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Mary E Lidstrom - One of the best experts on this subject based on the ideXlab platform.
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genomics of Methylotrophy in gram positive methylamine utilizing bacteria
Microorganisms, 2015Co-Authors: Tami L Mctaggart, Mary E Lidstrom, David A C Beck, Usanisa Setboonsarng, Nicole Shapiro, Tanja Woyke, Marina G Kalyuzhnaya, Ludmila ChistoserdovaAbstract:Gram-positive Methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of Methylotrophy pathways/enzymes within this group. However, genome-based knowledge of Methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive Methylotrophs limits our global understanding of Methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive Methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the Methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of Methylotrophy in Gram-positive Methylotrophic bacteria. This study is a major contribution to the field of Methylotrophy, aimed at closing the gap in the genomic knowledge of Methylotrophy within this diverse group of bacteria.
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Printed in Great Britain
2013Co-Authors: Ludmila V. Chistoserdova, Mary E LidstromAbstract:Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyasenegative Methylotroph Methylobacterium extorquens AM
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Functioning in situ: gene expression in Methylotenera mobilis in its native environment as assessed through transcriptomics
The ISME Journal, 2010Co-Authors: Marina G Kalyuzhnaya, Mary E Lidstrom, David A C Beck, Dominic Suciu, Alexander Pozhitkov, Ludmila ChistoserdovaAbstract:Methylotrophs, organisms able to gain energy and carbon from compounds containing no carbon–carbon bonds, such as methane, methanol and methylated amines, are widespread in nature. However, knowledge of their nutrient preference and their metabolism is mostly based on experiments with cultures grown in defined laboratory conditions. Here, we use transcriptomics to explore the activity of one Methylotroph, Methyotenera mobilis in its natural environment, lake sediment from which it has been previously isolated. Cells encapsulated in incubation cassettes were exposed to sediment conditions, with or without supplementation with a carbon/energy source (methylamine), and gene-expression patterns were compared for those cells to patterns for cells incubated in a defined medium supplemented with methylamine. A few specific trends in gene expression were observed at in situ conditions that may be of environmental significance, as follows. Expression of genes for the linear formaldehyde oxidation pathway linked to tetrahydromethanopterin increased, suggesting an important role for this pathway in situ , in contrast to laboratory condition culture, in which the cyclic ribulose monophosphate pathway seemed to be the major route for formaldehyde oxidation. Along with the ribulose monophosphate cycle that is also a major pathway for assimilating C_1 units, the methylcitric acid cycle seemd to be important in situ , suggesting that multicarbon compounds may be the natural carbon and/or energy substrates for M. mobilis , challenging the notion of an obligately Methylotrophic lifestyle for this bacterium. We also detected a major switch in expression of genes responsible for the mode of motility between different conditions: from flagellum-enabled motility in defined medium to in situ expression of pili known to be involved in twitching motility and adherence. Overall, this study offers a novel approach for gaining insights into the lifestyle of individual microbes in their native environments.
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cell to cell heterogeneity in growth rate and gene expression in methylobacterium extorquens am1
Journal of Bacteriology, 2007Co-Authors: Tim J Strovas, Xiaofeng Guo, Linda M Sauter, Mary E LidstromAbstract:Cell-to-cell heterogeneity in gene expression and growth parameters was assessed in the facultative Methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized Methylotrophy promoter (PmxaF) and gfpuv (encoding a variant of green fluorescent protein [GFPuv]) was used to assess single-cell gene expression. Using a flowthrough culture system and laser scanning microscopy, data on fluorescence and cell size were obtained over time through several growth cycles for cells grown on succinate or methanol. Cells were grown continuously with no discernible lag between divisions, and high cell-to-cell variability was observed for cell size at division (2.5-fold range), division time, and growth rate. When individual cells were followed over multiple division cycles, no direct correlation was observed between the growth rate before a division and the subsequent growth rate or between the cell size at division and the subsequent growth rate. The cell-to-cell variability for GFPuv fluorescence from the PmxaF promoter was less, with a range on the order of 1.5-fold. Fluorescence and growth rate were also followed during a carbon shift experiment, in which cells growing on succinate were shifted to methanol. Variability of the response was observed, and the growth rate at the time of the shift from succinate to methanol was a predictor of the response. Higher growth rates at the time of the substrate shift resulted in greater decreases in growth rates immediately after the shift, but full induction of PmxaF-gfpuv was achieved faster. These results demonstrate that in M. extorquens, physiological heterogeneity at the single-cell level plays an important role in determining the population response to the metabolic shift examined.
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bacterial populations active in metabolism of c1 compounds in the sediment of lake washington a freshwater lake
Applied and Environmental Microbiology, 2005Co-Authors: Olivier Nercessian, Marina G Kalyuzhnaya, Mary E Lidstrom, Emma Noyes, Ludmila ChistoserdovaAbstract:Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C 1 utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the 13 C-labeled C 1 substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active Methylotroph population, while only one other known Methylotrophic lineage, Methylophilaceae , was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae , allowed detection of alphaproteobacterial ( Methylosinus ) and gammaproteobacterial ( Methylobacter , Methylomonas , and Methylomicrobium ) methanotrophs, Methylotrophs of the genus Methylobacterium , and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C 1 compounds. Comparisons between 16S rRNA gene pools amplified from [ 13 C]DNA and from [ 12 C]DNA revealed that the proportion of Methylophilus -related sequences increased in the presence of [ 13 C]methanol, [ 13 C]methylamine, and [ 13 C]formaldehyde; Novosphingobium -related sequences were enriched in the presence of [ 13 C]methanol; Gemmatimonadaceae -related sequences were enriched in the presence of [ 13 C]formaldehyde and [ 13 C]formate; and Xanthomonadaceae -related sequences were enriched in the presence of [ 13 C]formate. Analysis of fae genes amplified from [ 13 C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C 1 compound: Methylosinus sequences dominated the [ 13 C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [ 13 C]methylamine microcosm pool. The [ 13 C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [ 13 C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active Methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.
Wendisch, Volker F. - One of the best experts on this subject based on the ideXlab platform.
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Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi
'Frontiers Media SA', 2021Co-Authors: Schultenkämper Kerstin, Gil Lopez Marina, Gütle, Desirée D., Keller, Laura B., Zhang Lin, Einsle Oliver, Jacquot Jean-pierre, Wendisch, Volker F.Abstract:Schultenkämper K, Gütle DD, Gil Lopez M, et al. Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPRi. Front Microbiol. 2021;12: 669220.The Gram-positive Bacillus methanolicus shows plasmid-dependent Methylotrophy. This facultative ribulose monophosphate (RuMP) cycle Methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBAC is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBAP is found on the natural plasmid pBM19 and is induced during Methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBAC and FBAP differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBAP. However, both FBAC and FBAP maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBAC in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBAC and FBAP concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fbaC RNA levels were reduced by CRISPRi, but concomitantly the fbaP RNA levels were increased. Vice versa, a similar compensatory increase of the fbaC RNA levels was observed when fbaP was repressed by CRISPRi. In addition, targeting fbaP decreased tktP RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tktP RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tktC
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Methanol-essential growth of Corynebacterium glutamicum: Adaptive laboratory evolution overcomes limitation due to methanethiol assimilation pathway
'MDPI AG', 2020Co-Authors: Hennig Guido, Haupka Carsten, Heux Stephanie, Rückert Christian, Fernandes De Brito, Luciana, Cahoreau Edern, Wendisch, Volker F.Abstract:Hennig G, Haupka C, Fernandes de Brito L, et al. Methanol-essential growth of Corynebacterium glutamicum: Adaptive laboratory evolution overcomes limitation due to methanethiol assimilation pathway. International Journal of Molecular Sciences. 2020;21(10): 3617.Methanol is a sustainable substrate for biotechnology. In addition to natural Methylotrophs, metabolic engineering has gained attention for transfer of Methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes
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Charting the metabolic landscape of the facultative Methylotroph Bacillus methanolicus
'American Society for Microbiology', 2020Co-Authors: Delépine Baudoin, Wendisch, Volker F., Gil Lopez Marina, Carnicer Marc, Vicente Claúdia, Heux StephanieAbstract:Delépine B, Gil Lopez M, Carnicer M, Vicente C, Wendisch VF, Heux S. Charting the metabolic landscape of the facultative Methylotroph Bacillus methanolicus. mSystems. 2020;5: e00745-20.Bacillus methanolicus MGA3 is a thermotolerant and relatively fast-growing Methylotroph able to secrete large quantities of glutamate and lysine. These natural characteristics make B. methanolicus a good candidate to become a new industrial chassis organism, especially in a methanol-based economy. This has motivated a number of omics studies of B. methanolicus at the genome, transcript, protein and metabolic levels. Intriguingly, the only substrates known to support B. methanolicus growth as sole source of carbon and energy are methanol, mannitol, and to a lesser extent glucose and arabitol. We hypothesized that comparing Methylotrophic and non-Methylotrophic metabolic states at the flux level would yield new insights into MGA3 metabolism. 13C metabolic flux analysis (13C-MFA) is a powerful computational method to estimate carbon flows from substrate to biomass (i.e. the in vivo reaction rates of the central metabolic pathways) from experimental labeling data. In this study, we designed and performed a 13C-MFA of the facultative Methylotroph B. methanolicus MGA3 growing on methanol, mannitol and arabitol to compare the associated metabolic states. The results obtained validate previous findings on the Methylotrophy of B. methanolicus, allowed us to characterize the assimilation pathway of one of the studied carbon sources, and provide a better overall understanding of this strain. IMPORTANCE Methanol is cheap, easy to transport and can be produced both from renewable and fossil resources without mobilizing arable lands. As such, it is regarded as a potential carbon source to transition toward a greener industrial chemistry. Metabolic engineering of bacteria and yeast able to efficiently consume methanol is expected to provide cell factories that will transform methanol into higher-value chemicals in the so-called methanol economy. Toward that goal, the study of natural Methylotrophs such as B. methanolicus is critical to understand the origin of their efficient Methylotrophy. This knowledge will then be leveraged to transform such natural strains into new cell factories, or to design Methylotrophic capability in other strains already used by the industry
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Synthetic Methylotrophy: past, present, and future
Editions Springer, 2018Co-Authors: Heux Stephanie, Vorholt, Julia A., Brautaset Trygve, Wendisch, Volker F., Portais Jean-charlesAbstract:Methane and methanol are regarded as alternative and highly attractive nonfood raw materials for the biotechnology sector. The supply of methane and methanol comes from both fossil and renewable resources, rendering them flexible and sustainable raw materials. Reduced one-carbon (C1) compounds are used by specialized groups of microorganisms, i.e., the Methylotrophs, as their sole source of carbon and energy. While progress to engineer and use natural Methylotrophs in biotechnology is ongoing, synthetic Methylotrophs only recently have gained interest as a parallel approach both in academia and private industry. Synthetic Methylotrophy refers to the design and rational engineering of Methylotrophy to established non-Methylotrophic production hosts for access to methane and methanol as feedstock while maintaining their biotechnological production potential. In this chapter, we will illustrate how combined systems and synthetic biology approaches capitalize on the metabolic versatility and engineered production pathways of industrially well-established microorganisms, such as Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum, for biotransformation from methane and methanol. Challenges and current prospects for designing and engineering the next generation of synthetic Methylotrophs are also discussed
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Transcriptome analysis of thermophilic Methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape
'Springer Science and Business Media LLC', 2015Co-Authors: Irla Marta, Brautaset Trygve, Rückert Christian, Neshat Armin, Kalinowski Jörn, Wendisch, Volker F.Abstract:Background Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle Methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5′-ends. Results Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5′-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5′-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to Methylotrophy had highly abundant transcripts. Conclusion The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism. Keywords: Bacillus methanolicus ; Methylotrophy; RNA-sequencing; Transcriptome analysis; Conserved sequence motifs; Operon structures; Regulatory RNA; Transcript abundances; Transcriptional start sites; Ribosome binding sitespublishedVersio
Ludmila Chistoserdova - One of the best experts on this subject based on the ideXlab platform.
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Table_2_Lanthanide-Dependent Methanol Dehydrogenases of XoxF4 and XoxF5 Clades Are Differentially Distributed Among Methylotrophic Bacteria and They Reveal Different Biochemical Properties.XLSX
2018Co-Authors: Jing Huang, Ludmila ChistoserdovaAbstract:Lanthanide-dependent alcohol dehydrogenases have recently emerged as environmentally important enzymes, most prominently represented in Methylotrophic bacteria. The diversity of these enzymes, their environmental distribution, and their biochemistry, as well as their evolutionary relationships with their calcium-dependent counterparts remain virtually untapped. Here, we make important advances toward understanding lanthanide-dependent Methylotrophy by assessing the distribution of XoxF4 and XoxF5 clades of lanthanide methanol dehydrogenases among, respectively, Methylophilaceae and non-Methylophilaceae Methylotrophs, and we carry out comparative biochemical characterization of XoxF4 and XoxF5 enzymes, demonstrating differences in their properties, including catalytic efficiencies. We conclude that one subtype of the XoxF4 enzyme, XoxF4-1 is the dominant type in nature while other XoxF4 subtypes appear to be auxiliary, representatives of this clade only found in the Methylophilaceae (Betaproteobacteria). In contrast, we demonstrate that XoxF5 enzymes are widespread among Alpha-, Beta-, and Gammaproteobacteria. We purified and biochemically characterized two XoxF4 enzymes (XoxF4-1 and XoxF4-2), both from Methylotenera mobilis, and one XoxF5 enzyme, from Methylomonas sp., after expressing their His-tagged versions in respective natural hosts. All three enzymes showed broad specificities toward alcohols and aldehydes and strict dependence on lighter lanthanides. However, they revealed differences in their properties in terms of optimal pH for in vitro activity, ammonia dependence, the range of lanthanides that could serve as cofactors, and in kinetic properties. Overall, our data advance the understanding of the biochemistry and environmental distribution of these recently discovered enzymes that appear to be key enzymes in lanthanide-dependent Methylotrophy.
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Presentation_2_Lanthanide-Dependent Methanol Dehydrogenases of XoxF4 and XoxF5 Clades Are Differentially Distributed Among Methylotrophic Bacteria and They Reveal Different Biochemical Properties.PDF
2018Co-Authors: Jing Huang, Ludmila ChistoserdovaAbstract:Lanthanide-dependent alcohol dehydrogenases have recently emerged as environmentally important enzymes, most prominently represented in Methylotrophic bacteria. The diversity of these enzymes, their environmental distribution, and their biochemistry, as well as their evolutionary relationships with their calcium-dependent counterparts remain virtually untapped. Here, we make important advances toward understanding lanthanide-dependent Methylotrophy by assessing the distribution of XoxF4 and XoxF5 clades of lanthanide methanol dehydrogenases among, respectively, Methylophilaceae and non-Methylophilaceae Methylotrophs, and we carry out comparative biochemical characterization of XoxF4 and XoxF5 enzymes, demonstrating differences in their properties, including catalytic efficiencies. We conclude that one subtype of the XoxF4 enzyme, XoxF4-1 is the dominant type in nature while other XoxF4 subtypes appear to be auxiliary, representatives of this clade only found in the Methylophilaceae (Betaproteobacteria). In contrast, we demonstrate that XoxF5 enzymes are widespread among Alpha-, Beta-, and Gammaproteobacteria. We purified and biochemically characterized two XoxF4 enzymes (XoxF4-1 and XoxF4-2), both from Methylotenera mobilis, and one XoxF5 enzyme, from Methylomonas sp., after expressing their His-tagged versions in respective natural hosts. All three enzymes showed broad specificities toward alcohols and aldehydes and strict dependence on lighter lanthanides. However, they revealed differences in their properties in terms of optimal pH for in vitro activity, ammonia dependence, the range of lanthanides that could serve as cofactors, and in kinetic properties. Overall, our data advance the understanding of the biochemistry and environmental distribution of these recently discovered enzymes that appear to be key enzymes in lanthanide-dependent Methylotrophy.
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lanthanide dependent methanol dehydrogenases of xoxf4 and xoxf5 clades are differentially distributed among Methylotrophic bacteria and they reveal different biochemical properties
Frontiers in Microbiology, 2018Co-Authors: Jing Huang, Ludmila ChistoserdovaAbstract:Lanthanide-dependent alcohol dehydrogenases have recently emerged as environmentally important enzymes, most prominently represented in Methylotrophic bacteria. The diversity of these enzymes, their environmental distribution and their biochemistry, as well as their evolutionary relationships with their calcium-dependent counterparts remain virtually untapped. Here we make important advances toward understanding lanthanide-dependent Methylotrophy by assessing the distribution of XoxF4 and XoxF5 clades of lanthanide methanol dehydrogenases among, respectively, Methylophilaceae and non-Methylophilaceae Methylotrophs, and we carry out comparative biochemical characterization of XoxF4 and XoxF5 enzymes, demonstrating differences in their properties, including catalytic efficiencies. We conclude that one subtype of the XoxF4 enzyme, XoxF4-1 is the dominant type in nature while other XoxF4 subtypes appear to be auxiliary, representatives of this clade only found in the Methylophilaceae (Betaproteobacteria). In contrast, we demonstrate that XoxF5 enzymes are widespread among alpha-, beta-, and gammaproteobacteria. We purified and biochemically characterized two XoxF4 enzymes (XoxF4-1 and XoxF4-2), both from Methylotenera mobilis, and one XoxF5 enzyme, from Methylomonas sp., after expressing their His-tagged versions in respective natural hosts. All three enzymes showed broad specificities toward alcohols and aldehydes and strict dependence on lighter lanthanides. However, they revealed differences in their properties in terms of optimal pH for in vitro activity, ammonia dependence, the range of lanthanides that could serve as cofactors, and in kinetic properties. Overall, our data advance the understanding of the biochemistry and environmental distribution of these recently discovered enzymes that appear to be key enzymes in lanthanide-dependent Methylotrophy.
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genomics of Methylotrophy in gram positive methylamine utilizing bacteria
Microorganisms, 2015Co-Authors: Tami L Mctaggart, Mary E Lidstrom, David A C Beck, Usanisa Setboonsarng, Nicole Shapiro, Tanja Woyke, Marina G Kalyuzhnaya, Ludmila ChistoserdovaAbstract:Gram-positive Methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of Methylotrophy pathways/enzymes within this group. However, genome-based knowledge of Methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive Methylotrophs limits our global understanding of Methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive Methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the Methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of Methylotrophy in Gram-positive Methylotrophic bacteria. This study is a major contribution to the field of Methylotrophy, aimed at closing the gap in the genomic knowledge of Methylotrophy within this diverse group of bacteria.
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modularity of Methylotrophy revisited
Environmental Microbiology, 2011Co-Authors: Ludmila ChistoserdovaAbstract:Methylotrophy is a metabolic capability possessed by microorganisms that allows them to build biomass and to obtain energy from organic substrates containing no carbon-carbon bonds (C1 compounds, such as methane, methanol, etc.). This phenomenon in microbial physiology has been a subject of study for over 100 years, elucidating a set of well-defined enzymatic systems and pathways enabling this capability. The knowledge gained from the early genetic and genomic approaches to understanding Methylotrophy pointed towards the existence of alternative enzymes/pathways for the specific metabolic goals. Different combinations of these systems in different organisms suggested that Methylotrophy must be modular in its nature. More recent insights from genomic analyses, including the genomes representing novel types of Methylotrophs, seem to reinforce this notion. This review integrates the new findings with the previously developed concept of modularity of Methylotrophy.
Volker Wendisch - One of the best experts on this subject based on the ideXlab platform.
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Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway
International Journal of Molecular Sciences, 2020Co-Authors: Guido Hennig, Carsten Haupka, Luciana Brito, Christian Rückert, Edern Cahoreau, Stephanie Heux, Volker WendischAbstract:Methanol is a sustainable substrate for biotechnology. In addition to natural Methylotrophs, metabolic engineering has gained attention for transfer of Methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cutoffs , i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.
Volker F Wendisch - One of the best experts on this subject based on the ideXlab platform.
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characterization of fructose 1 6 bisphosphatase and sedoheptulose 1 7 bisphosphatase from the facultative ribulose monophosphate cycle Methylotroph bacillus methanolicus
Journal of Bacteriology, 2013Co-Authors: Jessica Stolzenberger, Steffen N Lindner, Marcus Persicke, Trygve Brautaset, Volker F WendischAbstract:The genome of the facultative ribulose monophosphate (RuMP) cycle Methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX(C)) and one on plasmid pBM19 (GlpX(P)), which is required for Methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum Δfbp mutant could be phenotypically complemented with glpX(C) and glpX(P) from B. methanolicus. GlpX(P) and GlpX(C) share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn(2+) ions and inhibited by Li(+), but differed in terms of the kinetic parameters. GlpX(C) showed a much higher catalytic efficiency and a lower Km for fructose 1,6-bisphosphate (86.3 s(-1) mM(-1) and 14 ± 0.5 μM, respectively) than GlpX(P) (8.8 s(-1) mM(-1) and 440 ± 7.6 μM, respectively), indicating that GlpX(C) is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX(P) but not for GlpX(C). Based on these in vitro data, GlpX(P) is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.