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1.
Yi Chuan ; 41(9): 863-874, 2019 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-31549684

RESUMO

Membrane proteins play important functions not only as receptors and transporters, but also in many other important intracellular functions such as photosynthetic and respiratory electron transport. Identification of membrane proteins is a necessary step to understand their functions. Membrane proteins are generally highly hydrophobic and difficult to be resolved by aqueous solutions, and large-scale proteomic identification of membrane proteins has been a great technical challenge. Significant efforts have been invested in the field to improve the solubility of membrane proteins in aqueous solutions that are compatible for mass spectrometry analysis. This review summarizes the main technological achievements in the field of membrane proteomics particularly for the improvement of membrane protein identification, and uses the photosynthetic model cyanobacterium Synechocystis sp. PCC6803 as an example to illustrate how technology advances push forward the field in terms of the increased coverage of membrane proteome identification.


Assuntos
Proteoma , Proteômica/tendências , Synechocystis/genética , Proteínas de Bactérias/genética , Espectrometria de Massas
2.
Rev Soc Bras Med Trop ; 52: e20190237, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508785

RESUMO

INTRODUCTION: The increased use of colistin against infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa has resulted in colistin resistance. The purpose of this study was to detect plasmid-mediated mcr-1 gene in colistin-resistant A. baumannii and P. aeruginosa isolates. METHODS: A total of 146 clinical isolates of A. baumannii (n = 62) and P. aeruginosa (n = 84) were collected from the four largest tertiary care hospitals in Peshawar, Pakistan. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby-Baur disc diffusion method. The minimum inhibitory concentration (MIC) of colistin in all isolates was phenotypically performed using dilution methods. mcr-1 gene was detected through polymerase chain reaction and the nucleotide sequence of amplicon was determined using Sanger sequencing. RESULTS: Approximately 96.7% A. baumannii and 83.3% P. aeruginosa isolates were resistant to multiple antibiotics. Colistin resistance was found in 9.6% (6/62) of A. baumannii and 11.9% (10/84) of P. aeruginosa isolates. Among 16 colistin resistant isolates, the mcr-1 gene was detected in one A. baumannii (1.61% of total isolates; 16.6% of colistin resistant isolates) and one P. aeruginosa strain (1.19% of total isolates; 10% of colistin resistant isolates). Nucleotide BLAST showed 98-99% sequence similarity to sequences of the mcr-1 gene in GenBank. CONCLUSIONS: Our study reports, for the first time, the emergence of plasmid-mediated mcr-1-encoded colistin resistance in multidrug resistant strains of A. baumannii and P. aeruginosa. Further large scales studies are recommended to investigate the prevalence of this mode of resistance in these highly pathogenic bacteria.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Paquistão , Plasmídeos/genética , Pseudomonas aeruginosa/efeitos dos fármacos
3.
Ann Agric Environ Med ; 26(3): 405-408, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559794

RESUMO

INTRODUCTION: Carbapenemase-producing Enterobacteriaceae have spread rapidly through the countries and continents to become a global concern. One of the main reservoirs of NDM-1 positive strains from the Enterobacteriaceae family is the Indian subcontinent (Bangladesh, Pakistan, India). MATERIAL AND METHODS: During June 2017 - June 2018, rectal swab samples were collected routinely in all patients returning to Poland from South and South-East Asia. During molecular examinations gene blaNDM-1 encoding NDM-1 carbapenemase was detected. RESULTS: 31 patients were examined after returning to Poland from a trip to South and South-East Asia. The presence of New Delhi Metallo-ß-lactamase-1 producing Escherichia coli and Klebsiella pneumoniae was confirmed in three patients (9.7%) returning to Poland from travels to India. All the positive patients were hospitalized during the trip in a New Delhi hospital. CONCLUSIONS: Digestive tract carriage of NDM in a group of Polish travelers is a significant health and epidemiological problem. The study confirms the necessity for screening for carbapenemase-producing Enterobacteriaceae (CPE), particularly among travellers. Rectal swabs should be collected in every case of patients returning from international trips, and the possibility of environment-associated infections should be emphasized.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/enzimologia , Klebsiella pneumoniae/enzimologia , Viagem , beta-Lactamases/metabolismo , Adulto , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Humanos , Índia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Polônia , beta-Lactamases/genética
4.
Gene ; 720: 144082, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476406

RESUMO

The enzyme ß-Ketoacyl ACP synthase I (KasA) is a potent drug target in mycolic acid pathway of Mycobacterium tuberculosis (Mtb). In the present study, we investigated the structural dynamics of wild-type (WT) and mutants KasA (D66N, G269S, G312S, and F413L) in both monomer and dimer form to provide insight into protein structural stability. To gain better understanding of structural flexibility of KasA, combined molecular dynamics and essential dynamics were employed to analyze the conformational changes induced by non-active site mutations. The results confirm that non-active site mutations lower the structural stability in dimer KasA as compared to WT. The protein network topology and close residue interactions of WT and mutant residues of KasA have been predicted through residue interaction network analysis (RIN). Non-active site mutations distort RIN architecture and subsequently affect the drug binding landscape. T-pad associated with mode vector analysis comprehensively pronounces the structural impact caused by non-active site mutations. It also identified the critical fluctuating residues present in the gate segment (GS) region (115-147). The non-active site mutations altered the structural stability of the mutant protein structures, and these mutations may be a cause for the resistance mechanism of KasA against anti-tuberculosis drugs. Further, it is observed that dimer mutant KasA proteins display much more structural flexibility than WT at the ligand binding site which is evident from the binding site analysis and hydrogen bond interaction patterns. This study provides a better understanding of the structural dynamic behaviour of KasA mutants, thereby facilitating the need to find a novel and potent inhibitor against Mtb.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteínas de Bactérias/química , Isoenzimas/química , Proteínas Mutantes/química , Mutação , Mycobacterium tuberculosis/enzimologia , Tuberculose/microbiologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteínas de Bactérias/genética , Isoenzimas/genética , Simulação de Dinâmica Molecular , Proteínas Mutantes/genética , Conformação Proteica , Tuberculose/genética , Tuberculose/metabolismo
5.
Gene ; 720: 144094, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476407

RESUMO

Fourteen different insertion sequences belonging to seven families were identified in the genome of Streptococcus agalactiae. Among them, IS1548, a mobile element of the ISAs1 family, was linked to clonal complex (CC) 19 strains associated with neonatal meningitis and endocarditis. IS1548 impacts S. agalactiae in two reported ways: i) inactivation of virulence genes by insertion in an open reading frame (e.g. hylB or cpsD), ii) positive modulation of the expression of a downstream gene by insertion in an intergenic region (e.g. lmb). We previously identified an unknown integration site of IS1548 in the intergenic region between the folK and the murB genes involved in folate and peptidoglycan biosynthesis, respectively. In this work, we analyzed the prevalence of IS1548 in a large collection of nine hundred and eleven S. agalactiae strains. IS1548 positive strains belong to twenty-nine different sequence types and to ten CCs. The majority of them were, however, clustered within sequence type 19 and sequence type 22, belonging to CC19 and CC22, respectively. In contrast, IS1548 targets the folK-murB intergenic region exclusively in CC19 strains. We evaluated the impact of the insertion of IS1548 on the expression of murB by locating transcriptional promoters influencing its expression in the presence or absence of IS1548 and by comparative ß-galactosidase transcriptional fusion assays. We found that in the absence of IS1548, genes involved in folate biosynthesis are co-transcribed with murB. As it was postulated that a folic acid mediated reaction may be involved in cell wall synthesis, this co-transcription could be necessary to synchronize these two processes. The insertion of IS1548 in the folK-murB intergenic region disrupt this co-transcription. Interestingly, we located a promoter at the right end of IS1548 that is able to initiate additional transcripts of murB. The insertion of IS1548 in this region has thus a dual and divergent impact on the expression of murB. By comparative ß-galactosidase transcriptional fusion assays, we showed that, consequently, the overall impact of the insertion of IS1548 results in a minor decrease of murB gene transcription. This study provides new insights into gene expression effects mediated by IS1548 in S. agalactiae.


Assuntos
Proteínas de Bactérias/genética , DNA Intergênico , Regulação Bacteriana da Expressão Gênica , Sequências Repetitivas Dispersas , Mutagênese Insercional , Peptidoglicano/biossíntese , Streptococcus agalactiae/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Regiões Promotoras Genéticas , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/crescimento & desenvolvimento , Streptococcus agalactiae/metabolismo
6.
Pestic Biochem Physiol ; 159: 1-8, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400771

RESUMO

We examined the molecular regulation of porphyrin biosynthesis and protective responses in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum Fe-chelatase (BjFeCh) after treatment with acifluorfen (AF). During the photodynamic stress imposed by AF, transcript levels of BjFeCh in transgenic plants increased greatly; moreover, transcript levels of OsFeCh2 remained almost constant, whereas in wild type (WT) plants they were considerably down-regulated. In the heme branch, transgenic plants exhibited greater levels of OsFC and HO transcripts than WT plants in the untreated stems as well as in the AF-treated leaves and stems. Both WT and transgenic plants treated with AF substantially decreased transcript levels for all the genes in the chlorophyll branch, with less decline in transgenic plants. After AF treatment, ascorbate (Asc) content and the redox Asc state greatly decreased in leaves of WT plants; however, in transgenic plants both parameters remained constant in leaves and the Asc redox state increased by 20% in stems. In response to AF, the leaves of WT plants greatly up-regulated CatA, CatB, and GST compared to those of transgenic plants, whereas, in the stems, transgenic plants showed higher levels of CatA, CatC, APXb, BCH, and VDE. Photochemical quenching, qP, was considerably dropped by 31% and 18% in WT and transgenic plants, respectively in response to AF, whereas non-radiative energy dissipation through non-photochemical quenching increased by 77% and 38% in WT and transgenic plants, respectively. Transgenic plants treated with AF exhibited higher transcript levels of nucleus-encoded photosynthetic genes, Lhcb1 and Lhcb6, as well as levels of Lhcb6 protein compared to those of WT plants. Our study demonstrates that expression of BjFeCh in transgenic plants influences not only the regulation of porphyrin biosynthesis through maintaining higher levels of gene expression in the heme branch, but also the Asc redox function during photodynamic stress caused by AF.


Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/enzimologia , Ferroquelatase/metabolismo , Nitrobenzoatos/farmacologia , Oryza/metabolismo , Porfirinas/biossíntese , Proteínas de Bactérias/genética , Ferroquelatase/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Plantas Geneticamente Modificadas
7.
Epidemiol Mikrobiol Imunol ; 68(2): 99-102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31398983

RESUMO

The increasing incidence of multiresistant bacterial strains is currently a serious health concern. These pathogens are often the cause of nosocomial infections with limited treatment options and high fatality rates. A case report is presented of an uncommon detection of four different species (Citrobacter freundii, Klebsiella pneumoniae, Escherichia coli, and Morganella morganii) producing the same type of carbapenemase, KPC-2, in a female patient during her complicated long-term hospital stay. Resistance was probably spread to other species by horizontal transmission of plasmids carrying the blaKPC-2 genes. The implementation of strict anti-epidemic measures prevented further spread of these carbapenem-resistant bacteria.


Assuntos
Antibacterianos , Bactérias , Infecções Bacterianas , Infecção Hospitalar , beta-Lactamases , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Coinfecção/microbiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana/genética , Feminino , Transferência Genética Horizontal , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , beta-Lactamases/genética
8.
World J Microbiol Biotechnol ; 35(8): 127, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375931

RESUMO

Aeromonas hydrophila is a Gram-negative bacterium that causes serious infections in aquaculture and exhibits significant multidrug resistance. The LysR-type transcriptional regulator (LTTR) family proteins are a well-known group of transcriptional regulators involved in diverse physiological functions. However, the role of LTTRs in the regulation of bacterial resistance to antibiotics is still largely unknown. In this study, to further investigate the role of four putative LTTR family proteins (A0KIU1, A0KJ82, A0KPK0, and A0KQ63) in antibiotic resistance in A. hydrophila, their genes were cloned and overexpressed in engineered Escherichia coli. After the optimization of experimental conditions including incubation time, temperature, and IPTG concentration, these proteins were successfully purified, and their specific antibodies against mice were obtained. Using western blot analysis, we found that these LTTR family proteins were downregulated in A. hydrophila following antibiotic treatment, indicating that they may be involved in the regulation of antibiotic resistance. Additionally, minimum inhibitory concentration (MIC) assays of chloramphenicol (CM), chlortetracycline (CTC), ciprofloxacin (CF), furazolidone (FZ), and balofloxacin (BF) in E. coli showed that overexpression of these LTTRs led to increased sensitivity to several antibiotics. To further validate their functional role in antibiotic resistance, we demonstrated that bacteria with loss of A0KQ63 (ΔAHA_3980) exhibited multi-drug resistance properties. Our results indicate that these LTTR family proteins may play an important role in the antibiotic resistance of A. hydrophila, and the that underlying mechanisms controlling antibiotic resistance should be further investigated.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Reguladores , Fatores de Transcrição/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Perfilação da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Fatores de Transcrição/análise , Fatores de Transcrição/genética
9.
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
10.
Microbiol Res ; 227: 126309, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421713

RESUMO

The phosphorus availability in soil ranged from <0.01 to 1 ppm and found limiting for the utilization by plants. Hence, phosphate solubilizing bacteria (PSB) proficiently fulfill the phosphorus requirement of plants in an eco-friendly manner. The PSB encounter dynamic and challenging environmental conditions viz., high temperature, osmotic, acid, and climatic changes often hamper their activity and proficiency. The modern trend is shifting from isolation of the PSB to their genetic potentials and genome annotation not only for their better performance in the field trials but also to study their ability to cope up with stresses. In order to withstand environmental stress, bacteria need to restructure its metabolic network to ensure its survival. Pi starving condition response regulator (PhoB) and the mediator of stringent stress response alarmone (p)ppGpp known to regulate the global regulatory network of bacteria to provide balanced physiology under various stress condition. The current review discusses the global regulation and crosstalk of genes involved in phosphorus homeostasis, solubilization, and various stress response to fine tune the bacterial physiology. The knowledge of these network crosstalk help bacteria to respond efficiently to the challenging environmental parameters, and their physiological plasticity lead us to develop proficient long-lasting consortia for plant growth promotion.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Estresse Fisiológico , Bactérias/genética , Plasticidade Celular , Redes Reguladoras de Genes , Homeostase , Redes e Vias Metabólicas , Anotação de Sequência Molecular , Nitrogênio , Fosfatos/metabolismo , Desenvolvimento Vegetal , Plantas , Solo , Estresse Fisiológico/genética
11.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
12.
BMC Infect Dis ; 19(1): 678, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370804

RESUMO

BACKGROUND: Fecal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is a risk factor for bacterial translocation resulting in subsequent endogenous infections. The purpose of this study is to investigate the prevalence of CRE strains colonization in stool samples of outpatient in a tertiary pediatric hospital of Shanghai, China. METHODS: In a retrospective study, fecal samples were consecutively obtained from patients in 2016 and screening test for CRE was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and ß-lactamases were characterized by polymerase chain reaction (PCR) assays and DNA sequencing. Multilocus sequence typing (MLST) was performed for the genetic relationships of the isolates. RESULTS: A total of 880 fecal samples were included for this screening test and 32 CRE strains were identified in 32 non-duplicate fecal samples from 32 children (1.3 ± 1.5 years), with a carriage rate of 3.6%. These strains mainly distributed in Klebsiella pnuemoniae (37.5%) and Escherichia coli (37.5%). All CRE strains showed high resistance to most of the routinely used antibiotics (> 90%) except for polymyxin B and tigecycline. The blaNDM gene was the major carbapenemase gene harbored by gastrointestinal CRE strains, followed by blaKPC-2, blaIMP-26, and blaIMP-4. Other ß-Lactamase genes including blaCTX-M, blaSHV, blaTEM-1, and blaDHA-1 were also detected. MLST analysis revealed that various sequence types (STs) were detected in these strains, with ST11 and ST37 being more prevalent in K.pneumoniae and ST101 in E.coli. CONCLUSIONS: This study revealed the prevalence of CRE fecal carriage in children from outpatient and urgent implementation of infection control measure should be conducted to limit the spread of CRE strains.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Infecções por Enterobacteriaceae/epidemiologia , Fezes/microbiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Pré-Escolar , China/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/genética , Feminino , Humanos , Lactente , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Pacientes Ambulatoriais/estatística & dados numéricos , Prevalência , Estudos Retrospectivos , beta-Lactamases/genética
13.
J Agric Food Chem ; 67(35): 9868-9876, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389242

RESUMO

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.


Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Thermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Alinhamento de Sequência , Thermus/química , Thermus/genética
14.
Rev Bras Parasitol Vet ; 28(3): 451-457, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390434

RESUMO

The msp4 gene of A. marginale is unicodon, stable and mostly homogeneous, being considered as a useful marker for phylogeographic characterization of this bacterium. The objective of this work was to analyze the phylogeography of A. marginale based on the msp4 gene in beef cattle from the Brazilian Pantanal, compared to those found in other regions worldwide. The blood samples investigated were collected from 400 animals (200 cows and 200 calves) reared in five extensive breeding farms in this region. The results indicated that of the evaluated samples, 56.75% (227/400) were positive for A. marginale based on the msp1ß gene by quantitatitve PCR (qPCR), while 8.37% (19/227) were positive for the msp4 gene in the conventional PCR. In the Network distance analysis, 14 sequences from the Brazilian Pantanal were grouped into a single group with those from Thailand, India, Spain, Colombia, Parana (Brazil), Mexico, Portugal, Argentina, China, Venezuela, Australia, Italy and Minas Gerais (Brazil). Among 68 sequences from Brazil and the world, 15 genotypes were present while genotype number one (#1) was the most distributed worldwide. Both Splitstree and network analyses showed that the A. marginale msp4 sequences detected in beef cattle from the Brazilian Pantanal showed low polymorphism, with the formation of one genogroup phylogenetically related to those found in ruminants from South and Central America, Europe, and Asia.


Assuntos
Anaplasma marginale/genética , Proteínas de Bactérias/genética , Bovinos/microbiologia , Proteínas de Membrana/genética , Filogeografia/métodos , Américas , Sequência de Aminoácidos , Anaplasma marginale/isolamento & purificação , Animais , Ásia , Brasil , DNA Bacteriano/genética , Europa (Continente) , Feminino , Genótipo , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase
15.
Klin Mikrobiol Infekc Lek ; 25(1): 12-15, 2019 Mar.
Artigo em Tcheco | MEDLINE | ID: mdl-31266088

RESUMO

OBJECTIVE: Clostridioides difficile (formerly Clostridium difficile) is one of the main pathogens causing nosocomial infections today. It colonizes the intestines of patients receiving antibiotic therapy, causing unpleasant or even life-threatening conditions (diarrhea, toxic megacolon). Rapid and correct detection of strain toxigenicity is essential for treatment and isolation of patients. Simplexa C. difficile Direct Kit is a real-time PCR kit detecting the tcdB gene of C. difficile. The kit does not require DNA isolation; stool eluates are directly used for the reaction. The study aimed to verify the analytical properties of the kit by its comparison with culture and in-house multiplex PCR methods. MATERIAL AND METHODS: A total of 164 stool samples were prospectively tested using two immunoenzymatic kits (C. diff Quik Chek Complete and LIAISON C. difficile GDH, Toxins AandB). In 39 samples, the results were discrepant or unclear (GDH+TOX-). These samples were tested using in-house multiplex PCR and the Simplexa kit. RESULTS: The Simplexa kit had 94.7% sensitivity, 100% specificity, 100% positive predictive value and 95.2% negative predictive value. These parameters were calculated from the numbers of true-/false-positive and true-/false-negative results. True results were determined based on the consensus of culture and in-house multiplex PCR results. Another outcome of the study was comparison of the Quik Chek and LIAISON kits. CONCLUSION: The analytical properties of the Simplexa kit were tested on 39 samples. These samples were selected for their unclear (GDH+TOX-) or discrepant results yielded by immunoenzymatic methods. Compared with culture and subsequent in-house PCR detection of the tcdB gene, the Simplexa kit showed properties declared by the manufacturer. An important advantage of the kit was the absence inhibitions when stool eluates were directly used for PCR reactions.


Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Infecções por Clostridium , Clostridium difficile , Kit de Reagentes para Diagnóstico , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Infecções por Clostridium/diagnóstico , Clostridium difficile/genética , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade
16.
Gene ; 715: 144008, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31362038

RESUMO

Deinococcus radiodurans is a model microorganism used for studies on DNA repair and antioxidation due to its extraordinary tolerance to ionizing radiation and other DNA-damaging agents. Various transcriptome analyses have revealed that hundreds of genes are induced and that many other genes are repressed during recovery of D. radiodurans following irradiation, suggesting that gene regulation is of great importance for the extreme resistance of this microorganism to ionizing radiation. In this article, we focus on some reported strategies that are employed by D. radiodurans to regulate the genes implicated in its extreme tolerance to ionizing radiation for a comprehensive understanding of the reasons this bacterium can survive such extraordinary stress. We expect this review to shed light on potential radioprotective agents and applications for use in a range of fields.


Assuntos
Reparo do DNA/efeitos da radiação , DNA Bacteriano/metabolismo , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Tolerância a Radiação , Radiação Ionizante , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , DNA Bacteriano/genética , Deinococcus/genética
17.
J Agric Food Chem ; 67(31): 8548-8558, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31266305

RESUMO

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.


Assuntos
Proteínas de Bactérias/química , Esterases/química , Ésteres/química , Gordonia (Bactéria)/enzimologia , Ácidos Ftálicos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biodegradação Ambiental , Dietilexilftalato/química , Dietilexilftalato/metabolismo , Esterases/genética , Esterases/metabolismo , Ésteres/metabolismo , Gordonia (Bactéria)/genética , Gordonia (Bactéria)/isolamento & purificação , Gordonia (Bactéria)/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Alinhamento de Sequência , Microbiologia do Solo
18.
J Agric Food Chem ; 67(31): 8527-8535, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298526

RESUMO

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.


Assuntos
Proteínas de Bactérias/química , Corynebacterium glutamicum/enzimologia , Cetol-Ácido Redutoisomerase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Cristalografia por Raios X , Cetol-Ácido Redutoisomerase/genética , Cetol-Ácido Redutoisomerase/metabolismo , Metais/química , Metais/metabolismo , Simulação de Acoplamento Molecular , NADP/química , NADP/metabolismo
19.
J Agric Food Chem ; 67(31): 8581-8589, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31321975

RESUMO

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.


Assuntos
4-Hidroxicumarinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Policetídeo Sintases/genética , Quinolinas/metabolismo , 4-Hidroxicumarinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Photorhabdus/enzimologia , Photorhabdus/genética , Policetídeo Sintases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Quinolinas/química , ortoaminobenzoatos/metabolismo
20.
J Med Microbiol ; 68(8): 1173-1188, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31268417

RESUMO

PURPOSE: Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. METHODOLOGY: A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. RESULTS: The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. CONCLUSIONS: These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.


Assuntos
Cápsulas Bacterianas/genética , Tipagem Molecular/métodos , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Genoma Bacteriano/genética , Humanos , Infecções Pneumocócicas/microbiologia , Proteínas Tirosina Fosfatases/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Sorogrupo , Sorotipagem/normas , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Fluxo de Trabalho
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