Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals

Abstract

Microbial strains with high substrate efficiency and excellent environmental tolerance are urgently needed for the production of platform bio-chemicals. Bacillus coagulans has these merits; however, little genetic information is available about this species. Here, we determined the genome sequences of five B. coagulans strains, and used a comparative genomic approach to reconstruct the central carbon metabolism of this species to explain their fermentation features. A novel xylose isomerase in the xylose utilization pathway was identified in these strains. Based on a genome-wide positive selection scan, the selection pressure on amino acid metabolism may have played a significant role in the thermal adaptation. We also researched the immune systems of B. coagulans strains, which provide them with acquired resistance to phages and mobile genetic elements. Our genomic analysis provides comprehensive insights into the genetic characteristics of B. coagulans and paves the way for improving and extending the uses of this species.

Figure 1. Circular representation of the Bacillus coagulans 2-6 chromosome.

The nine circles (from outside to inside) show the following: (i) the predicted ORFs on the plus and minus strands based on the COG database (colors were assigned according to the colors of the COG functional classes, which are listed on the bottom); (ii–vi) homology of B. coagulans 2-6 CDSs identified using BLAST in the strains XZL4, XZL9, DSM1, H-1 and 36D1 (red-to-blue were assigned according to the similarity of homologs); (vii) the genomic islands predicted by IslandViewer; (viii) the value of the GC skew (G − C/G + C); and (ix) the percentage of GC content with a 10-kb window size.

Figure 2. Maximum likelihood tree of Bacillus strains.

Genes that are conserved in all strains were aligned and concatenated for tree construction. The B. coagulans strains are highlighted in green. And strains from B. cereus group are highlighted in pink. A scale bar for the genetic distance is shown at the bottom.

Figure 3. Comparative analysis of xylose metabolism in the B. coagulans strains.

(A) The metabolic pathway for xylose fermentation in lactic acid bacteria. (B) Schematic gene maps of the xylose-utilization genes found in the B. coagulans strains examined in this study. Genes, that were not filled, are pseudogenes. xylR: DNA-binding transcriptional activator; xylAI: xylose isomerase Type I; xylB: xylulokinase; xylT: Xylose H⁺-symporter; xylAIII: xylose isomerase Type III; P1: quinolinate synthetase; P2: iron-containing alcohol dehydrogenase; P3: peptidase; P4: hypothetical protein; P5: PfkB domain-containing protein; P6: LacI family transcriptional regulator; P7: beta-ketoacyl reductase; P8: hypothetical protein; P9: breakpoint of contigs; P10: hypothetical protein; P11: hypothetical protein; P12: 6-phospho-3-hexuloisomerase; (C) Maximum likelihood tree of xylA genes. The genes marked by filled circles () are from B. coagulans strains and are homologs of xylA in B. subtilis. The genes marked with squares () are from B. coagulans strains, and are the novel xylose isomerases discovered in this study. (D) Maximum likelihood tree of xylB genes. The genes marked with filled circle () are from B. coagulans strains. The accession numbers of these genes downloaded from the NCBI database are shown in the parentheses. A scale bar for the genetic distance is shown at the bottom.

Figure 4. Multiple sequence alignment of d-lactate dehydrogenases.

d-Lactate dehydrogenases were aligned by using ClustalX. 2DLD and 1J4A are the accession numbers for d-Lactate dehydrogenase from Lactobacillus helveticus and Lactobacillus bulgaricus, respectively, in the PDB. Visualization of the multiple sequence alignment was performed by ESPript. Secondary structure elements were calculated based on the structure of 1J4A. The residues that are marked in green are the key active sites that may affect the function.

Figure 5. Overview of the CRISPR-Cas systems in B. coagulans strains 2-6 and 36D1.

(A). Genetic map of the CRISPR-Cas systems detected in two B. coagulans strains (2-6 and 36D1). Cas genes were detected around the CRISPR loci. Different colors show the different CRISPR loci and cas genes: cas1: light purple; cas2: red; cas3: dark red; cas4: purple; cas5: gold; cas6: pink; cas7: green; cas8: orange; CRISPR: blue; IS3: black; IS4: gray; XRE transcriptional regulator: green. (B). Overview of the five CRISPR loci in the two B. coagulans strains. The repeats are shown as gray rectangles and the spacers are shown as white diamonds. Spacers with similar sequences (>90% identity) in the studied genomes are shown as the same color.