The Bowen–Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome

Metabolite Profiling Uncovers Plasmid-Induced Cobalt Limitation under Methylotrophic Growth Conditions

BACKGROUND:The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases. METHODOLOGY/PRINCIPAL FINDINGS:We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12)-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import. CONCLUSIONS/SIGNIFICANCE:This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress

Comparative Investigations on Different β-Glucosidase Surrogate Substrates

β-glucosidases are hydrolyzing enzymes which can release many aroma-active compounds from their glycoside form. Several yeasts produce these enzymes and thus are applied during the wine production process. To be able to test specific organisms for the presence of β-glucosidases and to investigate this enzyme activity, four main surrogate substrates have been described. The properties and applicability of these compounds, named arbutin (hydroquinone-β-D-glucopyranoside), esculin (6-O-(-D-glucosyl)aesculetin), 4-nitrophenyl-β-D-glucopyranoside (pNPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG), are discussed after comparing their advantages and disadvantages. Although all four substrates were found suitable for photometric assays, 4-MUG has proven to be most appropriate due to high sensitivity, high robustness and simple processing. Furthermore, the investigation of β-glucosidase product accumulation is described, which could be used to give indications about β-glucosidase localization

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids

SDS-PAGE analysis of CPO protein sizes after deglycosylation.

<p>Culture supernatants from fructose minimal medium cultures grown for 10 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and deglycosylation was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. N+O: glycosidases were added to remove N- and O-linked oligosaccharides, O: glycosidases were added to remove O-linked oligosaccharides, N: glycosidases were added to remove N-linked oligosaccharides, ut: untreated sample, M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.</p

Colony color and growth of <i>C.</i><i>fumago</i> wild type and cpo1 mutant on solid media.

<p><i>C. fumago</i> wild type strain and the cpo1 mutant were plated on fructose minimal medium plates with a piece of mycelium grown on a glucose potato agar plate and incubated at 24°C for 7 days.</p

UV-Vis spectra of <i>C.</i><i>fumago</i> wild type and cpo1 CPO purified by aqueous biphasic systems.

<p>Culture supernatants from fructose minimal medium cultures grown for 11 days on a rotary shaker at 24°C each were purified using aqueous biphasic systems and protein concentration was equalized by dilution of the samples with 0.1 M citric acid buffer solution (pH 4). CPO activity of the diluted samples was determined as 246.46 U mL⁻¹ and 0.03 U mL⁻¹ for wild type and cpo1, respectively.</p

Comparison of CPO production characteristics of wild type and cpo1 strain.

<p><b>A:</b> Comparison of CPO activities in supernatant samples of <i>C. fumago</i> wild type and cpo1 strain during cultivation in fructose minimal medium. Strain cultivation and determination of enzyme activity via the MCD assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. Data is expressed as mean of values from two shake flasks ± standard deviation. <b>B:</b> Analysis of CPO protein levels in supernatant samples of <i>C. fumago</i> wild type and cpo1 strain by SDS-PAGE. SDS-PAGE analysis was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067857#s2" target="_blank">materials and methods</a> section. M: molecular weight marker. The numbers on the left indicate the sizes of the molecular weight marker.</p

Methylobacterium extorquens: methylotrophy and biotechnological applications

Methylotrophy is the ability to use reduced one-carbon compounds, such as methanol, as a single source of carbon and energy. Methanol is, due to its availability and potential for production from renewable resources, a valuable feedstock for biotechnology. Nature offers a variety of methylotrophic microorganisms that differ in their metabolism and represent resources for engineering of value-added products from methanol. The most extensively studied methylotroph is the Alphaproteobacterium Methylobacterium extorquens. Over the past five decades, the metabolism of M. extorquens has been investigated physiologically, biochemically, and more recently, using complementary omics technologies such as transcriptomics, proteomics, metabolomics, and fluxomics. These approaches, together with a genome-scale metabolic model, facilitate system-wide studies and the development of rational strategies for the successful generation of desired products from methanol. This review summarizes the knowledge of methylotrophy in M. extorquens, as well as the available tools and biotechnological applications