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...

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...

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ϳ50% identical to MxaF and ϳ90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.Methylobacterium extorquens AM1 is a methylotrophic bacterium ubiquitous in the environment and in particular on the undersides of leaves. This organism is able to metabolize both single-carbon compounds like methanol and multicarbon compounds like succinate and pyruvate (1,11,19). M. extorquens AM1 is a model organism for understanding the process of methylotrophic growth and has been studied biochemically and genetically for over 50 years (reviewed in reference 7). To utilize methanol as a sole source of carbon and energy, methanol is first oxidized to formaldehyde in the periplasm via methanol dehydrogenase, a soluble quinoprotein (2-4). This enzyme uses pyrroloquinoline quinone (PQQ) to sequentially transfer two electrons to cytochrome c L , which enters the electron transport chain, resulting in ϳ1 molecule of ATP per molecule of methanol oxidized (9). Methanol dehydrogenase is a heterotetrameric protein consisting of two 66-kDa large subunits (MxaF) and two small 8.5-kDa subunits (MxaI) (28, 37). The large subunit contains the active-site residues and the PQQ prosthetic group, which is coordinated to a calcium ion in the active site (37). The loss of this enzyme in M. extorquens

In this paper we investigate effects of the content of interruptions and of the time of interruption delivery on mobile phones. We review related work and report on a naturalistic quasi-experiment using experience-sampling that showed that the receptivity to an interruption is influenced by its content rather than by its time of delivery in the employed modality of delivery -SMS. We also examined the underlying variables that increase the perceived quality of content and found that the factors interest, entertainment, relevance and actionability influence people's receptivity significantly. Our findings inform system design that seeks to provide context-sensitive information or to predict interruptibility and suggest the consideration of receptivity as an extension to the way we think and reason about interruptibility.

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-dichlorophenolindophenol (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 of xox1 and downregulation of mxa genes, consistent with the Ln-switch, no differential expression of formaldehyde conversion genes, downregulation of pyrroloquinoline quinone (pQQ) biosynthesis genes, and upregulation of fdh4 formate dehydrogenase (FDH) genes. Additionally, the Ln-dependent ethanol dehydrogenase exaF reduces methanol sensitivity in the fae mutant strain when lanthanides are present, providing evidence for the capacity of an auxiliary role for exaF during Ln-dependent methylotrophy. A direct link between the Ln elements and microbial metabolism has been firmly established with the discovery of PQQ-dependent alcohol dehydrogenases (ADHs), from methylotrophic bacteria, that contain a Ln atom in the active site 1-5. Thus far, Ln-PQQ ADHs can be grouped by their phylogeny and primary substrate as either XoxF-MDHs or ExaF-type ethanol dehydrogenases (EtDHs). MxaFI MDH has been considered the canonical primary catalyst for methanol oxidation in Gram-negative methylotrophs 6,7. The heterotetramer MxaFI contains PQQ that coordinates the calcium (Ca) ion 8-10. The discovery that Ln is incorporated into the active site of XoxF MDH in place of Ca, allowing catalytic function, has prompted the reexamination of methanol oxidation in methylotrophic bacteria 3,5,11-18. To date, only a few XoxF MDHs have been kinetically characterized 1,3,4,19,20. Phylogenetic analyses show there are at least five distinct families of XoxF MDHs 11,21 , and while it has been suggested that all XoxF MDHs may exhibit similar kinetic properties, reported data for these enzymes are currently inadequate to support such a broad characterization. In fact, recent studies have begun to identify differences in kinetic properties, cofactor usage, and pH optima of phylogenetically distinct XoxF enzymes 20,22,23. Lack of genes encoding the