Characterization of a novel thermophilic cyanobacterium within Trichocoleusaceae, Trichothermofontia sichuanensis gen. et sp. nov., and its CO2-concentrating mechanism

Abstract

Thermophiles from extreme thermal environments have shown tremendous potential regarding ecological and biotechnological applications. Nevertheless, thermophilic cyanobacteria remain largely untapped and are rarely characterized. Herein, a polyphasic approach was used to characterize a thermophilic strain, PKUAC-SCTB231 (hereafter B231), isolated from a hot spring (pH 6.62, 55.5°C) in Zhonggu village, China. The analyses of 16S rRNA phylogeny, secondary structures of 16S-23S ITS and morphology strongly supported strain B231 as a novel genus within Trichocoleusaceae. Phylogenomic inference and three genome-based indices further verified the genus delineation. Based on the botanical code, the isolate is herein delineated as Trichothermofontia sichuanensis gen. et sp. nov., a genus closely related to a validly described genus Trichocoleus. In addition, our results suggest that Pinocchia currently classified to belong to the family Leptolyngbyaceae may require revision and assignment to the family Trichocoleusaceae. Furthermore, the complete genome of Trichothermofontia B231 facilitated the elucidation of the genetic basis regarding genes related to its carbon-concentrating mechanism (CCM). The strain belongs to β-cyanobacteria according to its β-carboxysome shell protein and 1B form of Ribulose bisphosphate Carboxylase-Oxygenase (RubisCO). Compared to other thermophilic strains, strain B231contains a relatively low diversity of bicarbonate transporters (only BicA for HCO₃^- transport) but a higher abundance of different types of carbonic anhydrase (CA), β-CA (ccaA) and γ-CA (ccmM). The BCT1 transporter consistently possessed by freshwater cyanobacteria was absent in strain B231. Similar situation was occasionally observed in freshwater thermal Thermoleptolyngbya and Thermosynechococcus strains. Moreover, strain B231 shows a similar composition of carboxysome shell proteins (ccmK1-4, ccmL, -M, -N, -O, and -P) to mesophilic cyanobacteria, the diversity of which was higher than many thermophilic strains lacking at least one of the four ccmK genes. The genomic distribution of CCM-related genes suggests that the expression of some components is regulated as an operon and others in an independently controlled satellite locus. The current study also offers fundamental information for future taxogenomics, ecogenomics and geogenomic studies on distribution and significance of thermophilic cyanobacteria in the global ecosystem.

Keywords: 16S rRNA; 16S-23S ITS; CO2-concentrating mechanism; Leptolyngbyaceae; Pinocchia; Trichocoleusaceae; Trichothermofontia; thermophilic cyanobacterium.

Figure 1

Bayesian inference of 16S rRNA gene sequences representing 98 cyanobacterial strains. Collapsed genera are indicated by black polygons, with a length corresponding to the distance from the most basal sequence to the most diverged sequence of the genus. The complete phylogram refers to Supplementary Figure 1. Posterior probabilities (%) are given above the nodes.

Figure 2

ML phylogenomic inference of concatenated protein alignment of 647 single-copy genes shared by all genomes. Strain no. in bold represent the strain identified in this study. Bootstrap values are indicated at nodes. Scale bar = 10% substitutions per site.

Figure 3

ML phylogenetic inference of RubisCO large subunit protein sequences. The thermophilic cyanobacterium investigated in this study and previously reported thermophiles are indicated in bold. The accession numbers underscored refer to the gene IDs in Supplementary Table 1 or the reference (Tang et al., 2022d).

Figure 4

Molecular components of CCM for Trichothermofontia sichuanensis B231. 2PGL, 2-phosphoglycolate; PGA, phosphoglyceric acid. Three-dimensional structures of proteins visualized using related structures of 2YBV, 6OWF, 5SWC, 5BS1, 6KI1. Figure created with BioRender.com.

Figure 5

ML phylogenetic inference of protein sequences of ccmK1/2 (A), ccmK3 (B), and ccmK4 (C). The thermophilic cyanobacterium investigated in this study is indicated in bold. The accession numbers underscored refer to the gene IDs in Supplementary Table 1 or the reference (Tang et al., 2022d).

Figure 6

Genomic organization of CCM-related genes for Trichothermofontia sichuanensis B231. Solid arrow boxes refer to genes and the direction of transcription.

Figure 7

Microscopic morphology of Trichothermofontia B231. (A) Light microscopy image of liquid-medium grown cultures; (B) light microscopy image of solid-medium grown cultures; (C) SEM image; (D–F) TEM images. Cb, carboxysome; Cg, cyanophycin granule; Ld, lipid droplets; Pg, polyphosphate granule; Sp, septum; Th, thylakoid membrane, Gv, gas vesicles. Magnifications were 1,000× (A,B); 5,000× (C,D); 20,000× (E,F).