Phylogenetic, microbiological, and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor

Abstract

Phylogenetic, microbiological, and comparative genomic analyses were used to examine the diversity among members of the genus Caldicellulosiruptor, with an eye toward the capacity of these extremely thermophilic bacteria to degrade the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii, C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA gene phylogeny and cross-species DNA-DNA hybridization to a whole-genome C. saccharolyticus oligonucleotide microarray, revealing that C. saccharolyticus was the most divergent within this group. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid-pretreated switchgrass, only C. saccharolyticus, C. bescii, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman no. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that the cellulolytic species also had diverse secretome fingerprints. The C. saccharolyticus secretome contained a prominent S-layer protein that appears in the cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interactions. Growth physiology also correlated with glycoside hydrolase (GH) and carbohydrate-binding module (CBM) inventories for the seven bacteria, as deduced from draft genome sequence information. These inventories indicated that the absence of a single GH and CBM family was responsible for diminished cellulolytic capacity. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and this argues for continued efforts to isolate new members from high-temperature terrestrial biotopes.

FIG. 1.

Neighbor-joining 16S rRNA phylogenetic tree of Caldicellulosiruptor species and members from related genera. The scale represents 0.02 substitution per nucleotide position. Bootstrap values are based on 1,000 replicates. T, type strain.

FIG. 2.

Log₂ ratios for genomic DNA based on M-CGH analysis of tested Caldicellulosiruptor species versus C. saccharolyticus. (A) C. bescii; (B) C. hydrothermalis; (C) C. kristjanssonii; (D) C. kronotskyensis; (E) C. lactoaceticus; (F) C. owensensis. Log₂ ratios indicate the fold hybridization differences between the tester and reference samples, converted to a log₂ format. r is the corresponding Pearson correlation coefficient, quantifying the linear relationship between LSM hybridization levels of each Caldicellulosiruptor species tested versus C. saccharolyticus.

FIG. 3.

Comparisons of growth of Caldicellulosiruptor spp. on selected substrates. All substrates were present at 5 g/liter in modified DSMZ 640 medium except for yeast extract, which was present at 1 g/liter in modified DSMZ 640 medium. The species order was ranked with respect to growth levels on Avicel. Species abbreviations: Cbes, C. bescii; Chyd, C. hydrothermalis; Ckri, C. kristjanssonii; Ckro, C. kronotskyensis; Clac, C. lactoaceticus; Cowe, C. owensensis; Csac, C. saccharolyticus.

FIG. 4.

2D SDS-PAGE gels of secretomes from Caldicellulosiruptor species. (A) Avicel induced, with 100 μg protein loaded; (B) Avicel induced, 150 μg protein loaded and silver stained; (C) xylose induced, 100 μg loaded; (D) filter paper deconstruction cultures. Species abbreviations: Cbes, C. bescii; Chyd, C. hydrothermalis; Ckri, C. kristjanssonii; Ckro, C. kronotskyensis; Clac, C. lactoaceticus; Cowe, C. owensensis; Csac, C. saccharolyticus. N/D, not determined.