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1.
PeerJ ; 7: e7228, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293839

RESUMO

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

2.
Zhongguo Zhong Yao Za Zhi ; 44(12): 2493-2498, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359716

RESUMO

The standard decoction of Chinese herbal decoction pieces is a standard reference substance to measure whether different dosage forms of Chinese medicine are basically consistent with those of clinical decoction,and provides new ideas and methods for effectively solving the problems of uneven quality in Chinese medicine dispensing granules. In this study,a systematic method for evaluating the quality of Scrophulariae Radix decoction was established from the perspective of " standard decoction",providing reference for the quality control of the Scrophulariae Radix dispensing granules. 15 batches of Scrophulariae Radix decoction pieces from different origins were collected,and 15 batches of standard decoctions were prepared according to the standardized process with water as solvent.Harpagide and harpagoside were used as quantitative detection indicators to determine the content,calculate the transfer rates and determine the extraction rate. The high performance liquid chromatography( HPLC) was used to establish a standard decoction fingerprint analysis method. The results showed that the transfer rates of harpagide and harpagoside in 15 batches of Scrophulariae Radix pieces standard decoction were( 70. 84±13. 39) % and( 48. 56±6. 40) % respectively; the extraction rate was( 57. 47±5. 89) %. Nine peaks were identified in the HPLC fingerprint,and the similarity was higher than 0. 97 between the fingerprints of 15 batches of standard decoction and the control fingerprint. In this study,the preparation process of standard decoction of Scrophulariae Radix pieces conformed to the traditional decoction preparation method. The sources of the samples were representative,and the established fingerprint method was stable and feasible,which can provide reference for the preparation and quality control of Scrophulariae Radix dispensing granules.


Assuntos
Medicamentos de Ervas Chinesas/normas , Raízes de Plantas/química , Scrophularia/química , Cromatografia Líquida de Alta Pressão , Controle de Qualidade
3.
FEMS Microbiol Lett ; 366(10)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125044

RESUMO

The bacteria drug resistance is not only associated with the gain of drug resistance gene but also relied on the adaptation of bacterial cells to antibiotics by transcriptional regulation. However, only a few transcription factors that regulate drug resistance have been characterized in mycobacteria. In this study, a TetR family transcriptional factor (OxiR), encoded by Rv0067c in Mycobacterium tuberculosis, was found to be an isoniazid (INH) resistance regulator. Comparing with the wild-type strain, the oxiR overexpressing strain is four times resistant to INH, whereas the oxiR knockout strain is eight times sensitive to INH. However, the rifamycin and ethambutol resistance were not influenced by oxiR. OxiR can bind to self-promoter at a 66 bp imperfect palindromic motifs. Interestingly, OxiR directly binds to INH, and thereby alleviate the self-repression. Furthermore, OxiR negatively regulated an oxidoreductase encoded by Rv0068. And the susceptibility of the Rv0068-overexpressing and oxiR knockout strains to all the three above-mentioned anti-tuberculosis drugs was equivalent, suggesting that the effect of oxiR to INH susceptibility is attributed to the derepression of Rv0068. In conclusion, we showed that OxiR can specifically modulate INH susceptibility by regulating an oxidoreductase encoding gene, both of which have not been associated with drug-resistance previously.

4.
J Biochem ; 166(3): 237-243, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30993320

RESUMO

The ferric uptake regulator A (FurA) plays an essential role in responding to oxidative stress in mycobacteria. The genome of Mycobacterium smegmatis harbours three FurA orthologs; however, the potential cross-talk and contribution to drug resistance of different furA operon remain underdetermined. In this study, we characterized the cross-regulation and effect in drug resistance of these orthologs from M. smegmatis. Cross-binding of FurA protein to furA promoter was observed. The binding of FurA1 to furA3p and FurA2 to furA1p or furA3p is even more pronounced than their self-binding. The three FurA proteins are all functional at repressing the expression of the peroxidase enzyme katG1/katG2 in vivo. When overexpressing any of the furA orthologs in M. smegmatis, the bacteria become more resistant to isoniazid (INH). This pattern is consistent with that in Mycobacterium bovis. However, the knockdown of furA does not affect the INH sensitivity. This is the first report of cross-talk and contribution to drug resistance of all three furA orthologs in M. smegmatis.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Isoniazida/farmacologia , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/metabolismo , Isoniazida/química , Testes de Sensibilidade Microbiana , Mycobacterium bovis/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas Repressoras/metabolismo
5.
Microb Ecol ; 77(1): 76-86, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29858645

RESUMO

Bacterial social interaction is a potential influencing factor in determining the fate of invading pathogens in diverse environments. In this study, interactions between two representative resident species (Bacillus subtilis and Pseudomonas putida) and a leading food-borne disease causative pathogen (Vibrio parahaemolyticus) were examined. An antagonistic effect toward V. parahaemolyticus was observed for B. subtilis but not for P. putida. However, the relative richness of the pathogen remained rather high in B. subtilis co-cultures and was, unexpectedly, not sensitive to the initial inoculation ratios. Furthermore, two approaches were found to be efficient at modulating the relative richness of the pathogen. (1) The addition of trace glycerol and manganese to Luria-Bertani medium (LBGM) reduced the richness of V. parahaemolyticus in the co-culture with B. subtilis and in contrast, increased its richness in the co-culture with P. putida, although it did not affect the growth of V. parahaemolyticus by its own. (2) The relative richness of V. parahaemolyticus on semisolid medium decreased significantly as a function of an agar gradient, ranging from 0 to 2%. Furthermore, we explored the molecular basis of bacterial interaction through transcriptomic analysis. In summary, we investigated the interactions between a pathogen invader and two resident bacteria species, showing that the different influences on a pathogen by different types of interactions can be modulated by chemicals and medium fluidity.


Assuntos
Bactérias/patogenicidade , Fenômenos Fisiológicos Bacterianos , Doenças Transmitidas por Alimentos/microbiologia , Interações Microbianas , Bacillus subtilis/patogenicidade , Bacillus subtilis/fisiologia , Técnicas de Cocultura , Meios de Cultura/química , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Glicerol/metabolismo , Manganês/metabolismo , Pseudomonas putida/patogenicidade , Pseudomonas putida/fisiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade
6.
Artigo em Inglês | MEDLINE | ID: mdl-28649405

RESUMO

Clay minerals and metal oxides, as important parts of the soil matrix, play crucial roles in the development of microbial communities. However, the mechanism underlying such a process, particularly on the formation of soil biofilm, remains poorly understood. Here, we investigated the effects of montmorillonite, kaolinite, and goethite on the biofilm formation of the representative soil bacteria Bacillus subtilis. The bacterial biofilm formation in goethite was found to be impaired in the initial 24 h but burst at 48 h in the liquid-air interface. Confocal laser scanning microscopy showed that the biofilm biomass in goethite was 3-16 times that of the control, montmorillonite, and kaolinite at 48 h. Live/Dead staining showed that cells had the highest death rate of 60% after 4 h of contact with goethite, followed by kaolinite and montmorillonite. Atomic force microscopy showed that the interaction between goethite and bacteria may injure bacterial cells by puncturing cell wall, leading to the swarming of bacteria toward the liquid-air interface. Additionally, the expressions of abrB and sinR, key players in regulating the biofilm formation, were upregulated at 24 h and downregulated at 48 h in goethite, indicating the initial adaptation of the cells to minerals. A model was proposed to describe the effects of goethite on the biofilm formation. Our findings may facilitate a better understanding of the roles of soil clays in biofilm development and the manipulation of bacterial compositions through controlling the biofilm in soils.

7.
Int J Med Microbiol ; 306(8): 686-696, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27600408

RESUMO

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR associated proteins [Cas]) system can provide prokaryote with immunity against invading mobile genetic elements (MGEs) such as phages and plasmids, which are the main sources of staphylococcal accessory genes. To date, only a few Staphylococcus aureus strains containing CRISPR-Cas systems have been identified, but no functional study in these strains has been reported. In this study, 6 clinical isolates of S. aureus with type III-A CRISPR-Cas systems were identified, and whole-genome sequencing and functional study were conducted subsequently. Genome sequence analysis revealed a close linkage between the CRISPR-Cas system and the staphylococcal cassette chromosome mec (SCCmec) element in five strains. Comparative sequence analysis showed that the type III-A repeats are conserved within staphylococci, despite of the decreased conservation in trailer-end repeats. Highly homologous sequences of some spacers were identified in staphylococcal MGEs, and partially complementary sequences of spacers were mostly found in the coding strand of lytic regions in staphylococcal phages. Transformation experiments showed that S. aureus type III-A CRISPR-Cas system can specifically prevent plasmid transfer in a transcription-dependent manner. Base paring between crRNA and target sequence, the endoribonuclease, and the Csm complex were proved to be necessary for type III-A CRISPR-Cas immunity.


Assuntos
Sistemas CRISPR-Cas , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Ordem dos Genes , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Transcrição Genética , Transformação Bacteriana
8.
Sci Rep ; 5: 13969, 2015 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-26353937

RESUMO

Isoniazid (INH), an anti-tuberculosis (TB) drug, has been widely used for nearly 60 years. However, the pathway through which Mycobacterium tuberculosis responds INH remain largely unclear. In this study, we characterized a novel transcriptional factor, InbR, which is encoded by Rv0275c and belongs to the TetR family, that is directly responsive to INH. Disrupting inbR made mycobacteria more sensitive to INH, whereas overexpressing inbR decreased bacterial susceptibility to the drug. InbR could bind specifically to the upstream region of its own operon at two inverted repeats and act as an auto-repressor. Furthermore, InbR directly bind with INH, and the binding reduced InbR's DNA-binding ability. Interestingly, susceptibilities were also changed by InbR for other anti-TB drugs, such as rifampin, implying that InbR may play a role in multi-drug resistance. Additionally, microarray analyses revealed a portion genes of the inbR regulon have similar expression patterns in inbR-overexpressing strain and INH-treated wild type strain, suggesting that these genes, for example iniBAC, may be responsible to the drug resistance of inbR-overexpressing strain. The regulation of these genes by InbR were further assessed by ChIP-seq assay. InbR may regulate multiple drug resistance of mycobacteria through the regulation of these genes.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Isoniazida/farmacologia , Família Multigênica , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Sequências Repetidas Invertidas , Testes de Sensibilidade Microbiana , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica
9.
FEBS J ; 281(12): 2726-37, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24725376

RESUMO

Toxin-antitoxin (TA) systems play significant roles in the regulation of bacterial growth and persistence, and their functions usually depend on protein-protein interaction between their constituent TA proteins. However, the regulatory mechanisms of these systems, particularly their interaction with other cellular components, are still poorly understood. This study investigated cross-talk between the TA module RelJ/K (Rv3357/Rv3358) and the transcriptional regulator staphylococcal iron regulator repressor (SirR, Rv2788) from Mycobacterium tuberculosis. We characterized the physical interaction of SirR with both RelJ and RelK using bacterial two-hybrid, pull-down and co-immunoprecipitation assays. Similarly to RelK, SirR regulates the DNA-binding activity of RelJ and alleviates its inhibitory effect on the activity of the Rv3357p promoter. Furthermore, SirR may replace RelJ to alleviate the inhibitory effect of the toxin RelK on bacterial growth. Conversely, both RelJ and RelK competitively inhibit the interaction between SirR and their respective promoters. Thus, our results show that SirR interacts with a pair of toxin and antitoxin proteins, and exhibits antitoxin-like function to neutralize the toxin. These findings demonstrate a novel function of the SirR regulator of M. tuberculosis as well as a novel mechanism of regulation of TA systems.


Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , DNA Bacteriano/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ligação Proteica
10.
PLoS One ; 7(4): e36255, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22558408

RESUMO

The genome of Mycobacterium tuberculosis, the causative agent of tuberculosis, encodes a large number of putative transcriptional regulators. However, the identity and target genes of only a few of them have been clearly identified to date. In a recent study, the ArsR family regulator Rv2034 was characterized as a novel positive regulator of phoP. In the current study, we characterized the auto-repressive capabilities of Rv2034 and identified several residues in the protein critical for its DNA binding activities. We also provide evidence that Rv2034 forms dimers in vitro. Furthermore, by using DNaseI footprinting assays, a palindromic sequence was identified as its binding site. Notably, we found that the dosR promoter region contains the binding motif for Rv2034, and that Rv2034 positively regulates the expression of the dosR gene. The potential roles of Rv2034 in the regulation of lipid metabolism and hypoxic adaptation are discussed.


Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Sequências Repetidas Invertidas , Metabolismo dos Lipídeos/genética , Dados de Sequência Molecular , Motivos de Nucleotídeos , Oxigênio/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Repressoras/química , Especificidade por Substrato , Fatores de Transcrição/química
11.
Biochem Biophys Res Commun ; 411(4): 726-31, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21782791

RESUMO

Transcriptional regulation plays a critical role during the infection of Mycobacterium tuberculosis, the causative agent of tuberculosis. A two-component system, PhoPR, is clearly involved in the regulation of pathogenic virulence and persistence. However, the regulatory mechanism, as well as the regulator, of the phoPR operon remains uncharacterized in M. tuberculosis and its related species thus far. In the present study, we characterize an ArsR transcriptional factor, corresponding to Rv2034 and Ms6762 in M. tuberculosis and Mycobacterium smegmatis, respectively, as the first regulator of phoP in both mycobacterial species. The interaction between ArsR regulator and target promoters is conserved in these two mycobacterial species, and an inverted repeat sequence motif is successfully mapped out for the recognition of ArsR. Utilizing lacZ reporter genes and overexpression analysis, the ArsR regulator is shown to positively regulate the expression of phoP in M. smegmatis, different from most ArsR family regulators generally as a repressor. The current study establishes a direct link between the ArsR transcriptional factor and the regulation of phoP in mycobacteria. Our findings imply that ArsR may be involved in the pathogenesis of M. tuberculosis through its regulation of the phoPR operon.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Sequência Conservada , Filogenia , Regiões Promotoras Genéticas , Transativadores/classificação , Transativadores/genética
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