Konstantin S. Shavkunov

By definition, chimeric RNAs consist of transcript fragments from different genes. In eukaryotes, they are formed as trans-splicing products, some of which have already been recognized as biomarkers of pathological states. Fused RNAs have also been detected in bacteria, where intermolecular recombination is rare, though it remains an open question whether they are biologically significant products or sampling artifacts. Recently, we identified unusual chimeric RNAs in Escherichia coli containing uniform 8(9)-mers GCCAAGGC(G) at the 5’-end. We hypothesized that such conjunctions may arise due to the ability of 8/9-mers to prime transcription at promoters or resume RNA synthesis from stalled transcription complexes. Here, we analyzed 34 RNA-seq datasets, obtained on different sequencing platforms, and identified 560 types of modified oligonucleotides, with nearly 50% varieties coming from rRNAs (23.1%) and tRNAs (23.2%). Assessment of recurrently and incidentally modified RNAs revealed non-template cgttCCCGTGG 5’-motifs. Although being significantly less frequent, their existence suggests that GCCAAGGC(G)-mediated terminal modifications are not an exceptional phenomenon. We also identified 804 canonical chimeras formed alternatively with the 14 selected targets of GCCAAGGC(G), 62.2% of which included rRNA fragments. Observing that exogenous 9-mers increase the percentage of terminal extensions, we suggest that E. coli implements a mechanism of co-transcriptional modifications distinct from classical chimerization with a predominant selectivity for the 5’-ends of tRNAs.

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.

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.

Beneficial properties of lactic acid bacteria have been known long ago, but particular interest in probiotics has arisen in the last two decades due to the understanding of the important role of intestinal microflora in human life. Thus, the ability of probiotics to support healthy homeostasis of gut microbiomes has received particular attention. Here, we evaluated the effect of a probiotic consisting of Bifidobacterium longum and Lacticaseibacillus paracasei on the gut microbiome of male rats, assessed their persistence in the fecal biota, and compared probiotic-mediated changes in vitro and in vivo. As expected, microbiomes of two enterotypes were identified in the feces of 21 animals, and it turned out that even a single dose of the probiotic altered the microbial composition. Upon repeated administration, the E1 biota temporarily acquired properties of the E2 type. Being highly sensitive to the intervention of probiotic bacteria at the phylum and genus levels, the fecal microbiomes retained the identity of their enterotypes when transferred to a medium optimized for gut bacteria. For the E2 biota, even similarities between probiotic-mediated reactions in vitro and in vivo were detected. Therefore, fecal-derived microbial communities are proposed as model consortia to optimize the response of resident bacteria to various agents.

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.