Functional Prediction and Assignment of Methanobrevibacter ruminantium M1 Operome Using a Combined Bioinformatics Approach

Abstract

Methanobrevibacter ruminantium M1 (MRU) is a rod-shaped rumen methanogen with the ability to use H₂ and CO₂, and formate as substrates for methane formation in the ruminants. Enteric methane emitted from this organism can also be influential to the loss of dietary energy in ruminants and humans. To date, there is no successful technology to reduce methane due to a lack of knowledge on its molecular machinery and 73% conserved hypothetical proteins (HPs; operome) whose functions are still not ascertained perceptively. To address this issue, we have predicted and assigned a precise function to HPs and categorize them as metabolic enzymes, binding proteins, and transport proteins using a combined bioinformatics approach. The results of our study show that 257 (34%) HPs have well-defined functions and contributed essential roles in its growth physiology and host adaptation. The genome-neighborhood analysis identified 6 operon-like clusters such as hsp, TRAM, dsr, cbs and cas, which are responsible for protein folding, sudden heat-shock, host defense, and protection against the toxicities in the rumen. The functions predicted from MRU operome comprised of 96 metabolic enzymes with 17 metabolic subsystems, 31 transcriptional regulators, 23 transport, and 11 binding proteins. Functional annotation of its operome is thus more imperative to unravel the molecular and cellular machinery at the systems-level. The functional assignment of its operome would advance strategies to develop new anti-methanogenic targets to mitigate methane production. Hence, our approach provides new insight into the understanding of its growth physiology and lifestyle in the ruminants and also to reduce anthropogenic greenhouse gas emissions worldwide.

Keywords: hypothetical proteins; methane mitigation; methanobrevibacter; molecular machinery; protein function.

FIGURE 1

Experimental workflow of a combined bioinformatics approach employed for functional annotation of operome from MRU.

FIGURE 2

Functional classification of MRU operome based on the protein fold (A), functional category (B), subpathway systems (C), and transmembrane topologies (D). AAB, Amino acid biosynthesis; AAT, Aminoacyl-tRNA charging metabolic clusters; ACD, Aromatic compounds degradation; C1UA, C1 Compounds utilization and assimilation; CHB, Carbohydrates biosynthesis; CSB, Cell structures biosynthesis; CPEB, Cofactors, prosthetic groups, electron carriers biosynthesis; FALB, Fatty acid and lipid biosynthesis; GPME, Generation of precursor metabolites and energy; INM, Inorganic nutrients metabolism; NNB, Nucleosides and nucleotides biosynthesis; PMR, Protein-modification reactions; RR, RNA-reactions; SMD, Secondary metabolites degradation; tRR, tRNA reactions; OTR, Other reactions.

FIGURE 3

Detection of gene clusters from MRU operome responsible for protein folding (A), cold adaptation (B), sulfite tolerance (C), binding with adenosyl groups (D), degradation of the labile antitoxin (E), and defense/virulence system (F). The green arrow represents a gene with a known function. hsp, Heat shock protein; TRAM, RNA modification protein; dsr, Dissimilatory sulfate reductase; cbs, cystathionine beta-synthase; cas, CRISPR-associated gene.

FIGURE 4

The proposed D-gluconate catabolic pathway in MRU was discovered from the functional annotation of its operome. D-Gluconate is imported into the cytoplasm by the predicted gluconate transporter (gntP) gene. It can be phosphorylated to D-gluconate-6-phosphate by D-gluconate kinase (gntK), which is then converted to D-ribulose-5-phosphate by the catalytic action of NAD⁺-dependent phosphogluconate dehydrogenase (gntZ). D-Ribulose-5-phosphate is next oxidized to hexulose-6-phosphate by 3-hexulose phosphate synthase (hxlA) and converted into β-D-fructofuranose 6-phosphate with phospho-3-hexuloisomerase (phi1). The 6-phosphofructose 2-kinase phosphorylates β-D-fructofuranose 6-phosphate into β-D-fructose 2, 6-bisphosphate, which then interconverted from D-fructose-6-phosphate to β-D-fructofuranose 6-phosphate by fructose-2, 6-bisphosphate 2-phosphatase. In an alternative way, β—D-fructofuranose 6-phosphate is phosphorylated to D-glucopyranose 6-phosphate by 6-phosphofructo-2-kinase. Glucopyranose 6-phosphate is converted to 1D-myo-inositol 3-monophosphate by D-glucose 6-phosphate cycloaldolase (ino1) and reduced to myo-inositol by inositol-phosphate phosphatase (suhB).