Restriction–modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

Stromatolites are organosedimentary build-ups that have formed as a result of the sediment trapping, binding and precipitating activities of microbes. Today, extant systems provide an ideal platform for understanding the structure, composition, and interactions between stromatolite-forming microbial communities and their respective environments. In this study, we compared the metagenomes of three prevalent stromatolite-forming microbial mat types in the Spaven Province of Hamelin Pool, Shark Bay located in Western Australia. These stromatolite-forming mat types included an intertidal pustular mat as well as a smooth and colloform mat types located in the subtidal zone. Additionally, the metagenomes of an adjacent, non-lithifying mat located in the upper intertidal zone were also sequenced for comparative purposes. Taxonomic and functional gene analyses revealed distinctive differences between the lithifying and non-lithifying mat types, which strongly correlated with water depth. Three distinct populations emerged including the upper intertidal non-lithifying mats, the intertidal pustular mats associated with unlaminated carbonate build-ups, and the subtidal colloform and smooth mat types associated with laminated structures. Functional analysis of metagenomes revealed that amongst stromatolite-forming mats there was an enrichment of photosynthesis pathways in the pustular stromatolite-forming mats. In the colloform and smooth stromatolite-forming mats, however, there was an increase in the abundance of genes associated with those heterotrophic metabolisms typically associated with carbonate mineralization, such as sulfate reduction. The comparative metagenomic analyses suggest that stromatolites of Hamelin Pool may form by two distinctive processes that are highly dependent on water depth. These results provide key insight into the potential adaptive strategies and synergistic interactions between microbes and their environments that may lead to stromatolite formation and accretion.

Thrombolites are buildups of carbonate that exhibit a clotted internal structure formed through the interactions of microbial mats and their environment. Despite recent advances, we are only beginning to understand the microbial and molecular processes associated with their formation. In this study, a spatial profile of the microbial and metabolic diversity of thrombolite-forming mats of Highborne Cay, The Bahamas, was generated by using 16S rRNA gene sequencing and predictive metagenomic analyses. These molecular-based approaches were complemented with microelectrode profiling and in situ stable isotope analysis to examine the dominant taxa and metabolic activities within the thrombolite-forming communities. Analyses revealed three distinctive zones within the thrombolite-forming mats that exhibited stratified populations of bacteria and archaea. Predictive metagenomics also revealed vertical profiles of metabolic capabilities, such as photosynthesis and carboxylic and fatty acid synthesis within the mats that had not been previously observed. The carbonate precipitates within the thrombolite-forming mats exhibited isotopic geochemical signatures suggesting that the precipitation within the Bahamian thrombolites is photosynthetically induced. Together, this study provides the first look at the spatial organization of the microbial populations within Bahamian thrombolites and enables the distribution of microbes to be correlated with their activities within modern thrombolite systems. Key Words: Thrombolites—Microbial diversity—Metagenome—Stable isotopes—Microbialites. Astrobiology 17, 413–430.

Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant health and production. To that end, we developed a methodology to investigate the transcriptional activities of the microbiome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host–microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results show that this enrichment approach was highly reproducible and could be an effective approach for the on-site detection of microbial transcriptional activity. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential method for on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.