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2.
Methods Mol Biol ; 2317: 135-153, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028766

RESUMO

The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.


Assuntos
Genoma de Cloroplastos , Genomas de Plastídeos , Resistência a Canamicina/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Transgenes , Marcadores Genéticos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Espectinomicina/farmacologia , Tabaco/crescimento & desenvolvimento
3.
Antimicrob Agents Chemother ; 65(7): e0250220, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33903113

RESUMO

Eis promoter mutations can confer reduced Mycobacterium tuberculosis kanamycin susceptibility. GenoType MTBDRsl, a widely used assay evaluating this region, wrongly classified 17/410 isolates as eis promoter wild type. Six out of seventeen isolates harbored mutations known to confer kanamycin resistance, and the remainder harbored either novel eis promoter mutations (7/11) or disputed mutations (4/11). GenoType MTBDRsl can miss established and new variants that cause reduced susceptibility. These data highlight the importance of reflex phenotypic kanamycin testing.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Testes Diagnósticos de Rotina , Farmacorresistência Bacteriana Múltipla , Genótipo , Humanos , Canamicina/farmacologia , Resistência a Canamicina/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/genética
4.
PLoS Pathog ; 17(4): e1009537, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33930099

RESUMO

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.


Assuntos
Microbioma Gastrointestinal/genética , Intestinos/microbiologia , Klebsiella/genética , Plasmídeos/genética , Animais , Bacteriemia/genética , Proteínas de Bactérias/genética , Feminino , Aptidão Genética/fisiologia , Loci Gênicos/fisiologia , Genoma Bacteriano , Interações Hospedeiro-Patógeno/genética , Resistência a Canamicina/genética , Infecções por Klebsiella/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óperon/genética , Especificidade de Órgãos/genética , Virulência/genética , beta-Lactamases/genética
5.
Appl Environ Microbiol ; 87(9)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33608284

RESUMO

When subjected to nutritional stress, bacteria modify their amino acid metabolism and cell division activities by means of the stringent response, which is controlled by the Rsh protein in alphaproteobacteria. An important group of alphaproteobacteria are the rhizobia, which fix atmospheric N2 in symbiosis with legume plants. Although nutritional stress is common for rhizobia while infecting legume roots, the stringent response has scarcely been studied in this group of soil bacteria. In this report, we obtained a mutant with a kanamycin resistance insertion in the rsh gene of Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean. This mutant was defective for type 3 secretion system induction, plant defense suppression at early root infection, and nodulation competition. Furthermore, the mutant produced smaller nodules, although with normal morphology, which led to lower plant biomass production. Soybean (Glycine max) genes GmRIC1 and GmRIC2, involved in autoregulation of nodulation, were upregulated in plants inoculated with the mutant under the N-free condition. In addition, when plants were inoculated in the presence of 10 mM NH4NO3, the mutant produced nodules containing bacteroids, and GmRIC1 and GmRIC2 were downregulated. The rsh mutant released more auxin to the culture supernatant than the wild type, which might in part explain its symbiotic behavior in the presence of combined N. These results indicate that the B. diazoefficiens stringent response integrates into the plant defense suppression and regulation of nodulation circuits in soybean, perhaps mediated by the type 3 secretion system.IMPORTANCE The symbiotic N2 fixation carried out between prokaryotic rhizobia and legume plants performs a substantial contribution to the N cycle in the biosphere. This symbiotic association is initiated when rhizobia infect and penetrate the root hairs, which is followed by the growth and development of root nodules, within which the infective rhizobia are established and protected. Thus, the nodule environment allows the expression and function of the enzyme complex that catalyzes N2 fixation. However, during early infection, the rhizobia find a harsh environment while penetrating the root hairs. To cope with this nuisance, the rhizobia mount a stress response known as the stringent response. In turn, the plant regulates nodulation in response to the presence of alternative sources of combined N in the surrounding medium. Control of these processes is crucial for a successful symbiosis, and here we show how the rhizobial stringent response may modulate plant defense suppression and the networks of regulation of nodulation.


Assuntos
Bradyrhizobium/genética , Soja/microbiologia , Farmacorresistência Bacteriana/genética , Fertilizantes , Resistência a Canamicina/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Nitratos , Fixação de Nitrogênio , Proteínas de Plantas/genética , Nodulação , Soja/genética , Simbiose , Sistemas de Secreção Tipo III
6.
Nucleic Acids Res ; 49(2): e12, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33270888

RESUMO

The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.


Assuntos
Resistência Microbiana a Medicamentos/genética , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia/genética , Sistemas CRISPR-Cas , Cloranfenicol/farmacologia , Células Clonais , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Canamicina/farmacologia , Resistência a Canamicina/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Lactatos/metabolismo , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas/genética , Ácido Pirúvico/metabolismo , Seleção Genética , Tetraciclina/farmacologia , Resistência a Tetraciclina/genética , Fatores de Tempo , Transformação Bacteriana
7.
PLoS One ; 15(10): e0241058, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104745

RESUMO

Many epidemiological studies provide us with the evidence of horizontal gene transfer (HGT) contributing to the bacterial genomic diversity that benefits the bacterial populations with increased ability to adapt to the dynamic environments. Campylobacter jejuni, a major cause of acute enteritis in the U.S., often linked with severe post-infection neuropathies, has been reported to exhibit a non-clonal population structure and comparatively higher strain-level genetic variation. In this study, we provide evidence of the HGT of chromosomally encoded genetic markers between C. jejuni cells in the biphasic MH medium. We used two C. jejuni NCTC-11168 mutants harbouring distinct antibiotic-resistance genes [chloramphenicol (Cm) and kanamycin (Km)] present at two different neutral genomic loci. Cultures of both marker strains were mixed together and incubated for 5 hrs, then plated on MH agar plates supplemented with both antibiotics. The recombinant cells with double antibiotic markers were generated at the frequency of 0.02811 ± 0.0035% of the parental strains. PCR assays using locus-specific primers confirmed that transfer of the antibiotic-resistance genes was through homologous recombination. Also, the addition of chicken cecal content increased the recombination efficiency approximately up to 10-fold as compared to the biphasic MH medium (control) at P < 0.05. Furthermore, treating the co-culture with DNase I decreased the available DNA, which in turn significantly reduced recombination efficiency by 99.92% (P < 0.05). We used the cell-free supernatant of 16 hrs-culture of Wild-type C. jejuni as a template for PCR and found DNA sequences from six different genomic regions were easily amplified, indicating the presence of released chromosomal DNA in the culture supernatant. Our findings suggest that HGT in C. jejuni is facilitated in the chicken gut environment contributing to in vivo genomic diversity. Additionally, C. jejuni might have an active mechanism to release its chromosomal DNA into the extracellular environment, further expediting HGT in C. jejuni populations.


Assuntos
Campylobacter jejuni/genética , Resistência ao Cloranfenicol/genética , Transferência Genética Horizontal , Resistência a Canamicina/genética , Animais , Infecções por Campylobacter/microbiologia , Galinhas , DNA Bacteriano , Marcadores Genéticos , Genoma Bacteriano , Recombinação Homóloga
8.
J Med Microbiol ; 69(8): 1049-1061, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32602832

RESUMO

Introduction. Metal exposure is an important factor for inducing antibiotic resistance in bacteria. Dandelion extracts have been used for centuries in traditional Chinese and Native American medicine.Aim. We assessed the effects of dandelion water extracts and taraxasterol on heavy metal-induced antibiotic resistance in Escherichia coli as well as the underlying mechanisms.Methodology. Dandelion extracts were obtained through 4 h of boiling in distilled water. Bacterial growth was monitored with a spectrophotometer. Biochemical assays were performed to assess the activities and gene transcriptions of ß-lactamase and acetyltransferase. Oxidative stress was determined using an oxidation-sensitive probe, H2DCFDA.Results. The present study demonstrated that higher concentrations of nickel (>5 µg ml-1), cadmium (>0.1 µg ml-1), arsenic (>0.1 µg ml-1) and copper (>5 µg ml-1) significantly inhibited the growth of E. coli. Lower concentrations of nickel (0.5 µg ml-1), cadmium (0.05 µg ml-1) and arsenic (0.05 µg ml-1) had no effect on bacterial growth, but helped the bacteria become resistant to two antibiotics, kanamycin and ampicillin. The addition of dandelion root extracts and taraxasterol significantly reversed the antibiotic resistance induced by these heavy metals. The supplements of antibiotics and cadmium generated synergistic effects on the activities of ß-lactamase and acetyltransferase (two antibiotic resistance-related proteins), which were significantly blocked by either dandelion root extract or taraxasterol. In contrast, oxidative stress was not involved in the preventative roles of dandelion root extracts and taraxasterol in heavy metal-induced antibiotic resistance.Conclusion. This study suggests that heavy metals induce bacterial antibiotic resistance and dandelion root extracts and taraxasterol could be used to help reverse bacterial resistance to antibiotics.


Assuntos
Resistência Microbiana a Medicamentos/efeitos dos fármacos , Metais Pesados/efeitos adversos , Extratos Vegetais/farmacologia , Esteróis/farmacologia , Taraxacum/química , Triterpenos/farmacologia , Resistência a Ampicilina/efeitos dos fármacos , Arsênio/efeitos adversos , Cádmio/efeitos adversos , Cobre/efeitos adversos , Escherichia coli/efeitos dos fármacos , Humanos , Índios Norte-Americanos , Resistência a Canamicina/efeitos dos fármacos , Medicina Tradicional Chinesa , Medicina Tradicional , Níquel/efeitos adversos , Raízes de Plantas/química
9.
Biotechnol Lett ; 42(11): 2223-2230, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32500473

RESUMO

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.


Assuntos
Escherichia coli/genética , Formaldeído/farmacologia , Resistência a Canamicina/efeitos dos fármacos , Plasmídeos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Vacinas contra Escherichia coli/efeitos adversos , Vacinas contra Escherichia coli/genética , Transferência Genética Horizontal/efeitos dos fármacos , Plasmídeos/genética , Vacinas de Produtos Inativados , Vacinas Sintéticas
10.
ACS Chem Biol ; 15(6): 1581-1594, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421305

RESUMO

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.


Assuntos
Acetiltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Desenho de Fármacos , Resistência a Canamicina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Methods Mol Biol ; 2133: 31-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32144662

RESUMO

In recent years, split inteins have seen widespread use as molecular platforms for the design of a variety of peptide and protein chemistry technologies, most notably protein ligation. The development of these approaches is dependent on the identification and/or design of split inteins with robust activity, stability, and solubility. Here, we describe two approaches to characterize and compare the activities of newly identified or engineered split inteins. The first assay employs an E. coli-based selection system to rapidly screen the activities of many inteins and can be repurposed for directed evolution. The second assay utilizes reverse-phase high-performance liquid chromatography (RP-HPLC) to provide insights into individual chemical steps in the protein splicing reaction, information that can guide further engineering efforts. These techniques provide useful alternatives to common assays that utilize SDS-PAGE to analyze splicing reaction progress.


Assuntos
Clonagem Molecular/métodos , Inteínas , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas , Expressão Gênica , Inteínas/genética , Resistência a Canamicina , Processamento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas de Síntese em Fase Sólida/métodos , Trans-Splicing
12.
Sci Adv ; 6(7): eaay4453, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32095527

RESUMO

The postreplicative mismatch repair (MMR) is an almost ubiquitous DNA repair essential for maintaining genome stability. It has been suggested that Mycobacteria have an alternative MMR in which NucS, an endonuclease with no structural homology to the canonical MMR proteins (MutS/MutL), is the key factor. Here, we analyze the spontaneous mutations accumulated in a neutral manner over thousands of generations by Mycobacterium smegmatis and its MMR-deficient derivative (ΔnucS). The base pair substitution rates per genome per generation are 0.004 and 0.165 for wild type and ΔnucS, respectively. By comparing the activity of different bacterial MMR pathways, we demonstrate that both MutS/L- and NucS-based systems display similar specificity and mutagenesis bias, revealing a functional evolutionary convergence. However, NucS is not able to repair indels in vivo. Our results provide an unparalleled view of how this mycobacterial system works in vivo to maintain genome stability and how it may affect Mycobacterium evolution.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Mutagênese/genética , Mutação/genética , Mycobacterium/genética , Proteínas de Bactérias/genética , Pareamento de Bases/genética , DNA Bacteriano/genética , Genes Reporter , Genoma Bacteriano , Mutação INDEL/genética , Resistência a Canamicina/genética , Taxa de Mutação , Plasmídeos/genética
13.
Braz J Microbiol ; 51(3): 919-929, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32078730

RESUMO

Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.


Assuntos
Bacillus cereus/genética , Plasmídeos/genética , Transfecção/métodos , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Meios de Cultura/química , Meios de Cultura/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Canamicina/análise , Canamicina/farmacologia , Resistência a Canamicina/genética , Transformação Bacteriana
14.
J Pharm Biomed Anal ; 181: 113108, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31981830

RESUMO

Persistent abuse and overuse of antibiotics induces a widespread bloom of antibiotic resistance genes (ARGs) and the emergence of superbugs. A method designed to rapidly quantify ARGs in real-world scenarios is urgently needed. Here, we present an orthogonal test of heavy water and kanamycin exposure, namely, a "clover-assay", to reveal the capability of state-of-the-art Raman microspectroscopy to identify ARGs within microbial communities. This assay successfully recognizes the discriminating spectral alterations from two genetically identical strains that differ only in terms of the expression of one kanamycin resistance gene. In addition to the previously reported Raman shift at carbon-deuterium vibration bands (2,040-2,300 cm-1), we identify two new peak shifts (970-990 cm-1) and (1,110-1,130 cm-1) associated with deuterium labelling. Notably, the spectral alterations from 1,110-1,130 cm-1 strongly correlate with kanamycin exposure. By introducing dispersion index (DI) and clover assay index (CAI) as indicators, this assay is able to quantify the abundance of kanamycin resistance genes within artificial microbiotas. Based on our results, the biospectral clover assay is a powerful tool for the in situ interrogation of the occurrence of ARGs within microbial communities, which displays great potential to eliminate the need for culture protocols in the future. Due to the non-destructive and non-intrusive features, this approach may therefore potentially be able to diagnose horizontal gene transfer (HGT) in real time.


Assuntos
Óxido de Deutério/química , Técnicas Genéticas , Resistência a Canamicina/genética , Microbiota/genética , Análise Espectral Raman/métodos
15.
Microbiologyopen ; 9(1): e00953, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31638342

RESUMO

Marine purple photosynthetic bacteria are ideal organisms for the production of useful materials at reduced costs and contributing to a sustainable society because they can utilize sunlight, seawater, and components of air, including carbon dioxide and nitrogen gases, for their growth. However, conjugation is the only applicable method for the transformation of marine purple photosynthetic bacteria so far. Here, we examined a calcium chloride-mediated method for the transformation of marine purple photosynthetic bacteria. Plasmid DNAs containing the kanamycin resistance gene were successfully transferred into chemically competent cells of two strains of marine purple photosynthetic bacteria (Rhodovulum sulfidophilum and Roseospira marina). Heat shock treatment increased the transformation efficiency in R. sulfidophilum, whereas the addition of cell-penetrating peptide did not improve it. We also found that prolonged incubation in agar plates containing kanamycin led to spontaneous mutation of the 16S rRNA, resulting in kanamycin resistance in R. marina. Thus, we developed an efficient and facile transformation method using chemically competent cells of marine purple photosynthetic bacteria with calcium chloride.


Assuntos
Técnicas de Transferência de Genes , Resistência a Canamicina/genética , Rhodospirillaceae/genética , Rhodovulum/genética , Transformação Bacteriana/genética , Cloreto de Cálcio/química , DNA Bacteriano/química , DNA Bacteriano/genética , Resposta ao Choque Térmico/fisiologia , Plasmídeos/genética , Água do Mar/microbiologia , Microbiologia da Água
16.
Plasmid ; 102: 62-70, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30825470

RESUMO

Two B/O plasmids were sequenced. The kanamycin resistance plasmid R805a was found in a Salmonella Typhi strain from a 1972 typhoid fever outbreak in Mexico City. pCERC6, which confers resistance to ampicillin, streptomycin and sulphamethoxazole, was found in a commensal E. coli isolated from a healthy adult in Sydney in 2008. Both plasmids contain the same gene for RNAI, the incompatibility determinant, as the reference B/O plasmid pMU707 and the recently reported plasmid p838B-R, which came from a commensal E. coli isolated in Sydney in 2004. However, three different repA alleles are associated with the IncB/O rnaI. Whereas the repA gene of p838B-R is identical to that of pMU707, R805a and pCERC6 each carry a different repA. Comparison of the plasmid backbones revealed that R805a has a different oriT-nikAB region to that in pCERC6 and p838B-R, encoding different NikA relaxosome accessory factors and NikB relaxases. The different nikA genes were associated with oriT sites that differed in the sequences of their long, imperfect inverted repeats. A further 11 publicly available complete plasmid sequences contain the same B/O rnaI, with either the repA of pMU707/p838B-R or of R805a, and the oriT-nikAB region of p838B-R/pCERC6 or of R805a. All four possible combinations were seen. Extensive variation was also observed in the traY-excA entry exclusion region, and in the locations of mobile elements, including ones carrying antibiotic resistance genes. Plasmids that contain the same rnaI gene would be considered the same by a number of typing methods, but this study has unveiled previously unnoticed mosaicism in the backbones of one such group. This highlights the importance of considering entire plasmid backbones for the typing and tracking of specific lineages.


Assuntos
Mosaicismo , Plasmídeos/genética , Sequência de Bases , Mapeamento Cromossômico , Replicação do DNA/genética , Elementos de DNA Transponíveis/genética , Resistência a Canamicina/genética , Mapeamento por Restrição
17.
PLoS One ; 14(3): e0213933, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30908529

RESUMO

Eis (Enhanced Intracellular Survival) is an important aminoglycoside N-acetyltransferase enzyme contributing to kanamycin resistance in Mtb clinical isolates. Eis proteins from M. tuberculosis (RvEis) and M. smegmatis (MsEis) have 58% identical and 69% similar amino acid sequences and acetylate aminoglycosides at multiple amines. Both the Eis proteins are hexameric and composed of two symmetric trimers. RvEis has remarkable structural stability and heat-stable aminoglycoside acetyltransferase activity. Although the structure and biochemical properties of MsEis have been studied earlier, the detailed characterization of its acetyltransferase activity and structural stability is lacking. In this study, we have performed comparative analysis of structural stability and aminoglycoside acetyltransferase activity of RvEis and MsEis proteins. Unlike RvEis, MsEis undergoes a three-state unfolding induced by heat or chemical denaturants and involves self-association of partially unfolded oligomers to form high molecular weight soluble aggregates. MsEis is highly susceptible to chemical denaturants and unfolds completely at lower concentrations of GdmCl and urea when compared to RvEis. In contrast to RvEis, the oligomeric forms of MsEis are SDS sensitive. However, SDS treatment resulted in increased helix formation in MsEis than RvEis. MsEis shows lesser thermostable activity with a decreased efficiency of kanamycin acetylation in comparison to RvEis. Furthermore, overexpression of MsEis does not provide thermal resistance to M. smegmatis unlike RvEis. Collectively, this study reveals that homologous proteins from pathogenic and nonpathogenic mycobacteria follow different modes of unfolding and demonstrate differential structural stability and activity despite highly similar sequences and oligomeric organization.


Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Acetiltransferases/genética , Proteínas de Bactérias/genética , Humanos , Resistência a Canamicina/genética , Resistência a Canamicina/fisiologia , Cinética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Especificidade da Espécie , Espectrometria de Fluorescência , Termodinâmica , Resposta a Proteínas não Dobradas
18.
Biochem Biophys Res Commun ; 505(1): 333-337, 2018 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-30245132

RESUMO

Escherichia coli ß-lactamase TEM-1 is potentially useful in the antibody-directed enzyme/prodrug therapy (ADEPT), converting nontoxic prodrugs to toxic agents. The produced toxin would kill cancer cells, when the enzyme is attached to a tumor-antigen-specific antibody. However, the off-site reaction possibly occurring in the blood or normal tissues raises safety concern. In the present study, we engineered TEM-1 variants preferentially active at pH 5.8-6.2, near the pH of the acidic microenvironment of tumor. A library of randomly mutagenized variants was screened for the ability to confer an antibiotic resistance on E. coli cells in acidic growth media and not in neutral media, to isolate a variant with a Thr-to-Ile substitution at position 160. An extensive mutagenesis study was then conducted in the proximity of this position, to show that a Leu162Glu mutation also causes the acid preference. Kinetic analyses indicated that the overall activity of the wild-type TEM-1 hardly changes over a pH range from 5.8 to 7.0, whereas TEM-1(T160I) is 1.5-times as active at pH 6.2 than pH 7.0, and TEM-1(T160I) is 3.1-fold as active at pH 5.8 than pH 7.0. A further mutagenesis study suggested that a change in the overall structure of the enzyme underlies the pH dependency of the variants.


Assuntos
Substituição de Aminoácidos , Proteínas de Escherichia coli/genética , Resistência a Canamicina/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Biocatálise , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Canamicina/farmacologia , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios Proteicos , Engenharia de Proteínas/métodos , beta-Lactamases/química , beta-Lactamases/metabolismo
19.
PLoS One ; 13(3): e0193435, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29513730

RESUMO

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.


Assuntos
Resistência a Canamicina , Carne/microbiologia , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/isolamento & purificação , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos/microbiologia , Galinhas/microbiologia , Monitoramento Epidemiológico , Escherichia coli , Canamicina/farmacologia , Resistência a Canamicina/genética , Plasmídeos , RNA Bacteriano/metabolismo , Salmonella enterica/genética , Alinhamento de Sequência , Sus scrofa/microbiologia , Perus/microbiologia
20.
Biosci Biotechnol Biochem ; 82(7): 1169-1171, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29521166

RESUMO

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.


Assuntos
Alelos , Biodegradação Ambiental , Genes Bacterianos , Vetores Genéticos , Mutagênese , Mycobacterium/metabolismo , Naftóis/metabolismo , Fenantrenos/metabolismo , Plasmídeos , Sequência de Bases , Cloranfenicol/farmacologia , Cromossomos Bacterianos , Troca Genética , Resistência a Canamicina/genética , Mycobacterium/genética , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Recombinação Genética , Rhodococcus/genética , Poluentes do Solo/metabolismo , Resistência a Tetraciclina/genética
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