Proteome and strain analysis of cyanobacterium Candidatus "Phormidium alkaliphilum" reveals traits for success in biotechnology

Abstract

Cyanobacteria encompass a diverse group of photoautotrophic bacteria with important roles in nature and biotechnology. Here we characterized Candidatus "Phormidium alkaliphilum," an abundant member in alkaline soda lake microbial communities globally. The complete, circular whole-genome sequence of Ca. "P. alkaliphilum" was obtained using combined Nanopore and Illumina sequencing of a Ca. "P. alkaliphilum" consortium. Strain-level diversity of Ca. "P. alkaliphilum" was shown to contribute to photobioreactor robustness under different operational conditions. Comparative genomics of closely related species showed that adaptation to high pH was not attributed to specific genes. Proteomics at high and low pH showed only minimal changes in gene expression, but higher productivity in high pH. Diverse photosystem antennae proteins, and high-affinity terminal oxidase, compared with other soda lake cyanobacteria, appear to contribute to the success of Ca. "P. alkaliphilum" in photobioreactors and biotechnology applications.

Keywords: Cyanobacteria; Genomics; Microbial biotechnology; Proteomics.

Graphical abstract

Figure 1

Light microscopic images of Ca. P. alkaliphilum (A) Differential interference contrast microscopy at 1,000× magnification. (B) Bright-field microscopy at 400× magnification. The images were taken from stirred cultures grown at pH (10.4–11.2) and alkalinity (0.5 mol/L) with 1 mM NH₄Cl, 3 mM NaNO₃ as nitrogen source.

Figure 2

Genome assembly and structure (A) GC skew of the Ca. P. alkaliphilum genome. (B) GC skew of a Wenzhouxiangella genome, sequenced and assembled in the same laboratory with the same methods. The insets in (A and B) show the nanopore read length distribution and the percentage of each genome occupied by repetitive elements. (C) Genome synteny between Ca. P. alkaliphilum and Microcoleus sp. IPPAS B-353.

Figure 3

Maximum likelihood phylogenetic tree based on rpoABC amino acid sequences Organism names are according to the NCBI taxonomy database. Taxa names in blue and orange are associated with Metagenome Assembled Genomes (MAGs) obtained from Cariboo Plateau and Kulunda Steppe soda lakes, respectively. Two subclades of Phormidium and Nodosilinea are shown in teal boxes labeled as C5 and C1, consistent with Zorz et al. (2019). Lifestyle and habitat information is encoded to the right of the taxa. Nodes are color coded based on the % of bootstrap support. Melainabacteria species were used as outgroup. Table S4 contains all genomes with full NCBI and GTDB names.

Figure 4

Maximum likelihood phylogenetic tree Based on a concatenated alignment of 125 conserved marker genes used by the Genome Taxonomy Database (GTDB). The tree shows members with >84% average nucleotide identity (ANI) to Ca. P. alkaliphilum. Organism names are according to the NCBI taxonomy database. Bootstrap support is 100% for every branch.

Figure 5

Presence of orthologous genes by functional category among Phormidium (C5) and Nodosilinea (C1) from different habitats Organism names are according to the NCBI taxonomy database. “C5 Alkaline” refers to Phormidium species collected from alkaline soda lakes. “C5 Neutral” refers to Phormidium species from marine and salt lake habitats. “C1 Alkaline” refers to Nodosilinea species collected from alkaline soda lakes. Table S2 contains full list of Ca. P. alkaliphilum genes with annotations and proteomics values.

Figure 6

Bubble plot comparing protein expression of Ca. P. alkaliphilum and its close relative Phormidium sp. GEM2.Bin31 (C5) from Cariboo soda lakes Ca. P. alkaliphilum protein expression is shown for four different conditions (low pH with NH₄⁺ or NO₃^-, high pH with NO₃^- or urea). Size of the bubble is the average of normalized spectral abundance factor (NSAF) from quadruplet samples across each condition normalized against the summed NSAF of ribosomal proteins, translation factors, and protein chaperones in the MAG's proteome. See also Tables S5 and S6.