carbon, and nitrogen metabolism. We found that the thiocyanate dehydrogenase gene is present in 10 different Thioalkalivibrio strains, in two distinct genomic contexts/genotypes. The first genotype is defined by having genes for flavocytochrome c sulfide dehydrogenase upstream from the thiocyanate dehydrogenase operon (present in two strains including the type strain of Tv. paradoxus), whereas in the second genotype these genes are located downstream, together with two additional genes of unknown function (present in eight strains, including the type strains of Tv. thiocyanoxidans). Additionally, we found differences in the presence/absence of genes for various sulfur oxidation pathways, such as sulfide:quinone oxidoreductase, dissimilatory sulfite reductase, and sulfite dehydrogenase. One strain (Tv. thiocyanodenitrificans) lacks genes encoding a carbon concentrating mechanism and none of the investigated genomes were shown to contain known bicarbonate transporters. This study gives insight into the genomic variation of thiocyanate oxidizing bacteria and may lead to improvements in the application of these organisms in the bioremediation of industrial waste streams.

Thiocyanate (N=C-S-) is a moderately toxic, inorganic sulfur compound. It occurs naturally as a by-product of the degradation of glucosinolatecontaining plants and is produced industrially in a number of mining processes. Currently, two pathways for the primary degradation of thiocyanate in bacteria are recognized, the carbonyl sulfide pathway and the cyanate pathway, of which only the former has been fully characterized. Use of the cyanate pathway has been shown in only 10 strains of Thioalkalivibrio, a genus of obligately haloalkaliphilic sulfuroxidizing Gammaproteobacteria found in soda lakes. So far, only the key enzyme in this reaction, thiocyanate dehydrogenase (TcDH), has been purified and studied. To gain a better understanding of the other genes involved in the cyanate pathway, we conducted a transcriptomics experiment comparing gene expression during the growth of Thioalkalivibrio thiocyanoxidans ARh 2T with thiosulfate with that during its growth with thiocyanate. Triplicate cultures were grown in continuous substratelimited mode, followed by transcriptome sequencing (RNA-Seq) of the total mRNA. Differential expression analysis showed that a cluster of genes surrounding the gene for TcDH were strongly upregulated during growth with thiocyanate. This cluster includes genes for putative copper uptake systems (copCD, ABC-type transporters), a putative electron acceptor (fccAB), and a two-component regulatory system (histidine kinase and a σ54-responsive Fis family transcriptional regulator). Additionally, we observed the increased expression of RuBisCO and some carboxysome shell genes involved in inorganic carbon fixation, as well as of aprAB, genes involved in sulfite oxidation through the reverse sulfidogenesis pathway.

Thioalkalivibrio thiocyanodenitrificans strain ARhD 1^T is a motile, Gram-negative bacterium isolated from soda lakes that belongs to the Gammaproteobacteria. It derives energy for growth and carbon fixation from the oxidation of sulfur compounds, most notably thiocyanate, and so is a chemolithoautotroph. It is capable of complete denitrification under anaerobic conditions. The draft genome sequence consists of 3,746,647 bp in 3 scaffolds, containing 3558 proteincoding and 121 RNA genes. T. thiocyanodenitrificans ARhD 1^T was sequenced as part of the DOE Joint Genome Institute Community Science Program.