Complete Genome Sequence of Geobacillus thermodenitrificans T12, A Potential Host for Biotechnological Applications

Abstract

In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.

Fig. 1

Scanning electron micrographs of G. thermodenitrificans T12

Fig. 2

Acidification of MMy medium due to fermentation of various pectic substrates by G. thermodenitrificans strains T12 and DSM 465. Cultures were incubated at 65 °C for 24 h in a rotary shaker at 150 RPM. Xylan: beechwood xylan, Apple: apple pectin; Citrus: citrus pectin, RGI: rhamnogalacturonan I, Poly GA: poly galactic acid

Fig. 3

CRISPR-Cas Type II-C system architecture of G. thermodenitrificans T12. Rectangles: repeats; diamonds: spacers; dashed arrow: predicted promoter

Fig. 4

Neighbor-Joining tree of Cas9 protein sequences. The evolutionary history was inferred using the Neighbor-Joining method [29]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [17]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA6 [32]. All sequences found in Geobacillus spp. were included, as well as currently well-characterized sequences (Open circles: S. pyogenes, S. thermophiles, and A. naeslundii), as well as the closest non-thermophilic species Bacillus cereus (closed diamond). Non-Geobacillus strains capable of thermophilic growth have been included (closed squares). For all sequences, the percentage of amino acid sequence identity to T12 is indicated after the strain name between brackets