Enterotype-Dependent Probiotic-Mediated Changes in the Male Rat Intestinal Microbiome In Vivo and In Vitro.

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.

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.

Lacticaseibacillus paracasei: Occurrence in the Human Gut Microbiota and K-Mer-Based Assessment of Intraspecies Diversity.

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.

Suppression of Escherichia coli Growth Dynamics via RNAs Secreted by Competing Bacteria.

With the discovery of secreted RNAs, it has become apparent that the biological role of regulatory oligonucleotides likely goes beyond the borders of individual cells. However, the mechanisms of their action are still comprehended only in general terms and mainly for eukaryotic microRNAs, which can interfere with mRNAs even in distant recipient cells. It has recently become clear that bacterial cells lacking interference systems can also respond to eukaryotic microRNAs that have targets in their genomes. However, the question of whether bacteria can perceive information transmitted by oligonucleotides secreted by other prokaryotes remained open. Here we evaluated the fraction of short RNAs secreted by Escherichia coli during individual and mixed growth with Rhodospirillum rubrum or Prevotella copri, and found that in the presence of other bacteria E. coli tends to excrete oligonucleotides homologous to alien genomes. Based on this observation, we selected four RNAs secreted by either R. rubrum or P. copri, together with one E. coli-specific oligonucleotide. Both fragments of R. rubrum 23S-RNA suppressed the growth of E. coli. Of the two fragments secreted by P. copri, one abolished the stimulatory effect of E. coli RNA derived from the 3'-UTR of ProA mRNA, while the other inhibited bacterial growth only in the double-stranded state with complementary RNA. The ability of two RNAs secreted by cohabiting bacteria to enter E. coli cells was demonstrated using confocal microscopy. Since selected E. coli-specific RNA also affected the growth of this bacterium, we conclude that bacterial RNAs can participate in inter- and intraspecies signaling.

Excessive Promoters as Silencers of Genes Horizontally Acquired by Escherichia coli.

Horizontally acquired genes are usually transcriptionally inactive, although most of them are associated with genomic loci enriched with promoter-like sequences forming "promoter islands." We hypothesized that lateral DNA transfer induces local mutagenesis, accumulating AT base pairs and creating promoter-like sequences, whose occupancy with RNA polymerase and a specific silencer H-NS suppresses the transcription of foreign genes. Error-prone mutagenesis was implemented for the "promoter island" of a foreign gene appY and the promoter region of an inherent gene dps. Derivatives with changed transcriptional activity were selected using a reporter plasmid pET28_eGFP. Only one cycle of mutagenesis with negative selection suppressed the activity of the main dps promoter to the background level due to a single substitution in its -10 element, while positive selection gave a sequence with improved -35 element, thus testifying feasibility of the approach. The same suppression for appY was achieved by three cycles, while eightfold transcription activation required nine iterations of mutagenesis. In both cases, the number of potential start points decreased resulting in an ordinary regulatory region with only one dominant promoter in the case of positive selection. Efficiency of H-NS binding remained virtually unchanged in all mutant constructs. Based on these findings we conclude that excessive promoters can adversely affect transcription by providing a platform for interference between several RNA polymerase molecules, which can act as a silencer at promoter-dense regions.

A cohabiting bacterium alters the spectrum of short RNAs secreted by Escherichia coli.

Recently, it has been found that bacteria secrete short RNAs able to affect gene expression in eukaryotic cells, while certain mammalian microRNAs shape the gut microbiome altering bacterial transcriptome. The involvement of bacterial RNAs in communication with other bacteria is also expected, but has not been documented yet. Here, we compared the fractions of extremely short (12-22 nucleotides) RNAs secreted by Escherichia coli grown in a pure culture and jointly with bacteria of the Paenibacillus genus. Besides fragments of rRNAs and tRNAs, abundant in all samples, secreted oligonucleotides (exoRNAs) predominantly contained GC-rich fragments of messenger and antisense RNAs processed from regions with stable secondary structures. They differed in composition from oligonucleotides of intracellular fraction, where fragments of small regulatory RNAs were prevalent. Both fractions contained RNAs capable of forming complementary duplexes, while for exoRNA samples a higher percentage of 3΄-end modified RNAs and different endonuclease cleavage were detected. The presence of a cohabiting bacterium altered the spectrum of E. coli exoRNAs, indicating a population-dependent control over their composition. Possible mechanisms of this effect are discussed.

Promoter islands as a platform for interaction with nucleoid proteins and transcription factors.

Seventy-eight promoter islands with an extraordinarily high density of potential promoters have been recently found in the genome of Escherichia coli. It has been shown that RNA polymerase binds internal promoters of these islands and produces short oligonucleotides, while the synthesis of normal mRNAs is suppressed. This quenching may be biologically relevant, as most islands are associated with foreign genes, which expression may deplete cellular resources. However, a molecular mechanism of silencing with the participation of these promoter-rich regions remains obscure. It has been demonstrated that all islands interact with histone-like protein H-NS--a specific sentinel of foreign genes. In this study, we demonstrated the inhibitory effect of H-NS using Δhns mutant of Escherichia coli and showed that deletion of dps, encoding another protein of bacterial nucleoid, tended to decrease rather than increase the amount of island-specific transcripts. This observation precluded consideration of promoter islands as sites for targeted heterochromatization only and a computer search for the binding sites of 53 transcription factors (TFs) revealed six proteins, which may specifically regulate their transcriptional output.

Intragenic promotor-like sites in the genome of Escherichia coli discovery and functional implication.

Mapping of putative promoters within the entire genome of Escherichia coli (E. coli) by means of pattern-recognition software PlatProm revealed several thousand of sites having high probability to perform promoter function. Along with the expected promoters located upstream of coding sequences, PlatProm identified more than a thousand potential promoters for antisense transcription and several hundred very similar signals within coding sequences having the same direction with the genes. Since recently developed ChIP-chip technology also testified the presence of intragenic RNA polymerase binding sites, such distribution of putative promoters is likely to be a general biological phenomenon reflecting yet undiscovered regulatory events. Here, we provide experimental evidences that two internal promoters are recognized by bacterial RNA polymerase. One of them is located within the hns coding sequence and may initiate synthesis of RNA from the antisense strand. Another one is found within the overlapping genes htgA/yaaW and may control the production of a shortened mRNA or an RNA-product complementary to mRNA of yaaW. Both RNA-products can form secondary structures with free energies of folding close to those of small regulatory RNAs (sRNAs) of the same length. Folding propensity of known sRNAs was further compared with that of antisense RNAs (aRNAs), predicted in E. coli as well as in Salmonella typhimurium (S. typhimurium). Slightly lower stability observed for aRNAs assumes that their structural compactness may be less significant for biological function.