Mechanisms of ionizing radiation resistance in Arthrospira sp. PCC 8005

The multicellular cyanobacterium Arthrospira has been studied for many years because of its excellent nutritive value as a food- and feedstock and its many applications in biomedical sciences. The Microbiology Research Unit (MIC) at the Belgian Nuclear Research Center (SCK CEN) studies Arthrospira sp. PCC 8005 that was chosen by the European Space Agency as a principal organism in the Micro- Ecological Life Support System Alternative (MELiSSA) for efficient O2 production and recycling of CO2, and the production of biomass as a highly nutritional end product. In Chapter 1, we give a general overview on the properties of ionizing radiation (IR) and the effects it can have on living systems. In Chapter 2, we describe the cyanobacterium Arthrospira and highlight important aspects such as oxygenic photosynthesis and the ecology and taxonomy of Arthrospira. Please note that a recent phylogenetic study by Nowicka-Krawczyk and co-workers, 2019 has firmly placed Arthrospira sp. PCC 8005 into the novel genus Limnospira and was renamed as Limnospira indica PCC 8005, now being the species type strain. We included this taxonomic adjustment from Chapter 5 onwards yet for the rest of the thesis, we retained the previous non-allocating name Arthrospira sp. for strain PCC 8005. While investigating the molecular biology of radiation resistance in Arthrospira sp. PCC 8005, we noted variations in Arthrospira trichome geometry and morphology as well as radiation resistance between the new morphotype (designated P2, with linear trichomes and floating behavior) and the original morphotype (designated P6, with helical trichomes and sedimenting behavior). Henceforth, we set out to study the additional differences between the helical form and the new linear form before and after exposure to acute -radiation at the RITA facility (in the dark, using a doserate of 600 Gy.h-1) in terms of growth recovery, antioxidant activities, glutathione content, pigment production, trehalose concentration, and transmission electron microscopy ultrastructural analysis (Chapter 3). In addition, we obtained the full genome sequences for the P2 and P6 strains (both of vintage 2018) and performed a comparative sequence analysis between these sequences with the genome sequence of the Arthrospira sp. PCC 8005 parental strain, previously determined and annotated by the MIC group in 2010-2014, as reference (Chapter 3). Comparison of the P2 and P6 genomes revealed a difference of 168 SNPs, 48 indels, and four large insertions affecting 41 coding regions across both genomes with only nine of these regions encoded proteins with a known function. In Chapter 4, we focused on the arh genes as five genes of this locus were previously shown to be highly expressed in response to -radiation (up to 60-fold) and so, in an attempt to determine their function and to study their tentative role in radiation resistance, we cloned and heterologously expressed the arhABCDE gene set into E. coli. As a follow-up study of Chapter 3, we also monitored the genetic responses of the P2 and P6 strains when exposed to  -radiation at similar cumulative doses as before but at a lower doserate and a longer exposure time (i.e., with 3 days exposure in the GEUSE facility applying a lower doserate of 80 Gy.h-1), and with a continuous exposure to light i.e. in fully metabolically active cells (Chapter 5). In this work P6 and P2 cells exposed to the intermediate cumulative dose of chronic  -radiation (3200 Gy) were analyzed for differential gene expression using RNA-seq transcriptomic analysis for the discovery of genotype-phenotype associations and to verify whether any non-coding RNAs might be at play in Arthrospira radiation resistance mechanisms. From our studies, some interesting observations emerged. With high-dose acute exposure to 60Co .- radiation in the dark, both strains P2 and P6 recovered equally well from cumulative doses of up to 2100 Gy. However, whereas P2 fully recovered from the highest cumulative dose of 5000 Gy, the helical strain P6 did not. This was in contrast to chronic exposure to -radiation at a much lower dose rate in the presence of light, where both strains eventually recovered from a cumulative dose of 5700 Gy albeit that P6 took about 13 days to regain normal growth while the P2 strain recovered considerably faster i.e., within 6 days. The two strains, highly related to each other yet with distinct phenotypic and genotypic differences, clearly display a strain-specific behavior and genetic response to ionizing radiation-inflicted stress: total antioxidant activity, glutathione content, and pigments showed statistically significant (but minor) dose-specific differences between P2 and P6, while the distinct difference in trehalose content indicated that the P2 strain metabolizes trehalose much faster than the P6 strain, presumably as a dedicated response to cellular radiation damage. Although genetic changes in some genes such as psbD, ycf4, and gvpC were observed, a genotype-phenotype association could not be made solely on the basis of genome sequence. We therefor decided to measure gene expression of .-ray irradiated versus nonirradiated cells for both strains P2 and P6 and for the first-time used RNA-seq methodology (whereas previously microarray tiling procedures were used) allowing us to cover also genes transcribed into noncoding RNA. In these experiments we choose for a lower doserate in -radiation so that the same cumulative doses could be reached as in our previous studies but the exposure to -radiation proceeded under light and was prolonged in time to roughly one biomass doubling time (~72 hours). From the chronic exposure gene expression analyzes, it became clear that upon exposure to -radiation the P2 strain (straight trichomes, higher IR resistance) pursued cell stability via the enhanced expression of genes involved in cellular protection and protein repair. At the same time, a substantial number of genes were uniquely down regulated in P2 upon exposure to IR, including genes controlling circadian rhythm, dam methylation, and high-intensity light responses. On the other hand, IR-induced genes unique to the P6 strain (helical trichomes, slower post-irradiation recovery) included those involved in cellular protection while IR-repressed P6 genes were indirectly involved in shutting down processes to conserve cellular energy. In addition, some transcriptional regulators and sigma factors as well as some crucial regulatory non-coding RNAs (IsiR, NsiR, Yfr2, SsaA) were found differently expressed in P2 versus P6. Although the two Arthrospira sp. PCC 8005 morphotypes P2 and P6 have 352 differentially expressed genes (DEGs) in common (out of a total of 887 and 666 DEGs for P2 and P6, respectively), pointing to a significant overlapping response to -radiation, each substrain seems to follow slightly different routes of priority when it comes down to the use of certain metabolic and regulatory pathways and gene networks.