The abundance, genetic diversity, and crucial ecological and evolutionary roles of marine phages have prompted a large number of metagenomic studies. However, obtaining a thorough understanding of marine phages has been hampered by the low number of phage isolates infecting major bacterial groups other than cyanophages and pelagiphages. Therefore, there is an urgent requirement for the isolation of phages that infect abundant marine bacterial groups. In this study, we isolated and characterized HMO-2011, a phage infecting a bacterium of the SAR116 clade, one of the most abundant marine bacterial lineages. HMO-2011, which infects “ Candidatus Puniceispirillum marinum” strain IMCC1322, has an ∼55-kb dsDNA genome that harbors many genes with novel features rarely found in cultured organisms, including genes encoding a DNA polymerase with a partial DnaJ central domain and an atypical methanesulfonate monooxygenase. Furthermore, homologs of nearly all HMO-2011 genes were predominantly found in marine metagenomes rather than cultured organisms, suggesting the novelty of HMO-2011 and the prevalence of this phage type in the oceans. A significant number of the viral metagenome sequences obtained from the ocean surface were best assigned to the HMO-2011 genome. The number of reads assigned to HMO-2011 accounted for 10.3%–25.3% of the total reads assigned to viruses in seven viromes from the Pacific and Indian Oceans, making the HMO-2011 genome the most or second-most frequently assigned viral genome. Given its ability to infect the abundant SAR116 clade and its widespread distribution, Puniceispirillum phage HMO-2011 could be an important resource for marine virus research.

The acI lineage of the phylum Actinobacteria is the most abundant bacterial group in most freshwater lakes. However, due to difficulties in laboratory cultivation, only two mixed cultures and some incomplete single-amplified or metagenome-derived genomes have been reported for the lineage. Here, we report the initial cultivation and complete genome sequences of four novel strains of the acI lineage from the tribes acI-A1, -A4, -A7, and -C1. The acI strains, initially isolated by dilution-to-extinction culturing, eventually failed to be maintained as axenic cultures. However, the first complete genomes of the acI lineage were successfully obtained from these initial cultures through whole genome amplification applied to more than hundreds of cultured acI cells. The genome sequences exhibited features of genome streamlining and showed that the strains are aerobic chemoheterotrophs sharing central metabolic pathways, with some differences among tribes that may underlie niche diversification within the acI lineage. Actinorhodopsin was found in all strains, but retinal biosynthesis was complete in only A1 and A4 tribes.

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms. Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment. These heme auxotroph groups are largely limited to pathogens, symbionts, or microorganisms living in nutrient-replete conditions, whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms. Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme, based on the experimental or genomic evidence. We found that two recently cultivated acI isolates require exogenous heme for their growth. One of the cultured acI isolates also exhibited auxotrophy for riboflavin. According to whole-genome analyses, all (n = 20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and Marine groups IIb and III of Euryarchaeota. Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity. Significance Heme is essential for respiration. As a cofactor of cytochromes, heme functions as a main electron carrier in all respiratory electron transport chains. Therefore, it is natural to expect all respiring and free-living microorganisms to make heme. Against this expectation, here we show that the acI lineage, one of the most abundant bacterial groups in freshwater environments, is unable to biosynthesize heme and requires exogenous heme. Furthermore, we provide genomic evidence for putative heme auxotrophy among many not-yet-cultured aquatic microbial groups. Heme should be the focus of future research on the metabolic dependency among microorganisms and the role of exchangeable metabolites in structuring diverse ecosystems, and would also be a media component that must be considered when cultivating novel microbes.

Bioflocs are microbial aggregates that play a pivotal role in shaping animal health, gut microbiota, and water quality in biofloc technology (BFT)-based aquaculture systems. Despite the worldwide application of BFT in aquaculture industries, our comprehension of the community composition and functional potential of the floc-associated microbiota (FAB community; ≥3 µm size fractions) remains rudimentary. Here, we utilized genome-centric metagenomic approach to investigate the FAB community in shrimp aquaculture systems, resulting in the reconstruction of 520 metagenome-assembled genomes (MAGs) spanning both bacterial and archaeal domains. Taxonomic analysis identified Pseudomonadota and Bacteroidota as core community members, with approximately 93% of recovered MAGs unclassified at the species level, indicating a large uncharacterized phylogenetic diversity hidden in the FAB community. Functional annotation of these MAGs unveiled their complex carbohydrate-degrading potential and involvement in carbon, nitrogen, and sulfur metabolisms. Specifically, genomic evidence supported ammonium assimilation, autotrophic nitrification, denitrification, dissimilatory nitrate reduction to ammonia, thiosulfate oxidation, and sulfide oxidation pathways, suggesting the FAB community’s versatility for both aerobic and anaerobic metabolisms. Conversely, genes associated with heterotrophic nitrification, anaerobic ammonium oxidation, assimilatory nitrate reduction, and sulfate reduction were undetected. Members of Rhodobacteraceae emerged as the most abundant and metabolically versatile taxa in this intriguing community. Our MAGs compendium is expected to expand the available genome collection from such underexplored aquaculture environments. By elucidating the microbial community structure and metabolic capabilities, this study provides valuable insights into the key biogeochemical processes occurring in biofloc aquacultures and the major microbial contributors driving these processes. IMPORTANCE: Biofloc technology has emerged as a sustainable aquaculture approach, utilizing microbial aggregates (bioflocs) to improve water quality and animal health. However, the specific microbial taxa within this intriguing community responsible for these benefits are largely unknown. Compounding this challenge, many bacterial taxa resist laboratory cultivation, hindering taxonomic and genomic analyses. To address these gaps, we employed metagenomic binning approach to recover over 500 microbial genomes from floc-associated microbiota of biofloc aquaculture systems operating in South Korea and China. Through taxonomic and genomic analyses, we deciphered the functional gene content of diverse microbial taxa, shedding light on their potential roles in key biogeochemical processes like nitrogen and sulfur metabolisms. Notably, our findings underscore the taxa-specific contributions of microbes in aquaculture environments, particularly in complex carbon degradation and the removal of toxic substances like ammonia, nitrate, and sulfide.