Lanthanides (Ln) are a new group of life metals, and many questions remain regarding how they are acquired and used in biology. Methylotrophic bacteria can acquire, transport, biomineralize, and use Ln as part of a cofactor complex with pyrroloquinoline quinone (PQQ) in alcohol dehydrogenases. For most methylotrophic bacteria use is restricted to the light Ln, which range from lanthanum to samarium (atomic numbers 57–62). Understanding how the cell differentiates between light and heavy Ln, and the impacts of these metals on the metabolic network, will advance the field of Ln biochemistry and give insights into enzyme catalysis, stress homeostasis, and metal biomineralization and compartmentalization. We report robust methanol growth with the heavy Ln gadolinium by a genetic variant of the model methylotrophic bacterium Methylorubrum extorquens AM1, named evo -HLn, for “ evo lved for H eavy L antha n ides.” A non-synonymous single nucleotide polymorphism in a cytosolic hybrid histidine kinase/response regulator allowed for sweeping transcriptional alterations to heavy metal stress response, methanol oxidation, and central metabolism. Increased expression of genes for Ln acquisition and uptake, production of the Ln-chelating lanthanophore, PQQ biosynthesis, and phosphate transport and metabolism resulted in gadolinium hyperaccumulation of 36-fold with a trade-off for light Ln accumulation. Gadolinium was hyperaccumulated in an enlarged acidocalcisome-like compartment. This is the first evidence of a bacterial intracellular Ln-containing compartment that we name the “lanthasome.” Carotenoid and toblerol biosynthesis were also upregulated. Due to its unique capabilities, evo -HLn can be used to further magnetic resonance imaging (MRI) and bioremediation technologies. In this regard, we show that gadolinium hyperaccumulation was sufficient to produce MRI contrast in whole cells, and that evo -HLn was able to readily acquire the metal from the MRI contrast agent gadopentetic acid. Finally, hyperaccumulation of gadolinium, differential uptake of light and heavy Ln, increased PQQ levels, and phosphate transport provide new insights into strategies for Ln recovery.
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EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde
Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.
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- Award ID(s):
- 1736253
- NSF-PAR ID:
- 10292777
- Date Published:
- Journal Name:
- PLoS biology
- ISSN:
- 1544-9173
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Lanthanide (Ln) elements are utilized as cofactors for catalysis by XoxF-type methanol dehydrogenases (MDHs). A primary assumption is that XoxF enzymes produce formate from methanol oxidation, which could impact organisms that require formaldehyde for assimilation. We report genetic and phenotypic evidence showing that XoxF1 (MexAM1_1740) from
Methylobacterium extorquens AM1 produces formaldehyde, and not formate, during growth with methanol. Enzyme purified with lanthanum or neodymium oxidizes formaldehyde. However, formaldehyde oxidation via 2,6-dichlorophenol-indophenol (DCPIP) reduction is not detected in cell-free extracts from wild-type strain methanol- and lanthanum-grown cultures. Formaldehyde activating enzyme (Fae) is required for Ln methylotrophic growth, demonstrating that XoxF1-mediated production of formaldehyde is essential. Addition of exogenous lanthanum increases growth rate with methanol by 9–12% but does not correlate with changes to methanol consumption or formaldehyde accumulation. Transcriptomics analysis of lanthanum methanol growth shows upregulation ofxox1 and downregulation ofmxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (PQQ) biosynthesis genes, and upregulation offdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase ExaF reduces methanol sensitivity in thefae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for ExaF during Ln-dependent methylotrophy. -
Zhang, George (Ed.)Abstract Understanding the evolution of novel physiological traits is highly relevant for expanding the characterization and manipulation of biological systems. Acquisition of new traits can be achieved through horizontal gene transfer (HGT). Here, we investigate drivers that promote or deter the maintenance of HGT-driven degeneracy, occurring when processes accomplish identical functions through nonidentical components. Subsequent evolution can optimize newly acquired functions; for example, beneficial alleles identified in an engineered Methylorubrum extorquens strain allowed it to utilize a “Foreign” formaldehyde oxidation pathway substituted for its Native pathway for methylotrophic growth. We examined the fitness consequences of interactions between these alleles when they were combined with the Native pathway or both (Dual) pathways. Unlike the Foreign pathway context where they evolved, these alleles were often neutral or deleterious when moved into these alternative genetic backgrounds. However, there were instances where combinations of multiple alleles resulted in higher fitness outcomes than individual allelic substitutions could provide. Importantly, the genetic context accompanying these allelic substitutions significantly altered the fitness landscape, shifting local fitness peaks and restricting the set of accessible evolutionary trajectories. These findings highlight how genetic context can negatively impact the probability of maintaining native and HGT-introduced functions together, making it difficult for degeneracy to evolve. However, in cases where the cost of maintaining degeneracy was mitigated by adding evolved alleles impacting the function of these pathways, we observed rare opportunities for pathway coevolution to occur. Together, our results highlight the importance of genetic context and resulting epistasis in retaining or losing HGT-acquired degenerate functions.more » « less
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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.more » « less
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Cotruvo Jr, Joseph A (Ed.)Recent work has revealed that certain lanthanides—in particular, the more earth-abundant, lighter lanthanides—play essential roles in pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases from methylotrophic and non-methylotrophic bacteria. More recently, efforts of several laboratories have begun to identify the molecular players (the lanthanome) involved in selective uptake, recognition, and utilization of lanthanides within the cell. In this chapter, we present protocols for the heterologous expression in Escherichia coli, purification, and characterization of many of the currently known proteins that comprise the lanthanome of the model facultative methylotroph, Methylorubrum extorquens AM1. In addition to the methanol dehydrogenase XoxF, these proteins include the associated c-type cytochrome, XoxG, and solute binding protein, XoxJ. We also present new, streamlined protocols for purification of the highly selective lanthanide-binding protein, lanmodulin, and a solute binding protein for PQQ, PqqT. Finally, we discuss simple, spectroscopic methods for determining lanthanide- and PQQ-binding stoichiometry of proteins. We envision that these protocols will be useful to investigators identifying and characterizing novel members of the lanthanome in many organisms.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/doi: 10.131/journal.pbio.3001208
