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Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

ABSTRACT: Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.

SUBMITTER: Liu C

PROVIDER: S-EPMC10260701 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

Liu Changqing C Angius Federica F Pol Arjan A Mesman Rob A RA Versantvoort Wouter W Op den Camp Huub J M HJM

Archives of microbiology 20230612 7

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acid ...[more]

PMID: 37306788

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Project description:BACKGROUND: Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture. RESULTS: In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5'-m6ACN4GT-3' and 5'-CCm6AN5CTC-3' methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif. CONCLUSIONS: Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

Project description:Industrial methanol production converts methane from natural gas into methanol through a multistep chemical process. Biological methane-to-methanol conversion under moderate conditions and using biogas would be more environmentally friendly. Methanotrophs, bacteria that use methane as an energy source, convert methane into methanol in a single step catalyzed by the enzyme methane monooxygenase, but inhibition of methanol dehydrogenase, which catalyzes the subsequent conversion of methanol into formaldehyde, is a major challenge. In this study, we used the thermoacidophilic methanotroph "Methylacidiphilum fumariolicum" SolV for biological methanol production. This bacterium possesses a XoxF-type methanol dehydrogenase that is dependent on rare earth elements for activity. By using a cultivation medium nearly devoid of lanthanides, we reduced methanol dehydrogenase activity and obtained a continuous methanol-producing microbial culture. The methanol production rate and conversion efficiency were growth-rate dependent. A maximal conversion efficiency of 63% mol methanol produced per mol methane consumed was obtained at a relatively high growth rate, with a methanol production rate of 0.88 mmol/g (dry weight)/h. This study demonstrates that methanotrophs can be used for continuous methanol production. Full-scale application will require additional increases in the titer, production rate, and efficiency, which can be achieved by further decreasing the lanthanide concentration through the use of increased biomass concentrations and novel reactor designs to supply sufficient gases, including methane, oxygen, and hydrogen.IMPORTANCE The production of methanol, an important chemical, is completely dependent on natural gas. The current multistep chemical process uses high temperature and pressure to convert methane in natural gas to methanol. In this study, we used the methanotroph "Methylacidiphilum fumariolicum" SolV to achieve continuous methanol production from methane as the substrate. The production rate was highly dependent on the growth rate of this microorganism, and high conversion efficiencies were obtained. Using microorganisms for the production of methanol might enable the use of more sustainable sources of methane, such as biogas, rather than natural gas.

Project description:The Solfatara volcano near Naples (Italy), the origin of the recently discovered verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was shown to contain ammonium ([Formula: see text]) at concentrations ranging from 1 to 28 mM. Ammonia (NH3) can be converted to toxic hydroxylamine (NH2OH) by the particulate methane monooxygenase (pMMO), the first enzyme of the methane (CH4) oxidation pathway. Methanotrophs rapidly detoxify the intermediate NH2OH. Here, we show that strain SolV performs ammonium oxidation to nitrite at a rate of 48.2 nmol [Formula: see text].h-1.mg DW-1 under O2 limitation in a continuous culture grown on hydrogen (H2) as an electron donor. In addition, strain SolV carries out nitrite reduction at a rate of 74.4 nmol [Formula: see text].h-1.mg DW-1 under anoxic condition at pH 5-6. This range of pH was selected to minimize the chemical conversion of nitrite ([Formula: see text]) potentially occurring at more acidic pH values. Furthermore, at pH 6, we showed that the affinity constants (K s ) of the cells for NH3 vary from 5 to 270 μM in the batch incubations with 0.5-8% (v/v) CH4, respectively. Detailed kinetic analysis showed competitive substrate inhibition between CH4 and NH3. Using transcriptome analysis, we showed up-regulation of the gene encoding hydroxylamine dehydrogenase (haoA) cells grown on H2/[Formula: see text] compared to the cells grown on CH4/[Formula: see text] which do not have to cope with reactive N-compounds. The denitrifying genes nirk and norC showed high expression in H2/[Formula: see text] and CH4/[Formula: see text] grown cells compared to cells growing at μmax (with no limitation) while the norB gene showed downregulation in CH4/[Formula: see text] grown cells. These cells showed a strong upregulation of the genes in nitrate/nitrite assimilation. Our results demonstrate that strain SolV can perform ammonium oxidation producing nitrite. At high concentrations of ammonium this may results in toxic effects. However, at low oxygen concentrations strain SolV is able to reduce nitrite to N2O to cope with this toxicity.

Dataset Information

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

Publications

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

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