Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter

Abstract

In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.

Keywords: CRISPR/cas; Methanothermobacter wolfeii; comparative analyses; fragment recruitment; genome mining; metabolic pathway reconstruction; metagenomics; metatranscriptomics; thermophilic biogas fermenter.

Figure 1

Circular genome plot and identified genomic islands (GIs) of the strain M. wolfeii SIV6. From the inner to the outer circle: Circle 1—GC skew; Circle 2—GC-content; Circle 3—predicted protein coding sequences transcribed anticlockwise colored according to the assigned COG (Clusters of Orthologous Group) categories; Circle 4—predicted protein coding sequences transcribed clockwise colored according to the assigned COG categories; Circle 5—genomic position in kb and identified genomic islands (GI 1–3).

Figure 2

Maximum-likelihood phylogenetic tree based on the 16S rRNA gene sequences of all known Methanothermobacter type species according to the ‘list of prokaryotic names with standing in nomenclature’ (LPSN [12]) in comparison with M. wolfeii SIV6 and one outgroup, namely Methanobacterium formicicum, generated with MEGA X [30].

Figure 3

ANI (average nucleotide identity) diagram of M. wolfeii SIV6 and the reference strains Methanothermobacter sp. CaT2, M. thermautotrophicus $\Delta$H and M. marburgensis str. Marburg based on calculations within the EDGAR [31] platform.

Figure 4

Functional classification of the M. wolfeii SIV6 genes and their corresponding TPM according to COG (Clusters of Orthologous Groups). Shown are the COG categories (X-axis), the number of genes belonging to each category (left Y-axis) colored according to the COG categories and the TPM belonging to each category (right Y-axis) colored in grey.

Figure 5

Comparison of a partly homologous CRISPR/cas system within the M. thermautotrophicus $\Delta$H (A), M. wolfeii SIV6 (B) and Methanothermobacter sp. CaT2 (C) genomes. Shown are the cas gene types in different colors and the CRISPR arrays with the number of repeats. CRISPR arrays were identified with CRISPRone [56].

Figure 6

Reconstructed hydrogenotrophic methanogenesis pathway of M. wolfeii SIV6. Colors indicate the percentage of TPM (transcripts per million) values belonging to these genes or operons. Subunits of enzymes are indicated by the capital letters next to the corresponding gene designation. Abbreviations are further explained in the text.