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.

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.

Alignment-free approaches employing short k-mers as barcodes for individual genomes have created a new strategy for taxonomic analysis and paved a way for high-resolution phylogeny. Here, we introduce this strategy for the Lacticaseibacillus paracasei species as a taxon requiring barcoding support for precise systematics. Using this approach for phylotyping of L. paracasei VKM B-1144 at the genus level, we identified four L. paracasei phylogroups and found that L. casei 12A belongs to one of them, rather than to the L. casei clade. Therefore, we propose to change the specification of this strain. At the genus level we found only one relative of L. paracasei VKM B-1144 among 221 genomes, complete or available in contigs, and showed that the coding potential of the genome of this “rare” strain allows its consideration as a potential probiotic component. Four sets of published metagenomes were used to assess the dependence of L. paracasei presence in the human gut microbiome on chronic diseases, dietary changes and antibiotic treatment. Only antibiotics significantly affected their presence, and strain-specific barcoding allowed the identification of the main scenarios of the adaptive response. Thus, suggesting bacteria of this species for compensatory therapy, we also propose strain-specific barcoding for selecting optimal strains for target microbiomes.

An emerging area of microbial biology focuses on oligonucleotides excised from functional RNAs and subsequently fulfilling an independent cellular role. Some of these products are subjected to modifications that may expand their functional inventory. Here, we applied a differential analysis of intra- and extracellular RNA fragments produced by wild-type Escherichia coli and its dps-null mutant and discovered leucine tRNA fragments with random 3′-terminal extensions among oligonucleotides with Dps-dependent secretion. We observed an exclusive intracellular enrichment of modified LeuT(VPQ) tRNA fragments compared to secretomes, with abundance level dependent on growth medium and the presence of competing bacteria. To assess the pervasiveness of this phenomenon, we developed a custom computational pipeline for detecting variable RNA termini in RNA-seq data. Beyond LeuT(VPQ) tRNA fragments, several other genomic loci yielded oligos with highly heterogeneous ends, indicating that terminal elongation, most prevalent in LeuT(VPQ), is not exclusive to these fragments. Ex vivo testing using synthetic LeuT(VPQ) analogs revealed their stimulatory effect on the persistence of multiple taxa in an artificial microbiome, which was attenuated by 3′-end elongation. We propose that non-template extensions may serve to broaden the spectrum of target molecules for elimination of unused mRNAs by an interference-like mechanism or to generate sequences absent from the E. coli genome as part of a primitive defense system.

The development of alignment-free approaches using short k-mers as barcodes has revo-lutionized taxonomic analysis, enabling bacterial identification at the phylogroup level even within natural communities. We applied this approach to characterize Escherichia coli intraspecific diversity in human gut microbiomes using publicly available datasets. By estimating the relative abundance of eight E. coli phylogroups defined by their 18-mer markers in 558 fecal samples, we employed correlation analysis and machine learning techniques to assess the discriminatory power of their patterns under different host phys-iological states. We compared E. coli phylogroup distribution between microbiomes of healthy individuals and patients with chronic colorectal disorders, tracked their dynamic alterations following prolonged antibiotic exposure with subsequent probiotic-assisted recovery, and evaluated changes induced by a physiologically beneficial dietary interven-tion for obesity. As a result, binary classification methods distinguished most compared datasets, while correlation analysis revealed shifts in phylogroups connectivity networks and their bimodal interactions with Prevotella, suggesting intra- and inerspecific epistatic relationships. Finally, a technique based on nonlinear dimensionality reduction visual-ized all distinct clusters of all sets, providing a chance to test the feasibility of using intra-specific phylotyping of E. coli for medical applications.

A recently emerging area of molecular biology is focused on the regulatory potential of bacterial RNA fragments that are excised from functional transcripts and acquire an in-dependent role in cells. During processing, some fragments are subject to modifications that may expand their functional inventory. Here we introduce elongation of the 5'-terminal fragments from leucine tRNAs with random sequences as a discriminative feature protecting them from Dps-dependent secretion. By analyzing 14 E. coli datasets we observed an exclusive enrichment of modified LeuT(VPQ) tRNA fragments in intracellular transcriptomes compared to secretomes and revealed dependence of their abundance on growth medium and the presence of competing bacteria. To assess the ordinariness of this modification, we developed a computational pipeline for detecting variable RNA termini in RNA-seq data. Beyond tRNALeuT(VPQ) fragments a few types of genomic loci produced oligos with heterogeneous ends, suggesting that random non-templat elongation, though most abundant in LeuT(VPQ), is not exclusive to these fragments. Regulatory potency of LeuT(VPQ) synthetic analogs tested ex vivo revealed predominantly their stimulatory ef-fect on persistence of multiple taxa in artificial microbiome, which was attenuated by 3'-end elongation. Terminal diversification of certain RNA fragments may, therefore, be considered as a process evolved to broaden the spectrum of regulatory molecules in the cell.