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

Summary 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.


INTRODUCTION
Cyanobacteria are responsible for a large portion of carbon and nitrogen fixation on the Earth (García- Pichel et al., 2003;Partensky et al. 1999;Zehr et al., 2008). Substantial genetic diversity within cyanobacteria enables them to occupy a wide range of ecosystems, including extreme environments, such as deserts, saline lakes, glaciers, hot lakes, and alkaline soda lakes (Jungblut et al., 2005, Lopez-Cortes et al., 2001, Paerl et al., 2000Sanchez-Baracaldo et al., 2005, Taton et al., 2006, Ward et al., 1998. The ability of cyanobacteria to fix carbon dioxide (CO 2 ) through oxygenic photosynthesis, with minimal nutritional requirements, has led to their adoption for bioproduction of pharmaceuticals and food ingredients (Beal et al., 2018;Gronenberg et al., 2013). Although cyanobacteria-based biotechnologies hold much potential for the production of bioproducts, there remain several challenges for the efficient and cost-effective implementation of this technology at scale. The high cost of supplying cyanobacteria with CO 2 for growth is a major drawback to the economic feasibility of these technologies. At scale, CO 2 is typically supplied to production systems, such as open raceway ponds, in the form of sodium bicarbonate, up to 150 mmol/L (Vonshak and Richmond 1988). During growth, CO 2 assimilation increases pond pH and leads to depletion of bicarbonate. The resulting spent medium is discarded and replaced. Another drawback of current technologies is the need to seed the ponds with cyanobacterial monocultures. This increases costs because seed cultures need to be maintained under aseptic conditions. The monoculture additionally leaves the ponds susceptible to invasion by other microbes, such as protists or cyanophages, causing collapse of the culture, and can result in loss of the crop (Cauchie et al., 1995;Forehead & O'Kelly 2013;Oswald 1980). Alkaline soda lakes represent natural analogs of raceway ponds, with comparatively higher pH and carbonate concentration. Indeed, these conditions can be so extreme that CO 2 from the air spontaneously dissolves into the lake water. This process has driven planetary-scale carbon capture in Earth's geological past (Tutolo and Tosca 2018). Apparently, nature does not need a monoculture to realize robust and rapid growth. High productivity is enabled by the high concentration of dissolved bicarbonate. Carbonate (CO 3 2À cyanobacteria through the carbon concentrating mechanism (CCM), which provides a high local concentration of CO 2 for optimal carbon fixation by the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). This mechanism includes the biogenesis of carboxysome compartments within the cell where HCO 3 is converted to CO 2 by the enzyme carbonic anhydrase. The CCM also includes transporters for active import of HCO 3 across the cell membrane. Five different inorganic carbon uptake systems have been shown in cyanobacteria. Three are active HCO 3 transport systems: two Na + -dependent transport facilitators (BitA and SbtA) and one ATP-dependent transporter (BCT1 encoded by the cmpABCD operon). Two additional CO 2 uptake systems are based on modified NADPH dehydrogenase complexes (NDH-I 3 and NDH-I 4 ) (Omata et al., 1999;Price 2011;Price et al., 2007;Shibata et al. 2001Shibata et al. , 2002. The uptake systems generate a cytosolic pool of HCO 3 -, which allows for higher CO 2 concentration in the carboxysomes where (RuBisCO) carries out the initial steps of the Calvin-Benson-Bassham cycle for carbon fixation (Kerfeld and Melnicki 2016;Rae et al., 2013b). In soda lakes found on the Cariboo plateau in British Colombia, Canada, the CCM supports the growth of high-cell-density cyanobacterial mats (Brady et al., 2013). A core microbiome of <100 shared bacterial lineages was recently described for geographically distant alkaline soda lakes (Vavourakis et al., 2018;Zorz et al., 2019). This shared alkaline soda lake microbiome included nine cyanobacterial populations, each represented by multiple metagenome assembled genomes (MAGs). Two filamentous cyanobacteria from different lineages were highly abundant in soda lakes in British Colombia, one affiliated with Nodosilinea (NCBI accession GCA_007692715.1) and one with Phormidium (GCA_007693465.1) (Zorz et al., 2019).
Previously, we enriched a microbial consortium from alkaline soda lakes on the Cariboo Plateau (BC, Canada) in photobioreactors at high pH and alkalinity (Sharp et al., 2017). This consortium consisted of a cyanobacterium, which makes up >80% of the community. Heterotrophic bacteria and archaea made up the remainder. The consortium displayed rapid and robust growth. We also demonstrated that the spent medium (pH > 11) could be recharged by spontaneous capture of CO 2 from air while maintaining high and robust biomass productivity (15.2 G 1.0 g/m 2 /day) (Ataeian et al., 2019). These innovations could potentially reduce operational costs of cyanobacterial biotechnology.
Within cyanobacterial biotechnology applications, there is need for high-quality whole-genome sequences.
They enable the precise identification and characterization of strains acquired for regulatory and intellectual property applications. They support approval for use in food and pharmaceuticals and the assessment of biosafety. Many cyanobacteria produce toxins, and the potential for toxin production can be assessed if a complete genome is available. However, the high frequency of repetitive elements present within cyanobacterial genomes make them notoriously difficult to assemble even from pure culture (Mazel et al., 1990;Stucken et al., 2010), resulting in a dearth of complete, closed-circular cyanobacterial genomes available.
In the present study we combined Nanopore and Illumina sequencing to obtain the first complete, circular genome for the genus Phormidium so far. Using inStrain analysis we show evidence for evolution of the most abundant population as well as selection of different closely related strains in different conditions. We use comparative genomics to understand the ecological success of this species, both in alkaline soda lakes and in photobioreactors for biotechnology. We also provide experimental proteomes under different growth conditions, as well as morphological characterization.

Cultivation and morphology
In June 2017, microbial mats from four Cariboo Plateau (Canada, BC) soda lakes (Brady et al., 2013;Zorz et al., 2019) were sampled, mixed, and inoculated into planar photobioreactors with red cellophane filters (Sharp et al., 2017) ( Figure S1). After 3 months, the enrichment culture was transferred to and maintained in tubular photobioreactors (Ataeian et al., 2019) ( Figure S1). Between 5 and 7 months after transfer, the culture was used to study the effect of pH and nitrogen source on growth, as previously described (Ataeian et al., 2019). During that time, four metagenomes and four proteomes, with technical quadruplicates, were obtained. These were associated with four different conditions. The first two cultures were grown at an initial pH of 8.3 and received either ammonium or nitrate as the nitrogen source (low pH). The second two cultures were grown at an initial pH of 10.4 with either nitrate or urea as the nitrogen source (high pH). Unfortunately, despite very high sequencing depth of the cyanobacterial genome in the metagenomes, assembly of the cyanobacterial genome was challenging, resulting in a fragmented whole-genome sequence with hundreds of contigs. Downsampling of reads did not solve these assembly problems.  Table S1, only a single whole-genome sequence has been closed. This has been explained by a high frequency of repetitive sequences in these cyanobacterial genomes (Mazel et al., 1990;Stucken et al., 2010). Alternatively, because the target cyanobacterium was part of a microbial consortium and not available as a pure culture, presence of multiple, closely related strains may have compromised the assembly.
After 1 year in tubular bioreactors, the culture was transferred to stirred photobioreactors for ease of maintenance and sampling ( Figure S1). The stirred cultures consisted of illuminated 10-L glass bottles stirred with a magnetic stirrer. In these cultures, the consortium grew as loose flocs or aggregates. When the stirrer stopped, biomass spontaneously coagulated and amassed both at the top and the bottom of the bottle. Inspection of the aggregates with microscopy showed abundance of a filamentous cyanobacterium, with cells of 5 mm average width, appearing as cylindrical, unbranched filaments in green shades under bright-field illumination ( Figure 1). Observed morphology was consistent with the Phormidium previously enriched from the same soda lakes in planar photobioreactors (Sharp et al., 2017). In addition, filaments displayed motility by gliding along each other and the surface of the slide (Video S1).
Although culture methods were changed as described, the culture medium always remained the same and contained 0.5 mol/L combined bicarbonate and carbonate at a pH of 8.3-11.2. No culture ''crashes'' were ever observed, and a high growth rate was reported in both previous studies (Ataeian et al., 2019;Sharp et al., 2017).

Genomic sequencing and phylogenetic analysis
During summer 2020, 3 years after sampling the biomass from the soda lakes, DNA was extracted for parallel Nanopore and Illumina sequencing. Assembly and base-calling of the Nanopore reads yielded a circular whole-genome sequence associated with a Phormidium species. The whole-genome sequence was further polished and perfected with corresponding short reads generated by Illumina sequencing of DNA from the same biomass. Remaining assembly problems, manifesting themselves as regions with anomalous sequencing depth, were manually resolved. The complete, circular genome was 5,000,054 bp in size. Sequencing depth was $6003 for Nanopore reads and 803 for Illumina reads. For the whole-genome sequence, 4,261 protein coding genes were predicted. The coding density was 88%, fairly low, which was attributed to the prolific presence of repeat sequences. We found two rRNA operons, both containing 16S rRNA, 23S rRNA, 5S rRNA genes. The two operons were not identical, showing 99.12%, 99.65%, and 100% sequence identities respectively. No genes or gene clusters encoding production of any known toxin were detected in the genome.
A typical origin of replication (oriC) for the complete Phormidium genome was not found with Ori-Finder (Gao and Zhang 2008). A plot of the GC skew did not show the conventional symmetrical pattern with a peak at the midpoint of the genome, but rather a much lower skew without a smooth peak (Figure 2A). iScience Article As such the start of the genome was set at the dnaA gene. To determine if the lack of GC skew was the result of assembly artifacts caused by repeat sequences, we compared the Phormidium assembly to the assembly of a Gammaproteobacterium also sequenced in our laboratory using combined Nanopore and Illumina sequencing (Figures 2B;Sorokin et al., 2020). Even though the Nanopore sequencing depth for the Gammaproteobacterium was lower and repetitive elements were also abundant ( Figure 2B, inset), the GC skew showed clear trends with a clear peak and a clear origin of replication. To further investigate the fidelity of the assembly process, we compared the synteny (gene order) of the Phormidium with the closely related Microcoleus sp IPPAS B-353 (Kupriyanova et al., 2016). The two genomes shared 92.6% average nucleotide identity (ANI), and the quality of the Microcoleus genome was excellent, as it had only a single scaffold with four contigs. It appeared that gene order was preserved extremely well, with only four large-scale rearrangements setting the two species apart ( Figure 2C). Thus, the lack of canonical GC skew trends and oriC did not appear to be caused by faulty assembly or genome rearrangements by hyperactive mobile elements. Instead, it might result from a different mechanism of replication recently proposed for filamentous cyanobacteria (Ohbayashi et al., 2020;Watanabe 2020). Indeed, irregular GC skew profiles have been shown in several other cyanobacteria lacking oriC that likely initiate replication at multiple points within their genome (Nikolaou and Almirantis 2005;Ohbayashi et al., 2016;Worning et al., 2006).
To determine whether the genome underwent evolutionary changes during the 2.5 years between the previous tubular photobioreactor experiments (Ataeian et al., 2019) and the stirred photobioreactor Nanopore sequencing, we carried out analysis with inStrain (Olm et al., 2021). Using inStrain with the Nanoporesequenced Phormidium whole-genome sequence as a reference and reads from the four tubular photobioreactors and stirred photobioreactor metagenomes, we assessed the proportion of variant sites under each condition. Mapping results indicated that the same sequenced Phormidium was abundant in the four tubular and the stirred PBRs, recruiting between 42% (low-pH nitrate) and 90% (high-pH urea) of all metagenomic reads passing quality control (Table S3). The analysis revealed a low level of strain-level diversity present in the Phormidium population throughout all the conditions. A low number of variant sites was found ranging from 0.2 sites per kb (817 sites in total, high-pH urea) to 11 sites per kb (53,399 sites in total, low-pH nitrate) ( Figure S2, Table S3). Interestingly, more variant sites were shared between the two low-pH conditions than were found in the higher pH conditions ( Figure S2), indicating a higher level of variation in the Phormidium populations grown at low pH. iScience Article Over 85% of all variant sites across conditions occurred with <50% of the reads containing a variant base (single nucleotide variants in Table S3). These same low levels of variation were also found within the stirred bioreactor (0.4 variant sites per kb), showing that this natural variation persisted during multiple years of laboratory cultivation. Analysis of the abundance of unique k-mers present in the metagenome for each condition ( Figure S2 inset) provides insight into Phormidium populations under different conditions. Both high-pH experiments display a symmetrical peak at 600-1,0003 sequencing depth corresponding to a $4.7-Mb genome. This corresponds well to the size of the Phormidium whole-genome sequence, excluding repeat sequences (4.8 Mb). The low-pH ammonium condition displays the same peak with a shoulder and slightly larger estimated genome size, indicating the presence of a mixture of two strains, consistent with an intermediate number of reads mapped (71%) to the Phormidium whole-genome sequence. In the low-pH nitrate condition, two peaks are seen and the Phormidium genome could not be clearly recognized. This is consistent with a much lower percentage of reads mapped (only $50%) and considerable strain-level diversity detected by inStrain analysis in the low-pH nitrate condition.
To assess the possibility of strain evolution we looked at three areas in the genome where single-nucleotide substitutions (SNS, with R95% reads with variants) and consensus variants (with 50%-94% reads with variants) were more frequent ( Figure S2). The first area, from 754,780 bp to 765,901 bp in the genome, encompassed 60 SNSs across 12 predicted open reading frames (pha|00884-pha|00899, Table S2). The encoded gene products appeared to have regulatory functions. One of the genes appeared to be truncated and might be a pseudogene, which was no longer functional. Another encoded the iron uptake protein OprB.  Table S2) encoding a subunit of a urea ABC importer. For all SNS and consensus variant sites occurring in multiple conditions, the variant base was the same in all conditions. This would be consistent with the presence of different strains, not just random mutations. Furthermore, the higher frequency of variants within these regions, some of which have a probable pseudogene, suggests evolution by way of gene loss for genes no longer beneficial within the photobioreactors. These combined results support two processes at work underlying the observed diversity of the Phormidium population within the photobioreactors. One, a diversity of strains present emerges under different conditions and contributes to robustness with regard to changes in operating and environmental conditions. This is especially clear in the low-pH nitrate bioreactor, where the strain represented in the sequenced Phormidium whole genome appears to perform less well. Two, the Phormidium population is evolving to eliminate gene content that is no longer useful for life within the photobioreactors. In demonstrating how strain-level diversity within the cyanobacterial population offers robust responses to changing conditions and evolution toward the loss of non-beneficial genes, we demonstrate the value of maintaining strain-level diversity from natural environments within cyanobacteria-based biotechnologies.
Given that the enriched Phormidium originated from microbial mats in Cariboo soda lakes, we compared the whole-genome sequence to the MAGs previously obtained from these mats.  (Table S1). These close similarities indicated that the enriched, genome-sequenced Phormidium represents a cyanobacterial genus that is widespread and abundant in soda lakes worldwide.
Additional related cyanobacteria were identified through a BLASTp search of the RpoB protein (DNAdirected RNA polymerase subunit B) against the NCBI non-redundant protein sequence (nr) database.
The three most closely related species included Microcoleus sp IPPAS B-353 [GCF_009846485], from Kulunda soda lakes (Kupriyanova et al., 2016); Phormidium OSCR [GCA_001314905.1], from hypersaline, Hot Lake, Washington (Nelson et al., 2016); and Phormidium lacuna HE10JO [GCA_900,149,785.2], from North Sea rock pools (Nies et al., 2017). The whole-genome sequences of these organisms showed 93%, 86%, and 84% ANI to our newly sequenced genome, respectively. As these ANI numbers are less than the often used species threshold of 95% (Olm et al. 2021 iScience Article new species within the genus Phormidium. Owing to its ability to grow at high pH and alkalinity and increased relative abundance and productivity in high-pH experiments (Ataeian et al., 2019), we propose to provisionally name this species Candidatus ''Phormidium alkaliphilum.'' Isolation into pure culture will be required for this name to become officially recognized. Figure 3 shows the phylogenetic relationship of Ca. ''P. alkaliphilum'' with other cyanobacteria based on a concatenated alignment of RpoABC2 (Table S4). The tree also includes the cyanobacterial MAGs obtained from Cariboo mats and Kulunda Steppe sediments. Three species from Melainabacteria were used as the outgroup. These share a common ancestor with cyanobacteria but are not photosynthetic (Soo et al., 2017). The two most abundant cyanobacteria found in the Cariboo soda lakes, Phormidium (clade C5) and Nodosilinea (clade C1) (Zorz et al., 2019), are from quite distant phylogenetic lineages within the cyanobacteria (Shih et al., 2013). We compared the topology of our rpoABC tree with the GTDBtk reference tree as well as with a previously published tree (Shih et al., 2013) and found the same topology in all trees. According to iScience Article the Genome Taxonomy Database (GTDB), clade C5 including Ca. P. alkaliphilum is affiliated with the order Cyanobacteriales, family Geitlerinemaceae, and genus Phormidium. C1 is, counter-intuitively, affiliated with the order Phormidesmiales, family Phormidesmiaceae, and genus Nodosilinea. The historical naming of cyanobacteria, based on morphology, has unfortunately resulted in confusing taxonomic names.
To explore the evolutionary relationships of C5 Phormidium in more detail, a phylogenetic tree with higher resolution was constructed using the 125 GTDB conserved marker genes ( Figure 4). This phylogenetic tree showed that within C5, Phormidium from soda lakes form a separate evolutionary branch from cyanobacteria originating from marine environments and salt lakes. Ca. P. alkaliphilum clustered with other Phormidia from alkaline soda lake environments, including GEM2.Bin31, Microcoleus sp. IPPAS B-353, Cyanobacterium T3Sed10, and Geitlerinema sp. P-1104, at 96.7%, 92.6%, 88.6%, and 88.4% ANI, respectively. For comparison, the four Phormidium genomes from marine and inland salt lake environments displayed lower ANI to Ca. P. alkaliphilum at 84%-86%. Bacteria living in soda lakes have to adapt to sodium (bi)carbonate brines with high pH, whereas marine/salt lake bacteria live in pH-neutral habitats containing mainly sodium chloride (Jones et al., 1977). Comparing C5 genome content may show which adaptations contribute to the ecological success at high pH and high alkalinity.

Comparative genomics and orthologue analysis
To investigate the relationship between the soda lake and marine/salt lake Phormidium species in clade C5 we identified and analyzed sets of orthologous genes found in the five soda lake and four marine/salt lake Phormidium genomes (Tables S1 and S2).
Orthologue analysis revealed approximately 1,376 core genes shared between both groups. Shared genes included those required for DNA, RNA, and protein biosynthesis; homeostasis of the gram-negative cell envelope; and lipid biosynthesis. Genes encoding photosystem I (psaABCDEFIJGLMXK), photosystem II (psbADBCEFOHNPQUVZ), and cytochrome b6f (petADBCEJ) were also part of the core genome, as well as genes needed for production of chlorophyll a (chlELNB). Both groups have genes encoding superoxide dismutase, peroxiredoxin, glutaredoxin, peroxidase, and tocopherol for scavenging reactive oxygen   (Latifi et al., 2009). Genes for orange carotenoid protein, which is involved in photoprotection by dissipating excess energy, were also detected in both groups (Wilson et al., 2006). All genomes contained a complete set of genes for carbon fixation via the Calvin-Benson-Bassham cycle. Genes of the CCM, including carbonic anhydrases, bicarbonate transport system, CO 2 uptake systems, and genes required for carboxysome assembly (ccaA, bicA, ictB, NDH-I 3 , NDH-I 4 , and ccmKMNOL) were present in both groups ( Figure S3). The CCM is responsible for transport of bicarbonate into cells and providing a steady supply of carbon dioxide (CO 2 ) to the RuBisCO enzyme, to maximize the rate of CO 2 fixation into biomass (Kerfeld and Melnicki 2016;Price et al., 2007;Raven et al., 2008). Genes for dark carbon catabolism, including glycolysis, the citric acid cycle, the pentose phosphate pathway, as well as a respiratory electron transport chain (ETC) were observed in all genomes. All the genes required for pyruvate fermentation to lactate, acetate, ethanol, formate, and acetoin ( Figure S4) were shared, indicating the capability for energy generation under anoxic or low-oxygen conditions. Fermentation pathways may also support managing the overflow of metabolic intermediates as a result of high metabolic rates at high CO 2 availability in intense sunlight (Cano et al., 2018). Shared genes for production of osmotic solutes indicated the capability to produce glycerol, sucrose, trehalose, and glucosyl-glycerol ( Figure 5). These genes are used for maintaining cellular ionic and osmotic balance in response to environmental fluctuations in salt concentration. These osmotic solutes can also serve as substrates for energy storage and fermentation.
Genes required for a variety of nitrogen acquisition pathways were found in all genomes including nitrogen fixation, nitrate/nitrite assimilation, urea uptake and hydrolysis, ammonium transport and assimilation, and genes for transport of neutral, branched, and polar amino acids ( Figure 5). In the Cariboo Plateau soda lakes, ammonium and nitrate concentrations were very low (<23 mM ammonium and <0.16 mM nitrate) (Zorz et al., 2019). Genes for nitrogen fixation via nitrogenase and urea uptake were highly expressed in Cariboo soda lakes (Zorz et al., 2019). Cyanobacteria use different strategies to protect the oxygen-sensitive nitrogenase. One strategy is the physical separation of nitrogen fixation enzymes from oxygenic photosynthesis via the formation of heterocysts. Heterocysts are specialized cells dedicated to nitrogen fixation, protecting the nitrogenase from oxygen (Berman-Frank et al., 2003). Two genes required for initiation of heterocyst formation (hetR and ntcA) were found in all genomes, but other essential genes, including hetP, hetC, hetF, hetL, and patA were missing. Additionally, no visual evidence of heterocyst development was ever observed for Ca. P. alkaliphilum (Figure 1). It was suggested that hetR may be involved in other forms of regulation in nonheterocyst-forming filamentous bacteria  and ntcA is known to be involved in the regulation of nitrogen uptake as well as heterocyst formation (Herrero and Flores 2019). Therefore, these 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. iScience Article cyanobacteria may protect nitrogenase through a different mechanism, such as temporal separation of photosynthesis (day) and nitrogen fixation (night). Nitrogen fixation produces hydrogen (H 2 ) as a byproduct (Almon and Bö ger 1988;Bothe et al., 2010). To salvage this hydrogen, nitrogen-fixing cyanobacteria often possess hydrogenases. Genes for uptake hydrogenase (hypABCD) as well as bidirectional hydrogenase (hoxEUS, hndC) were found in all genomes. H 2 recycling is an important process for non-heterocystous cyanobacteria, as it can provide an anoxic environment for nitrogenase via the respiratory ''oxyhydrogen reaction'' (uptake and aerobic respiration of H 2 in the dark) (Bothe et al., 2010;Dixon 1972).
Genes required for formation and utilization of the storage molecules glycogen and cyanophycin were detected in both groups ( Figure 5). In cyanobacteria, glycogen is the major carbon reserve molecule for excess photosynthetic carbon. It is then used as a carbon and energy source in the dark, for survival and homeostasis (Cano et al., 2018;Lehmann and Wober 1976;Preiss 1984). Cyanophycin (multi-L-arginylpoly-L-aspartate) serves as both nitrogen and carbon storage. In non-heterocystous cyanobacteria, cyanophycin is synthesized at night when nitrogen fixation occurs and there is excess nitrogen available. During the day when photosynthesis resumes and nitrogen fixation is inhibited, cyanophycin is used as a nitrogen source (Finzi-Harta et al., 2009, Sherman et al., 1998. Genes for polyhydroxybutyrate biosynthesis were not present in any of the genomes. Surprisingly, C5 soda lake bacteria shared only eight orthologous genes not present in C5 marine/salt lake bacteria. Only 30 additional genes were present in all but one soda lake genome and missing in all but one C5 marine/salt lake genome. Apart from a single gene, a phytoene/squalene synthetase, none of these genes had a clear association with a known phenotype relevant to survival in alkaline conditions. Two toxin-antitoxin gene pairs were found specific to the C5 alkaliphiles. These may be involved in regulation of cell growth versus persistence, biofilm formation, and tolerance to antibiotics and radical oxygen species (Khomutovska et al., 2020;Marsan et al., 2017;Wen et al., 2014). Previous studies identified marker genes for adaptation to alkaline conditions. For example, multiple studies have implicated production of hopanoids, such as squalene, in survival under alkaline conditions by inhibiting cation leakage through the membrane bilayers (Haines 2001;Haub et al., 2002). Genomes of alkaliphilic but not neutrophilic gammaproteobacteria Wenzhouxiangella encode specific outer membrane porins, squalene biosynthesis, as well as the sodium-dependent NADH:ubiquinone oxidoreductase complex (Sorokin et al., 2020). Alkaliphilic Bacillus lentus synthesize a secondary acidic cell membrane consisting of peptidoglycan and teichuronopeptide to support the generation of the proton motive force in the hydroxide-rich alkaline environment (Aono et al., 1999). Genomes of Bacillus halodurans encode a multi-subunit Mrp Na + /H + antiporter for alkaline pH homeostasis (Hamamoto et al., 1994). Apart from the single phytoene/squalene synthetase gene, no gene involved in any conceivable form of adaptation was found to set the alkaliphilic Phormidia apart from their neutrophilic relatives. One explanation for lack of differences in gene content could be that the neutrophilic Phormidia are already innately adapted to high pH. These filamentous cyanobacteria are often found in close physical association, such as microbial mats in Cariboo soda lakes. In such arrangements, photosynthesis will quickly increase local pH, due to consumption of CO 2 . Fast-growing filamentous cyanobacteria would likely need mechanisms to manage this local pH increase even in neutral environments.
Lack of distinguishing gene content does not preclude evolutionary adaptation. The observation that alkaliphilic C5 Phormidia form a separate monophyletic clade (Figure 4) hints at the presence of some kind of adaptation. For example, variations of amino acid sequences and structural diversification in cell envelope proteins could result from differences between habitats (Panja et al., 2020). After all, without such adaptations, we would have expected the alkaliphilic species to be scattered phylogenetically among their neutrophilic relatives.

Photobioreactor selection of Candidatus ''Phormidium alkaliphilum''
Cultivation of the Cariboo soda lake mats in the photobioreactors at high pH and high alkalinity repeatedly enriched for a cyanobacterial consortium with high abundance of the same Phormidium species. Previous studies have reported the selection of related (>84% ANI) neutrophilic Phormidium species in photobioreactor environments (Cole et al., 2014;Nies et al., 2017). This suggests the capabilities of this genus to adapt to new conditions and outcompete other, naturally coexisting cyanobacteria in the laboratory or photobioreactor environments. In other words, the genus has potential in biotechnology. To investigate the genetic basis of this repeated selection we explored gene content specific to C5 Phormidium when ll OPEN ACCESS iScience 24, 103405, December 17, 2021 9 iScience Article compared with another, distantly related, abundant cyanobacteria from the same soda lakes, C1 Nodosilinea ( Figure 5 and Table S1).
A shared core genome of 892 genes was detected between C1 and C5 species and included genes involved in DNA, RNA, and protein biosynthesis as well as carbon and nitrogen metabolism. Genes for the production of the phycobilisome antenna complex differed between the Phormidium and Nodosilinea lineages. The phycobilisome complex contains a suite of pigment proteins to absorb light between 550 and 680 nm. The efficient fluorescence of this light energy from the phycobilisome to chlorophyll a of photosystem II, provides access to wavelengths of light otherwise inaccessible to chlorophyll (Ting et al., 2002). Although genes for biosynthesis of the phycobilisome protein allophycocyanin (maximum absorbance 650 nm) and phycocyanin (maximum absorbance 620 nm) were found in both lineages, C1 had at least three times more copies of allophycocyanin genes. C5, however, contained genes for two additional pigments: phycoerythrin (maximum absorbance at 495 and 560 nm) and phycoerythrocyanin (maximum absorbance at 575 nm) (Figure 4). Having additional pigments to harvest a wider spectrum of light could likely result in higher overall efficiency of photosynthesis and growth, which could provide selective advantage in a photobioreactor.
Additionally, differences in the genes involved in oxygen respiration were observed (Figure 4). Genes for aa3-type cytochrome c oxidoreductase (cox genes) existed in both groups. C5 species contained additional genes for the bd-type quinol oxidase complex. The high-affinity bd-type quinol oxidase is generally found only in bacteria that experience low-O 2 conditions (D'mello et al., 1996;Gong et al., 2018). In the photobioreactors, this feature would benefit Ca. P. alkaliphilum in competing for oxygen during dark cycles by outcompeting other species, while simultaneously enabling nitrogen fixation.
The ability to grow as a biofilm may also contribute to the enrichment of Ca. P. alkaliphilum in photobioreactors. Cyanobacterial biofilm formation has been attributed to type II secretion/type IV pilus assembly systems (Schatz et al., 2013), and these genes are present in all C5 Phormidium and C1 Nodosilinea species ( Figure 5 and Table S1). Interestingly, Ca. P. alkaliphilum contained three additional groups of proteins with potential roles in cell adhesion and biofilm formation. The first group consisted of nine von Willebrand factor type A (VWA)-domain-containing proteins (Konto-Ghiorghi et al., 2009;Kuchma et al., 2015;Ponting et al., 1999), present in all C5 species. The VWA domain in the pilus-associated adhesin (PilA) in Streptococcus agalactiae is required for adherence to epithelial cells and biofilm formation (Konto-Ghiorghi et al., 2009). The second group consisted of 17 proteins with RTX calcium binding domains. The RTX proteins are transported out of the cell in an unfolded state using ABC transporters. Later, by binding to calcium (Ca 2+ ions they form a folded, stable structure that could function in biofilm formation (Pé rez et al., 2010;Sá nchez-Magraner et al., 2007). Last, seven proteins with hemagglutination-like domains, also with a potential role in cell adhesion (Syed et al., 2009), were detected. Having a higher number of mechanisms to assist in cell adhesion and biofilm formation in C5 species could enable them to form microbial mats in their natural habitat and biofilms or flocs in photobioreactors. Aggregation provides, for example, effective self-shading and protection from direct sunlight, protection from predation by protists, and may enable nitrogen fixation by depletion of oxygen during dark cycles.
Comparison of genes involved in signal response mechanisms indicated that C5 bacteria were likely more responsive to their environment than C1 bacteria. Within the Ca. P. alkaliphilum genome, approximately 94 genes encoded for two-component signal transduction systems (Table S2). These genes consisted of sensor histidine kinases and response regulators. Genomes of C1 species encoded less than 40 of those genes. In this mechanism, the response regulator is phosphorylated by a histidine kinase in response to specific environmental stimuli, functioning as a molecular switch, activating downstream processes such as transcription, translation, or enzyme activity (Dikiy et al., Galperin 2010). Proteomic analysis showed that these genes were relevant to growth of C5 in bioreactors, as expression was demonstrated for 40 genes at high-pH compared with only 11 genes at low pH (Tables S5 and S6).
Coping with change in salinity is important in many natural habitats. Differences between the type of osmolytes used by C1 and C5 species were observed (Figure 4). Genes for synthesis of glucosylglycerol (Figure S5) was observed in both groups. Genes of sucrose biosynthesis ( Figure S5) were only detected in C5 species. Genes for two trehalose biosynthesis ( Figure S5) pathways (maltooligosyl-trehalose-synthase and trehalose synthase) were also only present in C5 species. In contrast, only C1 species had the genes for ll OPEN ACCESS 10 iScience 24, 103405, December 17, 2021 iScience Article maltooligosyl-trehalose-synthesis. Even though both groups had genes required for glycine betaine (GB) degradation ( Figure S5), C1 but not C5 species had genes for import of GB into the cell. As soda lakes are often shallow, their microbial inhabitants are impacted by desiccation and rehydration cycles. Survival in these ecosystems likely depends on effective responses to changes in salt concentration, for example, when brines get diluted during rainfall. It remains unclear whether the observed differences in osmolyte production also contribute to success in photobioreactors.
Ca. P. alkaliphilum proteome under different pH and N source and within the soda lakes To further explore the capacity of Ca. P. alkaliphilum to tolerate changing environments we carried out experiments within the tubular photobioreactors with different pH and N sources, namely, low pH (initial pH of 8.3) with either ammonia or nitrate and high pH (initial pH of 10.4) with either nitrate or urea. Analysis of the four tubular photobioreactors showed expression of 2,658 Ca. P. alkaliphilum genes under the four different pH and nitrogen conditions. The most abundant proteins were phycobilisome pigments, such as phycocyanin, making up $20% of the cyanobacterial proteome. Surprisingly, proteomes were very similar across the four different conditions. To identify proteins that differed significantly in expression between different conditions, Student's t test was applied and corrected for multiple testing with permutation-based false discovery rate (FDR) to all replicates. The pH significantly affected the expression of 121 genes (shown by log Student's t test p value >2, FDR of 0.05, Tables S5 and S6).
In low-pH bioreactors, where the bicarbonate concentration was higher, Ca. P. alkaliphilum allocated more resources toward increased expression of the Calvin cycle proteins and of the large chain and small chain of RuBisCO, to assimilate the abundant CO 2 (Figure 6, Tables S5 and S6). Correspondingly, increased expression of CcmK and CcmM, the structural components of the carboxysome (Badger and Price 2003), was seen, allowing for accommodation of higher amounts of RuBisCO (Rae et al., 2013a). Conversely, in high-pH bioreactors, where bicarbonate concentrations were lower, higher expression of the bicarbonate transporter (BicA) and the photosystem I reaction center subunit IV (PsaE), which powers bicarbonate transport (Sü ltemeyer et al., 1997), was observed. Additionally, expression of the CO 2 hydration protein (ChpX), also involved in CO 2 uptake and hydration (Price 2011), was increased. The increased expression of BicA, PsaE, and ChpX likely works to increase the concentration of cytosolic bicarbonate, relative to a lower external bicarbonate concentration ( Figure 6, Table S6). Additionally, the high pH led to a significant increase in the expression of photosynthetic ferredoxin-NADP reductase ( Figure 6, Tables S5 and S6). At the end of the photosynthetic ETC, ferredoxin-NADP reductase transfers the energized electrons from ferredoxin to NADPH (Chitnis 2001). The conversion of CO 2 to HCO 3 by ChpXY is driven by electron transport and proton translocation by dedicated NADPH dehydrogenase complexes (NDH-I 3 and NDH-I 4 ) (Badger and Price 2003;Maeda et al., 2002). This way, excess NADPH is used to retain CO 2 inside the cell by hydration to bicarbonate. A significant increase was also observed in expression of the ETC complexes including NdhFKH ( Figure 6, Tables S5 and S6). Higher nighttime energy demands for cytoplasmic pH homeostasis of Ca. P. alkaliphilum under high pH could be a possible explanation.
Expression of genes for nitrogen acquisition was dependent on the source of nitrogen. When provided with nitrate, expression of nitrate uptake and assimilation genes increased, regardless of the pH of the medium. A significant increase was observed for NrtA, the nitrate/nitrite binding protein involved in nitrate/nitrite uptake, and ferredoxin-nitrite reductase (NirA) converting nitrite to ammonium. When provided with urea, urea import proteins (Urt) as well as urease subunits were highly expressed ( Figure 6, Table S6). Nitrogenase genes were expressed in all experiments, despite the presence of fixed nitrogen in the form of ammonia, nitrate, or urea. The form of nitrogen source did not show any significant change in the expression of central carbon metabolism genes, as was previously discussed (Anderton et al., 2020). This observation could be due to different timing of sampling between these two studies, particularly as sample for this study were taken at the point of depletion of nitrogen in culture media.
Proteomes acquired for Ca. P. alkaliphilum in photobioreactors were compared with proteomes obtained of their close relatives living in soda lakes Phormidium sp. GEM2.Bin31 (C5). The relative gene expressions were very similar, with a few exceptions ( Figure 6, Tables S4 and S5). Phycocyanin and phycoerythrocyanin subunits were expressed 2 times more in the lakes, potentially enabling harvesting of more light on both the longer and shorter end of the spectrum. Orange carotenoid protein was expressed 10 times more in the lakes, potentially indicating a higher need for protection against photodamage. These differences might result from adaptation to growth on light-emitting diode lights instead of natural sunlight. Conversely,

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iScience 24, 103405, December 17, 2021 11 iScience Article allophycocyanin was expressed 5 times lower in the soda lakes GEM2.Bin31 and expression of phycoerythrin subunits, with maximum absorbance at 495 and 560 nm, was not detected in the lakes, potentially due to the light attenuation profile of lakes containing of high dissolved organic matter (Butturini et al., 2020;Croce and Van Amerongen 2014;Markager and Vincent 2000). CcmK was highly expressed in the lakes, contributing to the acquisition of bicarbonate. However, RuBisCO subunits showed higher expression in the photobioreactors compared with the lake (Figure 6, Table S6), possibly due to increased energy demands for homeostasis in the more dynamic lake environment, leaving less energy for carbon fixation.
Expression of nitrate uptake and assimilation genes could not be detected in the lakes, consistent with the absence of detectable nitrate in the lakes (Zorz et al., 2019). Urea transporters were detected in both lake and bioreactors; however, no expression for the urease subunits was detected in the lakes. Expression of nitrogenase was 10 times higher in the lakes, indicating the importance of dinitrogen as a nitrogen source for GEM2.Bin31 (Figure 6, Tables S5 and S6). The apparent capability of this group of cyanobacteria to allocate resources to the acquisition of a wide range of nitrogen sources could contribute to their survival and ecological success both in natural and engineered environments.

Conclusion
A whole-genome sequence was obtained for an alkaliphilic cyanobacterium affiliated with the genus Phormidium. This newly identified species was enriched from alkaline soda lake microbial mats in high-pH photobioreactors. Genome assembly challenges caused by prolific repeat sequences were overcome with Nanopore sequencing. This led to a closed, circular, near-perfect whole-genome sequence, the first of its kind for this genus. The species was provisionally named Candidatus ''Phormidium alkaliphilum.'' It iScience Article occurs in soda lakes worldwide and is the most abundant microbial species overall in Lake Goodenough, a soda lake in Canada. It also belongs to a clade of closely related Phormidia with potential for biotechnology, because of their rapid and robust growth. Evolutionary changes in the genome during 3 years of laboratory cultivation consisted of the elimination of genes that might not be useful in the photobioreactor environment. Selection of different strain variants contributed to photobioreactor performance under different operating conditions. Comparative genomics showed that these favorable assets could be explained by genes for harvesting sunlight across a wide portion of the solar spectrum, respiratory systems tuned to low-oxygen conditions, as well as an expanded set of genes involved in biofilm formation and response mechanisms.
We show that within the genus Phormidium, soda lake cyanobacteria form a separate clade from their relatives adapted to neutral pH environments. However, a lack of clear differences in gene content between alkaliphilic and neutral pH species suggested that both Phormidium lineages inherently have coping strategies for high-pH conditions. Consistently, comparison of proteomes of Ca. P. alkaliphilum grown at pH 9 and 11 showed only modest differences in gene expression in response to pH.

Limitations of study
Portions of this study rely on asynchronous datasets, such that the proteome and strain variation data were obtained from the tubular photobioreactors, and were not contemporaneous to the sample used for the long-read Nanopore assembled genome. As such, it is possible that the protein expression may be different under the stirred photo bioreactor conditions. However, given the consistency in expressed proteins under different tubular photobioreactor conditions (i.e., pH and nitrogen) this is unlikely. There is a difference in sequencing depth with short-read Illumina platform between the tubular photobioreactor, very deeply sequenced, and the stirred photobioreactor, not deeply sequenced. This difference could mean that the low level of strain heterogeneity observed in the stirred conditions may not completely capture all the variant bases within the population. However, the lower level of sequence variation observed for the deeply sequenced high pH-urea conditions indicates that the amount of strain variation is not simply a function of sequencing depth.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   iScience Article on these filtering criteria a total of 2127 unique cyanobacterial proteins were identified across all samples. Protein quantification was performed using the normalized spectral abundance factors (NSAFs) method (Florens et al., 2006). The relative abundance of proteins was calculated based on number of peptide spectral matches (PSMs) per protein that were normalized for length of the protein and the total number of spectra. NSAFs values were loaded into Perseus software (version 1.5.8.5, https://link.springer.com/ protocol/10.1007%2F978-1-4939-7493-1_7) and a cutoff number of proteins was reduced by only including proteins that had at least four NSAF values greater than 0 in all replicates of one condition. All NSAF values were transformed by taking log2. Missing values produced by log2(0) were replaced by sampling from a normal distribution assuming that the missing values were on the lower end of abundance. A student ttest was carried out on transformed NSAF to identify proteins with significant difference in expression level between samples using a permutation-based FDR calculation to account for multiple hypothesis testing, with parameters: groupings were not preserved for randomizations, both sides, 250 randomizations, FDR of 0.05 and s0 of 0.

Phylogenetic analysis
Phylogenetic tree of Ca. P. alkaliphilum genome was generated using amino acid sequences of DNAdirected RNA polymerase subunit A, B, and C2 (rpo A, B, C2). Sequences were aligned using Mafft (Katoh and Standley 2013) with -maxiterate 1000 -localpair options. Poorly aligned regions were identified with jalview and removed and remaining sequences were concatenated. Maximum-likelihood phylogenetic tree was constructed in MEGA7 (Kumar et al. 2016) using the LG+G model with 100 bootstraps. Three species from Melainabacteria were selected as the outgroup. The inset tree (Figure 3) was made as previously described (Sorokin et al., 2020).

Microscopy imaging
All microscopy was performed using the upright Axio Imager.A2 (Zeiss, Germany). Grayscale images were direct contrast interference microscopy at 100 X magnification and captured using the Axiocam 506 mono camera (Zeiss, Germany) with ZEN 2012 blue edition software (Zeiss, Germany). Coloured images were brightfield microscopy at 400 X magnification and captured using the DFK MKU226-10x22USB 3.0 Color Microscope Camera (The Imaging Source, Germany) with IC Capture 2.5 software (The Imaging Source, Germany).