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1.
BMC Vet Res ; 17(1): 348, 2021 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-34772402

RESUMO

BACKGROUND: Herbal tea residue (HTR) is generally considered to be the waste of herbal tea beverage production while it still retains rich nutrients and active substances. The main aim of the present study was to investigate the effect of fermentation technology on improving the quality of HTRs, and focus on the fermented HTR-induced alleviation of summer heat stress in fattening cattle. RESULTS: In this study, the waste HTR was fermented and then fed to a total of 45 fattening cattle that were divided into 3 groups (fermented HTR replaced 0, 15, 30% of the forage component of the diet), and the feeding experiment was lasted for 40 days. The physiological indexes, growth performance and fecal microbiota of fattening cattle were evaluated and results showed that fermented HTR could effectively reduce the respiratory rate and rectal temperature of fattening cattle under heat stress, increase the daily feed intake and daily gain, and improve the antioxidant content and blood immune index. In addition, we studied the fecal microbiota composition of 6 fattening cattle in control and 30% HTR substitution groups and found fermented HTR significantly changed the composition of fecal microbiota and increased microbial diversity, and correlation analysis suggested that the bacteria were closely related to fecal SCFA levels of fattening cattle under heat stress. CONCLUSIONS: In this study, fermented HTR replaced 30% of the forage component of the diet that can change the intestine microorganisms, maintain health and alleviate the heat stress of fattening cattle.


Assuntos
Bebidas , Doenças dos Bovinos/terapia , Dieta/veterinária , Indústria Alimentícia , Transtornos de Estresse por Calor/veterinária , Resíduos Industriais , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Bactérias/classificação , Bactérias/genética , Bovinos , Doenças dos Bovinos/prevenção & controle , Fezes/microbiologia , Feminino , Fermentação , Transtornos de Estresse por Calor/prevenção & controle , Transtornos de Estresse por Calor/terapia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética
2.
Enzymes ; 49: 305-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696836

RESUMO

RNA polymerase (RNAP) is the central enzyme of gene expression, which transcribes DNA to RNA. All cellular organisms synthesize RNA with highly conserved multi-subunit DNA-dependent RNAPs, except mitochondrial RNA transcription, which is carried out by a single-subunit RNAP. Over 60 years of extensive research has elucidated the structures and functions of cellular RNAPs. In this review, we introduce a brief structural feature of bacterial RNAP, the most well characterized model enzyme, and a novel experimental approach known as "Time-dependent soak-trigger-freeze X-ray crystallography" which can be used to observe the RNA synthesis reaction at atomic resolution in real time. This principle methodology can be used for elucidating fundamental mechanisms of cellular RNAP transcription.


Assuntos
RNA Polimerases Dirigidas por DNA , Transcrição Genética , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/genética
3.
Nat Commun ; 12(1): 5856, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615874

RESUMO

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.


Assuntos
Pirimidinonas/metabolismo , Pirróis/metabolismo , Transcriptoma , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Pirimidinonas/química , Pirróis/química , RNA Bacteriano/genética , Riboswitch
4.
PLoS One ; 16(10): e0256324, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34710139

RESUMO

Because of the continuous rise of foodborne illnesses caused by the consumption of raw fruits and vegetables, effective post-harvest anti-microbial strategies are necessary. The aim of this study was to evaluate the anti-microbial efficacy of ozone (O3) against two common causes of fresh produce contamination, the Gram-negative Escherichia coli O157:H7 and Gram-positive Listeria monocytogenes, and to relate its effects to potential mechanisms of xenobiosis by transcriptional network modeling. The study on non-host tomato environment correlated the dose × time aspects of xenobiosis by examining the correlation between bacterial survival in terms of log-reduction and defense responses at the level of gene expression. In E. coli, low (1 µg O3/g of fruit) and moderate (2 µg O3/g of fruit) doses caused insignificant reduction in survival, while high dose (3 µg/g of fruit) caused significant reduction in survival in a time-dependent manner. In L. monocytogenes, moderate dose caused significant reduction even with short-duration exposure. Distinct responses to O3 xenobiosis between E. coli and L. monocytogenes are likely related to differences in membrane and cytoplasmic structure and components. Transcriptome profiling by RNA-Seq showed that primary defenses in E. coli were attenuated after exposure to a low dose, while the responses at moderate dose were characterized by massive upregulation of pathogenesis and stress-related genes, which implied the activation of defense responses. More genes were downregulated during the first hour at high dose, with a large number of such genes getting significantly upregulated after 2 hr and 3 hr. This trend suggests that prolonged exposure led to potential adaptation. In contrast, massive downregulation of genes was observed in L. monocytogenes regardless of dose and exposure duration, implying a mechanism of defense distinct from that of E. coli. The nature of bacterial responses revealed by this study should guide the selection of xenobiotic agents for eliminating bacterial contamination on fresh produce without overlooking the potential risks of adaptation.


Assuntos
Antibacterianos/farmacologia , Escherichia coli O157/efeitos dos fármacos , Doenças Transmitidas por Alimentos/prevenção & controle , Listeria monocytogenes/efeitos dos fármacos , Lycopersicon esculentum/microbiologia , Ozônio/farmacologia , Carga Bacteriana/efeitos dos fármacos , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Frutas/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Estudo de Prova de Conceito , RNA Bacteriano/genética , RNA-Seq , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Verduras/microbiologia
5.
Invest Ophthalmol Vis Sci ; 62(12): 13, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34524384

RESUMO

Purpose: To investigate the ocular surface (OS) commensal bacteria profiles of patients with diabetes mellitus (DM) and dry eye disease (DED). Methods: In the present study, subjects were assigned to four groups: 37 to the diabetic mellitus with dry eye disease (DM with DED) group, 22 to the diabetes mellitus (DM)-only group, 34 to the dry eye disease (DED)-only group, and 22 to the control group. Tear fluid was collected using Schirmer's tear secretion test paper. 16S ribosomal ribonucleic acid (rRNA) gene sequencing was used to analyze the bacterial microbiota. Results: The DM with DED group showed the highest operational taxonomic unit (OTU) numbers and alpha diversity and the most different beta diversity. The groups shared the four most abundant phyla, accounting for over 96% of the total abundance. At the genus level, there were 10 types of overlap in the core microbiota in the groups. They showed significant differences between the groups. Additionally, the DM with DED group and the control group showed four unique core genera, respectively. Unclassified Clostridiales and Lactobacillus were the core microbiota members of the DM with DED group, the DM-only group, and the DED-only group, but not the control group. Conclusions: In the present study, our results showed that the patients in the DM with DED group had a more complex and comprehensive ocular surface microbial composition. To the best of our knowledge, this is the first study to reveal the microbial profile of dry eye disease in patients with diabetes mellitus.


Assuntos
Bactérias/genética , Diabetes Mellitus/metabolismo , Síndromes do Olho Seco/metabolismo , Microbiota , RNA Bacteriano/análise , Lágrimas/microbiologia , Síndromes do Olho Seco/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
6.
Nat Commun ; 12(1): 5706, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588441

RESUMO

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.


Assuntos
Anticódon/metabolismo , Códon/metabolismo , Evolução Molecular Direcionada , Escherichia coli/genética , RNA de Transferência/genética , Aminoácidos/genética , Aminoacil-tRNA Sintetases/metabolismo , Clonagem Molecular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/biossíntese , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo
7.
J Med Microbiol ; 70(9)2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34590996

RESUMO

The S. Typhi leuO gene, which codes for the LysR-type transcriptional regulator LeuO, contains five forward promoters named P3, P1, P2, P5 and P4, and two reverse promoters, P6 and P7. The activity of the forward promoters was revealed by primer extension using gene reporter fusions in an S. Typhi hns lrp mutant strain. Likewise, the activity of the reverse promoters was revealed in an hns background. Derepression of the transcription of the chromosomal gene was confirmed by RT-PCR in the hns lrp mutant. The leuOP1 transcriptional reporter fusion, which contained only the major P1 promoter, had a lower expression in a relA spoT mutant strain, indicating that the steady-state levels of the (p)ppGpp alarmone positively regulate it. In contrast, the leuOP3, leuOP5P4, leuOP6 and leuOP7 transcriptional fusions were derepressed in the relA spoT background, indicating that the alarmone has a negative effect on their expression. Thus, the search for genetic regulators and environmental cues that would differentially derepress leuO gene expression by antagonizing the action of the H-NS and Lrp nucleoid-associated proteins, or that would fine-tune the expression of the various promoters, will further our understanding of the significance that multiple promoters have in the control of LeuO expression.


Assuntos
Proteínas de Bactérias/genética , Regiões Promotoras Genéticas , Salmonella typhi/genética , Fatores de Transcrição/genética , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , RNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Biochemistry (Mosc) ; 86(8): 942-951, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488571

RESUMO

Translation of the genetic information into proteins, performed by the ribosome, is a key cellular process in all organisms. Translation usually proceeds smoothly, but, unfortunately, undesirable events can lead to stalling of translating ribosomes. To rescue these faulty arrested ribosomes, bacterial cells possess three well-characterized quality control systems, tmRNA, ArfA, and ArfB. Recently, an additional ribosome rescue mechanism has been discovered in Bacillus subtilis. In contrast to the "canonical" systems targeting the 70S bacterial ribosome, this latter mechanism operates by first splitting the ribosome into the small (30S) and large (50S) subunits to then clearing the resultant jammed large subunit from the incomplete nascent polypeptide. Here, I will discuss the recent microbiological, biochemical, and structural data regarding functioning of this novel rescue system.


Assuntos
Biossíntese de Proteínas , RNA Bacteriano/química , Ribossomos/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Bioquímica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Conformação de Ácido Nucleico , Peptídeos/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
9.
Biochemistry (Mosc) ; 86(8): 952-961, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34488572

RESUMO

A-minor motifs are RNA tertiary structure motifs that generally involve a canonical base pair and an adenine base forming hydrogen bonds with the minor groove of the base pair. Such motifs are among the most common tertiary interactions in known RNA structures, comparable in number with the non-canonical base pairs. They are often found in functionally important regions of non-coding RNAs and, in particular, play a central role in protein synthesis. Here, we review local variations of the A-minor geometry and discuss difficulties associated with their annotation, as well as various structural contexts and common A-minor co-motifs, and diverse functions of A-minors in various processes in a living cell.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Pareamento de Bases , Ligação de Hidrogênio , Modelos Moleculares , RNA Bacteriano/metabolismo , RNA Catalítico/química , Ribossomos , Software
10.
Int J Mol Sci ; 22(17)2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34502523

RESUMO

The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Elongação Traducional da Cadeia Peptídica , Fator Tu de Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Thermus thermophilus , Fator G para Elongação de Peptídeos/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo
11.
Nutrients ; 13(9)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34579113

RESUMO

Childhood is a critical stage of development during which diet can have profound influence on the microbiota-host interactions, leading to potentially lifelong impacts. This study aimed to investigate whether the consumption of cafeteria diet (CAFD) and sugary drinks during early rat life alters the structure of the gut microbial community and the metabolic activity. Four-week-old male Wistar rats (n = 27) were fed a standard chow diet with ad libitum access to water (CD) or to sucrose solution (HSD), and a third group was fed with CAFD and a sucrose solution for 14 weeks. HSD and CAFD consumption induced alterations in Firmicutes to Bacteroidetes ratio, Proteobacteria, and Verrucomicrobia. HSD increased the abundance of Barnesiella, whereas CAFD induced a depletion of Saccharibacteria. CAFD increased total white adipose tissue (WAT) weight (p < 0.0005) compared to CD. When CAFD was compared to HSD, a significant difference was found only for retroperitoneal WAT (p < 0.0005). Unhealthy diet-fed groups presented higher glucose (p < 0.0005), total cholesterol and creatinine serum levels (p < 0.005) compared to the CD rats. Early-life consumption of HSD, and of CAFD even more so, can have long-lasting negative effects on metabolic function. The gut microbiota communities were distinctively perturbed by diet composition.


Assuntos
Bactérias/classificação , Bactérias/efeitos dos fármacos , Dieta/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Ração Animal , Animais , Composição Corporal/efeitos dos fármacos , Fezes/microbiologia , Masculino , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ratos , Ratos Wistar
12.
Biochemistry ; 60(37): 2781-2794, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34472844

RESUMO

RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg2+ and Sr2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg2+. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.


Assuntos
Cátions Bivalentes/metabolismo , Conformação de Ácido Nucleico/efeitos dos fármacos , Riboswitch/genética , Aptâmeros de Nucleotídeos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sítios de Ligação/genética , Cátions Bivalentes/química , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligantes , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/química , Riboswitch/fisiologia , Transcrição Genética/genética
13.
Environ Toxicol ; 36(12): 2562-2577, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34520102

RESUMO

The crucial roles of the long noncoding RNAs (lncRNAs) in the development of ovarian cancer (OC) have been extensively studied. According to the prediction result from the Kaplan-Meier Plotter database, high expression of lncRNA proteasome subunit α type-3 antisense RNA1 (PSMA3-AS1) is associated with the poor prognosis in patients with OC. Thus, the study aimed to investigate the role of lncRNA PSMA3-AS1 in OC. Reverse transcription quantitative polymerase chain reaction analysis revealed that PSMA3-AS1 expression was significantly upregulated in OC cells and tissues. PSMA3-AS1 silencing inhibited OC cell proliferation, migration, and invasion, as shown by results of cell counting kit-8, colony formation, wound healing, and Transwell assays, respectively. Additionally, PSMA3-AS1 deficiency suppressed tumor growth in vivo. Mechanistically, luciferase reporter and RNA pulldown assays implied that PSMA3-AS1 served as a competing endogenous RNA for miR-378a-3p to upregulate the expression of polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). GALNT3 was a target gene of miR-378a-3p in OC. Moreover, PSMA3-AS1 activated the PI3K/Akt pathway by upregulating GALNT3 expression. Overall, PSMA3-AS1 promotes OC cell proliferation, migration, invasion, and xenograft tumor growth by activating the PI3K/Akt pathway via the miR-378a-3p/GALNT3 axis.


Assuntos
MicroRNAs , Neoplasias Ovarianas , RNA Longo não Codificante , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias Ovarianas/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Bacteriano , RNA Longo não Codificante/genética
14.
Biomolecules ; 11(7)2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34356622

RESUMO

BACKGROUND: The human intestinal microbiome plays a central role in overall health status, especially in early life stages. 16S rRNA amplicon sequencing is used to profile its taxonomic composition; however, multiomic approaches have been proposed as the most accurate methods for study of the complexity of the gut microbiota. In this study, we propose an optimized method for bacterial diversity analysis that we validated and complemented with metabolomics by analyzing fecal samples. METHODS: Forty-eight different analytical combinations regarding (1) 16S rRNA variable region sequencing, (2) a feature selection approach, and (3) taxonomy assignment methods were tested. A total of 18 infant fecal samples grouped depending on the type of feeding were analyzed by the proposed 16S rRNA workflow and by metabolomic analysis. RESULTS: The results showed that the sole use of V4 region sequencing with ASV identification and VSEARCH for taxonomy assignment produced the most accurate results. The application of this workflow showed clear differences between fecal samples according to the type of feeding, which correlated with changes in the fecal metabolic profile. CONCLUSION: A multiomic approach using real fecal samples from 18 infants with different types of feeding demonstrated the effectiveness of the proposed 16S rRNA-amplicon sequencing workflow.


Assuntos
Bactérias , Fezes/microbiologia , Microbioma Gastrointestinal , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Feminino , Humanos , Lactente , Recém-Nascido , Masculino
15.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385369

RESUMO

Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen Pseudomonas aeruginosa, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.


Assuntos
Pseudomonas aeruginosa/genética , Transcriptoma , Biofilmes/crescimento & desenvolvimento , Proteínas de Fímbrias/genética , Flagelina/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Hibridização in Situ Fluorescente , Fenótipo , Plâncton/genética , Plâncton/crescimento & desenvolvimento , Plâncton/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Piocinas/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , Análise Espaço-Temporal , Virulência/genética
16.
Science ; 373(6556): 768-774, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385391

RESUMO

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Cianobactérias/química , Elementos de DNA Transponíveis , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/metabolismo , Transposases/química , Transposases/metabolismo
17.
Biomed Res Int ; 2021: 6637617, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395621

RESUMO

Staphylococcus aureus is a major human pathogen present on a third of the healthy population. The bacterium possesses an extensive arsenal of virulence factors. The pathogenicity is linked with S. aureus high plasticity and its exceptional ability to incorporate foreign genetic material. The aim of the present study was to perform molecular characterization of Staphylococcus aureus strains isolated from the clinical environment of the CHU-Z Abomey-Calavi/Sô-Ava. Isolation of Staphylococcus aureus bacterium was performed on Chapman agar. Toxin production by isolated S. aureus strains was investigated using the radial immunoprecipitation technique. A colorimetric assay was used to evaluate Staphylococcus aureus lipase (SA-Lipase) production. Finally, the expression of antibiotic resistance genes and genes encoding toxins production was investigated. Our data suggest that none of the isolated Staphylococcus aureus strains expressed the investigated toxin genes. Interestingly, SA-Lipase was produced by 14.28% of our isolated S. aureus strains. The mecA gene was present in 57.14% of the isolated strains, while PVL and TSST-1 genes were identified in 2.85 and 7.14% of S. aureus, respectively. Significant genetic diversity was observed along the hospital environment S. aureus strains. The present study reveals the level of virulence of S. aureus strains isolated in the different units of CHU-Z Abomey Calavi/Sô-Ava through the production of lipase, PVL, and epidermolysins. The molecular study has favored a genetic characterization within the isolated strains.


Assuntos
Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Benin , Enterotoxinas/genética , Exotoxinas/genética , Hospitais Universitários , Humanos , Leucocidinas/genética , Lipase/genética , Proteínas de Ligação às Penicilinas/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA Ribossômico 16S/genética , Staphylococcus aureus/genética , Superantígenos/genética , Virulência
18.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
19.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
20.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360611

RESUMO

Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Bacteriano/química , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Proteínas de Bactérias/genética , Transcrição Genética
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