Neuromodulators as Interdomain Signaling Molecules Capable of Occupying Effector Binding Sites in Bacterial Transcription Factors.

Hormones and neurotransmitters are important components of inter-kingdom signaling systems that ensure the coexistence of eukaryotes with their microbial community. Their ability to affect bacterial physiology, metabolism, and gene expression was evidenced by various experimental approaches, but direct penetration into bacteria has only recently been reported. This opened the possibility of considering neuromodulators as potential effectors of bacterial ligand-dependent regulatory proteins. Here, we assessed the validity of this assumption for the neurotransmitters epinephrine, dopamine, and norepinephrine and two hormones (melatonin and serotonin). Using flexible molecular docking for transcription factors with ligand-dependent activity, we assessed the ability of neuromodulators to occupy their effector binding sites. For many transcription factors, including the global regulator of carbohydrate metabolism, CRP, and the key regulator of lactose assimilation, LacI, this ability was predicted based on the analysis of several 3D models. By occupying the ligand binding site, neuromodulators can sterically hinder the interaction of the target proteins with the natural effectors or even replace them. The data obtained suggest that the direct modulation of the activity of at least some bacterial transcriptional factors by neuromodulators is possible. Therefore, the natural hormonal background may be a factor that preadapts bacteria to the habitat through direct perception of host signaling molecules.

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.

Sense and antisense RNA products of the uxuR gene can affect motility and chemotaxis acting independent of the UxuR protein.

Small non-coding and antisense RNAs are widespread in all kingdoms of life, however, the diversity of their functions in bacteria is largely unknown. Here, we study RNAs synthesised from divergent promoters located in the 3'-end of the uxuR gene, encoding transcription factor regulating hexuronate metabolism in Escherichia coli. These overlapping promoters were predicted in silico with rather high scores, effectively bound RNA polymerase in vitro and in vivo and were capable of initiating transcription in sense and antisense directions. The genome-wide correlation between in silico promoter scores and RNA polymerase binding in vitro and in vivo was higher for promoters located on the antisense strands of the genes, however, sense promoters within the uxuR gene were more active. Both regulatory RNAs synthesised from the divergent promoters inhibited expression of genes associated with the E. coli motility and chemotaxis independent of a carbon source on which bacteria had been grown. Direct effects of these RNAs were confirmed for the fliA gene encoding σ28 subunit of RNA polymerase. In addition to intracellular sRNAs, promoters located within the uxuR gene could initiate synthesis of transcripts found in the fraction of RNAs secreted in the extracellular medium. Their profile was also carbon-independent suggesting that intragenic uxuR transcripts have a specific regulatory role not directly related to the function of the protein in which gene they are encoded.

Differential Impact of Hexuronate Regulators ExuR and UxuR on the Escherichia coli Proteome.

ExuR and UxuR are paralogous proteins belonging to the GntR family of transcriptional regulators. Both are known to control hexuronic acid metabolism in a variety of Gammaproteobacteria but the relative impact of each of them is still unclear. Here, we apply 2D difference electrophoresis followed by mass-spectrometry to characterise the changes in the Escherichia coli proteome in response to a uxuR or exuR deletion. Our data clearly show that the effects are different: deletion of uxuR resulted in strongly enhanced expression of D-mannonate dehydratase UxuA and flagellar protein FliC, and in a reduced amount of outer membrane porin OmpF, while the absence of ExuR did not significantly alter the spectrum of detected proteins. Consequently, the physiological roles of proteins predicted as homologs seem to be far from identical. Effects of uxuR deletion were largely dependent on the cultivation conditions: during growth with glucose, UxuA and FliC were dramatically altered, while during growth with glucuronate, activation of both was not so prominent. During the growth with glucose, maximal activation was detected for FliC. This was further confirmed by expression analysis and physiological tests, thus suggesting the involvement of UxuR in the regulation of bacterial motility and biofilm formation.

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.

Between computational predictions and high-throughput transcriptional profiling: in depth expression analysis of the OppB trans-membrane subunit of Escherichia coli OppABCDF oligopeptide transporter.

Bacterial oligopeptide transporters encoded by arrays of opp genes are implicated in a wide variety of physiological functions including nutrient acquisition, cell-to-cell communication, host-pathogen interaction. Combining the five opp genes in one oppABCDF operon of Escherichia coli assumes unified principle of their transcriptional regulation, which should provide a comparable amounts of translated products. This, however, contradicts the experimentally detected disproportion in the abundance of periplasmic OppA and the trans-membrane subunits OppB and OppC. As a first step towards understanding differential regulation of intraoperonic genes we examined genomic region proximal to oppB for its competence to initiate RNA synthesis using in silico promoter predictions, data of high-throughput RNA sequencing and targeted transcription assay. A number of transcription start sites (TSSs), whose potency depends on the presence of cationic oligopeptide protamine in cultivation medium, was found at the end of oppA and in the early coding part of oppB. We also show that full-size OppB conjugated with EGFP is produced under the control of its own genomic regulatory region and may be detected in analytical quantities of bacterial cell culture.

Overproduction and purification of the Escherichia coli transcription factors "toxic" to a bacterial cell.

Transcription factors play a crucial role in control of life of a bacterial cell, working as switchers to a different life style or pathogenicity. To reconstruct the network of regulatory events taking place in changing growth conditions, we need to know regulons of as many transcription factors as possible, and motifs recognized by them. Experimentally this can be attained via ChIP-seq in vivo, SELEX and DNAse I footprinting in vitro. All these approaches require large amounts of purified proteins. However, overproduction of transcription factors leading to their extensive binding to the regulatory elements on the DNA make them toxic to a bacterial cell thus significantly complicating production of a soluble protein. Here, on the example of three regulators from Escherichia coli, UxuR, ExuR, and LeuO, we show that stable production of toxic transcription factors in a soluble fraction can be significantly enhanced by holding the expression of a recombinant protein back at the early stages of bacterial growth. This can be achieved by cloning genes together with their regulatory regions containing repressor sites, with subsequent growth in a very rich media where activity of excessive regulators is not crucial, followed by induction with a very low concentration of an inducer. Schemes of further purification of these proteins were developed, and functional activity was confirmed.

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.

Control of hexuronate metabolism in Escherichia coli by the two interdependent regulators, ExuR and UxuR: derepression by heterodimer formation.

Two homologous proteins, UxuR and ExuR, were previously predicted to repress synthesis of enzymes required for hexuronic acid metabolism, but little is known about the relative roles of these proteins in gene regulation. We confirmed the previous report that UxuR is essential for rapid growth with d-glucuronate as the primary source of carbon and energy. In contrast, an exuR mutant grew more rapidly on d-glucuronate than the parent. Transcription of exuR is initiated at a σ70-dependent promoter predicted in silico. Purified ExuR bound to the exuR regulatory region in the presence, but not in the absence, of d-glucuronate. Apparently weaker UxuR binding in the presence of glucuronate was also detected, and its addition decreased ExuR binding by forming ExuR-UxuR heterodimers. Glucuronate induced exuR transcription in the parental strain, but not in the exuR mutant. No evidence was obtained for cAMP-dependent regulation of exuR by the catabolite repressor protein (CRP). A previous study reported that the divergent yjjM and yjjN genes, essential for l-galactonate metabolism, are repressed by UxuR. We showed that ExuR binds to the yjjM-yjjN regulatory region, and that the binding is also glucuronate-dependent. As for the exuR promoter, UxuR appeared to decrease ExuR binding. ExuR is required for glucuronate induction of yjjM and yjjN, and CRP is required for their transcription. The combined data established that UxuR and ExuR fulfil contrasting roles in regulating hexuronic acid metabolism and indicate that ExuR can function as a transcription activator, possibly by inactivating the repressor function of UxuR by heterodimer formation.

Translatomics combined with transcriptomics and proteomics reveals novel functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).

BACKGROUND: Genomes of E. coli, including that of the human pathogen Escherichia coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which, apparently, have escaped annotation due to their small size and non-essential function. To find such genes, global gene expression of EHEC EDL933 was examined, using strand-specific RNAseq (transcriptome), ribosomal footprinting (translatome) and mass spectrometry (proteome). RESULTS: Using the above methods, 72 short, non-annotated protein-coding genes were detected. All of these showed signals in the ribosomal footprinting assay indicating mRNA translation. Seven were verified by mass spectrometry. Fifty-seven genes are annotated in other enterobacteriaceae, mainly as hypothetical genes; the remaining 15 genes constitute novel discoveries. In addition, protein structure and function were predicted computationally and compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. Based on this comparison, 61 of the 72 novel proteins exhibit predicted structural and functional features similar to those of annotated proteins. Many of the novel genes show differential transcription when grown under eleven diverse growth conditions suggesting environmental regulation. Three genes were found to confer a phenotype in previous studies, e.g., decreased cattle colonization. CONCLUSIONS: These findings demonstrate that ribosomal footprinting can be used to detect novel protein coding genes, contributing to the growing body of evidence that hypothetical genes are not annotation artifacts and opening an additional way to study their functionality. All 72 genes are taxonomically restricted and, therefore, appear to have evolved relatively recently de novo.

Structural modeling of the ExuR and UxuR transcription factors of E. coli: search for the ligands affecting their regulatory properties.

Gammaproteobacteria get energy for their growth from different carbon sources using either glycolysis or alternative metabolic pathways induced in stress conditions. These metabolic switches are coordinated by complex interplay of regulatory proteins sensing concentrations of available metabolites by mechanisms yet to be understood. Here, we use two transcriptional regulators, ExuR and UxuR, controlling d-galacturonate (d-gal) and d-glucuronate metabolism in Escherichia coli, as the targets for computational search of low-molecular compounds capable to bind their ligand-binding domains. Using a flexible molecular docking, we modeled the interactions of these proteins with substrates and intermediates of glycolysis, Ashwell and Entner-Doudoroff pathways. For UxuR, the two preferred sites of ligand binding were found: one is located within the C-terminal domain, while another occupies the interdomain space. For ExuR, the only one preferred site was detected in the interdomain area. Availability of this area to different ligands suggests that, similar to the Lac repressor, the DNA-binding properties of UxuR and ExuR may be changed by repositioning of their domains. Experimental assays confirmed the ability of ligands with highest affinities to bind the regulatory proteins and affect their interaction with DNA. d-gal that is carried into the cell by the ExuT transporter appeared to be the best ligand for repressor of the exuT transcription, ExuR. For UxuR, the highest affinity was found for d-fructuronate transported by GntP, which biosynthesis is repressed by UxuR. Providing a feedback loop to balance the concentrations of different nutrients, such ligand-mediated modulation can also coordinate switching between different metabolic pathways in bacteria.

Visualizing the activity of Escherichia coli divergent promoters and probing their dependence on superhelical density using dual-colour fluorescent reporter vector.

Mosaic pattern of transcription in alternating directions is a common feature of prokaryotic and eukaryotic genomes which rationality and origin remain enigmatic. In Escherichia coli approximately 25% of genes comprise pairs of topologically linked divergently transcribed units. Given that transcriptional complex formation at each promoter in the pair induces topological changes and is itself sensitive to DNA structural perturbations, study of the functional anatomy in such areas requires special approaches. Here we suggested the dual-colour promoter probe vector which may become an ideal tool for divergent transcription profiling. The vector was used to characterize the specific genomic region nearby appY with multiple bidirectional promoters predicted in silico. Only three promoters of this region were shown to be engaged in the transcription initiation resulting in the expression of reporter genes. RNA product transcribed in antisense direction is suggested as a novel RNA. Nalidixin-induced topological modulation differentially affected transcription in sense and antisense directions thus exemplifying anticooperative mode in the response to topological alterations.

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy.

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

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.

Promoters of Escherichia coli versus promoter islands: function and structure comparison.

Expression of bacterial genes takes place under the control of RNA polymerase with exchangeable σ-subunits and multiple transcription factors. A typical promoter region contains one or several overlapping promoters. In the latter case promoters have the same or different σ-specificity and are often subjected to different regulatory stimuli. Genes, transcribed from multiple promoters, have on average higher expression levels. However, recently in the genome of Escherichia coli we found 78 regions with an extremely large number of potential transcription start points (promoter islands, PIs). It was shown that all PIs interact with RNA polymerase in vivo and are able to form transcriptionally competent open complexes both in vitro and in vivo but their transcriptional activity measured by oligonucleotide microarrays was very low, if any. Here we confirmed transcriptional defectiveness of PIs by analyzing the 5'-end specific RNA-seq data, but showed their ability to produce short oligos (9-14 bases). This combination of functional properties indicated a deliberate suppression of transcriptional activity within PIs. According to our data this suppression may be due to a specific conformation of the DNA double helix, which provides an ideal platform for interaction with both RNA polymerase and the histone-like nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen sites optimized by evolution for staying in a heterochromatin-like state. Since almost all promoter islands are associated with horizontally acquired genes, we offer them as specific components of bacterial evolution involved in acquisition of foreign genetic material by turning off the expression of toxic or useless aliens or by providing optimal promoter for beneficial genes. The putative molecular mechanism underlying the appearance of promoter islands within recipient genomes is discussed.

High-density transcriptional initiation signals underline genomic islands in bacteria.

Genomic islands (GIs), frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of "alien" elements which probably undergo special temporal-spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these "exotic" regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs) in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased "non-optimal" codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for "alien" regions, but also provide hints to the special evolutionary course and transcriptional regulation of GI regions.

Comparative genomic analysis of the hexuronate metabolism genes and their regulation in gammaproteobacteria.

The hexuronate metabolism in Escherichia coli is regulated by two related transcription factors from the FadR subfamily of the GntR family, UxuR and ExuR. UxuR controls the d-glucuronate metabolism, while ExuR represses genes involved in the metabolism of all hexuronates. We use a comparative genomics approach to reconstruct the hexuronate metabolic pathways and transcriptional regulons in gammaproteobacteria. We demonstrate differences in the binding motifs of UxuR and ExuR, identify new candidate members of the UxuR/ExuR regulons, and describe the links between the UxuR/ExuR regulons and the adjacent regulons UidR, KdgR, and YjjM. We provide experimental evidence that two predicted members of the UxuR regulon, yjjM and yjjN, are the subject of complex regulation by this transcription factor in E. coli.

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.

Predicting antisense RNAs in the genomes of Escherichia coli and Salmonella typhimurium using promoter-search algorithm PlatProm.

A pattern recognition software PlatProm, which takes into consideration both sequence-specific and structure-specific features in the genetic environment of the promoter sites and identifies transcription start points with a very high accuracy was used to reveal potentially transcribed regions in the genomes of two bacterial species. Along with the expected promoters located upstream from coding sequences PlatProm identified several hundred of very similar signals in other intergenic regions and within coding sequences. Homologous genes of Escherichia coli and Salmonella typhimurium, containing potential promoters on the template strand are suggested as putative targets for regulations by antisense RNA-products (aRNAs).