RESUMO
Impact of high-pressure processing (HP-P) on microbial inactivation, protein oxidation, collagen fiber, and muscle structure of the edible portion (EP) of blood clams (BC) was investigated. Aerobic plate count, Vibrio parahaemolyticus, V. vulnificus, other Vibrio spp. and Shewanella algae counts were not detectable when HP-P pressure of ≥300 MPa was applied. Carbonyl, disulphide bond content, and surface hydrophobicity upsurged as HP-P with augmenting pressure was employed. Protein with â¼53 kDa appeared when HP-P at 100 and 200 MPa was implemented. Increased pressure enhanced gap formation and abnormal muscle cell structure arrangements. HP-P also affected connective tissue, causing size reduction and disruption of the collagen filament fibers. However, firmness and toughness of BC-EP with HP-P ≤ 300 MPa were comparable to those of the control. HP-P at 300 MPa was therefore appropriate for treatment of BC with maintained textural properties, while less protein oxidation, collagen fiber and muscle structure disruption occurred.
Assuntos
Bivalves , Colágeno , Animais , Bivalves/química , Bivalves/microbiologia , Colágeno/química , Pressão , Shewanella/química , Shewanella/metabolismo , Manipulação de Alimentos , Frutos do Mar/análise , Frutos do Mar/microbiologia , Vibrio/química , Músculos/químicaRESUMO
Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.
Assuntos
Percepção de Quorum , Transativadores , Vibrio , Percepção de Quorum/efeitos dos fármacos , Vibrio/efeitos dos fármacos , Vibrio/química , Vibrio/metabolismo , Vibrio/genética , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/metabolismo , Transativadores/química , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/química , Sulfonamidas/farmacologia , Sulfonamidas/química , Tiofenos/química , Tiofenos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/químicaRESUMO
Vibrio species are common inhabitants of aquatic environments and have been described in connection with fish and human diseases. Six Vibrio species were isolated from diseased freshwater and ornamental fish in Poland. The strains were identified based on morphological and biochemical characteristics and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as V. albensis (n=3) from Gymnocephalus cernua, Sander lucioperca, Paracheirodon innesi, and Xiphophorus hellerii; V. mimicus (n=1) from Xiphophorus maculatus; and V. vulnificus (n=1) from Nematobrycon palmeri. This is the first time that Vibrio species have been isolated and described from ornamental fish in Poland. The isolates were resistant to ampicillin (83.3%), gentamicin (16.6%), ciprofloxacin (16.6%), sulfamethoxazole-trimethoprim (16.6%), and chloramphenicol (16.6%). The multiple antibiotic resistance (MAR) index was 0.00-0.08 for V. albensis, 0.17 for V. mimicus, and 0.33 for V. vulnificus. Our study confirmed the presence of potentially pathogenic Vibrio species in freshwater and ornamental fish. Therefore, further monitoring of the presence of Vibrio species, mainly in ornamental fish, is necessary.
Assuntos
Vibrioses , Vibrio , Humanos , Animais , Polônia/epidemiologia , Vibrioses/epidemiologia , Vibrioses/veterinária , Vibrio/química , Peixes , Água DoceRESUMO
Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican.
Assuntos
Proteínas de Bactérias , Bacteroides , Polissacarídeo-Liases , Polissacarídeos Bacterianos , Vibrio , Oligossacarídeos/química , Polissacarídeo-Liases/química , Polissacarídeos Bacterianos/química , Vibrio/química , Proteínas de Bactérias/química , Ressonância Magnética Nuclear Biomolecular , Bacteroides/enzimologiaRESUMO
Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax â¼ 490 nm) and green-absorbing PR (GPR; λmax â¼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.
Assuntos
Rodopsinas Microbianas/química , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Bases de Schiff/química , Vibrio/químicaRESUMO
The marine bacterium Vibrio campbellii expresses a chitooligosaccharide-specific outer-membrane channel (chitoporin) for the efficient uptake of nutritional chitosugars that are externally produced through enzymic degradation of environmental host shell chitin. However, the principles behind the distinct substrate selectivity of chitoporins are unclear. Here, we employed black lipid membrane (BLM) electrophysiology, which handles the measurement of the flow of ionic current through porins in phospholipid bilayers for the assessment of porin conductivities, to investigate the pH dependency of chitosugar-chitoporin interactions for the bacterium's natural substrate chitohexaose and its deacetylated form, chitosan hexaose. We show that efficient passage of the N-acetylated chitohexaose through the chitoporin is facilitated by its strong affinity for the pore. In contrast, the deacetylated chitosan hexaose is impermeant; however, protonation of the C2 amino entities of chitosan hexaose allows it to be pulled through the channel in the presence of a transmembrane electric field. We concluded from this the crucial role of C2-substitution as the determining factor for chitoporin entry. A change from N-acetylamino- to amino-substitution effectively abolished the ability of approaching molecules to enter the chitoporin, with deacetylation leading to loss of the distinctive structural features of nanopore opening and pore access of chitosugars. These findings provide further understanding of the multistep pathway of chitin utilization by marine Vibrio bacteria and may guide the development of solid-state or genetically engineered biological nanopores for relevant technological applications.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Quitosana/metabolismo , Oligossacarídeos/metabolismo , Porinas/metabolismo , Vibrio/metabolismo , Acetilação , Proteínas da Membrana Bacteriana Externa/química , Quitosana/química , Modelos Moleculares , Oligossacarídeos/química , Porinas/química , Conformação Proteica , Vibrio/químicaRESUMO
Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.
Assuntos
Luciferases Bacterianas/química , Simulação de Dinâmica Molecular , Photobacterium/química , Vibrio/química , Domínios Proteicos , Espectrometria de FluorescênciaRESUMO
The quorum-sensing signaling systems in Vibrio bacteria converge to control levels of the master transcription factors LuxR/HapR, a family of highly conserved proteins that regulate gene expression for bacterial behaviors. A compound library screen identified 2-thiophenesulfonamide compounds that specifically inhibit Vibrio campbellii LuxR but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on Vibrio quorum sensing. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), inhibits quorum sensing in multiple strains of V. vulnificus, V. parahaemolyticus, and V. campbellii at nanomolar concentrations. However, thiophenesulfonamide inhibition efficacy varies significantly among Vibrio species: PTSP is most inhibitory against V. vulnificus SmcR, but V. cholerae HapR is completely resistant to all thiophenesulfonamides tested. Reverse genetics experiments show that PTSP efficacy is dictated by amino acid sequence in the putative ligand-binding pocket: F75Y and C170F SmcR substitutions are each sufficient to eliminate PTSP inhibition. Further, in silico modeling distinguished the most potent thiophenesulfonamides from less-effective derivatives. Our results revealed the previously unknown differences in LuxR/HapR proteins that control quorum sensing in Vibrio species and underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.
Assuntos
Percepção de Quorum/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Vibrio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/química , Especificidade da Espécie , Relação Estrutura-Atividade , Sulfonamidas/química , Transativadores/química , Vibrio/química , Vibrio/genéticaRESUMO
Vibrio species are prevalent in the aquatic environments and can infect humans and aquatic organisms. Vibrio parahaemolyticus counteracts ß-lactam antibiotics and enhances virulence using a regulation mechanism mediated by a two-component regulatory system (TCS) consisting of the VbrK histidine kinase and the VbrR response regulator. The periplasmic sensor domain of VbrK (VbrKSD) detects ß-lactam antibiotics or undergoes S-nitrosylation in response to host nitrites. Although V. parahaemolyticus VbrKSD (vpVbrKSD) has recently been characterized through structural studies, it is unclear whether its structural features that are indispensable for biological functions are conserved in other VbrK orthologs. To structurally define the functionally critical regions of VbrK and address the structural dynamics of VbrK, we determined the crystal structures of Vibrio rotiferianus VbrKSD (vrVbrKSD) in two crystal forms and performed a comparative analysis of diverse VbrK structures. vrVbrKSD folds into a curved rod-shaped two-domain structure as observed in vpVbrKSD. The membrane-distal end of the vrVbrKSD structure, including the α3 helix and its neighboring loops, harbors both S-nitrosylation and antibiotic-sensing sites and displays high structural flexibility and diversity. Noticeably, the distal end is partially stabilized by a disulfide bond, which is formed by the cysteine residue that is S-nitrosylated in response to nitrite. Therefore, the distal end of VbrKSD plays a key role in initiating the VbrK-VbrR TCS pathway activation, and it is involved in the nitrosylation-mediated regulation of the structural dynamics of VbrK.
Assuntos
Proteínas de Bactérias/química , Histidina Quinase/química , Vibrio/química , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Histidina Quinase/metabolismo , Modelos Moleculares , Nitritos/metabolismo , Domínios Proteicos , Vibrio/metabolismoRESUMO
Biosynthesis of silver nanoparticles (AgNPs) by marine bacteria especially luminescent Vibrio species is least investigated. In this study, AgNPs were first synthesized by the culture supernatant of a luminescent bacterium (Vibrio sp. B4L) and then, the prepared samples were characterized employing several techniques. The antibacterial activity of the AgNPs was investigated against Escherichia coli and Staphylococcus aureus using disk diffusion agar and broth microdilution methods. The growth curve, Reactive Oxygen Species (ROS) formation, and Lactate Dehydrogenase (LDH) activity of the samples were measured along with Field Emission Scanning Electron Microscopy (FESEM) observation and inhibition of biofilm formation. Dynamic light scattering (DLS) analysis showed that the average particle size of the synthesized AgNPs was in the range of about 32.67-107.18 nm and the polydispersity index (PDI) of 0.1120 indicated the formation of monodispersed particles. The average zeta potential of AgNPs obtained -36.15 mV, showing the high stability of biosynthetic nanoparticles. Antibacterial studies indicated that not only the AgNPs had antibacterial activity but also increased the antibacterial properties of tetracycline when used in combination. ROS production was enhanced in a dose-dependent manner. A high difference in LDH activities was found between AgNPs treated cells and the control group. FESEM images revealed membrane disruption and lysis in AgNPs treated cells. The formation of E. coli biofilm was 100% inhibited at 62.5 µg/ml showing that our bacteriogenic AgNPs can be a potential alternative remedies for controlling antibiotic-resistant pathogens.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Vibrio/química , Antibacterianos/biossíntese , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Relação Dose-Resposta a Droga , Luminescência , Testes de Sensibilidade Microbiana , Estrutura Molecular , Prata/química , Prata/metabolismo , Relação Estrutura-Atividade , Vibrio/metabolismoRESUMO
This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo2H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(-) bacteria membranes. We also investigated the effect of marennine using 31P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.
Assuntos
Microalgas/química , Fenóis/química , Vibrio/química , Deutério , Espectroscopia de Ressonância Magnética , FósforoRESUMO
Methionine aminopeptidases (MetAPs) have been recognized as drug targets and have been extensively studied for discovery of selective inhibitors. MetAPs are essential enzymes in all living cells. While most prokaryotes contain a single gene, some prokaryotes and all eukaryotes including human have redundancy. Due to the similarity in the active sites of the MetAP enzyme between the pathogens and human limited the success of discovering selective inhibitors. We recently have discovered that MetAPs with small inserts within the catalytic domain to have different susceptibilities against some inhibitors compared to those that do not have. Using this clue we used bioinformatic tools to identify new variants of MetAPs with inserts in pathogenic species. Two new isoforms were identified in Vibrio species with two and three inserts in addition to an isoform without any insert. Multiple sequence alignment suggested that inserts are conserved in several of the Vibrio species. Two of the three inserts are common between two and three insert isoforms. One of the inserts is identified to have "NNKNN" motif that is similar to well-characterized quorum sensing peptide, "NNWNN". Another insert is predicted to have a posttranslational modification site. Three Vibrio proteins were cloned, expressed, purified, enzyme kinetics established and inhibitor screening has been performed. Several of the pyridinylpyrimidine derivatives selectively inhibited MetAPs with inserts compared to those that do not have, including the human enzyme. Crystal structure and molecular modeling studies provide the molecular basis for selective inhibition.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metionil Aminopeptidases/antagonistas & inibidores , Vibrio/enzimologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Metionil Aminopeptidases/química , Metionil Aminopeptidases/metabolismo , Simulação de Acoplamento Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Vibrio/química , Vibrio/metabolismoRESUMO
Quorum sensing is a cell density-dependent form of cellular communication among bacteria. This signaling process has been heavily studied in vibrios due to their diverse and complex phenotypes and relevance to human and aquaculture disease. Mechanistic studies of Vibrio quorum sensing have required optimization of protein purification techniques to examine the role of key proteins, such as the LuxR/HapR family of transcription factors that control quorum-sensing gene expression. Protein purification is the cornerstone of biochemistry, and it is crucial to consistently produce batches of protein that are pure, active, and concentrated to perform various assays. The methods described here are optimized for purification of the Vibrio master quorum-sensing regulators, LuxR (Vibrio harveyi), HapR (Vibrio cholerae), and SmcR (Vibrio vulnificus). We anticipate that these methods can be applied to other proteins in this family of transcription factors.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Vibrio/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Percepção de Quorum , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Bacterial luciferase catalyzes a bioluminescent reaction by oxidizing long-chain aldehydes to acids using reduced FMN and oxygen as co-substrates. Although a flavin C4a-peroxide anion is postulated to be the intermediate reacting with aldehyde prior to light liberation, no clear identification of the protonation status of this intermediate has been reported. Here, transient kinetics, pH variation, and site-directed mutagenesis were employed to probe the protonation state of the flavin C4a-hydroperoxide in bacterial luciferase. The first observed intermediate, with a λmax of 385 nm, transformed to an intermediate with a λmax of 375 nm. Spectra of the first observed intermediate were pH-dependent, with a λmax of 385 nm at pH < 8.5 and 375 at pH > 9, correlating with a pKa of 7.7-8.1. These data are consistent with the first observed flavin C4a intermediate at pH < 8.5 being the protonated flavin C4a-hydroperoxide, which loses a proton to become an active flavin C4a-peroxide. Stopped-flow studies of His44Ala, His44Asp, and His44Asn variants showed only a single intermediate with a λmax of 385 nm at all pH values, and none of these variants generate light. These data indicate that His44 variants only form a flavin C4a-hydroperoxide, but not an active flavin C4a-peroxide, indicating an essential role for His44 in deprotonating the flavin C4a-hydroperoxide and initiating chemical catalysis. We also investigated the function of the adjacent His45; stopped-flow data and molecular dynamics simulations identify the role of this residue in binding reduced FMN.
Assuntos
Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Peróxido de Hidrogênio/química , Luciferases Bacterianas/química , Oxigênio/química , Vibrio/química , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Vibrio/enzimologiaRESUMO
Synthesis of bacterial cell surface l-glycero-d-manno-heptose (l,d-Hep)- and d-glycero-d-manno-heptose (d,d-Hep)-containing higher carbon sugars is a challenging task. Here, we report a convenient and efficient approach for the synthesis of the l,d-Hep and d,d-Hep building blocks. Using l-lyxose and d-ribose as starting materials, this approach features diastereoselective Mukaiyama-type aldol reactions as the key steps. On the basis of the synthetic l,d-Hep and d,d-Hep building blocks, we achieved the first stereoselective synthesis of the unique α-l,d-Hep-(1â3)-α-d,d-Hep-(1â5)-α-Kdo core trisaccharide of the lipopolysaccharide of Vibrio parahemolyticus O2.
Assuntos
Heptoses/síntese química , Lipopolissacarídeos/síntese química , Trissacarídeos/síntese química , Vibrio/química , Heptoses/química , Lipopolissacarídeos/química , Estrutura Molecular , Trissacarídeos/químicaRESUMO
A method has been established to map a bacterial colony to the ever-expanding database of publicly available bacterial genomes by means of matrix-assisted laser desorption/ionization (MALDI) spectra. To accomplish this, spectra are mapped to the predicted masses of â¼65 families of mostly ribosomal proteins. Each of the â¼40â¯000 bacterial strains in the database receives scores, together with tables listing identified protein sequences and how the highest ranking strains are related to one another. The approach was first confirmed with 16 distinct species of bacteria from the Vibrionales whose genome had been sequenced. Identifications of a few species of bacteria from environmental samples from compost, lakes, and streams in Massachusetts are also reported. Most of these organisms map to known species in the Gammaproteobacteria and Firmicutes. The clades of bacteria deducible from shared ribosomal protein sequences do not always correspond well to named bacterial species. Instead, the identifications made by this methodology indicate groupings of organisms that can readily be distinguished by MALDI-TOF and indicate which polymorphisms in highly conserved proteins demarcate the groupings. Successful identifications highlight organism interrelationships that can be deduced from the available genomes, sorting together genomes into new proposed clades typically consistent with relationships deduced from DNA sequence analysis. In contrast, if for a high-quality spectrum from a fresh colony, no group of related organisms receives high scores, one might infer that no closely related genome has yet been deposited into the database.
Assuntos
Proteínas de Bactérias/química , Bases de Dados de Ácidos Nucleicos , Genoma Bacteriano/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aeromonas/química , Aeromonas/genética , Aeromonas/isolamento & purificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Água Doce/microbiologia , Massachusetts , Reprodutibilidade dos Testes , Software , Vibrio/química , Vibrio/genéticaRESUMO
In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Fluorescência Verde/biossíntese , Vibrio/química , Sistema Livre de Células , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Plasmídeos , Vibrio/metabolismoRESUMO
The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2â6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.
Assuntos
Frutanos/química , Frutose/química , Gluconobacter oxydans/enzimologia , Hexosiltransferases/química , Oligossacarídeos/química , Sphingomonadaceae/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Burkholderiaceae/química , Burkholderiaceae/enzimologia , Frutanos/biossíntese , Frutose/metabolismo , Expressão Gênica , Gluconobacter oxydans/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Oligossacarídeos/biossíntese , Prebióticos/análise , Ligação Proteica , Conformação Proteica , Rafinose/química , Rafinose/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Sphingomonadaceae/química , Homologia Estrutural de Proteína , Especificidade por Substrato , Sacarose/química , Sacarose/metabolismo , Vibrio/química , Vibrio/enzimologiaRESUMO
Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a rapid, cost-effective and high-throughput method for bacteria characterization. However, most previous studies focused on clinical isolates. In this study, we evaluated the use of MALDI-TOF MS as a rapid screening tool for marine bacterial symbionts. A set of 255 isolates from different marine sources (corals, sponge, fish and seawater) was analyzed using cell lysates to obtain a rapid grouping. Cluster analysis of mass spectra and 16S rRNA showed 18 groups, including Vibrio, Bacillus, Pseudovibrio, Alteromonas and Ruegeria. MALDI-TOF distance similarity scores ≥ 60% and ≥ 70% correspond to ≥ 98.7% 16S rRNA gene sequence similarity and ≥ 95% pyrH gene sequence similarity, respectively. MALDI-TOF MS is a useful tool for Vibrio species groups' identification.
Assuntos
Microbiologia Ambiental , Biologia Marinha/métodos , Água do Mar/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vibrio/classificação , RNA Ribossômico 16S/genética , Simbiose , Vibrio/química , Vibrio/genéticaRESUMO
Simple, reliable and flexibly multiplexed genetic identification and quantification of microbial pathogens is in urgent need for early disease diagnosis and timely treatment. This study presented an isothermal amplification-based portable microfluidic system (iso-µmGene) with features of multi-well chips for convenient filling and reliable sealing, flexible detection throughput, and a stand-alone and well-performing point of care (POC) genetic testing device. Using disposable chips with two kinds of reaction wells (eighteen and ten wells) and a device prototype with independent four chip holders, the iso-µmGene enables on-demand analysis of different target genes in one sample per chip and one to four samples (chips) per run, requiring only a single pipetting step for dispensing per chip with dehydrated primers. To completely seal the loop-mediated isothermal amplification (LAMP) reaction system to minimize the risk of amplicon escape, a dedicated plastic shell is used to assemble the array-type chip and reliably close its openings. Meanwhile, to enhance the precision for flexibly multiplexed detection and decrease the size and cost of the device, we designed a thermoelectric cooler (TEC)-based temperature-control module including two separate units and a CCD-based fluorescence imaging module containing a linear translation stage for real-time LAMP assay. This work demonstrated applications for the parallel detection of 2-2000 CFU (colony forming units) per reaction well with good intra- and inter-chip reproducibility using the crude lysates of two aquaculture pathogens Edwardsiella tarda and Vibrio harveyi. Overall, the iso-µmGene presented here possesses both a sophisticated instrument's functionality and performance and POC device's portability and cost.