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1.
World J Microbiol Biotechnol ; 35(9): 140, 2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451938

RESUMO

Pseudomonas species are the most versatile of all known bacteria for metabolic flexibility and the extent of host range from plants to humans that remains unmatched. The evolution of diverse metabolic strategies in these species to adapt to the fluctuating environment guarantees high fitness as well as the ability to withstand stress at multiple levels. These abilities in Pseudomonas species are imprinted by an adaptable genetic repertoire through the integration of external and internal signals via complex regulatory networks. One of the main regulatory networks that lead to optimal growth, survival and cellular robustness is the phenomenon of carbon catabolite repression (CCR). Even though a large array of information is available, the molecular machinery and the mechanism of CCR in Pseudomonas are distinctly diverse from Escherichia coli and Bacillus subtilis. In Pseudomonas, the Crc and Hfq proteins, CbrAB two-component systems and the CrcZ/CrcY small RNA are key components of CCR. The main focus of this review is to elucidate the mechanism of CCR and the accessories involved in regulation of preferred carbon source utilisation over non-preferred ones and how CCR influences the virulence, antibiotic resistance, bioremediation and plant growth promotion pathways. Furthermore, we have also tried to shed some light on the "omics" approaches which can provide deep mechanistic insights into the regulation of CCR. Understanding the mechanistic picture of key regulatory entities and mechanism responsible for metabolic flexibility will create opportunities for exploitation of these versatile prokaryotes in several biotechnological processes.


Assuntos
Proteínas de Bactérias/metabolismo , Repressão Catabólica , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , Pseudomonas/metabolismo , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Carbono/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Pseudomonas/genética , RNA Bacteriano/metabolismo
2.
J Appl Oral Sci ; 27: e20180256, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365706

RESUMO

OBJECTIVE: The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. METHODOLOGY: Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). RESULTS: Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). CONCLUSIONS: The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.


Assuntos
DNA Ribossômico/isolamento & purificação , Cavidade Pulpar/microbiologia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , Humanos , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/genética , Reprodutibilidade dos Testes , Tratamento do Canal Radicular/métodos , Streptococcus/genética
3.
Nat Commun ; 10(1): 2544, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186424

RESUMO

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ribonucleases/metabolismo , Ruminococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Conformação de Ácido Nucleico , Domínios Proteicos , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia/genética , Ribonucleases/química , Ribonucleases/genética , Ruminococcus/enzimologia
4.
BMC Evol Biol ; 19(1): 124, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215393

RESUMO

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.


Assuntos
Genoma Bacteriano , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Aminoacil-tRNA Sintetases/genética , Bacteriófagos/genética , Tamanho do Genoma , Genômica , Anotação de Sequência Molecular , Mycobacterium/classificação , Filogenia , Plasmídeos/genética , RNA não Traduzido/química , Ribonuclease P/genética , Inversão de Sequência
5.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia/genética , Especificidade por Substrato , Vibrio cholerae/genética
6.
J Anim Sci ; 97(6): 2368-2375, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31144716

RESUMO

Disease incidence is intimately associated with an animal's commensal bacteria populations (microbiome), as microbes that are involved with morbidity and mortality are commonly found in animals with no sign of disease. An understanding of the animal's resident respiratory pathogens, in the upper nasal cavity prior to weaning, may help us to understand the impact of these pathogens on incidence of respiratory disease. For this research, the overall goal was to characterize bacterial populations associated with calves at an early age and through time periods prior to weaning in 3 herds at the U.S. Meat Animal Research Center. Nasal swabs from the upper nasal cavity were collected at initial vaccination (approximately 40 d of age), preconditioning (approximately 130 d of age), and weaning (approximately 150 d of age) in 2015 and 2016. DNA was extracted from nasal swabs and combined into 2 pools of 10 animals for each sampling time point, in each herd, for a total of 6 pools at each sampling time point and 18 pools for all sampling time points within each year. To evaluate and compare the microbiome of each pooled sample, hypervariable regions 1 through 3 along the 16S ribosomal RNA (rRNA) gene were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for identification of the bacterial taxa present. Alpha and beta diversity were also measured. Overall, microbial communities were different between combinations of sampling year, herd location, and sampling time prior to weaning as shown by beta diversity. Analysis of these specific respiratory pathogens prior to weaning will present a clearer picture of the distribution of microbial populations in animals prior to weaning and not exhibiting clinical signs of respiratory disease. Therefore, evaluation of the animal's resident bacterial populations in the upper nasal cavity during different phases of the beef production system may help us to understand the impact of the microbiome on incidence of respiratory disease in cattle.


Assuntos
Bactérias/classificação , Doenças dos Bovinos/epidemiologia , Bovinos/microbiologia , Microbiota , Infecções Respiratórias/veterinária , Animais , Bactérias/genética , Biodiversidade , Doenças dos Bovinos/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/veterinária , Incidência , Cavidade Nasal/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Análise de Sequência de DNA , Simbiose , Estados Unidos/epidemiologia , Desmame
7.
Gene ; 708: 38-48, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31128223

RESUMO

Riboswitches are gene control elements that directly bind to specific ligands to regulate gene expression without the need for proteins. They are found in all three domains of life, including Bacteria, Archaea, and Eukaryota. Riboswitches are mostly spread in bacteria and archaea. In this paper, we discuss the general distribution, structure, and function of 28 different riboswitch classes as we focus our attention on riboswitches in bacteria. Bacterial riboswitches regulate gene expression by four distinct mechanisms. They regulate the expression of a limited number of genes. However, most of these genes are responsible for the synthesis of essential metabolites without which the cell cannot function. Therefore, riboswitch distribution is also important for antibacterial drug development.


Assuntos
Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Bacteriano/genética , Riboswitch/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Desenvolvimento de Medicamentos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , RNA Bacteriano/antagonistas & inibidores , Riboswitch/efeitos dos fármacos
8.
Int J Med Microbiol ; 309(3-4): 225-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31054808

RESUMO

Brucella species are the causative agents of brucellosis, a worldwide zoonotic disease that affects a broad range of mammals and causes great economic losses. Small regulatory RNAs (sRNAs) are post-transcriptional regulatory molecules that participate in the stress adaptation and pathogenesis of Brucella. In this study, we characterized the role of a novel sRNA, BSR1141, in the intracellular survival and virulence of Brucella melitensis. The results show that BSR1141 was highly induced during host infections and under in vitro stress situations that simulated the conditions encountered within host phagocytes. In addition, a BSR1141 mutant showed reduced survival both under in vitro stress conditions and in mice, confirming the role of BSR1141 in Brucella intracellular survival. Bioinformatic and experimental approaches revealed that BSR1141 affects the expression of many target genes, including the Brucella virulence component virB2. These data indicate that BSR1141 could influence the expression of virB2, which is important for B. melitensis pathogenesis and intracellular survival. This work provides new insight into the mechanism of adaptation to environmental stress and into the pathogenesis of intracellular pathogens.


Assuntos
Brucella melitensis/fisiologia , Brucella melitensis/patogenicidade , Pequeno RNA não Traduzido/metabolismo , Fatores de Virulência/genética , Animais , Brucella melitensis/genética , Brucelose/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Mutação , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Baço/microbiologia , Estresse Fisiológico , Virulência/genética
9.
Acta Trop ; 194: 165-168, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978311

RESUMO

Porcine haemoplasmosis caused by Mycoplasma suis affects the global pig industry with significant economic losses. The main transmission route of M. suis is through the blood and some haematophagous arthropods, like flies and mosquitoes, could be the vectors to this pathogen. However, the presence of M. suis in pig haematophagous ectoparasites in natural conditions has not yet been studied. The most frequent ectoparasite in pigs is the blood-sucking louse Haematopinus suis, an obligate and permanent parasite. Therefore, this work aims to study the occurrence of M. suis in H. suis samples from both domestic and wild pig populations from Argentina; using the 16S rRNA gene. A total of 98 sucking lice, collected from domestic and wild pigs from Buenos Aires Province in central Argentina, were examined. We found M. suis DNA in 15 H. suis samples (15.30%). Positive lice were detected from all studied populations. This is the first report of M. suis presence in H. suis, being also the first detection in a pig ectoparasite species. We conclude that H. suis could serve as a mechanical vector for M. suis. This information not only extends the knowledge about the pathogen spectrum potentially transmitted by H. suis, but may be also useful in epidemiological studies about Mycoplasma.


Assuntos
Anoplura/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma/isolamento & purificação , Doenças dos Suínos/parasitologia , Animais , Argentina , Mosquitos Vetores , Mycoplasma/classificação , Mycoplasma/genética , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S , Suínos , Doenças dos Suínos/epidemiologia
10.
Microbiol Spectr ; 7(2)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-31004423

RESUMO

Regulatory RNAs, present in many bacterial genomes and particularly in pathogenic bacteria such as Staphylococcus aureus, control the expression of genes encoding virulence factors or metabolic proteins. They are extremely diverse and include noncoding RNAs (sRNA), antisense RNAs, and some 5' or 3' untranslated regions of messenger RNAs that act as sensors for metabolites, tRNAs, or environmental conditions (e.g., temperature, pH). In this review we focus on specific examples of sRNAs of S. aureus that illustrate how numerous sRNAs and associated proteins are embedded in complex networks of regulation. In addition, we discuss the CRISPR-Cas systems defined as an RNA-interference-like mechanism, which also exist in staphylococcal strains.


Assuntos
RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Staphylococcus aureus/genética , Animais , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Humanos , RNA Bacteriano/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo
11.
BMC Plant Biol ; 19(1): 122, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940073

RESUMO

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.


Assuntos
Proteínas de Bactérias/genética , Citrus/microbiologia , Genoma Bacteriano/genética , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Animais , Citrus/imunologia , Resistência à Doença , Genótipo , Hemípteros/microbiologia , Insetos Vetores/microbiologia , Floema/imunologia , Floema/microbiologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , RNA Bacteriano/genética , RNA Mensageiro/genética , Rhizobiaceae/fisiologia
12.
Methods Mol Biol ; 1969: 33-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877668

RESUMO

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.


Assuntos
Perfilação da Expressão Gênica/métodos , Fator Proteico 1 do Hospedeiro/metabolismo , Imunoprecipitação/métodos , Neisseria meningitidis/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Ligação Proteica , RNA Bacteriano/genética , RNA Mensageiro/genética
13.
BMC Res Notes ; 12(1): 138, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871640

RESUMO

OBJECTIVE: This study was designed to investigate the transcriptional response of OmpF and OmpC along with an antisense RNA, MicF under concentration gradient carbapenem exposure. RESULT: An elevation in the expression of OmpF gene under concentration gradient imipenem stress from a particular concentration was observed. For OmpC gene a significant decrease in the expression was noticed under concentration gradient imipenem and meropenem stress. The study showed reduction in the expression of OmpC gene against imipenem and meropenem possibly preventing the entry of carbapenem antibiotic inside the cell indicating a possible role in carbapenem resistance.


Assuntos
Carbapenêmicos/farmacologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Porinas/genética , Antibacterianos/farmacologia , Relação Dose-Resposta a Droga , Resistência Microbiana a Medicamentos/genética , Imipenem/farmacologia , Meropeném/farmacologia , Testes de Sensibilidade Microbiana , RNA Antissenso/genética , RNA Bacteriano/genética , Transcrição Genética/efeitos dos fármacos
14.
ACS Appl Mater Interfaces ; 11(14): 13140-13146, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30888786

RESUMO

In this work, we propose a novel methodology for electrical monitoring using nanoporous alumina membranes of virulence factors secreted by bacterial pathogens. Bacterial hyaluronidase (HYAL), which is produced by a number of invasive Gram-positive bacteria, is selected as a model compound to prove the concept. Our electrochemical setup takes advantage of the flat surface of indium tin oxide/poly(ethylene terephthalate) (ITO/PET) electrodes for their assembly with the nanoporous membrane. The proposed analytical method, based on the electrical monitoring of the steric/electrostatic nanochannels blocked upon formation of an antibody-HYAL immunocomplex, reached detection limits as low as 64 UI/mL (17.3 U/mg) HYAL. The inert surface of the ITO/PET electrodes together with the anti-biofilm properties of the 20 nm pore-sized alumina membranes allows for culturing the bacteria, capturing the secreted enzymes inside the nanochannels, and removing the cells before the electrochemical measurement. Secreted HYAL at levels of 1000 UI/mL (270 U/mg) are estimated in Gram-positive Staphylococcus aureus cultures, whereas low levels are detected for Gram-negative Pseudomonas aeruginosa (used as a negative control). Finally, HYAL secretion inhibition by RNAIII-inhibiting peptide (YSPWTNF-NH2) is also monitored, opening the way for further applications of the developed monitoring system for evaluation of the antivirulence potential of different compounds. This label-free method is rapid and cheap, avoiding the use of the time-consuming sandwich assays. We envisage future applications for monitoring of bacterial virulence/invasion as well as for testing of novel antimicrobial/antivirulence agents.


Assuntos
Técnicas Biossensoriais , Nanoporos , Infecções Estafilocócicas/tratamento farmacológico , Fatores de Virulência/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Humanos , Peptídeos/química , Peptídeos/farmacologia , RNA Bacteriano/antagonistas & inibidores , RNA Bacteriano/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Compostos de Estanho/química , Fatores de Virulência/química
15.
J Ind Microbiol Biotechnol ; 46(6): 819-830, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30887255

RESUMO

Small noncoding RNAs, a large class of ancient posttranscriptional regulators, are increasingly recognized and utilized as key modulators of gene expression in a broad range of microorganisms. Owing to their small molecular size and the central role of Watson-Crick base pairing in defining their interactions, structure and function, numerous diverse types of trans-acting RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for genetic engineering and synthetic biology. The trans-acting regulatory RNAs accelerate this ability to establish potential framework for genetic engineering and genome-scale engineering, which allows RNA structure characterization, easier to design and model compared to DNA or protein-based systems. In this review, we summarize recent advances in engineered trans-acting regulatory RNAs that are used in bacterial genome-scale engineering and in novel cellular capabilities as well as their implementation in wide range of biotechnological, biological and medical applications.


Assuntos
Engenharia Genética/métodos , Genoma Bacteriano , RNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , RNA Mensageiro , Pequeno RNA não Traduzido , Biologia Sintética , Transativadores/genética
16.
Methods Mol Biol ; 1922: 525-548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838598

RESUMO

Early childhood caries (ECC) is a biofilm-mediated disease. Social, environmental, and behavioral determinants as well as innate susceptibility are major influences on its incidence; however, from a pathogenetic standpoint, the disease is defined and driven by oral dysbiosis. In other words, the disease occurs when the natural equilibrium between the host and its oral microbiome shifts toward states that promote demineralization at the biofilm-tooth surface interface. Thus, a comprehensive understanding of dental caries as a disease requires the characterization of both the composition and the function or metabolic activity of the supragingival biofilm according to well-defined clinical statuses. However, taxonomic and functional information of the supragingival biofilm is rarely available in clinical cohorts, and its collection presents unique challenges among very young children. This paper presents a protocol and pipelines available for the conduct of supragingival biofilm microbiome studies among children in the primary dentition, that has been designed in the context of a large-scale population-based genetic epidemiologic study of ECC. The protocol is being developed for the collection of two supragingival biofilm samples from the maxillary primary dentition, enabling downstream taxonomic (e.g., metagenomics) and functional (e.g., transcriptomics and metabolomics) analyses. The protocol is being implemented in the assembly of a pediatric precision medicine cohort comprising over 6000 participants to date, contributing social, environmental, behavioral, clinical, and biological data informing ECC and other oral health outcomes.


Assuntos
Bactérias/genética , Biofilmes , Cárie Dentária/microbiologia , Metabolômica/métodos , Metagenômica/métodos , Dente Decíduo/microbiologia , Bactérias/isolamento & purificação , Bactérias/metabolismo , Pré-Escolar , DNA Bacteriano/genética , Cárie Dentária/etiologia , Perfilação da Expressão Gênica/métodos , Gengiva/microbiologia , Humanos , Microbiota , RNA Bacteriano/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Software , Manejo de Espécimes/métodos , Transcriptoma
17.
Mol Cell ; 74(1): 132-142.e5, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30872121

RESUMO

Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Mimetismo Molecular , Pseudomonas aeruginosa/enzimologia , RNA Bacteriano/metabolismo , RNA Guia/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia/química , RNA Guia/genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
18.
RNA ; 25(5): 573-589, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792229

RESUMO

Identification and characterization of base-multiplets, which are essentially mediated by base-pairing interactions, can provide insights into the diversity in the structure and dynamics of complex functional RNAs, and thus facilitate hypothesis driven biological research. The necessary nomenclature scheme, an extension of the geometric classification scheme for base-pairs by Leontis and Westhof, is however available only for base-triplets. In the absence of information on topology, this scheme is not applicable to quartets and higher order multiplets. Here we propose a topology-based classification scheme which, in conjunction with a graph-based algorithm, can be used for the automated identification and characterization of higher order base-multiplets in RNA structures. Here, the RNA structure is represented as a graph, where nodes represent nucleotides and edges represent base-pairing connectivity. Sets of connected components (of n nodes) within these graphs constitute subgraphs representing multiplets of "n" nucleotides. The different topological variants of the RNA multiplets thus correspond to different nonisomorphic forms of these subgraphs. To annotate RNA base-multiplets unambiguously, we propose a set of topology-based nomenclature rules for quartets, which are extendable to higher multiplets. We also demonstrate the utility of our approach toward the identification and annotation of higher order RNA multiplets, by investigating the occurrence contexts of selected examples in order to gain insights regarding their probable functional roles.


Assuntos
Algoritmos , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA Bacteriano/química , RNA Fúngico/química , Pareamento de Bases , Biologia Computacional/métodos , Ligações de Hidrogênio , Nucleotídeos/genética , Nucleotídeos/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Software , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
19.
RNA ; 25(4): 472-480, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705137

RESUMO

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.


Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/genética , Ribossomos/química , Pareamento de Bases , Sequência de Bases , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pinças Ópticas , Biogênese de Organelas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
20.
Food Funct ; 10(2): 1028-1037, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30706916

RESUMO

d-aspartate (d-Asp), an endogenous amino acid, occurs widely in animals and humans with d-enantiomers and plays an important role in the endocrine and nervous systems. However, very few studies are available on growth performance, microbial community, and the intestinal immune and inflammatory status in response to d- and l-aspartate (l-Asp). Thus, in this study, we mainly investigated the effects of dietary 1% d- and l-Asp on growth performance, inflammation, and microbial community in young pigs. Twenty-eight young pigs were randomly divided into four groups (n = 7): a control group, in which piglets were fed a basal diet, and other three groups, in which piglets received 1% d-Asp, 1% l-Asp, and 1% dl-aspartate (dl-Asp) for 35 days. The results showed that dietary 1% d-Asp significantly inhibited average daily feed intake and average daily weight gain. Gut microbes were tested and the results showed that l-Asp enhanced bacterial diversity (Shannon and Simpson). At the phylum level, l-Asp enhanced intestinal Actinobacteria and Bacteroidetes abundance but decreased Firmicutes abundance. In contrast, dl-Asp decreased intestinal Actinobacteria and Bacteroidetes abundance and increased Firmicutes abundance. At the genus level, d-Asp enhanced Clostridium sensu stricto 1 and Intestinibacter abundance. Metagenomic predictions by PICRUSt suggested that the altered microbiota were mainly involved in membrane transport, carbohydrate metabolism, amino acid metabolism, replication and repair, translation, and nucleotide metabolism. In addition, dl-Asp markedly increased the activities of serum alanine aminotransferase, aspartate transaminase and alkaline phosphatase. Also, dietary d- and dl-Asp down-regulated TLR 4, NOD1, and MyD88 in the jejunum to mediate the inflammatory response. Collectively, these results indicated that dietary d-Asp and l-Asp affect the growth performance and inflammation in piglets, which might be associated with gut microbiota.


Assuntos
Ácido Aspártico/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Inflamação/prevenção & controle , Suínos/crescimento & desenvolvimento , Ração Animal , Fenômenos Fisiológicos da Nutrição Animal , Animais , Ácido Aspártico/administração & dosagem , Bactérias/classificação , Bactérias/genética , Dieta/veterinária , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Distribuição Aleatória
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