Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone

Abstract

Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. "Methylacidiphilum" isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.

Fig. 1. Cluster analysis of the microbial community in Italian volcanic mud samples and enrichment cultures.

Compositional dissimilarities between samples were quantified using Bray-Curtis dissimilarity. The community composition was obtained by 16S rRNA gene amplicon sequencing analysis. ‘M’ denotes cultures with methane. ‘M/P’ denotes cultures with methane plus propane. ‘Mud’ denotes the original sample. The color scale (from white to blue) represents the natural logarithm transformed values of the percentage relative abundances +1. Genera with individual abundances less than 1% together comprised <3% of the total community and were excluded from the analysis.

Fig. 2. Phylogenetic positions of isolated strains in relation to other verrucomicrobial methanotrophs based on 16S rRNA gene sequences.

The tree was constructed with MEGA 7 using the Neighbor-joining method. The evolutionary distances were computed using the Kimura 2-parameter method and are in units of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 5). All positions with less than 95% site coverage were removed, leaving a total of 1461 positions in the final dataset. The gammaproteobacterial methanotroph Methylococcus geothermalis was used as the outgroup. ‘*’ indicates that “Methylacidimicrobium thermophilum” is the only known thermophilic strain of the genus, “Methylacidimicrobium”. The scale bar represents 0.02 changes per nucleotide position.

Fig. 3. The growth of strain IT6 on C1 and C3 substrates.

The cultivation was performed at pH 4.5 and a temperature of 50 °C with shaking at 200 rpm. The error bars represent ±1 standard deviation for n ≥ 2 biological replicates. For inoculation, 10% (v/v) of late log phase cells (starting optical density values at 600 nm (OD₆₀₀) < 0.05) were used. Substrates were: 10% methane (v/v), 30 mM methanol, and 10 mM each of 2-propanol, acetone, or acetol.

Fig. 4. Comparative gene expression pattern of strain IT6 grown on C1 and C3 substrates.

A A volcano plot showing the differential gene expression between 2-propanol-grown (left) versus methane-grown cells (right) under oxygen-replete conditions. The volcano plot was generated with the fold-change values (log₂FC) and false discovery rates (FDR) from three replicates of both conditions, with the methane grown-cells used as the reference condition. Genes with less than two-fold expression difference (FC ≤ 2 and FDR ≥ 0.05) are represented by small gray dots and other genes of interest with more than two-fold expression difference (FC ≥ 2 and FDR < 0.05) in 2-propanol and methane-grown cells are represented by large colored dots with the following description: (1) Purple, blue and black are genes in cluster IT6_09370-09425, TCA cycle genes, and other upregulated genes, respectively, in 2-propanol-grown cells, (2) Red, green and pink are genes involved in methane oxidation, formate oxidation, and other processes, respectively, in methane-grown cells. Genes in purple (cluster IT6_09370-09425) and red (Genes for methane oxidation) are labeled. B Organization of genes with more than two-fold expression difference (FC ≥ 2 and FDR < 0.05) in 2-propanol-grown and methane-grown cells. The genes colored in blue were expressed relatively more during growth on methane, while genes colored red were expressed relatively more during growth on 2-propanol. The color intensity indicates the relative fold change difference (log₂FC). The numbers at the beginning and end of the genomic region indicate the nucleotide positions of the genes in the strain IT6 genome. The zigzag line indicates more than 1.5 kb distance between two genes. Arrows show gene direction and relative size. Detailed information on the genes can be found in Supplementary Table S7.

Fig. 5. Expression and promoter analyses of the genes localized in the cluster 9370-9425 in strain IT6.

Each bar represents gene expression levels (in transcript per million reads, TPM) for cells grown on methane, 2-propanol, acetone, and acetol, as indicated by the color bars. Expression of the housekeeping genes gyrA and fusA are shown for comparison. Error bars represent ±1 standard deviation for n ≥ 3 biological replicates. Apart from the housekeeping genes, differences in expression are statistically significant for each gene (p < 0.05). The gene cluster arrangements are shown and represented by different colors (except for genes with hypothetical functions shown in red). Black arrows below the cluster represent the promoter prediction results (LDF score of 0.2) in the gene cluster. An LDF score of 0.2 indicates the presence of an ±RpoD (σ70) promoter with 80% accuracy and specificity.

Fig. 6. The proposed metabolic pathway for C3 substrate oxidation in strain IT6.

The methane monooxygenases (PMO1 and PMO2) are involved in the oxidation of methane and other SCA [8] to their respective alcohols. The lanthanide-dependent methanol dehydrogenase (XoxF) is involved in the conversion of methanol to formaldehyde or formate. The XoxF or other alcohol dehydrogenases (e.g., the GMC oxidoreductase complex transported to the periplasm via the TAT system; GMC large subunit (GmcA) and small subunit (GmcB)) encoded by the strain might be involved in converting 2-propanol and 1,2-propanediol to acetone and acetol, respectively. The reduced quinones can provide the reducing equivalents for PMO2 and PMO3 from methanol oxidation and 2-propanol oxidation pathways. Acetone generated from 2-propanol is oxidized to acetol through the action of the PMO3. The acetol produced is oxidized to methylglyoxal by GMC oxidoreductase. Transport of methylglyoxal into the cytoplasm can occur via passive diffusion or possibly via a transporter. The glyoxalase (I and II) enzyme system convert methylglyoxal to lactate, further converted to pyruvate by lactate dehydrogenase. The pyruvate generated can be converted to PEP by PEP-synthase, an essential step in gluconeogenesis during growth on the C3 substrates. The alternative complex III (Comp ACIII) transfers electrons from reduced quinones via a cytochrome c (Cyt-c) to complex IV, thereby regenerating the quinone pool. The respiratory complexes translocate protons across the cellular membrane. Pyruvate enters the TCA cycle for ATP/NADH production and anabolic processes. For selected predicted reactions for C1 and C3 metabolism, the gene identifiers are shown in blue for genes in cluster IT6_09370-09425 and red for others. Black dashed arrows represent reactions for which the candidate enzyme was not confidently predicted.

Fig. 7. Effect of ATU and copper concentration on the growth of strain IT6.

A shows the specific growth rate of strain IT6 under varying concentrations of allylthiourea when grown on C1 and C3 substrates. B shows the specific growth rate of strain IT6 in the presence and absence of copper (10 nM). Error bars represent ±1 standard deviation for n ≥ 3 biological replicates. ND indicates no detectable growth.