The Fate and Functionality of Alien tRNA Fragments in Culturing Medium and Cells of Escherichia coli.

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria.

Unique k-mers as Strain-Specific Barcodes for Phylogenetic Analysis and Natural Microbiome Profiling.

The need for a comparative analysis of natural metagenomes stimulated the development of new methods for their taxonomic profiling. Alignment-free approaches based on the search for marker k-mers turned out to be capable of identifying not only species, but also strains of microorganisms with known genomes. Here, we evaluated the ability of genus-specific k-mers to distinguish eight phylogroups of Escherichia coli (A, B1, C, E, D, F, G, B2) and assessed the presence of their unique 22-mers in clinical samples from microbiomes of four healthy people and four patients with Crohn's disease. We found that a phylogenetic tree inferred from the pairwise distance matrix for unique 18-mers and 22-mers of 124 genomes was fully consistent with the topology of the tree, obtained with concatenated aligned sequences of orthologous genes. Therefore, we propose strain-specific "barcodes" for rapid phylotyping. Using unique 22-mers for taxonomic analysis, we detected microbes of all groups in human microbiomes; however, their presence in the five samples was significantly different. Pointing to the intraspecies heterogeneity of E. coli in the natural microflora, this also indicates the feasibility of further studies of the role of this heterogeneity in maintaining population homeostasis.