Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes (SAGs) followed by restriction-site associated DNA (RAD) sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating stress responses in prokaryotes and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, while thermophiles have received merely limited attention. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using protein domain analysis tools. We also used the NCBI BLAST, Operon Mapper, ProOpDB, and sequence alignment tools to reveal the geobacilli TA features. We identified 28 putative TA pairs, distributed over eight TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins, belonging to the MazEF, MNT-HEPN, ParDE, RelBE, and XRE-COG2856 TA families. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase TA (GacTA) family, which potentially represents one of the unique TA families with a reverse gene order. Moreover, we are proposing a hypothesis on the xre-cog2856 gene expression regulation, which seems to involve the c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus and facilitating future experimental research.

The toxin-antitoxin (TA) systems have been attracting attention due to their role in regulating prokaryotic cell responses to stress and their biotechnological potential. Much recognition has been given to type II TA system of mesophiles, but so far, limited attention has been given to thermophiles. Here, we are presenting the putative type II TA families encoded on the genomes of four Geobacillus strains. We employed the TA finder tool to mine for TA-coding genes and manually curated the results using various tools. We identified 28 putative TA pairs, distributed over 8 TA families. Among the identified TAs, 15 represent putative novel toxins and antitoxins that have been overlooked or annotated as hypothetical proteins in their genome records. We also identified a potentially new TA composite, AbrB-ParE. Furthermore, we are suggesting the Geobacillus acetyltransferase toxin-antitoxin (GacTA) family which potentially represents one of the unique TA families that has a reverse gene order. Moreover, we are proposing a hypothesis on the regulation of the xre-cog2856 gene expression, which seems to involve c-di-AMP. This study aims for highlighting the significance of studying TAs in Geobacillus, since they have special features. It also aims for facilitating future experimental research.

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