New isolates and genomes allow for a comprehensive characterization of SAR11 subclade IIIa

The SAR11 clade (Pelagibacterales) of bacterioplankton are the most abundant heterotrophs in global oceans and comprise multiple subclades with unique spatiotemporal distributions. Subclade IIIa is poorly understood due to the lack of isolates and sequenced genomes. As part of a 3-year cultivation effort along the coastal northern Gulf of Mexico (nGOM), we isolated six new IIIa strains and have closed genomes for three via Illumina sequencing: LSUCC0261 (1.27 Mbp), LSUCC0723 (1.20 Mbp), and LSUCC0664 (1.17 Mbp). Our isolates, genomes, two existing isolate genomes, and 5 metagenome-assembled genomes (MAGs) from Tara Oceans provide an unprecedented opportunity to study the genomics and physiology of group IIIa. Phylogenomics of 91 SAR11 genomes and average amino acid identity groupings supports the subdivision of IIIa into 3 groups- IIIa.1, IIIa.2, and IIIa.3. Phylogenetic separation corresponds with overlapping but distinct salinity ranges in growth data and ecological distributions for IIIa.1 and IIIa.3. Salinity tolerance experiments indicate LSUCC0261 (IIIa.3) can grow in salinities of 0.36-34.8 and LSUCC0664 (IIIa.1) in 1.45-34.8. The IIIa.3 euryhaline physiology mirrors our nGOM 16S rRNA gene community amplicon data in which IIIa.3 abundances had no correlation with salinity while IIIa.1 had a positive correlation (R²= 0.81). Comparative genomics indicates IIIa shares much of the central carbon metabolism as other SAR11s, but is enriched with genes for Poly(3-hydroxyalkanoate) synthetase and superoxide dismutase. Variation within IIIa include genome-specific compatible solute production/transport variation due to mosaic gene indels. Unique genes to LSUCC0261 (IIIa.3) skew towards nitrogen-associated genes and include a duplication in the amtBammonium transporter, a taurine transporter, and the urease gene suite. Current studies include defining key distinctions in IIIa, metagenomic recruitment to global databases, and verifying urea metabolism in LSUCC0261.