Table_1_Concerted Metabolic Shifts Give New Insights Into the Syntrophic Mechanism Between Propionate-Fermenting Pelotomaculum thermopropionicum and Hydrogenotrophic Methanocella conradii.PDF

<p>Microbial syntrophy is a thermodynamically-based cooperation between microbial partners that share the small amounts of free energy for anaerobic growth. To gain insights into the mechanism by which syntrophic microorganisms coordinate their metabolism, we constructed cocultures of propionate-oxidizing Pelotomaculum thermopropionicum and hydrogenotrophic Methanocella conradii and compared them to monocultures. Transcriptome analysis was performed on these cultures using strand-specific mRNA sequencing (RNA-Seq). The results showed that in coculture both P. thermopropionicum and M. conradii significantly upregulated the expression of genes involved in catabolism but downregulated those for anabolic biosynthesis. Specifically, genes coding for the methylmalonyl-CoA pathway in P. thermopropionicum and key genes for methanogenesis in M. conradii were substantially upregulated in coculture compared to monoculture. The putative flavin-based electron bifurcation/confurcation systems in both organisms were also upregulated in coculture. Formate dehydrogenase encoding genes in both organisms were markedly upregulated, indicating that formate was produced and utilized by P. thermopropionicum and M. conradii, respectively. The inhibition of syntrophic activity by formate and 2-bromoethanesulphonate (2-BES) but not H₂/CO₂ also suggested that formate production was used by P. thermopropionicum for the recycling of intracellular redox mediators. Finally, flagellum-induced signal transduction and amino acids exchange was upregulated for syntrophic interactions. Together, our study suggests that syntrophic organisms employ multiple strategies including global metabolic shift, utilization of electron bifurcation/confurcation and employing formate as an alternate electron carrier to optimize their metabolisms for syntrophic growth.</p

Comparative characteristics of strain HZ254^T and <i>Methanocella paludicola</i> SANAE^T and <i>Methanocella arvoryzae</i> MRE50^T.

<p>Data for strain HZ254^T is from this study, and strain SANAE^T and MRE50^T were retrieved from Sakai <i>et al.</i>, 2008 and 2010.</p>*<p>The data in parentheses were determined by HPLC, other data were taken from genome information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Erkel1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-L1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Sakai4" target="_blank">[24]</a>.</p>†<p>pH for HZ254^T and other strains were determined at 55°C and 25°C, respectively. Abbreviations, −, negative; +, positive; N.A., not applicable.</p

Data_Sheet_1_Concerted Metabolic Shifts Give New Insights Into the Syntrophic Mechanism Between Propionate-Fermenting Pelotomaculum thermopropionicum and Hydrogenotrophic Methanocella conradii.XLSX

<p>Microbial syntrophy is a thermodynamically-based cooperation between microbial partners that share the small amounts of free energy for anaerobic growth. To gain insights into the mechanism by which syntrophic microorganisms coordinate their metabolism, we constructed cocultures of propionate-oxidizing Pelotomaculum thermopropionicum and hydrogenotrophic Methanocella conradii and compared them to monocultures. Transcriptome analysis was performed on these cultures using strand-specific mRNA sequencing (RNA-Seq). The results showed that in coculture both P. thermopropionicum and M. conradii significantly upregulated the expression of genes involved in catabolism but downregulated those for anabolic biosynthesis. Specifically, genes coding for the methylmalonyl-CoA pathway in P. thermopropionicum and key genes for methanogenesis in M. conradii were substantially upregulated in coculture compared to monoculture. The putative flavin-based electron bifurcation/confurcation systems in both organisms were also upregulated in coculture. Formate dehydrogenase encoding genes in both organisms were markedly upregulated, indicating that formate was produced and utilized by P. thermopropionicum and M. conradii, respectively. The inhibition of syntrophic activity by formate and 2-bromoethanesulphonate (2-BES) but not H₂/CO₂ also suggested that formate production was used by P. thermopropionicum for the recycling of intracellular redox mediators. Finally, flagellum-induced signal transduction and amino acids exchange was upregulated for syntrophic interactions. Together, our study suggests that syntrophic organisms employ multiple strategies including global metabolic shift, utilization of electron bifurcation/confurcation and employing formate as an alternate electron carrier to optimize their metabolisms for syntrophic growth.</p

T-RFLP patterns based on 16S rRNA genes for enrichment cultures of strain HZ254^T along with successive transfers.

<p>The analysis was performed using Ar109f/915r primer set and <i>TaqI</i> restriction enzymes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Peng1" target="_blank">[14]</a>. T-RFLP fingerprints were normalized to a total of 100 relative fluorescence units (RFU), and T-RF peaks with RFU less than 1 were discarded. The 254-bp T-RF was affiliated with <i>Methanocellales</i> (RC-I) as determined by cloning and sequencing of 16S rRNA genes, and the T-RF length calculated from the sequence was actually 258-bp (data not shown). All other T-RF peaks could be assigned correspondingly to <i>Methanomicrobiales</i> (Mm), <i>Methanobacteriales</i> (Mb), <i>Methanosarcinaceae</i> (Msr)/Crenarchaeotal group 1.1b (G1.1b), <i>Methanosaetaceae</i> (Msa) and RC-I/<i>Methanomicrobiales</i> (Mm), according to our previous studies in the same soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Peng1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Yuan1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Wu1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035279#pone.0035279-Yuan2" target="_blank">[36]</a>, respectively. The pre-incubation samples were sampled after 24 hours of incubation, and all other samples were sampled after that methane production ceased and/or hydrogen could not be detected in the headspace. After the 13^th transfer, the archaeal community was still frequently monitored by T-RFLP analysis along with subsequent transfers, but the 254-bp was always the sole T-RF product.</p

Image_1_Concerted Metabolic Shifts Give New Insights Into the Syntrophic Mechanism Between Propionate-Fermenting Pelotomaculum thermopropionicum and Hydrogenotrophic Methanocella conradii.PDF

<p>Microbial syntrophy is a thermodynamically-based cooperation between microbial partners that share the small amounts of free energy for anaerobic growth. To gain insights into the mechanism by which syntrophic microorganisms coordinate their metabolism, we constructed cocultures of propionate-oxidizing Pelotomaculum thermopropionicum and hydrogenotrophic Methanocella conradii and compared them to monocultures. Transcriptome analysis was performed on these cultures using strand-specific mRNA sequencing (RNA-Seq). The results showed that in coculture both P. thermopropionicum and M. conradii significantly upregulated the expression of genes involved in catabolism but downregulated those for anabolic biosynthesis. Specifically, genes coding for the methylmalonyl-CoA pathway in P. thermopropionicum and key genes for methanogenesis in M. conradii were substantially upregulated in coculture compared to monoculture. The putative flavin-based electron bifurcation/confurcation systems in both organisms were also upregulated in coculture. Formate dehydrogenase encoding genes in both organisms were markedly upregulated, indicating that formate was produced and utilized by P. thermopropionicum and M. conradii, respectively. The inhibition of syntrophic activity by formate and 2-bromoethanesulphonate (2-BES) but not H₂/CO₂ also suggested that formate production was used by P. thermopropionicum for the recycling of intracellular redox mediators. Finally, flagellum-induced signal transduction and amino acids exchange was upregulated for syntrophic interactions. Together, our study suggests that syntrophic organisms employ multiple strategies including global metabolic shift, utilization of electron bifurcation/confurcation and employing formate as an alternate electron carrier to optimize their metabolisms for syntrophic growth.</p

Isolation of Viable Type I and II Methanotrophs Using Cell-Imprinted Polyurethane Thin Films

Studies on methanotrophs utilizing methane as sole source of carbon and energy are meaningful for governing global warming; although, the isolation of methanotrophs from nature is challenging. Here, surface imprinted polyurethane films were fabricated to selectively capture living methanotrophs from paddy soil. Two tracks of molecularly imprinted film based on polyurethane (PU-MIF₁ and PU-MIF₂) were imprinted using type I or II methanotrophs as template, respectively, and then reacted with polyethylene glycol, castor oil, and hexamethylene diisocyanate. Results demonstrated these PU-MIFs hold low water absorption rate and superior biocompatibility, which was highly demanded for maintaining cell viability. Superior selectivity and affinity of PU-MIFs toward their cognate methanotroph cells was observed by fluorescent microscopy. Atomic force microscopy revealed the adhesion force of PU-MIFs with its cognate cells was much stronger in comparison with noncognate ones. Using the as-prepared PU-MIFs, within 30 min, methanotroph cells could be separated from rice paddy efficiently. Therefore, the PU-MIFs might be used as an efficient approach for cell sorting from environmental samples

Image_1_Stimulatory Effect of Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing Consortium.TIF

<p>Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe₃O₄) consistently enhanced butyrate oxidation and CH₄ production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe₃O₄ caused the similar stimulatory effect. Silica coating of nanoFe₃O₄ surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe₃O₄. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.</p

Image_4_Stimulatory Effect of Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing Consortium.TIF

<p>Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe₃O₄) consistently enhanced butyrate oxidation and CH₄ production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe₃O₄ caused the similar stimulatory effect. Silica coating of nanoFe₃O₄ surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe₃O₄. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.</p

Image_2_Stimulatory Effect of Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing Consortium.TIF

<p>Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe₃O₄) consistently enhanced butyrate oxidation and CH₄ production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe₃O₄ caused the similar stimulatory effect. Silica coating of nanoFe₃O₄ surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe₃O₄. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.</p

Image_3_Stimulatory Effect of Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing Consortium.TIF

<p>Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe₃O₄) consistently enhanced butyrate oxidation and CH₄ production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe₃O₄ caused the similar stimulatory effect. Silica coating of nanoFe₃O₄ surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe₃O₄. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.</p