Comparative analysis of the Geobacillus hemicellulose utilization locus reveals a highly variable target for improved hemicellulolysis

Abstract

Background: Members of the thermophilic genus Geobacillus can grow at high temperatures and produce a battery of thermostable hemicellulose hydrolytic enzymes, making them ideal candidates for the bioconversion of biomass to value-added products. To date the molecular determinants for hemicellulose degradation and utilization have only been identified and partially characterized in one strain, namely Geobacillus stearothermophilus T-6, where they are clustered in a single genetic locus.

Results: Using the G. stearothermophilus T-6 hemicellulose utilization locus as genetic marker, orthologous hemicellulose utilization (HUS) loci were identified in the complete and partial genomes of 17/24 Geobacillus strains. These HUS loci are localized on a common genomic island. Comparative analyses of these loci revealed extensive variability among the Geobacillus hemicellulose utilization systems, with only seven out of 41-68 proteins encoded on these loci conserved among the HUS+ strains. This translates into extensive differences in the hydrolytic enzymes, transport systems and metabolic pathways employed by Geobacillus spp. to degrade and utilize hemicellulose polymers.

Conclusions: The genetic variability among the Geobacillus HUS loci implies that they have variable capacities to degrade hemicellulose polymers, or that they may degrade distinct polymers, as are found in different plant species and tissues. The data from this study can serve as a basis for the genetic engineering of a Geobacillus strain(s) with an improved capacity to degrade and utilize hemicellulose.

Figure 1

Schematic diagram of the G. stearothermophilus T-6 hemicellulose utilization locus. Each arrow represents a gene in the locus, with genes encoding predicted transposons colored in black, while open reading frames interrupted by transposons are colored in red. Genes encoding glycosyl hydrolases are colored in blue. The G. stearothermophilus HUS locus was subdivided into thirteen gene clusters on the basis of their predicted function.

Figure 2

Comparative diagram of the Geobacillus hemicellulose loci. The aligned echD-npd islands of the twenty-four sequenced HUS⁺ and HUS^- Geobacillus strains and that of G. stearothermophilus T-6 are shown. The flanking echD and npd genes are colored in yellow, genes coding for transposons in black, and genes in which the reading frames are transposon-disrupted are in red. Those genes conserved among >70% of the HUS⁺ strains are colored in green, those conserved among >50% and <70% in light green, while those conserved among <50% of the HUS⁺ strains are colored in white.

Figure 3

Phylogeny of the sequenced Geobacillus strains showing the distribution of hemicellulose loci. A neighbour-joining phylogeny was constructed on the basis of the recN gene for the twenty-four sequenced Geobacillus strains as well as twelve Geobacillus spp. type strains. The presence or absence of HUS loci in the sequenced strains is indicated by green and red dots, respectively. The blue star next to G. stearothermophilus ATCC 12980^T indicates the predicted phylogenetic position of G. stearothermophilus T-6 for which a recN sequence is not available. The recN gene sequence of Bacillus subtilis 168 was used as outgroup. Bootstrap values (n = 1,000) are shown.

Figure 4

Phylogenetic comparison of the HUS ⁺ Geobacillus strains on the basis of RecN and XynDCEFG-XylAB. A neighbour-joining tree was constructed on the basis of the RecN amino acid sequences of the HUS⁺ Geobacillus strains as well as sixteen representatives of related genera in which orthologs of XynDCEFG-XylAB are present. Similarly a neighbour-joining tree was constructed based on alignment of the concatenated XynDCEFG-XylAB amino acid sequences. Desmospora sp. 8437 was used as outgroup and bootstrap values are shown (n = 1,000). The branches in red and in green, represent the phylogenetic positions of Geobacillus sp. C56-T2 and G. caldoxylolyticus CIC9, respectively, in both trees.