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 the taxonomic placement and the 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.

Obtaining the genome:

Bacteria growing over the surface of H. branconii CENA392 were removed by serial washing following a procedure reported by Delbaje et al. (2021). Total gDNA was extracted from washed cyanobacterial cells using an All-Prep DNA/RNA Mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions.  DNA stable Plus (Biomatrica, San Diego, USA) was added at a final volume of 25 %(v/v) to preserve integrity of the gDNA during lyophilization. The lyophilized gDNA was sent for whole-genome sequencing to the Joint Genome Institute (Berkley, CA, USA; 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.sh program 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). Completeness and quality assessments were measured using CheckM v1.0.13 (Parks et al., 2015) and Quast v5.0.2 (Gurevich et al., 2013). The genome file was uploaded to National Center for Biotechnology Information (NCBI) and will be publicaly available once it receives an accession number.

The bash script used for the assembly is shown here:

#!/bin/bash

#$ -q all.q

#$ -V

#$ -cwd

#$ -pe smp 8

module load Flye/2.9

flye --pacbio-hifi /Storage/data/JGI_data/CENA392/ccs.filter.fasta.gz -o flye_out392 -t 8 -i 4

The script starts with a shebang code (#!/bin/bash) that identifies the file as a bash script. Afterwards, the next 4 lines are resource requests identified by #$

-q all.q recognizes the location in which the work will be performed;

-V and -cwd permit that the output files generated are stored within the same location as the script;

-pe smp reserves the indicated cores in the computational module for running the script.

module load Flye2.9 loads the necessary program from the server.

flye --pacbio-hifi indicates the type of input data.

/Storage/data/JGI_data/CENA392/ccs.filter.fasta.gz shows the path in which the reads were stored in the server.

-o flye_out392 names the output file that will contain the results of the assembly.

-t exits after the script is done, setting the allocated cores free.

-i indicates that the script is interactive and X number of reserved cores are the minimum amount needed for completing the script.

Mycosporine-like amino acid quantification:

Identification and quantification of the MAAs palythine, shinorine and porphyra-334 used previously published procedures (Geraldes et al. 2019; 2020) from biomass that had been grown for 45 days in Z8 medium (Kotai 1972) under fluorescent light (45 µmol photons·μm-2·s-1) in a 14:10h light/dark cycle and temperature of 22 ± 1 ºC. Cells were not exposed to additional UV irradiance.  After 45 days of growth, the biomass generated in each replicate (n =3) was centrifuged and lyophilized at -80°C. Later, the biomass was submitted to the following extraction process:

1. Weighing 5.00 mg of lyophilized biomass in an Eppendorf;
2. Add 2.00 mL of acid solvent (0.1% (v/v) formic acid + 0.2 mM ammonium formate, pH ~2.55) and vortex for 5s;
3. Sample sonication with ultrasound probe for 1 min (amplitude 30%, impulse 10) and vortex again for 5s; 
4. Extract at room temperature (~22°C) for 1 hour;
5. Centrifuge samples (10,000 rpm for 10 min at 5°C) and pipette the supernatant in a syringe attached to a 0.45 µm filter;
6. Filtration to a HPLC 1.0 mL vial for sample reading.

The HPLC/MS-MS readings followed the parameters described in Geraldes et al. 2019. Blanks containing only growth media were used in between readings and standard patterns for shinorine, palythine and porphyra-334 were used. The standards were donated by Prof. Ernani Pinto. HPLC reading values are provided in an EXCEL file attached to this submission.

Chlorophyll a and carotenoids quantification:

On the first and last days of the experiment, 1.0 mL samples were taken from each replicate to evaluate photosynthetic-related pigments chlorophyll a and total carotenoids. These aliquots were filtered in glass membranes with 47 mm diameter in a setting coupled to a vacuum pump. The resulting filters were stored in amber Eppendorf’s in an ultra-freezer (-80°C) until extraction, performed as described here:

1. Addition of 1.1 mL of cold 90% acetone in each Eppendorf containing the frozen filters;
2. Sample vortexed for 30s and extraction in dark and refrigerated storage for 24h;
3. Centrifuge samples (3,000 rpm for 30 min at 5°C) and pipette the supernatant in a new Eppendorf for spectrometer readings.

The data concerning specific wavelengths were taken to be used in specific equations that allow the calculation of chlorophyll a and total carotenoids concentrations (Kirk and Allen, 1965). The readings are provided in an EXCEL file attached to this submission.

Chlorophyll a (μg mL-1) = (12,7 x A663) – (2,69 x A645)

Carotenoids (μg mL-1) = A480 + (0,114 x A663) – (0,638 x A645)

The data are available in .sh and .xlsx files. The first can be opened with any basic text reader (such as Notepad or File Reader) while the second is a spreadsheet from Office Excel.