Verrucomicrobial Methanotrophs

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Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp Strain SC2

Dam B, Dam S, Blom J, Liesack W. Genome Analysis Coupled with Physiological Studies Reveals a Diverse Nitrogen Metabolism in Methylocystis sp Strain SC2. Plos One. 2013;8(10): e74767.Background: Methylocystis sp. strain SC2 can adapt to a wide range of methane concentrations. This is due to the presence of two isozymes of particulate methane monooxygenase exhibiting different methane oxidation kinetics. To gain insight into the underlying genetic information, its genome was sequenced and found to comprise a 3.77 Mb chromosome and two large plasmids. Principal Findings: We report important features of the strain SC2 genome. Its sequence is compared with those of seven other methanotroph genomes, comprising members of the Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. While the pan-genome of all eight methanotroph genomes totals 19,358 CDS, only 154 CDS are shared. The number of core genes increased with phylogenetic relatedness: 328 CDS for proteobacterial methanotrophs and 1,853 CDS for the three alphaproteobacterial Methylocystaceae members, Methylocystis sp. strain SC2 and strain Rockwell, and Methylosinus trichosporium OB3b. The comparative study was coupled with physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N-2 fixation, strain SC2 was found to grow with atmospheric N-2 as the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol N-30(2)/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N-2 production is related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases. Conclusions/Perspectives: Presence of a complete denitrification pathway in strain SC2, including the plasmid-encoded nosRZDFYX operon, is unique among known methanotrophs. However, the exact ecophysiological role of this pathway still needs to be elucidated. Detoxification of toxic nitrogen compounds and energy conservation under oxygen-limiting conditions are among the possible roles. Relevant features that may stimulate further research are, for example, absence of CRISPR/Cas systems in strain SC2, high number of iron acquisition systems in strain OB3b, and large number of transposases in strain Rockwell

Optimized cryopreservation of mixed microbial communities for conserved functionality and diversity.

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at -80 °C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications

Physiology and Biochemistry of the Aerobic Methanotrophs

Methanotrophs are a widely distributed group of aerobic bacteria that use methane as their source of carbon and energy. They play key roles in the global carbon cycle, including controlling anthropogenic and natural emissions of the greenhouse gas methane. Methanotrophs oxidize methane using the unique enzyme methane monooxygenase which exists in two structurally and biochemically distinct forms. One form, the membrane-associated or particulate methane monooxygenase (pMMO), is found in most known methanotrophs and is located in the cytoplasmic membrane. Another form, the soluble methane monooxygenase (sMMO), is found in some methanotrophs and is located in the cytoplasm. Both forms of MMO can co-oxidize a range of hydrocarbons and chlorinated pollutants and hence are interesting with respect to the biotechnological potential of methanotrophs. Methanol is further oxidized to formaldehyde, formate, and CO2, by specific methylotrophic enzymes, while biomass is built from formaldehyde, formate, CO2, or a combination thereof via three cyclic biochemical pathways: the ribulose monophosphate (RuMP) cycle, the serine pathway, and the Calvin-Benson-Bassham (CBB) cycle. The availability of genome sequences of methanotrophs enables postgenomic studies to investigate the regulation of methane oxidation in the laboratory and in the environment by natural methanotrophs and in laboratory or industrial conditions by platform organisms. Recent studies have included synthetic biology approaches and in future may incorporate the design of new pathways