Copper (Cu) is an essential micronutrient required as a co-factor in the catalytic center of many enzymes. However, excess Cu can generate pleiotropic effects in the microbial cell. In addition, leaching of Cu from pipelines results in elevated Cu concentration in the environment, which is of public health concern. Sulfate-reducing bacteria (SRB) have been demonstrated to grow in toxic levels of Cu. However, reports on Cu toxicity towards SRB have primarily focused on the degree of toxicity and subsequent elimination. Here, Cu(II) stress-related effects on a model SRB, Desulfovibrio alaskensis G20, is reported. Cu(II) stress effects were assessed as alterations in the transcriptome through RNA-Seq at varying Cu(II) concentrations (5 µM and 15 µM). In the pairwise comparison of control vs. 5 µM Cu(II), 61.43% of genes were downregulated, and 38.57% were upregulated. In control vs. 15 µM Cu(II), 49.51% of genes were downregulated, and 50.5% were upregulated. The results indicated that the expression of inorganic ion transporters and translation machinery was massively modulated. Moreover, changes in the expression of critical biological processes such as DNA transcription and signal transduction were observed at high Cu(II) concentrations. These results will help us better understand the Cu(II) stress-response mechanism and provide avenues for future research.
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This content will become publicly available on March 1, 2025
Genetical and Biochemical Basis of Methane Monooxygenases of Methylosinus trichosporium OB3b in Response to Copper
Over the past decade, copper (Cu) has been recognized as a crucial metal in the differential expression of soluble (sMMO) and particulate (pMMO) forms of methane monooxygenase (MMO) through a mechanism referred to as the “Cu switch”. In this study, we used Methylosinus trichosporium OB3b as a model bacterium to investigate the range of Cu concentrations that trigger the expression of sMMO to pMMO and its effect on growth and methane oxidation. The Cu switch was found to be regulated within Cu concentrations from 3 to 5 µM, with a strict increase in the methane consumption rates from 3.09 to 3.85 µM occurring on the 6th day. Our findings indicate that there was a decrease in the fold changes in the expression of methanobactin (Mbn) synthesis gene (mbnA) with a higher Cu concentration, whereas the Ton-B siderophore receptor gene (mbnT) showed upregulation at all Cu concentrations. Furthermore, the upregulation of the di-heme enzyme at concentrations above 5 µM Cu may play a crucial role in the copper switch by increasing oxygen consumption; however, the role has yet not been elucidated. We developed a quantitative assay based on the naphthalene–Molisch principle to distinguish between the sMMO- and pMMO-expressing cells, which coincided with the regulation profile of the sMMO and pMMO genes. At 0 and 3 µM Cu, the naphthol concentration was higher (8.1 and 4.2 µM, respectively) and gradually decreased to 0 µM naphthol when pMMO was expressed and acted as the sole methane oxidizer at concentrations above 5 µM Cu. Using physical protein–protein interaction, we identified seven transporters, three cell wall biosynthesis or degradation proteins, Cu resistance operon proteins, and 18 hypothetical proteins that may be involved in Cu toxicity and homeostasis. These findings shed light on the key regulatory genes of the Cu switch that will have potential implications for bioremediation and biotechnology applications.
more » « less- Award ID(s):
- 1920954
- PAR ID:
- 10537354
- Publisher / Repository:
- Methane
- Date Published:
- Journal Name:
- Methane
- Volume:
- 3
- Issue:
- 1
- ISSN:
- 2674-0389
- Page Range / eLocation ID:
- 103 to 121
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bC c P)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a Cu I primary-sphere ligand, being positioned ∼2.9 Å away from the Cu I ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis.more » « less
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ABSTRACT Methane oxidation by aerobic methanotrophs is well known to be strongly regulated by the availability of copper, i.e., the “copper switch.” That is, there are two forms of methane monooxygenase: a cytoplasmic or soluble methane monooxygenase (sMMO) and a membrane-bound or particulate methane monooxygenase (pMMO). sMMO is only expressed and active in the absence of copper, while pMMO requires copper. Previous work has also shown that one gene in the operon of the soluble methane monooxygenase
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ABSTRACT Aerobic methanotrophs have long been known to play a critical role in the global carbon cycle, being capable of converting methane to biomass and carbon dioxide. Interestingly, these microbes exhibit great sensitivity to copper and rare-earth elements, with the expression of key genes involved in the central pathway of methane oxidation controlled by the availability of these metals. That is, these microbes have a “copper switch” that controls the expression of alternative methane monooxygenases and a “rare-earth element switch” that controls the expression of alternative methanol dehydrogenases. Further, it has been recently shown that some methanotrophs can detoxify inorganic mercury and demethylate methylmercury; this finding is remarkable, as the canonical organomercurial lyase does not exist in these methanotrophs, indicating that a novel mechanism is involved in methylmercury demethylation. Here, we review recent findings on methanotrophic interactions with metals, with a particular focus on these metal switches and the mechanisms used by methanotrophs to bind and sequester metals.more » « less
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