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Abstract
Understanding intricate microbial interactions in the environment is crucial. This is especially true for the relationships between nutrients and bacteria, as phosphorus, nitrogen and organic carbon availability are known to influence bacterial population dynamics. It has been suggested that low nutrient conditions prompt the evolutionary process of genome streamlining. This process helps conserve scarce nutrients and allows for proliferation. Genome streamlining is associated with genomic properties such as %GC content, genes encoding sigma factors, percent coding regions, gene redundancy, and functional shifts in processes like cell motility and ATP binding cassette transporters, among others. The current study aims to unveil the impact of nutrition on the genome size, %GC content, and functional properties of pelagic freshwater bacteria. We do this at finer taxonomic resolutions for many metagenomically characterized communities. Our study confirms the interplay of trophic level and genomic properties. It also highlights that different nutrient types, particularly phosphorus and nitrogen, impact these properties differently. We observed a covariation of functional traits with genome size. Larger genomes exhibit enriched pathways for motility, environmental interaction, and regulatory genes. ABC transporter genes reflect the availability of nutrients in the environment, with small genomes presumably relying more on metabolites from other organisms. We also discuss the distinct strategies different phyla adopt to adapt to oligotrophic environments. The findings contribute to our understanding of genomic adaptations within complex microbial communities.
ABSTRACT
Advancements in sequencing technologies have revolutionized environmental research, leading to a superior understanding of ecosystem functions. In recent years, high-throughput sequencing techniques evolved from short-read Illumina sequencing to long-read Oxford Nanopore Technologies (ONT) sequencing. While Illumina still dominates the sequencing market and offers high accuracy with low error rates, it is limited by shorter read lengths. ONT provides much longer reads, allowing for greater taxonomic resolution but at the cost of higher error rates. Since existing comparative amplicon analyses deal almost exclusively with prokaryotes, it is uncertain if ONT can serve as a reliable alternative to Illumina regarding protistic metabarcoding approaches. Therefore, this study focuses on comparing error rates, taxonomic classification accuracy, and the community composition of sequences obtained from Illumina and ONT sequencing of the 18S V9 rRNA gene region as well as the full-length 18S rRNA gene from sediment samples. In addition, the applicability of the recently updated Natrix2 amplicon processing pipeline for both sequencing approaches was evaluated. While higher error rates were observed for ONT reads, especially for short reads, the full-length ONT 18S sequences achieved greater taxonomic classification accuracy down to the species level. Illumina V9 sequencing, by contrast, provided reliable classification only down to the phylum level. An analysis of community compositions revealed significant taxonomic differences between the sequencing approaches, with ONT identifying more subtle responses of species to external influences. Nevertheless, the basic community patterns were detectable with all sequencing approaches. To conclude, this study showed for the first time that particularly full-length 18S ONT sequencing can serve as a reliable alternative to Illumina V9 sequencing for protist analysis and may even outperform Illumina depending on the specific research objectives.
The discharge of treated wastewater (TWW) into freshwater ecosystems poses a significant impact on microbial communities, particularly protists, which play a crucial role in nutrient cycling and ecosystem stability. While the ecological effects of TWW on microbial diversity have been studied, understanding the functional responses of protist communities remains limited. This study employs metatranscriptomics to unravel the temporal dynamics of protist community functions in response to TWW exposure. Using mesocosm experiment, water samples were analyzed over a ten-day period to monitor shifts in metabolic pathways and community interactions. Our results indicate that processed metatranscriptomic data, focusing on treatment-significant pathways, is more sensitive than traditional methods, such as meta-barcoding, and non-target screening, in detecting wastewater-induced perturbations. Early exposure to TWW significantly altered expression of pathways associated with signal transduction and environmental interaction, while general metabolic pathways showed resilience. Over time, the protist community showed signs of adaptation with expression levels stabilizing towards the end of the experiment. This study underscores the importance of focussing on functional shifts rather than just taxonomic changes for assessing wastewater impacts on freshwater ecosystems. Our findings advocate for the use of metatranscriptomics as a robust indicator for TWW detection, aiding in development of targeted environmental management strategies.

