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1.
Molecules ; 26(4)2021 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-33672726

RESUMEN

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.


Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/química , Biotecnología , Endopeptidasas/química , Aceite de Oliva/metabolismo , Temperatura , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Carbono/química , Carbono/metabolismo , Endopeptidasas/biosíntesis , Endopeptidasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Nitrógeno/química , Nitrógeno/metabolismo , Aceite de Oliva/química
2.
Methods Mol Biol ; 2259: 191-201, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687716

RESUMEN

Recent advances in MS/MS technology have made it possible to use proteomic data to predict protein-coding sequences. This approach is called proteogenomics, and it allows to correctly translate start and stop sites and to reveal new open reading frames. Here, we focus on using proteogenomics to improve the annotation of Mycobacteriumtuberculosis strains. We also describe detail procedures of the extraction of proteins and their further preparation for LC-MS/MS analysis and outline the main steps of data analysis.


Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Proteogenómica/métodos , Tuberculosis/microbiología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida/métodos , Genoma Bacteriano , Humanos , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/química , Espectrometría de Masas en Tándem/métodos
3.
Methods Mol Biol ; 2259: 205-213, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687717

RESUMEN

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.


Asunto(s)
Bacterias/aislamiento & purificación , Proteínas Bacterianas/análisis , Microbiología de Alimentos , Proteómica/métodos , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida/métodos , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos
4.
Nat Plants ; 7(3): 365-375, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33731920

RESUMEN

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min-1, a Michaelis-Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.


Asunto(s)
Proteínas Bacterianas/química , Burkholderiales/química , Clorofila/química , Metaloproteínas/química , Protoporfirinas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Burkholderiales/enzimología , Burkholderiales/genética , Clorofila/metabolismo , Clonación Molecular , Transporte de Electrón , Escherichia coli , Espectrometría de Masas , Metaloproteínas/genética , Metaloproteínas/aislamiento & purificación , Metaloproteínas/metabolismo , Protoporfirinas/metabolismo
5.
Food Chem ; 349: 129130, 2021 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-33540220

RESUMEN

The antifungal protein MG-3A was isolated from Bacillus amyloliquefaciens MG-3, and was purified and identified. The results showed that antifungal protein MG-3A was likely a serine protease with a molecular weight of ~48 kDa. The serine protease exhibited a broad antifungal spectrum and effectively extended the shelf-life of loquat fruit up to 25 d. The antifungal protein MG-3A showed good stabilities to temperature, pH and protease K. Primers were designed according to the mass spectrum of antifungal protein and the comparison with proteins in the NCBI database. The serine protease gene MG-3A from B. amyloliquefaciens genome was isolated and cloned using PCR. The prokaryotic expression plasmid pET28a-MG-3A was constructed and used to express the antimicrobial protein in vitro. The SDS-PAGE results showed that the recombinant protein expressed in Escherichia coli BL21 (DE3) was highly soluble. Affinity chromatography was used to purify the recombinant protein and its antifungal activity was evaluated.


Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Bacillus amyloliquefaciens/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Antifúngicos/farmacología , Proteínas Bacterianas/farmacología , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Plásmidos/genética
6.
BMC Infect Dis ; 21(1): 142, 2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33541274

RESUMEN

BACKGROUND: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data. METHODS: The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were "colonised" or "infected" with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes. RESULTS: Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was blaOXA-232, with blaNDM-1 additionally identified in 19 of them. All CPE isolates carrying either blaOXA-232 or blaNDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n = 18) and CPE-colonised patients who developed CPE infections during the study (n = 3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n = 15) and ST231 (n = 14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes. CONCLUSIONS: This study has revealed the genomic features of colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Enterobacteriaceae/genética , Klebsiella pneumoniae/aislamiento & purificación , Tipificación de Secuencias Multilocus , Secuenciación Completa del Genoma , beta-Lactamasas/aislamiento & purificación , Anciano , Anciano de 80 o más Años , Antibacterianos/farmacología , Colistina/farmacología , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Epidemiología Molecular , Filogenia , Plásmidos , Tailandia/epidemiología , Factores de Virulencia , beta-Lactamasas/genética
7.
Nat Commun ; 12(1): 790, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542233

RESUMEN

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.


Asunto(s)
Proteínas Bacterianas/metabolismo , Flavonas/metabolismo , Flavonoles/metabolismo , Microbioma Gastrointestinal/fisiología , Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Clostridiales/enzimología , Clostridiales/genética , Cristalografía por Rayos X , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/ultraestructura , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura
8.
Nat Commun ; 12(1): 796, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33542236

RESUMEN

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Desoxirribosa/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/ultraestructura , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Simulación del Acoplamiento Molecular , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Especificidad por Sustrato , Thermus thermophilus/enzimología , Thermus thermophilus/genética
9.
Int J Biol Macromol ; 169: 551-563, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-33385459

RESUMEN

Alginate lyases are essential tools for depolymerizing alginate into bioactive oligosaccharides and fermentable monosaccharides. Herein, we characterized a novel polysaccharide lyase AlgSH17 from marine bacterium Microbulbifer sp. SH-1. The recombinant enzyme exhibited the maximum activity at 30 °C, pH 7.0 and retained 86.20% and 65.43% of its maximum activity at 20 °C and 15 °C, respectively, indicating that AlgSH17 has an excellent cold-adapted property. The final products of AlgSH17 mainly consisted of monosaccharides with small amounts of oligosaccharides with degrees of polymerization (DP) 2-6, suggesting that AlgSH17 possesses both exolytic and endolytic activity. Degradation pattern analysis indicated that AlgSH17 could degrade DP ≥ 4 oligosaccharides into disaccharides and trisaccharides by cleaving the endo-glycosidic bonds and further digest disaccharides and trisaccharides into monosaccharides in an exolytic manner. Products distribution and molecular docking analysis revealed that AlgSH17 could cleave the glycosidic bonds between -1 and +2 within the substrate. Furthermore, The ABTS+, hydroxyl and DPPH radicals scavenging activity of the enzymatic hydrolysates prepared by AlgSH17 reached up to 91.53%, 81.23% and 61.06%, respectively, and the enzymatic hydrolysates displayed an excellent preservation effect on fresh-cut apples. The above results suggested that AlgSH17 could be utilized for the production of monosaccharides, antioxidants and food additives.


Asunto(s)
Polisacaridoliasas/aislamiento & purificación , Polisacaridoliasas/metabolismo , Alginatos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Gammaproteobacteria/enzimología , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Concentración de Iones de Hidrógeno , Peso Molecular , Monosacáridos/metabolismo , Oligosacáridos/metabolismo , Polisacaridoliasas/química , Especificidad por Sustrato
10.
Int J Biol Macromol ; 172: 360-370, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33460659

RESUMEN

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.


Asunto(s)
Proteínas Bacterianas/química , Cabello/efectos de los fármacos , Proteoglicanos/química , Serina Proteasas/química , Piel/efectos de los fármacos , Mataderos , Animales , Bacillus cereus/química , Bacillus cereus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Brevibacterium/química , Brevibacterium/enzimología , Pruebas de Enzimas , Cabras , Cabello/química , Cinética , Serina Proteasas/aislamiento & purificación , Piel/química , Especificidad por Sustrato
11.
Nat Commun ; 12(1): 460, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469029

RESUMEN

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.


Asunto(s)
Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/patología , Proteínas de Unión al GTP rab1/metabolismo , Regulación Alostérica , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Sitios de Unión/genética , Cristalografía por Rayos X , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/aislamiento & purificación , Factores de Intercambio de Guanina Nucleótido/ultraestructura , Guanosina Trifosfato/metabolismo , Humanos , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/microbiología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Fagocitosis , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/aislamiento & purificación , Proteínas de Unión al GTP rab1/ultraestructura
12.
Nat Commun ; 12(1): 459, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469030

RESUMEN

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteroides/enzimología , Esterasas/metabolismo , Microbioma Gastrointestinal/fisiología , Xilanos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Bacteroides/genética , Colon/microbiología , Ácidos Cumáricos/metabolismo , Cristalografía por Rayos X , Fibras de la Dieta/metabolismo , Pruebas de Enzimas , Esterasas/genética , Esterasas/aislamiento & purificación , Esterasas/ultraestructura , Humanos , Mucosa Intestinal/microbiología , Simulación de Dinámica Molecular , Familia de Multigenes/genética , Especificidad por Sustrato , Xilanos/química
13.
Nat Commun ; 12(1): 144, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420046

RESUMEN

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.


Asunto(s)
Proteínas Bacterianas/farmacología , Chromobacterium/metabolismo , Depsipéptidos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Depsipéptidos/biosíntesis , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Esterasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Hemípteros , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
14.
J Zoo Wildl Med ; 51(4): 733-744, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33480553

RESUMEN

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as the cause of a global pandemic in 2019-2020. In March 2020, New York City became the epicenter in the United States for the pandemic. On 27 March 2020, a Malayan tiger (Panthera tigris jacksoni) at the Bronx Zoo in New York City developed a cough and wheezing with subsequent inappetence. Over the next week, an additional Malayan tiger and two Amur tigers (Panthera tigris altaica) in the same building and three lions (Panthera leo krugeri) in a separate building also became ill. The index case was anesthetized for diagnostic workup. Physical examination and bloodwork results were unremarkable. Thoracic radiography and ultrasonography revealed a bronchial pattern with peribronchial cuffing and mild lung consolidation with alveolar-interstitial syndrome, respectively. SARS-CoV-2 RNA was identified by real-time, reverse transcriptase PCR (rRT-PCR) on oropharyngeal and nasal swabs and tracheal wash fluid. Cytologic examination of tracheal wash fluid revealed necrosis, and viral RNA was detected in necrotic cells by in situ hybridization, confirming virus-associated tissue damage. SARS-CoV-2 was isolated from the tracheal wash fluid of the index case, as well as the feces from one Amur tiger and one lion. Fecal viral RNA shedding was confirmed in all seven clinical cases and an asymptomatic Amur tiger. Respiratory signs abated within 1-5 days for most animals, although they persisted intermittently for 16 days in the index case. Fecal RNA shedding persisted for as long as 35 days beyond cessation of respiratory signs. This case series describes the clinical presentation, diagnostic evaluation, and management of tigers and lions infected with SARS-CoV-2 and describes the duration of viral RNA fecal shedding in these cases. This report documents the first known natural transmission of SARS-CoV-2 from humans to nondomestic felids.


Asunto(s)
/veterinaria , Heces/virología , Leones/virología , Tigres/virología , Animales , Animales de Zoológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , /epidemiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Ciudad de Nueva York/epidemiología , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación
15.
Int J Biol Macromol ; 169: 39-50, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-33316342

RESUMEN

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.


Asunto(s)
Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Actinobacteria/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Detergentes , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Estabilidad de Enzimas/fisiología , Concentración de Iones de Hidrógeno , Cinética , /metabolismo , Serina/química , Serina Proteasas/aislamiento & purificación , Tensoactivos , Temperatura , Termodinámica
16.
Methods Mol Biol ; 2220: 137-153, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975772

RESUMEN

Proteomics has become an essential tool to answer biologists' questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell "compartments" are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins: (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.


Asunto(s)
Proteínas Bacterianas/análisis , Listeria monocytogenes/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Proteínas Bacterianas/aislamiento & purificación , Biotinilación , Fraccionamiento Celular/métodos , Centrifugación/métodos , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Listeria monocytogenes/citología , Listeriosis/microbiología
17.
Carbohydr Polym ; 254: 117463, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33357922

RESUMEN

Better understanding through direct observation of the mechanisms involved in chemical and enzymatic hydrolysis of biomass is of great importance, to implement a substitute for the common cellulose standards. We report the hydrolysis of biomass, using exclusively the parenchyma, to isolate cellulose nanoplatelets using a less harsh pretreatment. Then, we show direct evidence of the effect of endoglucanase on the structure of cellulose nanoplatelets, finding that amorphous cellulose is exclusively digested, loosening the cellulose nanofibrils in the process. The analysis of micrographs demonstrates that when cellulose nanoplatelets are deposited on a silicon wafer, its thickness can be qualitatively measured by the interference color detected using an optical microscope. This finding facilitates further studies of mechanisms involved in lignin removal and cellulose nanofibrils production by specific enzymatic digestion.


Asunto(s)
Agave/química , Proteínas Bacterianas/química , Celulasa/química , Lignina/química , Nanofibras/química , Actinobacteria/química , Actinobacteria/enzimología , Proteínas Bacterianas/aislamiento & purificación , Biomasa , Celulasa/aislamiento & purificación , Humanos , Hidrólisis , Lignina/aislamiento & purificación , Nanofibras/ultraestructura , Ácidos Sulfúricos/química
18.
J Fish Dis ; 44(1): 53-61, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32959452

RESUMEN

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)-based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease-encoding (lldY) gene was selected as a target for the design of S. iniae-specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 101 amplicon copies per assay (equivalent to 2 × 10-9  ng/µl) using bacterial DNA and of 1.44 × 101 gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Enfermedades de los Peces/diagnóstico , Peces/microbiología , Proteínas de Transporte de Membrana/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Infecciones Estreptocócicas/veterinaria , Animales , Proteínas Bacterianas/genética , Lubina/microbiología , Cartilla de ADN , Enfermedades de los Peces/microbiología , Proteínas de Transporte de Membrana/genética , Oncorhynchus mykiss/microbiología , Sensibilidad y Especificidad , Infecciones Estreptocócicas/diagnóstico , Streptococcus iniae/genética , Streptococcus iniae/aislamiento & purificación
19.
Int J Biol Macromol ; 170: 164-177, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33352153

RESUMEN

Thermo-alkaline xylanases are widely applied in paper pulping industry. In this study, a novel thermostable and alkaline tolerant GH10 xylanase (Xyn30Y5) gene from alkaliphilic Bacillus sp. 30Y5 was cloned and the surface-layer homology (SLH) domains truncated enzyme (Xyn30Y5-SLH) was expressed in Escherichia coli. The purified Xyn30Y5-SLH was most active at 70 °C and pH 7.0 and showed the highest specific activity of 349.4 U mg-1. It retained more than 90% activity between pH 6.0 to 9.5 and was stable at pH 6.0-10.0. To improve the activity, 47 mutants were designed based on eight rational strategies and 21 mutants showed higher activity. By combinatorial mutagenesis, the best mutant 3B demonstrated specific activity of 1016.8 U mg-1 with a doubled catalytic efficiency (kcat/Km) and RA601/2h value, accompanied by optimal pH shift to 8.0. The molecular dynamics simulation analysis indicated that the increase of flexibility of α5 helix and loop7 located near to the catalytic residues is likely responsible for its activity improvement. And the decrease of flexibility of the most unstable regions is vital for the thermostablity improvement. This work provided not only a novel thermostable and alkaline tolerant xylanase with industrial application potential but also an effective mutagenesis strategy for xylanase activity improvement.


Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Endo-1,4-beta Xilanasas/aislamiento & purificación , Secuencia de Aminoácidos , Bacillus/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Desnaturalización Proteica , Estabilidad Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Xilanos/metabolismo
20.
Int J Biol Macromol ; 170: 94-106, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33358950

RESUMEN

Considering the need of new lactic acid bacteria (LAB) for the production of novel biosurfactant (BS) molecules, the current study brings out a new insight on the exploration of cheese samples for BS producers and process optimization for industrial applications. In view of this, Lactobacillus plantarum 60FHE, Lactobacillus paracasei 75FHE, and Lactobacillus paracasei 77FHE were selected as the most operative strains. The biosurfactants (BSs) described as glycolipoproteins via Fourier-transform infrared spectroscopy (FTIR) exhibited antimicrobial activity against the food-borne pathogens. L. plantarum 60FHE BS showed an anticancer activity against colon carcinoma cells and had a week antiviral activity against Hepatitis A virus. Furthermore, glycolipoprotein production was enhanced by 1.42-fold through the development of an optimized process using central composite design (CCD). Emulsifying activities were stable after 60-min incubation from 4 to 120 °C, at pH 2-12, and after the addition of NaCl (2-14%). Characterization by nuclear magnetic resonance spectroscopy (1H NMR) revealed that BS produced from strain 60FHE was glycolipoprotein. L. plantarum produced mixed BSs determined by Liquid Chromatography/Mass Spectrometry (LC-MS). Thus, indicating that BS was applied as a microbial food prevention and biomedical. Also, L. plantarum 60FHE BS was achieved with the use of statistical optimization on inexpensive food wastes.


Asunto(s)
Antiinfecciosos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Queso/microbiología , Glicoproteínas/aislamiento & purificación , Lactobacillus plantarum/química , Lipoproteínas/aislamiento & purificación , Tensoactivos/aislamiento & purificación , Antiinfecciosos/química , Antiinfecciosos/economía , Antiinfecciosos/farmacología , Antineoplásicos/química , Antineoplásicos/economía , Antineoplásicos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/economía , Proteínas Bacterianas/farmacología , Línea Celular Tumoral , Cromatografía Liquida , Neoplasias del Colon/patología , Glicoproteínas/química , Glicoproteínas/economía , Glicoproteínas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Virus de la Hepatitis A/efectos de los fármacos , Humanos , Lactobacillus paracasei/química , Lactobacillus paracasei/aislamiento & purificación , Lactobacillus plantarum/aislamiento & purificación , Lipoproteínas/química , Lipoproteínas/economía , Lipoproteínas/farmacología , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Filogenia , Ribotipificación , Espectroscopía Infrarroja por Transformada de Fourier , Tensoactivos/química , Tensoactivos/economía , Tensoactivos/farmacología , Residuos/análisis
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