Special Issue "Microbial C1 Metabolism"

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A special issue of Microorganisms (ISSN 2076-2607).

Deadline for manuscript submissions: closed (31 January 2015)

Special Issue Editors

Guest Editor
Dr. Marina G. Kalyuzhnaya

Department of Biology, San Diego State University, 5500 Campanile Drive, NLS rm 406A, San Diego, CA 92182-4614, USA
Guest Editor
Dr. Ludmila Chistoserdova

Department of Chemical Engineering, University of Washington, Box 355014, Seattle, WA 98195-5014, USA
Interests: methylotrophy; bacterial physiology; microbial ecology; microbial communities; genomics; metagenomics

Special Issue Information

Dear Colleagues,

One-carbon (C1) compounds, which include methane, methanol, and other methylated compounds, are important metabolites in the global carbon cycle. Recent years have seen a number of major breakthroughs in understanding the details of microbial C1 metabolism, including discoveries of novel metabolic strategies and novel taxa involved in global C1 cycling. This Special Issue aims to cover the modern aspects of the unique metabolic properties exhibited by microbes active in the conversion of C1 compounds, in both aerobic and anaerobic environments. We especially welcome studies describing insights from the omics approaches. Both original research and review type papers are invited. Potential topics include, but are not limited to, the following: Genomics and metagenomics of C1 metabolism; Systems level studies of microbes involved in C1 cycling; Ecology of natural and engineered communities with active C1 metabolism; C1-based biotechnological applications; Evolution of C1 metabolism; and C1 cycling in nature and climate change.

Dr. Ludmila Chistoserdova
Dr. Marina G. Kalyuzhnaya
Guest Editor
s

Submission

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Keywords

  • C1 metabolism
  • C1 transfer
  • methylotropy
  • methanogenesis
  • genomics
  • metagenomics
  • transcriptomics

Published Papers (10 papers)

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Research

Jump to: Review

Open AccessArticle Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer
Microorganisms 2015, 3(3), 327-343; doi:10.3390/microorganisms3030327
Received: 29 April 2015 / Revised: 9 June 2015 / Accepted: 18 June 2015 / Published: 2 July 2015
Cited by 1 | PDF Full-text (606 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were [...] Read more.
The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
Open AccessArticle Methane Oxidation and Molecular Characterization of Methanotrophs from a Former Mercury Mine Impoundment
Microorganisms 2015, 3(2), 290-309; doi:10.3390/microorganisms3020290
Received: 29 April 2015 / Revised: 1 June 2015 / Accepted: 11 June 2015 / Published: 23 June 2015
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Abstract
The Herman Pit, once a mercury mine, is an impoundment located in an active geothermal area. Its acidic waters are permeated by hundreds of gas seeps. One seep was sampled and found to be composed of mostly CO2 with some CH [...] Read more.
The Herman Pit, once a mercury mine, is an impoundment located in an active geothermal area. Its acidic waters are permeated by hundreds of gas seeps. One seep was sampled and found to be composed of mostly CO2 with some CH4 present. The δ13CH4 value suggested a complex origin for the methane: i.e., a thermogenic component plus a biological methanogenic portion. The relatively 12C-enriched CO2 suggested a reworking of the ebullitive methane by methanotrophic bacteria. Therefore, we tested bottom sediments for their ability to consume methane by conducting aerobic incubations of slurried materials. Methane was removed from the headspace of live slurries, and subsequent additions of methane resulted in faster removal rates. This activity could be transferred to an artificial, acidic medium, indicating the presence of acidophilic or acid-tolerant methanotrophs, the latter reinforced by the observation of maximum activity at pH = 4.5 with incubated slurries. A successful extraction of sterol and hopanoid lipids characteristic of methanotrophs was achieved, and their abundances greatly increased with increased sediment methane consumption. DNA extracted from methane-oxidizing enrichment cultures was amplified and sequenced for pmoA genes that aligned with methanotrophic members of the Gammaproteobacteria. An enrichment culture was established that grew in an acidic (pH 4.5) medium via methane oxidation. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
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Open AccessArticle Parallel and Divergent Evolutionary Solutions for the Optimization of an Engineered Central Metabolism in Methylobacterium extorquens AM1
Microorganisms 2015, 3(2), 152-174; doi:10.3390/microorganisms3020152
Received: 16 February 2015 / Revised: 30 March 2015 / Accepted: 1 April 2015 / Published: 9 April 2015
Cited by 2 | PDF Full-text (812 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Bioengineering holds great promise to provide fast and efficient biocatalysts for methanol-based biotechnology, but necessitates proven methods to optimize physiology in engineered strains. Here, we highlight experimental evolution as an effective means for optimizing an engineered Methylobacterium extorquens AM1. Replacement of the [...] Read more.
Bioengineering holds great promise to provide fast and efficient biocatalysts for methanol-based biotechnology, but necessitates proven methods to optimize physiology in engineered strains. Here, we highlight experimental evolution as an effective means for optimizing an engineered Methylobacterium extorquens AM1. Replacement of the native formaldehyde oxidation pathway with a functional analog substantially decreased growth in an engineered Methylobacterium, but growth rapidly recovered after six hundred generations of evolution on methanol. We used whole-genome sequencing to identify the basis of adaptation in eight replicate evolved strains, and examined genomic changes in light of other growth and physiological data. We observed great variety in the numbers and types of mutations that occurred, including instances of parallel mutations at targets that may have been “rationalized” by the bioengineer, plus other “illogical” mutations that demonstrate the ability of evolution to expose unforeseen optimization solutions. Notably, we investigated mutations to RNA polymerase, which provided a massive growth benefit but are linked to highly aberrant transcriptional profiles. Overall, we highlight the power of experimental evolution to present genetic and physiological solutions for strain optimization, particularly in systems where the challenges of engineering are too many or too difficult to overcome via traditional engineering methods. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
Open AccessArticle C1-Pathways in Methyloversatilis universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies
Microorganisms 2015, 3(2), 175-197; doi:10.3390/microorganisms3020175
Received: 5 January 2015 / Revised: 17 February 2015 / Accepted: 26 March 2015 / Published: 9 April 2015
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Abstract
Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate [...] Read more.
Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate pathway were induced during growth on methylamine. Among other functions responding to the aminated source of C1-carbon, are a heme-containing amine dehydrogenase (Qhp), a distant homologue of formaldehyde activating enzyme (Fae3), molybdenum-containing formate dehydrogenase, ferredoxin reductase, a set of homologues to urea/ammonium transporters and amino-acid permeases. Mutants lacking one of the functional subunits of the amine dehydrogenase (ΔqhpA) or Δfae3 showed no growth defect on C1-compounds. M. universalis FAM5 strains with a lesion in the H4-folate pathway were not able to use any C1-compound, methanol or methylamine. Genes essential for C1-assimilation (the serine cycle and glyoxylate shunt) and H4MTP-pathway for formaldehyde oxidation showed similar levels of expression on both C1-carbon sources. M. universalis FAM5 possesses three homologs of the formaldehyde activating enzyme, a key enzyme of the H4MTP-pathway. Strains lacking the canonical Fae (fae1) lost the ability to grow on both C1-compounds. However, upon incubation on methylamine the fae1-mutant produced revertants (Δfae1R), which regained the ability to grow on methylamine. Double and triple mutants (Δfae1RΔfae3, or Δfae1RΔfae2 or Δfae1RΔfae2Δfae3) constructed in the revertant strain background showed growth similar to the Δfae1R phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
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Open AccessArticle High Throughput Sequencing to Detect Differences in Methanotrophic Methylococcaceae and Methylocystaceae in Surface Peat, Forest Soil, and Sphagnum Moss in Cranesville Swamp Preserve, West Virginia, USA
Microorganisms 2015, 3(2), 113-136; doi:10.3390/microorganisms3020113
Received: 25 January 2015 / Revised: 23 February 2015 / Accepted: 26 March 2015 / Published: 2 April 2015
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Abstract
Northern temperate forest soils and Sphagnum-dominated peatlands are a major source and sink of methane. In these ecosystems, methane is mainly oxidized by aerobic methanotrophic bacteria, which are typically found in aerated forest soils, surface peat, and Sphagnum moss. We contrasted [...] Read more.
Northern temperate forest soils and Sphagnum-dominated peatlands are a major source and sink of methane. In these ecosystems, methane is mainly oxidized by aerobic methanotrophic bacteria, which are typically found in aerated forest soils, surface peat, and Sphagnum moss. We contrasted methanotrophic bacterial diversity and abundances from the (i) organic horizon of forest soil; (ii) surface peat; and (iii) submerged Sphagnum moss from Cranesville Swamp Preserve, West Virginia, using multiplex sequencing of bacterial 16S rRNA (V3 region) gene amplicons. From ~1 million reads, >50,000 unique OTUs (Operational Taxonomic Units), 29 and 34 unique sequences were detected in the Methylococcaceae and Methylocystaceae, respectively, and 24 potential methanotrophs in the Beijerinckiaceae were also identified. Methylacidiphilum-like methanotrophs were not detected. Proteobacterial methanotrophic bacteria constitute <2% of microbiota in these environments, with the Methylocystaceae one to two orders of magnitude more abundant than the Methylococcaceae in all environments sampled. The Methylococcaceae are also less diverse in forest soil compared to the other two habitats. Nonmetric multidimensional scaling analyses indicated that the majority of methanotrophs from the Methylococcaceae and Methylocystaceae tend to occur in one habitat only (peat or Sphagnum moss) or co-occurred in both Sphagnum moss and peat. This study provides insights into the structure of methanotrophic communities in relationship to habitat type, and suggests that peat and Sphagnum moss can influence methanotroph community structure and biogeography. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
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Open AccessArticle Genomics of Methylotrophy in Gram-Positive Methylamine-Utilizing Bacteria
Microorganisms 2015, 3(1), 94-112; doi:10.3390/microorganisms3010094
Received: 3 February 2015 / Revised: 27 February 2015 / Accepted: 6 March 2015 / Published: 20 March 2015
Cited by 2 | PDF Full-text (688 KB) | HTML Full-text | XML Full-text
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
Open AccessArticle Experimental Horizontal Gene Transfer of Methylamine Dehydrogenase Mimics Prevalent Exchange in Nature and Overcomes the Methylamine Growth Constraints Posed by the Sub-Optimal N-Methylglutamate Pathway
Microorganisms 2015, 3(1), 60-79; doi:10.3390/microorganisms3010060
Received: 19 December 2014 / Revised: 16 February 2015 / Accepted: 16 February 2015 / Published: 10 March 2015
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Abstract
Methylamine plays an important role in the global carbon and nitrogen budget; microorganisms that grow on reduced single carbon compounds, methylotrophs, serve as a major biological sink for methylamine in aerobic environments. Two non-orthologous, functionally degenerate routes for methylamine oxidation have been [...] Read more.
Methylamine plays an important role in the global carbon and nitrogen budget; microorganisms that grow on reduced single carbon compounds, methylotrophs, serve as a major biological sink for methylamine in aerobic environments. Two non-orthologous, functionally degenerate routes for methylamine oxidation have been studied in methylotrophic Proteobacteria: Methylamine dehydrogenase and the N-methylglutamate pathway. Recent work suggests the N-methylglutamate (NMG) pathway may be more common in nature than the well-studied methylamine dehydrogenase (MaDH, encoded by the mau gene cluster). However, the distribution of these pathways across methylotrophs has never been analyzed. Furthermore, even though horizontal gene transfer (HGT) is commonly invoked as a means to transfer these pathways between strains, the physiological barriers to doing so have not been investigated. We found that the NMG pathway is both more abundant and more universally distributed across methylotrophic Proteobacteria compared to MaDH, which displays a patchy distribution and has clearly been transmitted by HGT even amongst very closely related strains. This trend was especially prominent in well-characterized strains of the Methylobacterium extroquens species, which also display significant phenotypic variability during methylamine growth. Strains like Methylobacterium extorquens PA1 that only encode the NMG pathway grew on methylamine at least five-fold slower than strains like Methylobacterium extorquens AM1 that also possess the mau gene cluster. By mimicking a HGT event through the introduction of the M. extorquens AM1 mau gene cluster into the PA1 genome, the resulting strain instantaneously achieved a 4.5-fold increase in growth rate on methylamine and a 11-fold increase in fitness on methylamine, which even surpassed the fitness of M. extorquens AM1. In contrast, when three replicate populations of wild type M. extorquens PA1 were evolved on methylamine as the sole carbon and energy source for 150 generations neither fitness nor growth rate improved. These results suggest that the NMG pathway permits slow growth on methylamine and is widely distributed in methylotrophs; however, rapid growth on methylamine can be achieved quite readily through acquisition of the mau cluster by HGT. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
Open AccessArticle Role of NAD+-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b
Microorganisms 2015, 3(1), 47-59; doi:10.3390/microorganisms3010047
Received: 30 December 2014 / Revised: 21 January 2015 / Accepted: 5 February 2015 / Published: 27 February 2015
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Abstract
We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were [...] Read more.
We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD+-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa) enzyme displaying nearly equal reductive (malate formation) and oxidative (oxaloacetate formation) activities and higher affinity to malate (Km = 0.11 mM) than to oxaloacetate (Km = 0.34 mM). The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa), two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM) than to malate (Km = 1.28 mM). The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)
Open AccessArticle Rapid Reactivation of Deep Subsurface Microbes in the Presence of C-1 Compounds
Microorganisms 2015, 3(1), 17-33; doi:10.3390/microorganisms3010017
Received: 10 December 2014 / Revised: 26 January 2015 / Accepted: 29 January 2015 / Published: 5 February 2015
Cited by 2 | PDF Full-text (1286 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect [...] Read more.
Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect the long-term stability of the repository systems, Here, we studied how microorganisms present in fracture fluid from a depth of 500 m in Outokumpu, Finland, respond to simple carbon compounds (C-1 compounds) in the presence or absence of sulphate as an electron acceptor. C-1 compounds such as methane and methanol are important intermediates in the deep subsurface carbon cycle, and electron acceptors such as sulphate are critical components of oxidation processes. Fracture fluid samples were incubated in vitro with either methane or methanol in the presence or absence of sulphate as an electron acceptor. Metabolic response was measured by staining the microbial cells with fluorescent dyes that indicate metabolic activity and transcriptional response with RT-qPCR. Our results show that deep subsurface microbes exist in dormant states but rapidly reactivate their transcription and respiration systems in the presence of C-1 substrates, particularly methane. Microbial activity was further enhanced by the addition of sulphate as an electron acceptor. Sulphate- and nitrate-reducing microbes were particularly responsive to the addition of C-1 compounds and sulphate. These taxa are common in deep biosphere environments and may be affected by conditions disturbed by bedrock intrusion, as from drilling and excavation for long-term storage of hazardous waste. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)

Review

Jump to: Research

Open AccessReview Interactions of Methylotrophs with Plants and Other Heterotrophic Bacteria
Microorganisms 2015, 3(2), 137-151; doi:10.3390/microorganisms3020137
Received: 3 February 2015 / Revised: 18 March 2015 / Accepted: 27 March 2015 / Published: 2 April 2015
Cited by 1 | PDF Full-text (1142 KB) | HTML Full-text | XML Full-text
Abstract
Methylotrophs, which can utilize methane and/or methanol as sole carbon and energy sources, are key players in the carbon cycle between methane and CO2, the two most important greenhouse gases. This review describes the relationships between methylotrophs and plants, and [...] Read more.
Methylotrophs, which can utilize methane and/or methanol as sole carbon and energy sources, are key players in the carbon cycle between methane and CO2, the two most important greenhouse gases. This review describes the relationships between methylotrophs and plants, and between methanotrophs (methane-utilizers, a subset of methylotrophs) and heterotrophic bacteria. Some plants emit methane and methanol from their leaves, and provide methylotrophs with habitats. Methanol-utilizing methylotrophs in the genus Methylobacterium are abundant in the phyllosphere and have the ability to promote the growth of some plants. Methanotrophs also inhabit the phyllosphere, and methanotrophs with high methane oxidation activities have been found on aquatic plants. Both plant and environmental factors are involved in shaping the methylotroph community on plants. Methanotrophic activity can be enhanced by heterotrophic bacteria that provide growth factors (e.g., cobalamin). Information regarding the biological interaction of methylotrophs with other organisms will facilitate a better understanding of the carbon cycle that is driven by methylotrophs. Full article
(This article belongs to the Special Issue Microbial C1 Metabolism)

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