Comparative Genomic Analysis of a Methylorubrum rhodesianum MB200 Isolated from Biogas Digesters Provided New Insights into the Carbon Metabolism of Methylotrophic Bacteria

Abstract

Methylotrophic bacteria are widely distributed in nature and can be applied in bioconversion because of their ability to use one-carbon source. The aim of this study was to investigate the mechanism underlying utilization of high methanol content and other carbon sources by Methylorubrum rhodesianum strain MB200 via comparative genomics and analysis of carbon metabolism pathway. The genomic analysis revealed that the strain MB200 had a genome size of 5.7 Mb and two plasmids. Its genome was presented and compared with that of the 25 fully sequenced strains of Methylobacterium genus. Comparative genomics revealed that the Methylorubrum strains had closer collinearity, more shared orthogroups, and more conservative MDH cluster. The transcriptome analysis of the strain MB200 in the presence of various carbon sources revealed that a battery of genes was involved in the methanol metabolism. These genes are involved in the following functions: carbon fixation, electron transfer chain, ATP energy release, and resistance to oxidation. Particularly, the central carbon metabolism pathway of the strain MB200 was reconstructed to reflect the possible reality of the carbon metabolism, including ethanol metabolism. Partial propionate metabolism involved in ethyl malonyl-CoA (EMC) pathway might help to relieve the restriction of the serine cycle. In addition, the glycine cleavage system (GCS) was observed to participate in the central carbon metabolism pathway. The study revealed the coordination of several metabolic pathways, where various carbon sources could induce associated metabolic pathways. To the best of our knowledge, this is the first study providing a more comprehensive understanding of the central carbon metabolism in Methylorubrum. This study provided a reference for potential synthetic and industrial applications of this genus and its use as chassis cells.

Keywords: Methylorubrum rhodesianum MB200; carbon metabolism; comparative genomics; methanol.

Figure 1

Growth profile of the strain MB200 and strain AM1. (A) The cells were grown on methanol (125, 250, and 375 mM) as the sole carbon and energy source. (B) Growth curves and methanol (250 mM) consumption curves in the strain MB200. (C) The cells were grown on succinate (102, 153, and 104 mM) as the sole carbon and energy source. (D) The cells were grown on ethanol (86, 152, and 258 mM) as the sole carbon and energy source. (E) The cells were grown on formaldehyde (0.21, 0.42, and 0.84 mM) as the sole carbon and energy source. (F) Growth profiles of the strain MB200 cultured for RNA sequencing in the presence of six carbon sources. Data are represented as mean ± standard deviation calculated from three biological replicates.

Figure 2

Graphical map of the genome (A) and two plasmids (B,C) of the strain MB200. Successive circles from the center to outside: 1, GC content; 2, GC skew (positive in pink and negative in purple); 3, rRNA in green bars and tRNA in yellow bars; 4, all genes colored by functional categories according COG classification, listed in the lower left; 5, modification of bases (positive in red and negative in blue); 6, restrictive modification of system-related enzymes; and 7, scale marks of genomes. Plasmids are not shown to scale.

Figure 3

Comparative genomic study of 26 Methylobacterium strains with complete sequence. (A) Analysis of orthogroups of the 26 Methylobacterium strains. The 1656 core orthogroups shared by all strains are indicated in the center (red), and the unique orthogroups are indicated on each petal. (B) Correlation analysis of the orthogroups among 26 strains. The more the occurrence of same orthogroups, the higher the p-value. Phylogenetic tree on the right shows the evolutionary relationship among the 26 Methylobacterium strains.

Figure 4

Transcriptome analysis of M. rhodesianum strain MB200. (A) The differentially expressed genes under other carbon sources relative to those under succinate (SA). For each carbon source, up- and downregulated genes are given in red and blue bars, respectively. (B) Venn diagram representing the core and specific enrichment of all upregulated genes under the 5 carbon sources relative to SA. (C) Venn diagram representing the core and specific enrichment of all downregulated genes under the 5 carbon sources relative to SA. (D) The degree of GO enrichment in the common upregulated genes was marked; the bubble chart shows the top 20 features. (E) The degree of KEGG enrichment of the core downregulated genes was marked. The bubble chart indicates the top 20 features. The closer the p-value is to zero, the greater the rich factor is. The higher the gene number, the more significant the enrichment.

Figure 5

The central carbon metabolic pathways in M. rhodesianum strain MB200 and proposed EMC pathway containing partial propionate metabolic pathway based on our customized genomic and transcriptomic analyses. Genes encoding enzymes for each step are listed in Table S1. The common up– (red) and down– (blue) regulated genes are marked. SA: succinate; MA: methanol; FM: formaldehyde; MA_FM: methanol and formaldehyde; SA_FM: succinate and formaldehyde; EA: ethanol.

Figure 6

(A) The distribution of all genes in GSH on 26 strains and gene copies. The ordinate is the strain name: yellow square represents gfa gene, orange square represents hgd gene, orange circle represents fdh gene, and blue square represents fgh gene. The highlighted part in blue is the strain with complete GSH pathway, and the rest is the strain without fgh gene. (B) The FPKM values of genes from GSH pathway in strain MB200 grown under 6 different carbon sources. The abscissa is gene and number. Different blocks represent different carbon sources. (C) Phylogenetic tree of fgh gene in strains highlighted in blue.