Methanogen Genomes

Advances in next generation DNA sequencing technologies has made genome sequencing a rapid and economical research method (Grada and Weinbrecht 2013). Methanogen genomic information is useful in investigating ruminant CH4 mitigation strategies, and one of the strengths of this approach is the ability to compare between genomes to identify which genes are conserved among particular types of methanogens and also which genes define the unique features of each microorganism. Difficulties in the cultivation and purification of rumen methanogens has hindered their widespread genome sequencing. However, the genomes of several cultivated rumen methanogens have been sequenced (Table 1.1.). The methanogen genome sequences currently completed range in size from 1.5 Mb to 4.5 Mb, with most between 1.5 Mb and 3 Mb. The genome of Methanosarcina barkeri CM1 is larger than other methanogen genomes. The larger genome size of members of the Methanosarcinales is correlated with their ability to form CH4 via hydrogenotrophic, acetoclastic and methylotrophic pathways; and are thus capable of more complex metabolic capacities (Maeder et al. 2006).

Methanobrevibacter ruminantium M1T was the first genome sequence of a rumen methanogen

(Leahy et al. 2010). Like other hydrogenotrophic methanogens, it has a small genome of 2.9 Mb with a %G+C of 33%, and it encodes each of the seven steps in the methanogenesis pathway using H2 + CO2. In hydrogentrophic methanogens, the last two steps of the methanogenesis pathway are strongly conserved, which includes the CoM methyltransferase and the methyl CoM reductase genes. Several methanogenesis marker genes, currently without any ascribed function, are also highly conserved (Gao and Gupta 2007; Liu and Whitman 2008). The M1T genome sequence explains why it needs acetate, 2-methylbutyrate and CoM to survive, as it lacks several genes involved in the use or synthesis of these compounds. Insights from the analysis of the M1T genome have led to the optimization of Mbb. ruminantium growth. The presence of two copies of nicotinamide adenine dinucleotide phosphate (NADP)-dependent alcohol dehydrogenase within the genome suggested possible

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alcohol utilisation and it was found that methanol and ethanol could stimulate, but not support growth. Subsequently methanol has been added to media for growth of M1T. The M1T genome sequence also lead to the discovery of a prophage sequence, designated φmru, and two non- ribosomal peptide synthase (NRPS) genes, which have never been found previously in archaea (Leahy et al. 2010). Several more rumen Methanobacteriales genomes have been sequenced, including AbM4, Mbb. boviskoreani JH1T (JH1T), SM9, YLM1, Mbb. wolinii SHT (SHT), Mbb. millerae ZA-10T _(ZA-10T_{), Methanosphaera sp. ISO3-F5, Methanobacterium formicicum} BRM9. Several other rumen methanogen genome sequencing projects are underway as indicated in Table 1.1, including Mbb. millerae HW02 and Methanobrevibacter sp. YE315 (YE315).

17 Table 1.1. Rumen methanogen genomes from Genomes OnLine Database (GOLD).

Strain Name GOLD ID Order Genome Size

(Mb)

GC (%)

ORFs Completion Reference

Methanosphaera sp. ISO3-F5 Methanobacteriales 2.8 31 2354 In Progress

Methanobrevibacter ruminantium M1T _{Gc01203 Methanobacteriales 2.9 33 2283 Complete Leahy et al., 2010}

Methanobrevibacter millerae SM9 Gi06992 Methanobacteriales 2.5 32 2321 Complete Kelly et al., 2016

Methanobrevibacter sp. YE286 Methanobacteriales Draft

Methanobacterium formicicum BRM9 Gi06999 Methanobacteriales 2.4 41 2352 Complete Kelly et al., 2014

Methanobacterium sp YE299 Methanobacteriales Draft

Methanobrevibacter olleyae YLM1 Gi07000 Methanobacteriales 2.2 27 1862 Draft Kelly et al., 2016

Methanobrevibacter sp. AbM4 Gi17672 Methanobacteriales 2 29 1730 Complete Leahy et al., 2013

Methanobrevibacter millerae HW02 Gi0074693 Methanobacteriales In Progress

Methanobrevibacter sp. YE315 N/A Methanobacteriales In Progress

Methanobrevibacter wolinii SHT _{Gi0054485 Methanobacteriales 2 24 1736 Draft}

Methanobrevibacter millerae DSM 16643 Gi0070837 Methanobacteriales 2.7 37 2467 Draft

Methanobrevibacter boviskoreani JH1T _{Gi39333 Methanobacteriales 2.1 29 1774 Draft Lee et al., 2013}

Thermoplasmatales archaeonBRNA1 Gi0052360,

Gc0052360

Methanomassiliicoc cales

1.5 58 1577 Complete

Methanosarcina sp. CM1 Gi06991 Methanosarcinales 4.5 39 3655 Complete Lambie et al., 2015

Genomes with N/A (not available) are genome is in early stage of sequencing and no GOLD ID were assigned. Genomes without a GOLD ID were acquired from Morgavi et al. (2012).

18 1.8. Research questions

Genome sequencing of rumen methanogens provides detailed knowledge of their genetic makeup and provides insights into their metabolism via reconstruction of their metabolic pathways from bioinformatic analyses. However, many different types of methanogens exist within the rumen, and ruminant CH4 mitigation strategies should target all rumen methanogens and not just a few species. Therefore, in order to assess the genomic diversity of methanogens in the rumen, multiple methanogen genomes selected from all available taxonomic levels (order, family, genera, species and strain), should be sequenced and compared. My PhD project seeks to contribute to this effort by acquiring detailed knowledge of rumen methanogens through genome sequencing and comparative bioinformatic analyses of their genomes.

At the beginning of my PhD project, only the Mbb. ruminantium M1T _{genome had been} sequenced and was publicly available (Leahy et al 2010) and my interests were in a particular group of recently discovered methanogens called Rumen Cluster C (RCC, now classified as members of the newly formed methanogen order, Methanomassiliicoccales) and in strain level variations in Methanobrevibacter genome sequences. A pure culture of RCC had never been isolated from a ruminant, therefore our knowledge of the rumen RCCs was very limited. The aim of my PhD work was to obtain a wider representation of the rumen methanogen genomes by sequencing the DNA of a CH4-producing enrichment culture called ISO4-H5, containing a methanogenic archaeon belonging to the RCC group. In addition, the Methanobrevibacter sp. D5 sequence was also examined as part of a Methanobrevibacter ‘pan-genome’ analysis to assess strain level genomic variation in these methanogen species.

My specific research questions are:

 What is the genome composition of the methanogenic archaeon RCC ISO4-H5,

and how does this differ from other sequenced methanogens?

 What is the metabolic scheme of RCC ISO4-H5, how does it grow in the rumen, and can these features be used to isolate a pure culture of this organism?

 What is the genome composition of Methanobrevibacter sp. D5, how does this differ from other sequenced Methanobrevibacter spp. strains and how do these differences allow co-existence of multiple Methanobrevibacter spp. in the rumen?

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