High-quality genome of a novel Thermosynechococcaceae species from Namibia and characterization of its protein expression patterns at elevated temperatures

Abstract

Thermophilic cyanobacteria thrive in extreme environments, making their thermoresistant enzymes valuable for industrial applications. Common habitats include hot springs, which act as evolutionary accelerators for speciation due to geographical isolation. The family Thermosynechococcaceae comprises thermophilic cyanobacteria known for their ability to thrive in high-temperature environments. These bacteria are notable for their photosynthetic capabilities, significantly contributing to primary production in extreme habitats. Members of Thermosynechococcaceae exhibit unique adaptations that allow them to perform photosynthesis efficiently at elevated temperatures, making them subjects of interest for studies on microbial ecology, evolution, and potential biotechnological applications. In this study, the genome of a thermophilic cyanobacterium, isolated from a hot spring near Okahandja in Namibia, was sequenced using a PacBio Sequel IIe long-read platform. Cultivations were performed at elevated temperatures of 40, 50, and 55°C, followed by proteome analyses based on the annotated genome. Phylogenetic investigations, informed by the 16S rRNA gene and aligned nucleotide identity (ANI), suggest that the novel cyanobacterium is a member of the family Thermosynechococcaceae. Furthermore, the new species was assigned to a separate branch, potentially representing a novel genus. Whole-genome alignments supported this finding, revealing few conserved regions and multiple genetic rearrangement events. Additionally, 129 proteins were identified as differentially expressed in a temperature-dependent manner. The results of this study broaden our understanding of cyanobacterial adaptation to extreme environments, providing a novel high-quality genome of Thermosynechococcaceae cyanobacterium sp. Okahandja and several promising candidate proteins for expression and characterization studies.

Keywords: Thermosynechococcaceae; cyanobacteria; genomics; proteomics; taxonomy; thermophilic.

Figure 1

Microscopic images of Thermosynechococcaceae cyanobacterium sp. Okahandja using an electron microscope. (a) Secondary electron image using a low‐energy ionization (LEI) detector, (b) STEM image; (c) brightfield image using a light microscope.

Figure 2

Fluorescence microscopic images of Thermosynechococcaceae cyanobacterium sp. Okahandja, grown at (a) 40°C, (b) 50°C, and (c) 55°C. The autofluorescence of chlorophyll results in red emissions; magnification ×1000.

Figure 3

BUSCO 5.4.6 results for Thermosynechococcaceae cyanobacterium sp. Okahandja (this study) and the reference genome of P. lividus PCC 6715. Genome completeness assessment based on near‐universal single‐copy ortholog genes from the cyanobacteria lineage data set odb_10, comprising 141 cyanobacterial genomes. According to BUSCO 5.4.6 (Manni et al., 2021), the genome of this study exhibits less fragmented and overall more orthologous genes compared to the reference genome of P. lividus PCC 6715 (RefSeq NZ_CP018092.1). Both genomes were obtained from PacBio long‐read sequencing with genome assemblies resulting in a single, circular chromosome, respectively.

Figure 4

The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model (Nei & Kumar, 2000). Maximum Likelihood bootstrap values are given at the nodes together with Bayesian posterior probabilities. The tree with the highest log likelihood (−8988.74) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor‐Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 0.2925}). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.30% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 48 nucleotide sequences. There were a total of 2071 positions in the final data set. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Figure 5

Phylogenetic tree of representative members of the family Thermosynechococcaceae based on orthologs as identified by OrthoFinder 2.5.4 (Emms & Kelly, 2015). Throughout all investigated genomes, 451 common orthogroups encompassing 270 single‐copy genes could be identified. Between the depicted Thermosynechococcus and Parathermosynechococcus genomes, 1476 shared orthogroups were detected. Gene sequence searching was performed with DIAMOND 2.0.15 (Buchfink et al., 2014), unrooted gene trees were then inferred with DendroBLAST (Kelly & Maini, 2013), and unrooted species trees were inferred with STAG (Emms & Kelly, 2018). The latter were then rooted with STRIDE (Emms & Kelly, 2017) for ortholog inference by OrthoFinder. All tools were utilized as implemented in the OrthoFinder (Emms & Kelly, 2015) pipeline on the public galaxy.eu server of the Galaxy web platform (Afgan et al., 2016). Finally, the phylogenetic species tree was visualized on iTOL (Letunic & Bork, 2024) and modified with Inkscape.

Figure 6

Phylogenetic classification of Thermosynechococcaceae cyanobacterium sp. Okahandja (this study) genome‐encoded proteins on the basis of the database of Clusters of Orthologous Groups of proteins (COGs). Each COG includes proteins that are inferred to be orthologs from at least three lineages. Out of the 2,162 total proteins, 575 could not be assigned a function, resulting in an annotation rate of 73.4%. COGs were obtained with eggNOG‐mapper (Kelly & Maini, 2013).

Figure 7

Comparative circular genome plot of Thermosynechococcaceae cyanobacterium sp. Okahandja (this study) and the reference strain Parathermosynechococcus lividus PCC 6715. Depicted from the outermost to the innermost circle are: (Knoll, 2008) the singular, circular contigs with ticks indicating the genome lengths and gray bands, which represent inactive phage regions, as predicted with PHASTER, (Ward et al., 2012) Genome‐encoded gene labels, (Galtier & Lobry, 1997) GC‐skews, representing increased G/C nucleotide (blue) or increased A/T nucleotide (orange) abundance compared to the genome‐wide average, (Thompson & Eisenberg, 1999) plus strand‐encoded genes, (Van Noort et al., 2013) minus strand‐encoded genes, and (Sabath et al., 2013) locally collinear blocks (LCBs) derived from whole‐genome alignment with progressiveMauve, indicating conserved regions with a LCB length of above 4000 kb.

Figure 8

Biosynthetic gene clusters (BCGs) of Thermosynechococcaceae cyanobacterium sp. Okahandja (this study) predicted with antiSMASH (Blin et al., 2023). Three BCGs, all of which are involved in terpene biosynthesis, could be identified and localized within the genome: (a) Cluster I spans over 20,977 nucleotides on the plus strand (bases 1,274,895–1,275,951) and includes the genes 1208–1232. The biosynthetic core gene 1219 is annotated as 6‐carboxytetrahydropterin synthase. (b) Cluster II spans over 19,823 nucleotides on the minus strand (bases 1,936,532–1,937,468) and includes the genes 1843–1864. The biosynthetic core gene 1853 is annotated as phytoene synthase. (c) Cluster III spans over 21,923 nucleotides on the plus strand (bases 2,793,297–2,795,220) and includes the genes 2687–2710. The biosynthetic core gene 2698 is annotated as squalene‐hopene cyclase.

Figure 9

Heatmaps of differentially expressed proteins in Thermosynechococcaceae cyanobacterium sp. Okahandja (this study) at 40°C, 50°C, or 55°C. For each temperature condition, nine technical samples were taken and evaluated. Detected expression levels were normalized with the 40°C sample; thus, all depicted fold‐change counts at 50 or 55°C refer to the respective expression level at 40°C.

Figure 10

Selected differential protein expression levels of Thermosynechococcaceae cyanobacterium sp. Okahandja at 55°C in relation to 40°C. Proteins involved in ion homeostasis or redox chemistry, metabolism, photosynthesis, and regulation show tendencies toward downregulation, while CRISPR, transport, or genetic replication‐related proteins exhibit both increased and decreased expression levels.

Figure A1

Whole‐genome alignments of (a) Parathermosynechococcus lividus PCC 6715 and (b) Thermosynechococcaceae cyanobacterium sp. Okahandja (this study). Alignments were performed with progressive Mauve (Koren et al., 2017), applying a Locally Collinear Block (LCB) weight of 4000 bp, and therefore only considering conserved regions with lengths above 4 kb, resulting in 212 LCBs. T. lividus PCC 6175 was set as reference (A, top); thus, all LCBs are depicted on its plus gene strand. Inverted genes are depicted on the minus strand (B, lower row) for the strain of this study. Corresponding LCBs are framed and colored identically, as well as connected by a line. Peaks within an LCB‐box inform about respective sequence homology, with white gaps illustrating a lack of homology.