Cultivation of Dominant Freshwater Bacterioplankton Lineages Using a High-Throughput Dilution-to-Extinction Culturing Approach Over a 1-Year Period

Abstract

Although many culture-independent molecular analyses have elucidated a great diversity of freshwater bacterioplankton, the ecophysiological characteristics of several abundant freshwater bacterial groups are largely unknown due to the scarcity of cultured representatives. Therefore, a high-throughput dilution-to-extinction culturing (HTC) approach was implemented herein to enable the culture of these bacterioplankton lineages using water samples collected at various seasons and depths from Lake Soyang, an oligotrophic reservoir located in South Korea. Some predominant freshwater bacteria have been isolated from Lake Soyang via HTC (e.g., the acI lineage); however, large-scale HTC studies encompassing different seasons and water depths have not been documented yet. In this HTC approach, bacterial growth was detected in 14% of 5,376 inoculated wells. Further, phylogenetic analyses of 16S rRNA genes from a total of 605 putatively axenic bacterial cultures indicated that the HTC isolates were largely composed of Actinobacteria, Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Verrucomicrobia. Importantly, the isolates were distributed across diverse taxa including phylogenetic lineages that are widely known cosmopolitan and representative freshwater bacterial groups such as the acI, acIV, LD28, FukuN57, MNG9, and TRA3–20 lineages. However, some abundant bacterial groups including the LD12 lineage, Chloroflexi, and Acidobacteria could not be domesticated. Among the 71 taxonomic groups in the HTC isolates, representative strains of 47 groups could either form colonies on agar plates or be revived from frozen glycerol stocks. Additionally, season and water depth significantly affected bacterial community structure, as demonstrated by 16S rRNA gene amplicon sequencing analyses. Therefore, our study successfully implemented a dilution-to-extinction cultivation strategy to cultivate previously uncultured or underrepresented freshwater bacterial groups, thus expanding the basis for future multi-omic studies.

Adaptive genetic traits in pelagic freshwater microbes

“…Nanopelagicales’, Acidimicrobiia (also known as acIV; Newton et al, 2011) are commonly found in freshwater systems, albeit in lower numbers (Figure 8A). Although several strains have been isolated (Henson et al, 2020; Kim, Islam, et al, 2021), no genomes of cultures have so far been published, thus MAGs are the only source of genomic information for the acIV lineage. The families Ilumatobacteraceae, ‘ Ca .…”

Heliorhodopsin binds and regulates glutamine synthetase activity

“…The concentration of nitrate does not differ between the hypolimnion and the epilimnion layer in any season. Ammonium is only detectable in the lake hypolimnion during spring [ 61 ]. The lake hypolimnion is an extremely low-nutrient environment, and the nutrient levels decrease gradually over time from spring to winter.…”

Complete Genome Sequences of Polynucleobacter sp. Subcluster PnecA Strains SHI2 and SHI8, Isolated from an Oligotrophic-Dystrophic Lake in Japan

“…To date, only two species ( Polynucleobacter rarus and Polynucleobacter kasalickyi ) have been described ( 3 , 4 ). Isolation and cultivation of PnecA bacteria have rarely been successful ( 3 – 6 ). Thus, the genomic information on PnecA bacteria is very limited.…”