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1.
Inorg Chem ; 58(17): 11351-11363, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31433627

RESUMO

Iron storage in biology is carried out by cage-shaped proteins of the ferritin superfamily, one of which is the dodecameric protein Dps. In Dps, four distinct steps lead to the formation of metal nanoparticles: attraction of ion-aquo complexes to the protein matrix, passage of these complexes through translocation pores, oxidation of these complexes at ferroxidase centers, and, ultimately, nanoparticle formation. In this study, we investigated Dps from Listeria innocua to structurally characterize these steps for Co2+, Zn2+, and La3+ ions. The structures reveal that differences in their ion coordination chemistry determine alternative metal ion-binding sites on the areas of the surface surrounding the translocation pore that captures nine La3+, three Co2+, or three Zn2+ ions as aquo clusters and passes them on for translocation. Inside these pores, ion-selective conformational changes at key residues occur before a gating residue to actively move ions through the constriction zone. Ions upstream of the Asp130 gate residue are typically hydrated, while ions downstream directly interact with the protein matrix. Inside the cavity, ions move along negatively charged residues to the ferroxidase center, where seven main residues adapt to the three different ions by dynamically changing their conformations. In total, we observed more than 20 metal-binding sites per Dps monomer, which clearly highlights the metal-binding capacity of this protein family. Collectively, our results provide a detailed structural description of the preparative steps for amino acid-assisted biomineralization in Dps proteins, demonstrating unexpected protein matrix plasticity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Listeria/química , Metais Pesados/química , Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/biossíntese , Modelos Moleculares , Eletricidade Estática
2.
MMWR Morb Mortal Wkly Rep ; 68(30): 664-666, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31369523

RESUMO

Candida auris is an emerging drug-resistant yeast that causes outbreaks in health care facilities; cases have been reported from approximately 30 countries. U.S. cases of C. auris are likely the result of importation from abroad followed by extensive local transmission in health care settings (1). Early detection of Candida auris is key to preventing its spread. C. auris frequently co-occurs with carbapenemase-producing organisms (CPOs), like carbapenem-resistant Enterobacteriaceae (CRE), organisms for which testing and public health response capacity substantially increased beginning in 2017. In September 2018, the Maryland Department of Health (MDH) was notified of a hospitalized resident with CPO infection and colonization and recent hospitalization in Kenya. In light of this history, the patient was screened for C. auris and found to be colonized. Public health responses to CPOs can aid in the early identification of C. auris. As part of CPO investigations, health departments should assess whether the patient has risk factors for C. auris and ensure that patients at risk are tested promptly.


Assuntos
Proteínas de Bactérias/biossíntese , Candida/isolamento & purificação , Candidíase/diagnóstico , Hospitalização/estatística & dados numéricos , beta-Lactamases/biossíntese , Humanos , Quênia , Estados Unidos
3.
Vet Microbiol ; 233: 52-60, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176413

RESUMO

The spread of extended-spectrum ß-lactamases (ESBLs) in Escherichia coli is a major public health issue and ESBL-producing bacteria are frequently reported in livestock. For the assessment of the role of the foodborne transmission pathway in Germany, detailed data on the prevalence and characteristics of isolates of food origin are necessary. The objective of this study was to describe the prevalence of cefotaxime resistant E. coli as well as ESBL/pAmpC-producing E. coli and their characteristics in foods in Germany. Out of 2256 food samples, the highest prevalence of cefotaxime resistant E. coli was observed in chicken meat (74.9%), followed by turkey meat (40.1%). Prevalence in beef, pork and minced meat was considerably lower (4.2-15.3%). Whereas 18.0% of the raw milk samples, collected at farm level were positive, this was true only for few cheese samples (1.3%). In one out of 399 vegetable samples a cefotaxime-resistant E. coli was isolated. ESBL resistance genes of the CTX-M-group (10.1% of all samples) were most frequently detected, followed by genes of the pAmpC (2.6%), SHV (2.0%) and TEM (0.8%) families. Distribution of ESBL/AmpC-encoding E. coli resistance genes and E. coli phylogroups was significantly different between the chicken related food samples and all other food items. Our study results reflect that consumers might get exposed to ESBL/pAmpC-producing E. coli through several food chains. These results together with those collected at primary production and in the human population in other studies will allow more detailed analysis of the foodborne pathways, considering transmission from livestock populations to food at retail and to consumers in Germany.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Microbiologia de Alimentos , Carne/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cefotaxima/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/enzimologia , Infecções por Escherichia coli/transmissão , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Alemanha , Gado/microbiologia , Aves Domésticas/microbiologia , Prevalência , Carne Vermelha/microbiologia , Verduras/microbiologia , beta-Lactamases/biossíntese
4.
Future Microbiol ; 14: 691-704, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31148474

RESUMO

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.


Assuntos
Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , beta-Lactamases/biossíntese , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Paquistão/epidemiologia , Plasmídeos , Análise de Sequência de DNA
5.
Mar Drugs ; 17(5)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067786

RESUMO

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic strain Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.


Assuntos
Cianobactérias/genética , Cianobactérias/metabolismo , Microcistinas/biossíntese , Microcistinas/genética , Sequência de Aminoácidos , Aminoácidos/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Genes Bacterianos , Microcistinas/química , Família Multigênica , Filogenia , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA
6.
Pol J Microbiol ; 68(1): 43-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050252

RESUMO

Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.


Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Prodigiosina/biossíntese , Serratia marcescens/genética , Serratia marcescens/metabolismo , Fatores de Transcrição/genética , Transcrição Genética/genética , Aciltransferases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Temperatura Alta , Oxirredutases/genética , Ativação Transcricional/genética
8.
Vet Microbiol ; 231: 100-106, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955795

RESUMO

Extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli are found in the poultry production even without antibiotic use. The spread of these bacteria has been suggested to occur via imported parent birds, enabling transmission to production level broilers vertically via eggs. We studied transmission of ESBL/pAmpC-producing E. coli and E. coli without antibiotic selection by sampling imported parent birds (n = 450), egg surfaces prior to and after the incubation period (n = 300 and n = 428, respectively) and the laying house environment (n = 20). Samples were additionally taken from embryos (n = 422). To study the prevention of transmission, a competitive exclusion (CE) solution was added onto freshly laid eggs prior to incubation period (n = 150). Results showed carriage of ESBL/pAmpC-producing E. coli in parent birds (26.7%), the environment (5%) and egg surfaces before the incubation period (1.3%), but not from egg surfaces or embryos after the incubation period. Whole genome sequencing revealed ESBL/pAmpC-producing E. coli isolates belonging to clonal lineages ST429 and ST2040. However, the finding of E. coli cultured without antibiotic selection in two (2.2%) embryos strengthens the need to study E. coli transmission in poultry production in more depth. Since ESBL/pAmpC-producing E. coli seem not to persist on egg surfaces, there is no need to use CE solution ex ovo as a prevention method. The results indicate that other routes, such as for example transmission through fomites or horizontal gene transfer by other bacterial species, could be more important than vertical transmission in the spread of resistance in broiler production.


Assuntos
Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/isolamento & purificação , Transmissão Vertical de Doença Infecciosa/veterinária , Doenças das Aves Domésticas/transmissão , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Cloaca/microbiologia , Escherichia coli/enzimologia , Infecções por Escherichia coli/transmissão , Genoma Bacteriano , Óvulo/microbiologia , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Sequenciamento Completo do Genoma , beta-Lactamases/biossíntese
9.
Vet Microbiol ; 232: 58-64, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030845

RESUMO

This study was conducted to assess: (1) a change in between-herd prevalence of extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) between 2011 and 2013, the period during which the antimicrobial policy in animal husbandry in the Netherlands changed significantly, and (2) the prevalence of ESBL/AmpC-EC in individual calves, young stock, and dairy cows in the Netherlands. In 196 randomly selected conventional dairy herds, faecal samples were collected from calves (maximum n = 15), and randomly selected young stock (n = 5) and dairy cows (n = 15). Additionally, fresh faecal samples were collected from five different places on the floors where the dairy cows were housed. Samples were screened for E. coli with non-wild type susceptibility for cefotaxime and isolates were phenotypically confirmed as ESBL/AmpC-producing by disc diffusion, using cefotaxime and ceftazidime with and without clavulanic acid, and cefoxitin. Samples containing ESBL/AmpC-EC were examined semi-quantitatively. In 59.6% of the dairy herds one or more samples tested positive for ESBL/AmpC-EC. The between-herd prevalence based on floor samples in 2013 (18.0%) was significantly lower than the prevalence in 2011 based on comparable samples (32.7%). The individual animal prevalence of ESBL/AmpC-EC, with a minimum shedding level of 103 cfu/g of faeces, was 19.3% in calves, 0.9% in young stock, and 0.8% in dairy cows. Although ESBL/AmpC-EC was found in the majority of dairy herds, the herd prevalence declined significantly between 2011 and 2013. Calves were found to have both, a much higher individual animal prevalence and a higher level of shedding than young stock and cows.


Assuntos
Proteínas de Bactérias/biossíntese , Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/enzimologia , beta-Lactamases/biossíntese , Animais , Antibacterianos/farmacologia , Bovinos , Doenças dos Bovinos/epidemiologia , Cefotaxima/farmacologia , Cefoxitina/farmacologia , Ácido Clavulânico/farmacologia , Indústria de Laticínios , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Feminino , Países Baixos/epidemiologia , Prevalência
10.
Prep Biochem Biotechnol ; 49(5): 521-528, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017522

RESUMO

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.


Assuntos
Proteínas de Bactérias/biossíntese , Meios de Cultura/metabolismo , Lisostafina/biossíntese , Engenharia Metabólica/métodos , Proteínas Recombinantes/biossíntese , Arabinose/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Meios de Cultura/química , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Escherichia coli/genética , Fermentação , Lisostafina/isolamento & purificação , Engenharia Metabólica/economia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Staphylococcus/química , Staphylococcus/metabolismo , Temperatura Ambiente , Fatores de Tempo
11.
Microb Cell Fact ; 18(1): 72, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995928

RESUMO

BACKGROUND: In terms of protein production, the internal environment of the host influences the activity of expression elements, thus affecting the expression level of the target protein. Native expression elements from a specific strain always function well in the original host. In the present study, to enhance the endoxylanase (XynA) production level in Corynebacterium glutamicum CGMCC1.15647 with its native expression elements, approaches to reduce host expression obstacles and to promote expression were evaluated. RESULTS: We identified the signal peptide of CspB2 in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it along with its promoter for the production of endoxylanase (XynA) in this strain. The native cspB2 promoter and cspB2 signal peptide are superior to the well-used cspB1 promoter and cspA signal peptide for XynA expression in C. glutamicum CGMCC1.15647, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest XynA secretion efficiency level in deep 24-well plates level (2492.88 U/mL) was achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively. In fed-batch cultivation, the highest XynA accumulation (1.77 g/L) was achieved in the culture supernatant after 44 h of cultivation. CONCLUSION: Adaptation between the expression elements and the host is crucial for XynA production in C. glutamicum CGMCC1.15647. Strategies including host optimization, chromosomal integration, and coexistence of plasmids were useful for efficient protein production in C. glutamicum.


Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Engenharia Metabólica , Plasmídeos , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas
12.
Mar Drugs ; 17(4)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934874

RESUMO

Seaweeds are of significant interest in the food, pharmaceutical, and agricultural industries as they contain several commercially relevant bioactive compounds. Current extraction methods for macroalgal-derived metabolites are, however, problematic due to the complexity of the algal cell wall which hinders extraction efficiencies. The use of advanced extraction methods, such as enzyme-assisted extraction (EAE), which involve the application of commercial algal cell wall degrading enzymes to hydrolyze the cell wall carbohydrate network, are becoming more popular. Ascophyllum nodosum samples were collected from the Irish coast and incubated in artificial seawater for six weeks at three different temperatures (18 °C, 25 °C, and 30 °C) to induce decay. Microbial communities associated with the intact and decaying macroalga were examined using Illumina sequencing and culture-dependent approaches, including the novel ichip device. The bacterial populations associated with the seaweed were observed to change markedly upon decay. Over 800 bacterial isolates cultured from the macroalga were screened for the production of algal cell wall polysaccharidases and a range of species which displayed multiple hydrolytic enzyme activities were identified. Extracts from these enzyme-active bacterial isolates were then used in EAE of phenolics from Fucus vesiculosus and were shown to be more efficient than commercial enzyme preparations in their extraction efficiencies.


Assuntos
Ascophyllum/microbiologia , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Fracionamento Químico/métodos , Polissacarídeo-Liase/biossíntese , Polissacarídeo-Liase/química , Bactérias/enzimologia , Bactérias/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Fucus/química , Hidrólise , Microbiota , Fenóis/isolamento & purificação , Polissacarídeo-Liase/isolamento & purificação , Proteólise , Alga Marinha/microbiologia
13.
Molecules ; 24(7)2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30974879

RESUMO

Isomaltose-oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0-10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/metabolismo , Geobacillus/genética , Isomaltose/química , alfa-Glucosidases/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Geobacillus/enzimologia , Oligossacarídeos/síntese química , Oligossacarídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , alfa-Glucosidases/biossíntese , alfa-Glucosidases/genética
14.
Res Microbiol ; 170(4-5): 182-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30953690

RESUMO

This paper presents the effects of the composition of different media (i.e., Tryptic soy broth (TSB), Brain heart infusion (BHI), Listeria enrichment broth (LEB), Fraser broth (FB) and University of Vermont medium (UVM)) on the detection of a short peptide fragment PepD specific to the p60 protein (p60) of L. monocytogenes by a monoclonal antibody (anti-PepD mAb). Expression of the p60 obtained was demonstrated to be proportional to the cellular growth of Listeria monocytogenes regardless of the tested growth medium. However, the early growth of L. monocytogenes and the expression of the p60 were negatively affected by the presence of selective agents present in LEB, FB and UVM. Among those three selective enrichment media commonly used for L. monocytogenes, LEB allowed a better expression of L. monocytogenes p60 after an incubation period of 18 h. Optimization of the LEB revealed that the dextrose concentration was the critical factor for improving the expression of p60 and promotes the early expression of p60. Moreover, an optimal dextrose concentration of 0.5% (w/v) in LEB, coupled with anti-PepD mAb immobilized to solid support, reduced the detection of p60 from 18 h to 9 h for an initial concentration of L. monocytogenes of 108 CFU/ml.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/imunologia , Meios de Cultura/química , Lipoproteínas/biossíntese , Lipoproteínas/imunologia , Listeria monocytogenes/crescimento & desenvolvimento , Técnicas Bacteriológicas , Ensaio de Imunoadsorção Enzimática , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação
15.
Appl Microbiol Biotechnol ; 103(10): 4033-4043, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30937497

RESUMO

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.


Assuntos
Proteínas de Bactérias/biossíntese , Corynebacterium glutamicum/metabolismo , Ácido Glutâmico/metabolismo , Lisina/metabolismo , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Glutâmico/genética , Lisina/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Appl Microbiol Biotechnol ; 103(11): 4467-4481, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989253

RESUMO

Locillomycins are cyclic lipononapeptides assembled by a nonlinear hexamodular NRPS and have strong antibacterial activity. In this study, we genetically engineered Bacillus velezensis FZB42 as a surrogate host for the heterologous expression of the loc gene cluster for locillomycins. The fosmid N13 containing whole loc gene cluster was screened from the B. velezensis 916 genomic library. Subsequently, a spectinomycin resistance cassette, and the cassette fused with an IPTG inducible promoter Pspac, was introduced in the fosmid N13 using λ Red recombination system, respectively. The resulting fosmids, designated N13+Spec and N13+PSSpec, were used for the transformation of B. velezensis FZB42 to obtain derivative strains FZBNPLOC and FZBPSLOC. RT-PCR and qRT-PCR results revealed the efficient heterologous expression of the loc gene cluster in both derivative strains. Particularly, there was positive correlation between the derivative FZBPSLOC strain and the enhanced production of locillomycins upon addition of the inducer IPTG with the highest production of locillomycins at 15-fold more than that of B. velezensis 916. This overproduction of locillomycins was also related to the enhancement of antibacterial activity against methicillin-resistant Staphylococcus aureus, and exhibited moderate changes in its hemolytic activity. Together our findings demonstrate that the nonlinear hexamodular NRPS, encoded by the loc gene cluster from B. velezensis 916, is sufficient for the biosynthesis of cyclic lipononapeptide locillomycins in the surrogate host B. velezensis FZB42. Moreover, the FZBPSLOC strain will also be useful for further development of novel locillomycins derivatives with improved antibacterial activity.


Assuntos
Antibacterianos/biossíntese , Bacillus/metabolismo , Proteínas de Bactérias/biossíntese , Vias Biossintéticas/genética , Lipopeptídeos/biossíntese , Engenharia Metabólica/métodos , Peptídeos Cíclicos/biossíntese , Proteínas Recombinantes/biossíntese , Bacillus/genética , Proteínas de Bactérias/genética , Expressão Gênica , Lipopeptídeos/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Família Multigênica , Peptídeos Cíclicos/genética , Proteínas Recombinantes/genética
17.
J Med Microbiol ; 68(4): 560-565, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30869579

RESUMO

PURPOSE: This study evaluated in-house PCR testing for local identification of bacteria carrying the major carbapenemase genes (blaOXA-48-like, blaVIM, blaNDM, blaKPC and blaIMP). METHODOLOGY: Carbapenemase-producing organisms (CPOs) isolated from patients managed in two tertiary care hospitals in Scotland from September 2014-January 2017 were investigated. A combination of chromogenic screening agar (ChromID CARBA SMART), a carbapenem hydrolysis test (Rapidec Carba NP) and in-house real-time PCR for the blaOXA-48-like, blaVIM, blaNDM, blaKPC and blaIMP genes were utilized. All isolates were sent to the AMRHAI reference unit for confirmatory testing. RESULTS: During the 29-month study period 39 CPO were isolated from 34 patients. The average turnaround time for a workflow involving phenotypic and molecular testing was 4.2 days. PCR had a sensitivity and specificity of 100 %. The most common carbapenemase genes were blaOXA-48-like (31%), blaVIM (23%) and blaNDM (20%). Resistance to antimicrobials other than beta-lactams was common; the most active agents were colistin, amikacin and fosfomycin. Twenty-seven patients were considered to be colonized (although CPO detection influenced empiric antimicrobials in five) and a CPO was implicated in infection in seven patients (bacteraemia in immunocompromised patients, n=2; surgical site infections, n=2; osteomyelitis in a patient with diabetes mellitus, n=1; and urinary tract infections, n=2). All patients survived infection. CONCLUSION: In a lowincidence setting we demonstrate the efficacy of a combined local laboratory workflow for rapid detection of CPOs, incorporating phenotypic and molecular testing. In 7/34 patients the CPO was implicated as a pathogen and detection influenced antimicrobial decision-making in five colonized patients.


Assuntos
Proteínas de Bactérias/biossíntese , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/genética , beta-Lactamases/biossíntese , Idoso , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Carbapenêmicos/farmacologia , Enterobacteriaceae/enzimologia , Infecções por Enterobacteriaceae/diagnóstico , Feminino , Humanos , Hidrólise , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Escócia , Sensibilidade e Especificidade , Centros de Atenção Terciária
18.
Int J Antimicrob Agents ; 53(6): 774-780, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30831233

RESUMO

Pseudomonas aeruginosa is one of the most important pathogens in cystic fibrosis. This study was conducted to analyse the genetic basis and phylogenetic profile of resistance to ceftazidime/avibactam, ceftolozane/tazobactam and carbapenems in cystic fibrosis P. aeruginosa isolates. Whole genome sequence analysis was conducted of isolates resistant to piperacillin/tazobactam collected from seven hospitals in Scotland since the introduction of these two cephalosporin/ß-lactamase inhibitor combinations. Ceftazidime resistance was primarily related to AmpC induction, as tested by cloxacillin inhibition assays, while high-level ceftazidime resistance not reversed by cloxacillin was associated with amino acid variations in AmpC. Only isolates resistant to both ceftazidime/avibactam and ceftolozane/tazobactam carried AmpD mutations, likely resulting in ampC overexpression. All isolates resistant to ceftazidime/avibactam and/or ceftolozane/tazobactam were resistant to carbapenems and showed inactivating mutations in the chromosomal oprD gene. None of the isolates bore class A, B, D plasmid-encoded carbapenemases. This study showed that mutational resistance emerged in phylogenetically distant lineages, which indicates the mutations occur independently without conferring a selective advantage to any phylogenetic lineage. These findings confirm the strong contribution of mutation-driven evolution to the population structure of P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Carbapenêmicos/farmacologia , Ceftazidima/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana , Combinação Piperacilina e Tazobactam/farmacologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tazobactam/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Fibrose Cística/complicações , Combinação de Medicamentos , Feminino , Hospitais , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Porinas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Escócia , Sequenciamento Completo do Genoma , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/biossíntese , beta-Lactamases/genética
19.
Gene ; 701: 152-160, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30910556

RESUMO

Edwardsiella tarda belongs to the genera of Gram negative bacterium mainly associated with edwardsiellosis, the most commonly found infectious fish disease throughout the globe. E. tarda is also a widespread pathogen which cause infections such as cellulitis or gas gangrene and generalized infections in humans. To control the escalating infection of E. trada on various species, it is essential to decoded the mysterious mechanism behind the bacterial infection at transcript level. In this present study, we carry out a de novo E. tarda Whole transcriptome sequencing, isolated from infected fish intestine using SOLiD sequencing platform. RNA-Seq data analysis was performed using various bioinformatics pipelines. Protein-protein interaction study for pathway enrichment and gene ontology study were executed for further investigation. Assembly statistics for E. tarda dataset showed that the number of transcript contigs was 9657 out of which 6749 were GO annotated whereas 1528 were not assigned any GO terms. GO analysis showed that the expressed genes were enhanced with molecular function, cellular component and biological process. A KEGG enrichment study showed that pathway's that are directly linked with immune diseases like Rheumatoid arthritis (0.2%), Tuberculosis (0.3%) Endocytosis (0.6%) was considerably enriched. Protein-protein interaction study showed that most of the expressed proteins were involved in metabolic pathways, flagellar assembly, Propanoate metabolism, Microbial metabolism in diverse environments, Butanoate metabolism and Carbon. The present study provides novel E. tarda transcriptome sequence data, allowing us to identify biologically significant genes and their functional relationship with fish diseases, and will be useful in recognize the reliable therapeutic targets in near feature.


Assuntos
Proteínas de Bactérias , Cipriniformes/microbiologia , Edwardsiella tarda , Infecções por Enterobacteriaceae , Doenças dos Peixes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Edwardsiella tarda/genética , Edwardsiella tarda/isolamento & purificação , Edwardsiella tarda/metabolismo , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/veterinária , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Doenças dos Peixes/microbiologia
20.
MBio ; 10(2)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862746

RESUMO

Clostridioides difficile infection (CDI) is a toxin-mediated diarrheal disease. Several factors have been identified that influence the production of the two major C. difficile toxins, TcdA and TcdB, but prior published evidence suggested that additional unknown factors were involved in toxin regulation. Previously, we identified a C. difficile regulator, RstA, that promotes sporulation and represses motility and toxin production. We observed that the predicted DNA-binding domain of RstA was required for RstA-dependent repression of toxin genes, motility genes, and rstA transcription. In this study, we further investigated the regulation of toxin and motility gene expression by RstA. DNA pulldown assays confirmed that RstA directly binds the rstA promoter via the predicted DNA-binding domain. Through mutational analysis of the rstA promoter, we identified several nucleotides that are important for RstA-dependent transcriptional regulation. Further, we observed that RstA directly binds and regulates the promoters of the toxin genes tcdA and tcdB, as well as the promoters for the sigD and tcdR genes, which encode regulators of toxin gene expression. Complementation analyses with the Clostridium perfringens RstA ortholog and a multispecies chimeric RstA protein revealed that the C. difficile C-terminal domain is required for RstA DNA-binding activity, suggesting that species-specific signaling controls RstA function. Our data demonstrate that RstA is a transcriptional repressor that autoregulates its own expression and directly inhibits transcription of the two toxin genes and two positive toxin regulators, thereby acting at multiple regulatory points to control toxin production.IMPORTANCE Clostridioides difficile is an anaerobic, gastrointestinal pathogen of humans and other mammals. C. difficile produces two major toxins, TcdA and TcdB, which cause the symptoms of the disease, and forms dormant endospores to survive the aerobic environment outside the host. A recently discovered regulatory factor, RstA, inhibits toxin production and positively influences spore formation. Herein, we determine that RstA directly binds its own promoter DNA to repress its own gene transcription. In addition, our data demonstrate that RstA directly represses toxin gene expression and gene expression of two toxin gene activators, TcdR and SigD, creating a complex regulatory network to tightly control toxin production. This study provides a novel regulatory link between C. difficile sporulation and toxin production. Further, our data suggest that C. difficile toxin production is regulated through a direct, species-specific sensing mechanism.


Assuntos
Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Clostridium difficile/genética , Clostridium difficile/fisiologia , Enterotoxinas/biossíntese , Regulação Bacteriana da Expressão Gênica , Locomoção , Proteínas Repressoras/metabolismo , Clostridium perfringens/genética , Análise Mutacional de DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Teste de Complementação Genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética
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