Methylocystis sp. strain SC2

Strain SC2 can adapt to a wide range of methane concentrations. This is due to its ability to produce two isozymes of particulate methane monooxygenase (pMMO). These exhibit different methane oxidation kinetics and are encoded by pmoCAB1 (low-affinity pMMO1) and pmoCAB2 (high-affinity pMMO2). To gain insight into the underlying genetic information, the genome of strain SC2 was sequenced and found to comprise a 3.77 Mb circular chromosome and two large plasmids of 229.6 kb (pBSC2-1) and 143.5 kb (pBSC2-2) [1,2,3].

In addition to methane, nitrogen source and concentration are major determinants of methanotrophic activity. The application of ammonium fertilizers to various soils and sediments has been shown to inhibit methanotrophic activity. Long-term effects were observed particularly for atmospheric methane oxidation in various upland soils. Thus, besides methane, ammonia is a major factor determining methanotrophic activity in soil. The pMMO is evolutionarily related to the ammonia monooxygenase and methanotrophs, like ammonia oxidizers, are able to convert ammonia to hydroxylamine. Ammonia oxidizers can couple the oxidation of hydroxylamine with energy production and cellular growth, while methanotrophic bacteria cannot. Because hydroxylamine is a highly toxic intermediate, methanotrophs rely on the ability to remove it quickly by a detoxifying reaction.

We used Illumina RNA-Seq to identify strain SC2 genes that respond to standard (10 mM) and high (30 mM) NH4concentrations in the growth medium, compared to 10 mM NO3. The experimental treatments were named AMS (10 mM NH4+), NH4 (30 mM NH4+), and NMS (10 mM NO3). Strain SC2 cells were grown under high methane concentrations (20%, v/v) . High-quality non-rRNA reads were mapped against the concatenated sequence of the chromosome and the two plasmids of strain SC2; totaling 4,058 genes and referred to as the genome (Figure 1). RNA-Seq expression data were presented as RPKM (Reads Per Kilobase of CDS [coding sequence] model per Million mapped reads) values.

Figure 1. Whole-genome plot of Methylocystis sp. strain SC2. The circles represent from outside to inside: circle 1, DNA base position (bp), base 1 to 3,773,444 are for the chromosome, followed by plasmids pBSC2-1 and pBSC2-2; circle 2, protein-coding regions transcribed on the plus strand (clockwise); circle 3, protein-coding regions transcribed on the minus strand (anticlockwise); circle 4, tRNA genes; circle 5, G+C content plotted using a 10- kb window (sea green and magenta indicate values greater and less than the average G+C content, respectively); circle 6, GC skew ([G+C]/[G-C]) plotted using a 10-kb window (blue indicates values above average and red indicates values below average). The whole-genome plot was generated using DNAPlotter version 1.4 from Artemis 12.0, Sanger Institute (taken from Dam et al. 2013, DOI: 10.1371/journal.pone.0074767; but modified to include the plasmids pBSC2-1 and pBSC2-2 in the genome plot).

The majority of the strain SC2 genes showed no significant differential expression (Figure 2). Based on logfold change values of ≥ 2 or ≤ -2, a total of 198 genes were identified as differentially expressed between the different nitrogen conditions (NMS vs. AMS, NMS vs. NH4). Among these were pmoCAB2 and the genes (haoAB) encoding hydroxylamine oxidoreductase (HAO). In addition, a set of 67 genes was predicted to be differentially expressed in AMS/NH4, but not in NMS/AMS or NMS/NH4.

While the expression of pmoCAB1 was unaffected, pmoCAB2 was significantly downregulated (logfold changes of 5.0 to 6.0). Among nitrogen metabolism-related processes, genes involved in hydroxylamine detoxification (haoAB) were highly upregulated, while those for assimilatory nitrate/nitrite reduction, high-affinity ammonium uptake, and nitrogen regulatory protein PII were downregulated. The most likely explanation for the downregulation of pmoCAB2 is the ability of ammonia to competitively inhibit methane oxidation by pMMO. This would lead to the production of hydroxylamine. Competitive inhibition and poisoning of strain SC2 by hydroxylamine would be of ecophysiological relevance particularly in low-methane environments, where pMMO2 but not pMMO1 is functional [Baani and Liesack (2008) PNAS 105: 10203-10208]. The upregulation of haoABexpression with increasing ammonium concentration also fits well into such an ecophysiological perspective, assuming that the primary role of HAO is to detoxify hydroxylamine that has been produced by the pMMO isozymes.

Figure 2. Differential expression of genes in strain SC2 in response to different nitrogen conditions. The histogram indicates differential expression levels of the complete set of 4,058 genes identified in the genome of strain SC2. Logfold changes of RPKM values were compared for NMS/AMS (green), NMS/NH4 (red), and AMS/NH4 (blue). The inset shows the same graph with a y-axis zoomed in for the range 0 to 10 (taken from Dam et al. 2014, DOI: 10.1111/1462-2920.12367).

Thus, pmoCAB1 and pmoCAB2 in strain SC2 are differentially expressed but in response to two different key drivers of methanotrophic activity. While methane concentration is the factor controlling differential expression of pmoCAB1, it has no effect on pmoCAB2 expression (Baani and Liesack, 2008). Conversely, pmoCAB2 expression is affected by the nitrogen source and, in particular, the concentration of ammonia, but not the expression of pmoCAB1Methylocystis is a major component of the methanotrophic communities in upland and hydromorphic soils. Differential expression of pmoCAB2 thus provides some explanation as to why ammonium fertilizers have a strong inhibitory effect on atmospheric methane oxidation in such soils [4].

We also performed physiological experiments to verify that strain SC2 has diverse nitrogen metabolism capabilities. In correspondence to a full complement of 34 genes involved in N2fixation, strain SC2 was found to grow with atmospheric Nas the sole nitrogen source, preferably at low oxygen concentrations. Denitrification-mediated accumulation of 0.7 nmol30N2/hr/mg dry weight of cells under anoxic conditions was detected by tracer analysis. N2production was related to the activities of plasmid-borne nitric oxide and nitrous oxide reductases. The 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 [3].

References

[1] Dam, B., Kube, M., Dam, S., Reinhardt, R., Liesack, W. (2012) Methanotroph-specificrepABC-containing plasmids from Methylocystis sp. strain SC2. Appl. Environ. Microbiol. 78, 4373–4379.

[2] Dam, B., Dam, S., Kube, M., Reinhardt, R., Liesack, W. (2012) Complete genome sequence of Methylocystis sp. strain SC2, an aerobic methanotroph with high-affinity methane oxidation potential. J. Bacteriol. 21, 6008–6009.

[3] Dam, B., Dam, S., Blom, J., Liesack, W. (2013) Genome analysis coupled with physiological studies reveals a diverse nitrogen metabolism in Methylocystis sp. strain SC2. PLoS ONE 8(10): e74767. doi:10.1371/journal.pone.0074767.

[4] Dam, B., Dam, S., Kim, Y., Liesack, W. (2013) Ammonium induces differential expression of methane and nitrogen metabolism-related genes in Methylocystis sp. strain SC2. Environ. Microbiol., publication online ahead of print, doi: 10.1111/1462-2920.12367.

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