Environmental adaptations by the intertidal Antarctic cyanobacterium Halotia branconii CENA392 as revealed using long‐read genome sequencing

Antarctica poses numerous challenges to life such as cold shock, low nutrient concentrations, and periodic desiccation over a wide range of extreme temperatures. Cyanobacteria survive this harsh environment having evolved adaptive metabolic plasticity to become the dominant primary producers. The type strain cyanobacterium Halotia branconii CENA392 was isolated from an Antarctic intertidal seashore. The complete circular genome of this strain is presented herein, which was assembled using long‐sequence reads. The genome encoded some stress‐related genes associated with low‐temperature adaptation and biosynthesis of mycosporine‐like amino acid (MAA) photoprotective compounds. Empirical experimentation demonstrated constitutive production of the MAA porphyra‐334 and total carotenoids without exposure to low temperatures or ultraviolet radiation stress. Phylogenetic analysis provided insights on taxonomic placement and evolutionary history of some annotated genes. These data exemplify the importance of generating complete quality genome sequences of microorganisms isolated from extreme intertidal environments, facilitating in‐depth evaluation of ecological and taxonomic inferences.

evolutionary history of some annotated genes.These data exemplify the importance of generating complete quality genome sequences of microorganisms isolated from extreme intertidal environments, facilitating indepth evaluation of ecological and taxonomic inferences.
Cold environments present several extreme conditions for life.Exposure to freezing shock, desiccation, high solar irradiance, and fluctuations in temperature are some abiotic factors that affect survival (Zakhia et al. 2008;Jadhav et al. 2022).These ecosystems also tend to be nutrient poor (Wait et al. 2006).Cyanobacteria have evolved to become leading primary producers in these habitats, fulfilling several vital ecological roles such as atmospheric C and N 2 fixing (Velichko et al. 2021).Cyanobacteria survive in polar regions by forming biofilms or thin-layer mats in meltwater ponds, on rocks or in holes on ice (Zakhia et al. 2008;Williamson et al. 2019).Within these biofilms, cyanobacteria are dominant but commonly associated with other phototrophic microalgae and heterotrophic bacteria, establishing complex and diverse communities (Mueller et al. 2001).The intertidal Antarctic habitat adds specific challenges, with extreme seasonal thermal shifts associated with air and water temperatures, wind exposure, and salinity fluctuation during submerged periods (Obermüller et al. 2011).
Although microclimates amongst rocks and underneath the thin ice surface may afford some protection from physical adversities, polar cyanobacteria present numerous cold tolerance traits and UV-defensive mechanisms (Velichko et al. 2021).Specialized enzymes biosynthesized under lower temperatures, such as RNA helicase C (encoded by the ctrC gene) and specific cryoprotective molecules such as the des family of fatty acid desaturases, have been shown to be upregulated in cyanobacteria exposed to cold shock conditions (Chamot et al. 1999;Los and Murata 1999;Chintalapati et al. 2007).In addition, higher yields of carotenoids and photoprotective molecules, such as mycosporine-like amino acids (MAAs), have also been reported in Antarctic cyanobacteria (Quesada and Vincent 1997;George et al. 2001).
Assembling genomes from sequenced cyanobacterial DNA isolated from extreme environments provides genetic information for taxonomic and biotechnological exploitation (Hess 2011;Walter et al. 2017;Dextro et al. 2021).Platforms such as the Genome Taxonomy Database (GTDB), that uses 120 marker genes in a complete-genome based approach, provide taxonomic clarity that aids definition of bacterial families and genera (Parks et al. 2022).Functional annotation of complete genome sequences provide holistic overview of the biosynthetic potential of a strain, assisting bioprospection (Zotchev et al. 2012).The cyanobacterial strain Halotia branconii CENA392 was isolated from an intertidal Antarctic ecosystem (Genu ario et al. 2015).To explore some environmental adaptations related to this habitat, genes encoding cryoprotective proteins and enzymes associated with the biosynthesis of UV photoprotective MAAs were annotated on the complete circular genome sequence.Constitutive levels of MAAs and total carotenoids were measured to establish correlations between arrangements of biosynthetic genes and ecological adaptations associated with these compounds.

Cyanobacterium strain
H. branconii CENA392 is the type strain for this species, isolated from a biofilm growing on an exposed whale bone during low tide in Admiralty Bay, Antarctica (Fig. 1; Genu ario

Genome sequencing and assembly
Bacteria growth over the surface of H. branconii CENA392 trichomes were reduced by serial washing (as reported by Delbaje et al. 2021).Total gDNA was extracted from these washed cells using an All-Prep DNA/RNA Mini kit (Qiagen) according to manufacturer's instructions.DNA stable Plus (Biomatrica) was added at a final volume of 25% (vol/vol) to preserve the integrity of gDNA during lyophilization.The lyophilized gDNA was sent for whole-genome sequencing to the Joint Genome Institute (JGI Project ID: 1338759).A PacBio SMRTbell library was prepared for circular consensus sequencing using a PacBio RS platform.The reads were filtered using BBMap v38.90 (Bushnell et al. 2017) with the icecreamfinder.shprogram using default parameters.De novo genome assembly was performed with default parameters using Flye v2.9 (Kolmogorov et al. 2019).The assembled scaffolds were classified with Kaiju v1.7.2 (Menzel et al. 2016) to obtain only cyanobacterial sequences.Circularity of the assembled genome was confirmed with Bandage v0.8.1 (Wick et al. 2015).Sequences for 16S rRNA genes were automatically obtained using Barrnap v0.9 (Seemann 2018).Completeness and quality assessments were measured using CheckM v1.0.13 (Parks et al. 2015) and Quast v5.0.2 (Gurevich et al. 2013).A genome map, GC skew and GC content graphs were generated using the GBK file in the Prokka genome annotation program in GView Server v3.0 (Petkau et al. 2010).

Phylogenomics of H. branconii CENA392
The amino acid sequences of 120 bacterial single-copy conserved marker proteins were selected and aligned with GTDB-Tk v0.3.2 (Chaumeil et al. 2019).These protein sequences were used to infer the maximum-likelihood phylogenomic tree of H. branconii CENA392 with RAxML v8.0.0 (Stamatakis 2014) from 1000 bootstraps using the PROT-GAMMAIGTR model in ProtTest 3.4.2(Darriba et al. 2011).A maximum-likelihood phylogenomic tree was also constructed based on 16S rRNA gene encoding sequences from the order Nostocales.The 16S rRNA encoding gene sequence of H. branconii CENA392 used in the phylogeny was downloaded from GenBank (accession number KJ843312.1;Genu ario et al. 2015) and showed 99.9% identity (with no gaps) with one of the three copies from the genome.Phylogenetic analysis used the General Time Reversible (GTR) model (Nei and Kumar 2000), which allowed a tree to be drawn after 1000 bootstrap resampling, with branch lengths measured as the number of substitutions per site.This evolutionary analysis was performed in MEGA 11 (Tamura et al. 2021).

Gene annotation and antiSMASH analysis
The complete genome sequence was annotated to specifically identify genes associated with cold temperature adaptation and solar UV photoprotection.These genes included those encoding enzymes for the biosynthesis of MAAs (designated mysA, mysB, mysC, mysD, and NPRS-like; following Lim et al. 2021), cold-adapted desaturase proteins (designated desA, desB, desC, and desD; following Los and Murata 1999), and ctrC which encodes for RNA helicase C (Chamot et al. 1999).DNA sequences for homologs of these genes were downloaded from NCBI and aligned against the genome using BLAST 2.8.1+ (Boratyn et al. 2013).Potential genes encoded within the genome sequence were automatically annotated with Prokka (Seemann 2014) and manually curated using Artemis (Carver et al. 2012).A biosynthetic cluster analysis of the complete genome was performed using antiSMASH 6.0 (Blin et al. 2021).
MAA analysis, chlorophyll a, and carotenoids estimation HPLC/MS-MS identification and quantification of the MAAs palythine, shinorine and porphyra-334 used previously published procedures (Geraldes et al. 2019(Geraldes et al. , 2020) ) from biomass that had been grown for 45 d in Z8 medium (Kotai 1972) under the same cultivation parameters described in item 2.1.Cells were not exposed to additional UV irradiance.Aliquots of 1 mL were removed from the cultures at day 1 and at the end of the growth period (45 d), and filtered through a microfiber glass membrane (GE Life Sciences, diameter 47 mm).The filters were stored in amber Eppendorf tubes at À80 C until extraction and determination of chlorophyll a and total carotenoids (Kirk and Allen 1965;Strickland and Parson 1968).Further details for all methodology are provided in Supporting Information Material.The genome is publicly available at NCBI under the accession number GCF_029953635.1.Raw data can be found in a public-access dataset in Dryad (https://doi.org/10.5061/dryad.k98sf7mb1,Dextro et al. 2023a).

Results and discussion
The type strain H. branconii CENA392 has a morphology characterized by isopolar non-branched filaments with both terminal and intercalar heterocytes (Supporting Information Fig. S1).The hglE gene, commonly encoded within the genomes of heterocyst-forming cyanobacteria (Campbell et al. 1997), was also found in CENA392 using an antiSMASH analysis.The long-read genome sequencing of this strain generated 347,638 quality-filtered PacBio reads (2503.2Mb).The complete genome was assembled as a 6,866,024 bp single circular scaffold (Fig. 2) and a plasmid of 250,409 bp with 39.94% G + C content.There was 172Â coverage of the main chromosome, which was composed of 40.37% G + C content with areas of high GC composition and an uneven and asymmetric GC skewing.The genome completeness (99.31%) and contamination level (0.58%) attested to the quality of this assembly.The genome size and G + C content were similar to those reported in other strains of the Nostocaceae family (Leão et al. 2016;Moraes et al. 2017).Even though a similarity in genome size or GC may not necessarily imply taxonomic or evolutionary closeness, it might reflect conservation of shared adaptive traits (Larsson et al. 2011).The asymmetric GC skew of the H. branconii CENA392 genome agreed with previous observation that most cyanobacteria display irregular GC skew profiles (Ohbayashi et al. 2020).
Three genes previously demonstrated to be essential for the biosynthesis of MAAs, namely mysA (encoding for sedoheptulose 7-phosphate cyclase), mysB (encoding for Omethyltransferase), and mysC (encoding for ATP-grasp), were found as a colinear cluster juxtaposed to an NPRS-like gene.This cluster was also automatically located with the antiSMASH analysis, showing considerable sequence similarity (> 75%) to other Nostocales strains.The mysD gene (encoding for the recently renamed mycosporine-glycine-amine ligase, Dextro et al. 2023b) was located over 1,500,000 bp downstream from the cluster.This suggested that this gene homolog was probably unrelated to MAA biosynthesis, and more likely associated with other biochemical pathways such as peptidoglycan biosynthesis (Walsh 1989).Isolated compounds for three common MAAs (shinorine, porphyra-334, and palythine) were used as standards to quantify constitutive MAA biosynthesis.Only porphyra-334 could be detected (0.83 AE 0.02 μg mg À1 ) with the HPLC/MS-MS method used.This level of constitutive P334 production was consistent with those levels reported in other cyanobacteria not exposed to UV irradiance.For example, 0.32 μg mg À1 measured in the freshwater strain Sphaerospermopsis torques-reginae ITEP-024 and 0.50 μg mg À1 measured in Komarekiella atlantica CCIBt3307 isolated from a Brazilian rainforest (Geraldes et al. 2020;Dextro et al. 2023c).The photosynthesis related pigment chlorophyll a varied from 0.42 AE 0.02 μg mL À1 (day 1) to 3.46 AE 0.07 μg mL À1 (day 45).The total carotenoid content ranged from 0.031 AE 0.005 μg mL À1 (day 1) to 0.17 AE 0.01 μg mL À1 (day 45).The increased carotenoid content and detection of porphyra-334 in H. branconii CENA392 were metabolic attributes commonly found in Antarctic cyanobacteria previously credited to environmental cues (George et al. 2001).Research from McMurdo Ice Shelf Station has corroborated that pigments and MAAs are both UV-protective strategies employed by Antarctic cyanobacteria (Vincent et al. 1993;Quesada et al. 1998).In these studies, scytonemin (found in sheathed strains) and high carotenoid contents were identified in benthic films of filamentous strains like Pleurocapsa (1.7 μg carotenoid cm À2 ) collected during Antarctic summer (Vincent et al. 1993).In situ experiments in an alkaline pond (pH 9) in the McMurdo region used differential UV treatments to explore carotenoids and MAAs present in a mat community where Lyngbya sp. was the dominant genus (Quesada et al. 1998).High carotenoid content was detected after 11 d of experiments (25 μg carotenoid cm À2 ), along with the presence of porphyra-334, the same MAA found in H. branconii CENA392.
Cyanobacteria have evolved a diverse array of strategies to compensate harmful effects of obligatory solar UV exposure, including heat dissipation and gliding motility (Quesada and Vincent 1997;Karsten 2008;Castenholz and Garcia-Pichel 2012;Demay et al. 2019), as well as the production of photoprotective compounds.In this study, the total content of carotenoid pigments accumulated fivefold even without exposure to UV irradiation.It is, therefore, conceivable that H. branconii CENA392 might also combine the biosynthesis of MAAs and carotenoids pigments to enhance photoprotection.Such solar UV photoprotective strategies are essential for life in Antarctica, where there are extremely high levels of almost continuous exposure to solar UV irradiation.Considering that Antarctica experiences 24-h of total daylight in the summer, all lifeforms must endure extremely high doses of UV exposure (Bernhard et al. 2004).Ozone depletion significantly increases UV levels during the Antarctic spring (Bernhard et al. 2004).Despite some recovery, the springtime ozone hole    effect has caused a high UV index ($ 9) to be recorded in the most recent 2018-2019 measurements (Lakkala et al. 2020).
Genes encoding terpene cyclase and phytoene synthase were found in the antiSMASH analysis, but with low sequence identity (< 25%).A gene encoding a Schizokinen-based siderophore involved in the sequestration of iron was found in the same antiSMASH analysis.This gene had 100% sequence identity to the gene copy described in Anabaena variabilis NIES23, which is a well-known Schizokinen producer (Ito and Butler 2005).The biosynthesis of siderophores has previously been described in psychrotolerant cyanobacteria from Antarctica, where nutrient deficiency is the norm.The heterotrophic bacterium Pseudomonas sp.ANT_H12B was recently described as a siderophore producer (Musialowski et al. 2023).This strain was isolated from a soil sample collected at King George Island, the same location where CENA392 was sampled.
The intertidal zone where strain H. branconii CENA392 was isolated also poses a unique challenge for growth.The combined exposure to marine water and air creates a circadian regulation of metabolism associated with fluctuations in sunlight, salinity and desiccation.The production of UVabsorbing compounds might represent a prerequisite for survival in this habitat, especially in daytime low tide conditions.Accumulation of MAAs in cyanobacteria exposed to high salinity were linked with osmotic regulation as an additional biological function to UV photoprotection (Karsten 2002;Rath et al. 2014).Thus, biosynthesis of porphyra-334 by H. branconii CENA392 arguably may serve multiple purposes.Red algae collected from intertidal zones around the coast of Brazil (Briani et al. 2018) and subantarctic regions (Jofre et al. 2020) have also been described as MAA producers, displaying seasonal variation in the concentrations accumulated proportional to levels of solar UV radiation.Few intertidal cyanobacteria strains have been described as MAA producers (Sinha et al. 2007), especially from polar habitats other than mat communities (Quesada et al. 1998;Joshi et al. 2018).An ecological survey of the intertidal polar region on Kola Bay has shown higher species abundance in upper intertidal zones, with the majority of cyanobacterial strains (70%) as benthic on natural substrates (Miroshnichenko 2021), similar to H. branconii CENA392.Out of the 46 cyanobacterial genera identified by morphology alone, no Halotia strains were reported.Griffiths and Waller (2016) proposed that warming conditions are shifting the intertidal biodiversity of Antarctica from microalgal biofilms to macroalgal dominated shores.According to the authors, these alterations can be observed throughout the intertidal Southern Ocean, generating widely distributed but species poor communities.As such, sampling intertidal Antarctic cyanobacteria could be considered as archiving a unique range of microbial biodiversity that might become extinct in the near-future.
Survival and growth in Antarctic environments require additional protective mechanisms, such as resistance to cold shock.Cyanobacteria have genes that respond to cold stress, encoding specific proteins to maintain cellular integrity.The ATP-dependent motor protein RNA helicase C eliminates anomalies in RNA secondary structure (Chamot et al. 1999).This protein has been detected in cyanobacteria isolated from Antarctica and Artic polar regions, when these cyanobacteria have been grown under laboratory low-temperature conditions (Sinetova and Los 2016;Velichko et al. 2021).The ctrC found in H. branconii CENA392 had a high nucleotide sequence identity (> 96%) and coverage (100%) to the copies from Nostoc sp.PCC7120 and Trichormus variabilis NIES23.The ctrC gene was found encoded close to two of the desaturase genes (desA and desD) used for the query search.Desaturases are enzymes that create double bond unsaturated fatty acids commonly found in cellular membranes.These increase membrane fluidity, which can be considered a cold shock response.Chintalapati et al. (2007) described upregulation of desA and desB in the Antarctic strain Nostoc sp.SO-36 when grown at low temperatures.At 10 C, the content of tri-unsaturated fatty acids increased and was correlated with upregulation of desaturase gene expression.However, when cultivated at 25 C and subsequently subcultured at 10 C, expression levels of the des genes were unaltered.This suggested constitutive expression of these genes was an adaptive trait exhibited by cyanobacteria inhabiting cold ecosystems.
A phylogeny of DesA (Fig. 3) indicated that the homolog from H. branconii CENA392 had a high amino acid sequence identity (> 80%) with other homologs found in strains isolated from environments exposed to snowfall and colder average temperatures (Nostoc linckia NIES25 and Nostoc sp.NIES4103 from Japan, Nostoc sp.36 from Antarctica and Nostoc sp.ATCC53789 from Scotland).Conversely, the homolog of DesD from H. branconii CENA392 clustered as an external branch in the phylogeny (Fig. 4).As a more derived protein, it shared an ancestral state to a group formed by strains isolated in very distinct habitats and geographical locations, unlikely to be exposed to cold shock.This multiplicity might reflect the conserved nature of desaturase family domains in cyanobacteria mentioned in the literature (Sakamoto et al. 1994;Chintalapati et al. 2006;Los and Mironov 2015).Alternatively, this could signify an underrepresentation of desD from cyanobacteria adapted to cold environments in the NCBI database.The highest identity of H. branconii CENA392 desD (80.97%) was with the gene from Calothrix sp.NIES2098, isolated in Japan.These results reinforce a necessity to sequence cyanobacterial genomes from diverse environments, improving resolution of gene evolution and strengthening evolutionary inferences (Dextro et al. 2021).
The phylogenomic analysis highlighted a close taxonomic relationship between H. branconii CENA392 and Atlanticothrix silvestris CENA357, which formed a branch in the phylogeny with common ancestry (Fig. 5).The filamentous morphology, presence of heterocytes and biofilm growth on exposed surfaces are common traits shared between these genera.Nevertheless, this closeness might also be an artifact that reflects a paucity of available gene sequences since the 16S rRNA phylogenetic tree (Fig. S2) had a different topology.H. branconii CENA392 and A. silvestris CENA357 were isolated from Antarctica and Brazil, distant geographical locations with entirely different habitat settings.Within the clade where H. branconii CENA392 serves as an external branch, there are strains also isolated from harsh habitat, such as brackish water, tidal sediment, marine water and soda lakes.Remarkably, Nodularia spumigena CCY9414 is a part of this clade, and is also present in both DesA and DesD phylogenetic trees (Figs. 3,4).N. spumigena CCY9414 was isolated from blooms in brackish water bodies off the coast of Denmark, in the Baltic Sea (Voß et al. 2013).Kopf et al. (2015) correlated metabolic plasticity and environmental acclimation to multiple copies of stress-related genes encoded within the genome of N. spumigena CCY9414.Amongst the arsenal of potential adaptive genes were copies of desA, desB, and desD encoding desaturases (Voß et al. 2013).The 16S rRNA phylogenetic tree (Supporting Information Fig. S2) grouped the nine species that are currently described in the genus Halotia with several Nostocales strains.This phylogeny is updated from a previous 16S rRNA phylogeny of H. branconii CENA392 presented by Genu ario et al. (2015).A close evolutionary relationship between Halotia and other newly described genera such as Komarekiella, Dendronalium, and Atlanticothrix is now reported, and further strengthens phylogenomic inferences of the H. branconii CENA392 genome assembly (Fig. 5).
The evolutionary success of cyanobacteria can be attributed to a wide repertoire of adaptive traits.This study exploited next generation sequencing technologies to recover long-sequence reads from H. branconii CENA392 gDNA.Genes encoding specific putative adaptive traits were annotated from the genome, which was assembled in a single circular scaffold.Comparative phylogenetic analysis strongly supported the taxonomic placement of H. branconii CENA392 within the order Nostocales and associated this strain with other environmentally diverse species.Uncovering the evolutionary origins of adaptive traits (e.g., constitutive biosynthesis of photoprotective compounds and enzymes that alter fatty acid composition of cellular membranes) necessitates further exploration of cyanobacterial genomes isolated from extreme habitats.

Fig. 1 .
Fig. 1. Isolation site of the strain Halotia branconii CENA392 at Punta Plaza (red dot) with an actual photo of the location.Squares 1 and 2 are zoom-in cut-outs of King George Island's and Admiralty Bay's locations.

Fig. 2 .
Fig. 2. The circular assembled genome of Halotia branconii CENA392shows its GC content and skew and highlights the location of eight genes of interest and their respective size in base pairs (bp).

Fig. 3 .
Fig. 3. Maximum-likelihood phylogenetic tree of the desA gene displaying the relationship of the assembled Halotia branconii CENA392 genome (in bold) with other species of cyanobacteria from NCBI that presented a desA homologous.Colored circles represent the habitat of origin.A bootstrap test involving 1000 resampling was performed.Bootstrap values (> 0.50) are displayed at the relevant nodes.

Fig. 4 .
Fig. 4. Maximum-likelihood phylogenetic tree of the desD gene displaying the relationship of the assembled Halotia branconii CENA392 genome (in bold) with other species of cyanobacteria from NCBI that presented a desD homologous.Colored circles represent the habitat of origin.A bootstrap test involving 1000 resampling was performed.Bootstrap values (> 0.50) are displayed at the relevant nodes.

Fig. 5 .
Fig. 5. Maximum-likelihood phylogenetic tree based on 120 bacterial single-copy conserved marker proteins displaying the relationship of the assembled Halotia branconii CENA392 genome (in bold) with other species of cyanobacteria from NCBI.A bootstrap test involving 1000 resampling was performed.Bootstrap values (> 50%) are displayed at the relevant nodes.