Development of an Optimized Medium, Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Delaney, Nigel F.; Kaczmarek, Maria E.; Ward, Lewis M.; Swanson, Paige; Lee, Ming‐Chun; Marx, Christopher J.

doi:10.1371/journal.pone.0062957

Cited by 132 publications

(141 citation statements)

References 42 publications

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“…It was previously shown that xoxF (but not mxaF) is required for expression of mxaF and for repression of xoxF in hypho minimal medium lacking La (22). These studies were repeated here to determine if expression from the mxa and xox1 promoters behaves identically in the more robust MP medium (36) and to determine how loss of mxaF and xoxF1 affects expression in medium containing La. As mxaF and xoxF1 are required for methanol growth in the absence of lanthanides, succinate was added as an additional carbon source.…”

Section: Resultsmentioning

confidence: 91%

“…M. extorquens AM1 strains were grown at 29°C in Methylobacterium PIPES [piperazine-N,N=-bis(2-ethanesulfonic acid)] (MP) medium (36), except that FeSO 4 was omitted from the C7 metal mixture (citrate-chelated trace element solution containing 7 metals) and prepared separately. Succinate (0.4%) and/or methanol (0.5%) was added as a carbon source(s) to media containing calcium chloride (20 M) and/or lanthanide chlorides (2.5 nM to 20 M).…”

Section: Methodsmentioning

confidence: 99%

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Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

et al. 2016

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Methylobacterium extorquens AM1 has two distinct types of methanol dehydrogenase (MeDH) enzymes that catalyze the oxidation of methanol to formaldehyde. MxaFI-MeDH requires pyrroloquinoline quinone (PQQ) and Ca in its active site, while XoxFMeDH requires PQQ and lanthanides, such as Ce and La. Using MeDH mutant strains to conduct growth analysis and MeDH activity assays, we demonstrate that M. extorquens AM1 has at least one additional lanthanide-dependent methanol oxidation system contributing to methanol growth. Additionally, the abilities of different lanthanides to support growth were tested and strongly suggest that both XoxF and the unknown methanol oxidation system are able to use La, Ce, Pr, Nd, and, to some extent, Sm. Further, growth analysis using increasing La concentrations showed that maximum growth rate and yield were achieved at and above 1 M La, while concentrations as low as 2.5 nM allowed growth at a reduced rate. Contrary to published data, we show that addition of exogenous lanthanides results in differential expression from the xox1 and mxa promoters, upregulating genes in the xox1 operon and repressing genes in the mxa operon. Using transcriptional reporter fusions, intermediate expression from both the mxa and xox1 promoters was detected when 50 to 100 nM La was added to the growth medium, suggesting that a condition may exist under which M. extorquens AM1 is able to utilize both enzymes simultaneously. Together, these results suggest that M. extorquens AM1 actively senses and responds to lanthanide availability, preferentially utilizing the lanthanide-dependent MeDHs when possible. IMPORTANCEThe biological role of lanthanides is a nascent field of study with tremendous potential to impact many areas in biology. Our studies demonstrate that there is at least one additional lanthanide-dependent methanol oxidation system, distinct from the MxaFI and XoxF MeDHs, that may aid in classifying additional environmental organisms as methylotrophs. Further, our data suggest that M. extorquens AM1 has a mechanism to regulate which MeDH is transcribed, depending on the presence or absence of lanthanides. While the mechanism controlling differential regulation is not yet understood, further research into how methylotrophs obtain and use lanthanides will facilitate their cultivation in the laboratory and their use as a biomining and biorecycling strategy for recovery of these commercially valuable rare-earth elements. Methylotrophs have gained worldwide interest as platforms for the production of value-added chemicals from singlecarbon compounds, turning atmospheric pollutants like methane and methanol into green chemicals, including biofuels and biodegradable plastics (1-5). A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6, 7). Recently, it was discovered that some types of MeDHs require rareearth elements, specifically lantha...

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Section: Resultsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

et al. 2016

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“…For comparison, despite being unable to grow on DCM, AM1 can grow on 0.5 mM formaldehyde (data not shown). We next used an automated system to measure growth rates under various medium conditions (29). Somewhat unexpectedly, DM4 ⌬dcmA(pJM10) was the most sensitive to growth in high external chloride or low external pH (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cultures were then diluted 64ϫ into 640 l of fresh medium. Over 48 h, optical densities at 600 nm (OD 600 ) were measured every 30 to 45 min using an automated system (29). Growth rates were calculated using CurveFitter (29).…”

Section: Media and Chemicalsmentioning

confidence: 99%

Phylogeny Poorly Predicts the Utility of a Challenging Horizontally Transferred Gene in Methylobacterium Strains

Vuilleumier¹,

Bringel²,

Marx³

2014

Journal of Bacteriology

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cHorizontal gene transfer plays a crucial role in microbial evolution. While much is known about the mechanisms that determine whether physical DNA can be transferred into a new host, the factors determining the utility of the transferred genes are less clear. We have explored this issue using dichloromethane consumption in Methylobacterium strains. Methylobacterium extorquens DM4 expresses a dichloromethane dehalogenase (DcmA) that has been acquired through horizontal gene transfer and allows the strain to grow on dichloromethane as the sole carbon and energy source. We transferred the dcmA gene into six Methylobacterium strains that include both close and distant evolutionary relatives. The transconjugants varied in their ability to grow on dichloromethane, but their fitness on dichloromethane did not correlate with the phylogeny of the parental strains or with any single tested physiological factor. This work highlights an important limiting factor in horizontal gene transfer, namely, the capacity of the recipient strain to accommodate the stress and metabolic disruption resulting from the acquisition of a new enzyme or pathway. Understanding these limitations may help to rationalize historical examples of horizontal transfer and aid deliberate genetic transfers in biotechnology for metabolic engineering.

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“…For M. extorquens AM1 cultures, glassware was prepared as reported in reference 29. M. extorquens AM1 cultures were grown in minimal medium (43) in borosilicate culture tubes, polystyrene round-bottom tubes, or shake flasks at 30°C with 15 mM succinic acid, 34 mM ethanol, or 125 mM methanol added as the growth substrate. A 2 M concentration of LaCl 3 was added to the growth medium when stated.…”

Section: Methodsmentioning

confidence: 99%

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates

et al. 2016

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Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3؉ IMPORTANCEExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxFtype alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases. Methylotrophy is the capability of organisms to metabolize reduced carbon compounds lacking carbon-carbon bonds as the sole source of carbon and energy (1). The genus Methylobacterium is comprised of aerobic facultative methylotrophs that can metabolize single-carbon compounds, such as methanol and methylamine, as well as multicarbon substrates like ethanol, acetate, ethylamine, pyruvate, and succinate (2, 3). Members of the genus Methylobacterium are wide-spread plant epiphytes (4, 5) that utilize their metabolic flexibility to gain an advantage in the phyllosphere, an oligotrophic environment with transient substrate availability (6, 7).Methanol dehydrogenase (MDH) is an essential enzyme for the methylotrophic metabolism of methanol and methane (8). In Gram-negative methylotrophic bacteria, MDHs are soluble, periplasmic proteins with pyrroloquinoline quinone (PQQ) as the prosthetic group (9, 10). The best studied PQQ-containing MDHs are ␣ 2 ␤ 2 tetramers consisting of the MxaF and MxaI proteins (11-14) that contain calcium (Ca 2ϩ ) in the active site (15, 16). Studies have provided evidence for the physiological role of a second type of PQQ-dependent MDH, XoxF, which has ϳ50% amino acid identity to MxaF from MxaFI-type MDHs (17). Metagenomic and environmental proteomics studies have demonstrated that xoxF is more widespread than mxaF in environmental samples (18)(19)(20)(21). Phylogenetic analysis of putative PQQ-containing MDHs has shown that XoxF-type MDHs are genetically diverse with at least five distinct clades, and it has been suggested that MxaFI-type MDHs represent a minor fraction of these MDHs (8,22). It has been further proposed that MxaFI-type MDHs may be the result of a second evolutionary event, with an ancestral XoxF-type MDH prototype (22). Together, these suppositions suggest that XoxFtype MDHs may be the primary MDHs for meth...

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Development of an Optimized Medium, Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Cited by 132 publications

References 42 publications

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

Phylogeny Poorly Predicts the Utility of a Challenging Horizontally Transferred Gene in Methylobacterium Strains

Pyrroloquinoline Quinone Ethanol Dehydrogenase in Methylobacterium extorquens AM1 Extends Lanthanide-Dependent Metabolism to Multicarbon Substrates

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