Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. Shifts from N-limited to N-excess conditions were achieved by raising NH4Cl from 2.8 mM to the standard 10 mM. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. For shifts from P-limited to P-excess conditions, phosphate was raised from 0.18 mM to the standard 0.8 mM. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. For shifts from P-limited to P-excess conditions, phosphate was raised from 0.12 mM to the standard 0.8 mM. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. For shifts from P-limited to P-excess conditions, phosphate was raised from 0.13 mM to the standard 0.8 mM. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter and have applications in carbon-neutral fuel production. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 100 different steady-state and time course experiments that were performed in chemostats, or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel TFs in the regulation of phosphate-dependent repression of formate dehydorgenase M-bM-^@M-^S a key enzyme in the methanogenesis pathway. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). Growth conditions were carefully designed to separate the effects of different environmental factors. To ensure physico-chemical factors being constant, all cell cultures were performed using continuous cultivations with a constant dilution rate. The standard gassing regime was 110 mL/min H2, 40 mL/min CO2, 35 mL/min Ar, and 15 mL/min H2S/Ar mixture (1:99). For shifts from a H2 excess to a H2 limited condition, H2 was lowered from standard 110 mL/min to 21 mL/min and Ar was raised from standard 35 mL/min to 125 mL/min. For shifts from P-limited to P-excess conditions, phosphate was raised from 0.12 mM to the standard 0.8 mM. The dilution rate was held constant at 0.083 h-1. For time-series array data, cultures before perturbation were allowed to reach steady state. We rapidly changed concentration(s) of H2 and/or a nutrient, and sampled at intervals after the perturbation; right away, after 5 mins, 10, 20, 30, 45, 60, 90, 120, 180, and 300 mins. Culture samples (1.5 mL) were rapidly removed from the chemostat vessels by syringe and cell pellets collected by microcentrifugation, immediately frozen in an ethanol-dry ice bath, and stored at -80M-BM-0C. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Hydrogenotrophic methanogenic Archaea are defined by a H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. Hydrogenotrophs respond to H2 starvation both phenotypically and at the level of gene expression; however, the responses during growth on formate have not been characterized. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess, suggesting that energy spilling occurs. Using microarray analysis, we show that the expression of genes encoding F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation, but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). The medium contained 380 mM sodium formate in place of NaCl. The agitation rate was 250 revolutions per minute. The gas mixture initially supplied to the chemostat was H2/Ar/CO2/1% H2S (21/125/40/14 mLs min-1) (Proc Natl Acad Sci USA 104: 8930-8934, 2007). After the OD increased above 0.6 (24 h), the medium flow was turned on at 0.083 L/h. Medium was either non-limiting (described above), formate limiting (modified to contain 200 mM sodium formate and 180 mM NaCl), nitrogen limiting (2 mM NH4Cl), or phosphate limiting (0.08 mM K2HPO4). Also at this point, gas flow through the vessel was shut off, a 1 psi check valve was installed on the exhaust line to maintain positive pressure within the vessel while also allowing for exhaust of gaseous metabolic byproducts, and a 1% Na2S9H2O feed was started into the vessel at 0.83 mL/h. 48 hours later, samples were taken for physiological measurements or microarray analysis. Samples were collected for microarray analysis as follows: 1.5 mLs of chemostat culture (OD660 ~0.65) was centrifuged at 13,000 x g for 30 seconds and supernatant was discarded. The pellet was immediately placed in a dry ice/ethanol bath and stored at -80C until RNA extraction and microarrays could be performed. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Dataset Information

Steady-state samples of Methanococcus maripaludis MM901 grown on formate-excess medium

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