RESUMO
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.
Assuntos
Fezes , Microbioma Gastrointestinal , Probióticos , Animais , Masculino , Ratos , Fezes/microbiologia , Bifidobacterium longum , RNA Ribossômico 16S/genéticaRESUMO
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.
Assuntos
Bactérias , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Simulação de Acoplamento Molecular , Ligantes , Sítios de Ligação , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Neurotransmissores , HormôniosRESUMO
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.
Assuntos
Escherichia coli , Espécies Introduzidas , Escherichia coli/genética , Reprodutibilidade dos Testes , RNA de Transferência/genética , Meios de Cultura , RNARESUMO
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.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Ácidos Hexurônicos/metabolismo , Proteoma/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Doença de Crohn/genética , Código de Barras de DNA Taxonômico/métodos , Infecções por Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Microbiota , Algoritmos , Estudos de Casos e Controles , Biologia Computacional , Doença de Crohn/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Humanos , MetagenomaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/toxicidade , Regulação Bacteriana da Expressão Gênica , Óperon , Fatores de Transcrição/metabolismo , Fatores de Transcrição/toxicidadeRESUMO
The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr's salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe3O4, rather than the expected Fe2O3, in the sample. Assuming that Fe2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe2O3 with Dps, but the binding sites with putative functionality were predicted only for Fe2O3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ferro/metabolismo , Sítios de Ligação , Compostos Férricos/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Simulação de Acoplamento Molecular , Multimerização Proteica , Estabilidade ProteicaRESUMO
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.
Assuntos
Escherichia coli O157/genética , Evolução Molecular , Genes Bacterianos , Proteoma/genética , Transcriptoma , Animais , Bovinos , Biologia Computacional , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Fenótipo , RNA Bacteriano/genética , Análise de Sequência de RNARESUMO
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.
Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores/genética , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Sequência de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Regulação Bacteriana da Expressão Gênica , Genômica , Ácidos Hexurônicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Vias Biossintéticas , Gammaproteobacteria/química , Gammaproteobacteria/classificação , Dados de Sequência Molecular , Filogenia , Regulon , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Biologia Computacional , Escherichia coli/genética , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala , Biologia Computacional/métodos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reporter , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Sequências Reguladoras de Ácido Nucleico , Sítio de Iniciação de TranscriçãoRESUMO
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 3Î-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.
Assuntos
Escherichia coli/metabolismo , RNA Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico , Escherichia coli/química , Escherichia coli/genética , Genoma Bacteriano , Conformação de Ácido Nucleico , RNA Antissenso/química , RNA Antissenso/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , Escherichia coli/genética , Genoma Bacteriano/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética , Sequência de Bases , Dados de Sequência MolecularRESUMO
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).
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Escherichia coli/genética , Genoma Bacteriano/genética , Regiões Promotoras Genéticas/genética , RNA Antissenso/genética , Salmonella typhimurium/genética , Inteligência Artificial , Sequência de Bases , Dados de Sequência Molecular , Reconhecimento Automatizado de Padrão/métodos , Análise de Sequência de RNA/métodos , SoftwareRESUMO
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.