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1.
mBio ; 12(2)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653882

RESUMO

Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulcerans IMPORTANCE Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays. To facilitate mycobacterial research, we have created a biological and bioinformatic resource (https://msrdb.org/) using Mycobacterium smegmatis as a model organism. The resource focuses specifically on 1,153 proteins that are highly conserved across the mycobacterial genus and, therefore, likely perform conserved mycobacterial core functions. Thus, functional insights from the MSR will apply to all mycobacterial species. We believe that the availability of this mycobacterial systems resource will accelerate research throughout the mycobacterial research community.


Assuntos
Genes Bacterianos , Mycobacterium smegmatis/genética , Mycobacterium/genética , Pesquisa , Biologia Computacional , Biblioteca Gênica , Mycobacterium/classificação , Mycobacterium/patogenicidade , Mycobacterium smegmatis/crescimento & desenvolvimento
2.
Med Sci Monit ; 26: e920879, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31986127

RESUMO

BACKGROUND Debaryomyces hansenii exhibits a therapeutic effect on antibiotic-associated diarrhea (AAD) by promoting the growth of beneficial intestinal bacteria. Previous research has reported that AAD involves not only dysbacteriosis but also dysfunction of the activity of intestinal enzymes (such as lactase). Enzyme activities can be influenced by many other factors, such as gene expression. The present study showed that D. hansenii has a curative effect on AAD at the lactase gene level. MATERIAL AND METHODS The effect of D. hansenii on the lactase gene from intestinal bacteria in AAD mice was investigated. The diarrhea model was established with a gentamycin sulfate and cefradine capsule mixture. The antibiotic mixture (23.33 mL·kg⁻¹·day⁻¹) was intragastrically administered for 5 days. Subsequently, half of the diarrhea mice were treated with D. hansenii twice a day for 3 days while the other mice were intragastrically administered with the same volume of distilled water. Next, the intestinal contents were collected, and metagenomic DNA was extracted for high-throughput sequencing analysis. RESULTS The Chao1 and Shannon indices decreased significantly following treatment with D. hansenii (P<0.01 and P<0.05, respectively). Moreover, the clusters in the D. hansenii group mice were quite different from those in the normal group mice and model group mice. Following treatment with D. hansenii, the quantity of lactase genes in Enterobacter sp. 638 and Modestobacter increased markedly, and the richness of intestinal bacterial lactase genes in Fretibacterium recovered. CONCLUSIONS D. hansenii altered the lactase-producing bacterial community structure and promoted the growth of several critical lactase-producing bacteria, such as Enterobacter sp. 638 and Modestobacter.


Assuntos
Antibacterianos/uso terapêutico , Bactérias/genética , Biodiversidade , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Genes Bacterianos , Intestinos/microbiologia , Lactase/genética , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Sequência de Bases , Feminino , Masculino , Camundongos
3.
PLoS Negl Trop Dis ; 13(12): e0007946, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31881061

RESUMO

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.


Assuntos
Farmacorresistência Bacteriana , Técnicas de Genotipagem/métodos , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , China , Feminino , Genes Bacterianos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/isolamento & purificação , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Adulto Jovem
4.
PLoS One ; 14(12): e0225802, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31809511

RESUMO

AIM: The current study aimed to investigate the effects of Debaryomyces hansenii on the diversity of bacterial lactase gene in the intestinal mucosa of antibiotic-associated diarrhea (AAD) mice. METHODS: Eighteen mice were randomly divided into three groups (6 mice per group): healthy control group, diarrhea model group and D. hansenii treatment group. The antibiotic-associated diarrhea model was established by intragastric administration with a mixture of cephradine and gentamicin sulfate (23.33 mL·kg-1·d-1) twice a day for 5 days continuously. After establishing the AAD model, the mice in the D. hansenii treatment group were gavaged with D. hansenii for three days, while other groups were gavaged with distilled water. Then, the intestinal mucosa of all three groups was collected and DNA was extracted in an aseptic environment for the following analysis. RESULTS: The difference in the richness and homogeneity of the bacterial lactase gene among all samples were inapparent, as the difference in the Chao1, ACE, Simpson and Shannon indices among the three groups were insignificant (P>0.05). NMDS analysis also showed that the distance of the samples among the three groups was unobvious. Furthermore, the bacterial lactase gene in the mucosa mainly originated from Actinobacteria, Firmicutes and Proteobacteria. Compared with the healthy control group, the abundance of lactase genes originating from Cupriavidus, Lysobacter, Citrobacter, Enterobacter and Pseudomonas was increased in the D. hansenii treatment group, while the lactase gene from Acidovorax and Stenotrophomonas decreased (p < 0.01 or p < 0.05) in the diarrhea model group and the D. hansenii treatment group. CONCLUSION: D. hansenii was capable of improving the growth of some key lactase-producing bacteria like Deinococcus, Cupriavidus and Lysobacter for treating AAD.


Assuntos
Antibacterianos/efeitos adversos , Diarreia/induzido quimicamente , Diarreia/microbiologia , Genes Bacterianos , Variação Genética , Mucosa Intestinal/microbiologia , Lactase/genética , Saccharomycetales/fisiologia , Animais , Sequência de Bases , Biodiversidade , Feminino , Masculino , Camundongos , Filogenia , Análise de Componente Principal
5.
Anal Bioanal Chem ; 411(30): 7997-8009, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31732785

RESUMO

A common technique used to differentiate bacterial species and to determine evolutionary relationships is sequencing their 16S ribosomal RNA genes. However, this method fails when organisms exhibit high similarity in these sequences. Two such strains that have identical 16S rRNA sequences are Mycobacterium indicus pranii (MIP) and Mycobacterium intracellulare. MIP is of significance as it is used as an adjuvant for protection against tuberculosis and leprosy; in addition, it shows potent anti-cancer activity. On the other hand, M. intracellulare is an opportunistic pathogen and causes severe respiratory infections in AIDS patients. It is important to differentiate these two bacterial species as they co-exist in immuno-compromised individuals. To unambiguously distinguish these two closely related bacterial strains, we employed Raman and resonance Raman spectroscopy in conjunction with multivariate statistical tools. Phenotypic profiling for these bacterial species was performed in a kinetic manner. Differences were observed in the mycolic acid profile and carotenoid pigments to show that MIP is biochemically distinct from M. intracellulare. Resonance Raman studies confirmed that carotenoids were produced by both MIP as well as M. intracellulare, though the latter produced higher amounts. Overall, this study demonstrates the potential of Raman spectroscopy in differentiating two closely related mycobacterial strains. Graphical abstract.


Assuntos
Complexo Mycobacterium avium/classificação , Mycobacterium/classificação , Análise Espectral Raman/métodos , Genes Bacterianos , Mycobacterium/genética , Complexo Mycobacterium avium/genética , RNA Ribossômico 16S/genética , Especificidade da Espécie
6.
N Biotechnol ; 52: 60-68, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31096013

RESUMO

Bacterial nanocellulose (BNC) produced by Komagataeibacter hansenii has received significant attention due to its unique supernetwork structure and properties. It is nevertheless necessary to modify bacterial nanocellulose to achieve materials with desired properties and thus with broader areas of application. The aim here was to influence the 3D structure of BNC by genetic modification of the cellulose producing K. hansenii strain ATCC 53582. Two genes encoding proteins with homology to the MotA and MotB proteins, which participate in motility and energy transfer, were selected for our studies. A disruption mutant of one or both genes and their respective complementation mutants were created. The phenotype analysis of the disruption mutants showed a reduction in motility, which resulted in higher compaction of nanocellulose fibers and improvement in their mechanical properties. The data strongly suggest that these genes play an important role in the formation of BNC membrane by Komagataeibacter species.


Assuntos
Acetobacteraceae/citologia , Acetobacteraceae/genética , Celulose/química , Genes Bacterianos , Mutação/genética , Nanopartículas/química , Acetobacteraceae/ultraestrutura , Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Movimento , Homologia de Sequência de Aminoácidos , Espectroscopia de Infravermelho com Transformada de Fourier
7.
Tuberculosis (Edinb) ; 115: 63-66, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30948178

RESUMO

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Cisteína/biossíntese , Genes Bacterianos/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Vias Biossintéticas/genética , Cisteína/genética , Farmacorresistência Bacteriana/genética , Deleção de Genes , Hansenostáticos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estresse Oxidativo/efeitos dos fármacos
8.
Int J Mol Sci ; 20(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818787

RESUMO

In dealing with Mycobacterium tuberculosis, the causative agent of the deadliest human disease-tuberculosis (TB)-utilization of cholesterol as a carbon source indicates the possibility of using cholesterol catabolic genes/proteins as novel drug targets. However, studies on cholesterol catabolism in mycobacterial species are scarce, and the number of mycobacterial species utilizing cholesterol as a carbon source is unknown. The availability of a large number of mycobacterial species' genomic data affords an opportunity to explore and predict mycobacterial species' ability to utilize cholesterol employing in silico methods. In this study, comprehensive comparative analysis of cholesterol catabolic genes/proteins in 93 mycobacterial species was achieved by deducing a comprehensive cholesterol catabolic pathway, developing a software tool for extracting homologous protein data and using protein structure and functional data. Based on the presence of cholesterol catabolic homologous proteins proven or predicted to be either essential or specifically required for the growth of M. tuberculosis H37Rv on cholesterol, we predict that among 93 mycobacterial species, 51 species will be able to utilize cholesterol as a carbon source. This study's predictions need further experimental validation and the results should be taken as a source of information on cholesterol catabolism and genes/proteins involved in this process among mycobacterial species.


Assuntos
Proteínas de Bactérias/genética , Colesterol/metabolismo , Genes Bacterianos , Mycobacterium/genética , Animais , Proteínas de Bactérias/metabolismo , Colesterol/química , Genes Essenciais , Macrófagos/metabolismo , Macrófagos/microbiologia , Redes e Vias Metabólicas , Camundongos , Viabilidade Microbiana/genética , Mycobacterium/crescimento & desenvolvimento , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Especificidade da Espécie
9.
Biochem Biophys Res Commun ; 509(3): 779-783, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30616886

RESUMO

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Mycobacterium leprae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Alquilação/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano/efeitos dos fármacos , Humanos , Hanseníase/microbiologia , Modelos Moleculares , Mycobacterium leprae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
10.
Microbiologyopen ; 8(5): e00731, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30365246

RESUMO

Komagataeibacter species are well-recognized bionanocellulose (BNC) producers. This bacterial genus, formerly assigned to Gluconacetobacter, is known for its phenotypic diversity manifested by strain-dependent carbon source preference, BNC production rate, pellicle structure, and strain stability. Here, we performed a comparative study of nineteen Komagataeibacter genomes, three of which were newly contributed in this work. We defined the core genome of the genus, clarified phylogenetic relationships among strains, and provided genetic evidence for the distinction between the two major clades, the K. xylinus and the K. hansenii. We found genomic traits, which likely contribute to the phenotypic diversity between the Komagataeibacter strains. These features include genome flexibility, carbohydrate uptake and regulation of its metabolism, exopolysaccharides synthesis, and the c-di-GMP signaling network. In addition, this work provides a comprehensive functional annotation of carbohydrate metabolism pathways, such as those related to glucose, glycerol, acetan, levan, and cellulose. Findings of this multi-genomic study expand understanding of the genetic variation within the Komagataeibacter genus and facilitate exploiting of its full potential for bionanocellulose production at the industrial scale.


Assuntos
Acetobacteraceae/genética , Celulose/metabolismo , Genoma Bacteriano , Genômica , Acetobacteraceae/classificação , Acetobacteraceae/metabolismo , Genes Bacterianos , Variação Genética , Nanopartículas/metabolismo , Filogenia , Sintenia
11.
Syst Appl Microbiol ; 41(6): 581-592, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30177404

RESUMO

Strains T5K1 and AV446 isolated from apple cider vinegars during a submerged vinegar production in two separate vinegar facilities showed 94% 16S rRNA gene similarity to its closest neighbors Komagataeibacter maltaceti LMG 1529T and Gluconacetobacter entanii LTH 4560T. Further phylogenetic and phenotypic characterizations indicated that the isolates belonged to a novel species of the Komagataeibacter genus. Comparison based on 16S-23S rRNA gene ITS sequences and concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, grouped both strains to a single phylogenetic cluster well separated from the other species of the Komagataeibacter genus. Average nucleotide identity of T5K1 and AV446 draft genome sequences compared to other Komagataeibacter type strains was below 94% and at the same time, in-silico DNA-DNA hybridization was below 70%. Both strains on the other hand showed approximately 98% (average nucleotide identity) and 87% (in silico DNA-DNA hybridization) similarity to each other. Strains T5K1 and AV446 can be differentiated from other Komagataeibacter type strains based on their ability to produce 2-keto-d-gluconic acid and at the same time inability to produce 5-keto-d-gluconic acid. Furthermore, strains of the new species do not grow on Asai medium supplemented with d-glucose or d-mannitol. The growth is also absent (T5K1) or weak (AV446) on Hoyer-Frateur medium supplemented with afore mentioned sugars. Both strains produce cellulose. In addition, draft genome analysis revealed that strains T5K1 and AV446 possess genes involved in the synthesis of acetan-like extracellular heteropolysaccharide. We propose the name Komagataeibacter pomaceti sp. nov. for the new species with LMG 30150T [=CCM 8723T=ZIM B1029T] as the type strain. Data collected in this study and in a previous study also revealed that Komagataeibacter kombuchae is a later heterotypic synonym of Komagataeibacter hansenii.


Assuntos
Ácido Acético , Acetobacteraceae/classificação , Microbiologia de Alimentos , Filogenia , Acetobacteraceae/genética , Acetobacteraceae/isolamento & purificação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Técnicas de Tipagem Bacteriana , Sequência de Bases , DNA Bacteriano/genética , Genes Bacterianos , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Eslovênia
12.
Emerg Infect Dis ; 24(8): 1584-1585, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30016255

RESUMO

Skin biopsies from US leprosy patients were tested for mutations associated with drug resistance. Dapsone resistance was found in 4 of 6 biopsies from American Samoa patients. No resistance was observed in patients from other origins. The high rate of dapsone resistance in patients from American Samoa warrants further investigation.


Assuntos
Dapsona/uso terapêutico , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Samoa Americana , Biópsia , Clofazimina/uso terapêutico , Esquema de Medicação , Humanos , Hanseníase/diagnóstico , Hanseníase/microbiologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium leprae/classificação , Mycobacterium leprae/isolamento & purificação , Rifampina/uso terapêutico , Pele/efeitos dos fármacos , Pele/microbiologia
13.
Appl Environ Microbiol ; 83(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28159794

RESUMO

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.


Assuntos
Actinomycetales/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Rifamicinas/biossíntese , Transativadores/genética , Transativadores/metabolismo , Actinomycetales/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Elementos de DNA Transponíveis , Técnicas de Inativação de Genes , Recombinação Homóloga , Família Multigênica , Mutação , Fases de Leitura Aberta , Óperon , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos
14.
Enzyme Microb Technol ; 82: 58-65, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26672449

RESUMO

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.


Assuntos
Proteínas de Bactérias/química , Gluconacetobacter/enzimologia , Glucosiltransferases/química , Complexos Multienzimáticos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Celulose/biossíntese , Centrifugação , Clonagem Molecular , Dimerização , Genes Bacterianos , Gluconacetobacter/genética , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Glucosiltransferases/metabolismo , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mutagênese Insercional , Subunidades Proteicas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
J Antimicrob Chemother ; 70(9): 2507-10, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26045528

RESUMO

OBJECTIVES: Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. METHODS: We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. RESULTS: We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. CONCLUSIONS: Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mutação Puntual , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Genes Bacterianos , Genoma Bacteriano , Humanos , Análise de Sequência de DNA
17.
PLoS One ; 10(5): e0124282, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25970602

RESUMO

We have examined a 5th to 6th century inhumation from Great Chesterford, Essex, UK. The incomplete remains are those of a young male, aged around 21-35 years at death. The remains show osteological evidence of lepromatous leprosy (LL) and this was confirmed by lipid biomarker analysis and ancient DNA (aDNA) analysis, which provided evidence for both multi-copy and single copy loci from the Mycobacterium leprae genome. Genotyping showed the strain belonged to the 3I lineage, but the Great Chesterford isolate appeared to be ancestral to 3I strains found in later medieval cases in southern Britain and also continental Europe. While a number of contemporaneous cases exist, at present, this case of leprosy is the earliest radiocarbon dated case in Britain confirmed by both aDNA and lipid biomarkers. Importantly, Strontium and Oxygen isotope analysis suggest that the individual is likely to have originated from outside Britain. This potentially sheds light on the origins of the strain in Britain and its subsequent spread to other parts of the world, including the Americas where the 3I lineage of M. leprae is still found in some southern states of America.


Assuntos
Genes Bacterianos , Genoma Bacteriano , Hanseníase Virchowiana/história , Mycobacterium leprae/genética , Adulto , Radioisótopos de Carbono , Fíbula/microbiologia , Fíbula/patologia , Genótipo , História Medieval , Humanos , Hanseníase Virchowiana/microbiologia , Hanseníase Virchowiana/patologia , Lipídeos/isolamento & purificação , Masculino , Ossos do Metatarso/microbiologia , Ossos do Metatarso/patologia , Mycobacterium leprae/classificação , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/metabolismo , Osteologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Tálus/microbiologia , Tálus/patologia , Reino Unido
18.
Emerg Infect Dis ; 20(12): 2111-4, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25417797

RESUMO

Bovine nodular thelitis is a granulomatous dermatitis associated with infection with acid-fast bacteria. To identify the mycobacterium responsible for this infection, we conducted phylogenetic investigations based on partial sequencing of 6 genes. These bacteria were identified as an undescribed Mycobacterium species that was phylogenetically related to M. leprae and M. lepromatosis.


Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Mycobacterium/veterinária , Mycobacterium/classificação , Animais , Biópsia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/patologia , Genes Bacterianos , Tipagem de Sequências Multilocus , Mycobacterium/genética , Mycobacterium leprae/genética , Filogenia , Pele/microbiologia , Pele/patologia
19.
Proc Natl Acad Sci U S A ; 111(37): 13264-71, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25197070

RESUMO

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc(2)155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC-mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium fortuitum/metabolismo , Mycobacterium smegmatis/metabolismo , Plasmídeos/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Biologia Computacional , Deleção de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mutação/genética , Mycobacterium smegmatis/genética , Fenótipo , Homologia de Sequência de Aminoácidos , Transformação Genética
20.
Nihon Hansenbyo Gakkai Zasshi ; 83(1): 6-13, 2014 Mar.
Artigo em Japonês | MEDLINE | ID: mdl-25076760

RESUMO

Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Hansenostáticos/farmacologia , Mutação/genética , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sequência de Bases , Códon/genética , Primers do DNA/genética , DNA Bacteriano/genética , Dapsona/farmacologia , Método Duplo-Cego , Genes Bacterianos/genética , Dados de Sequência Molecular , Quinolonas/farmacologia , Rifampina/farmacologia
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