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1.
Methods Mol Biol ; 2392: 17-33, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34773612

RESUMO

Classical restriction fragment length polymorphism (RFLP) and sequencing are labor-intensive and expensive methods to study single base changes, whereas polymerase chain reaction amplification of specific alleles (PASA) or allele-specific polymerase chain reaction (ASPCR) is a PCR-based application that allows direct detection of any point mutation by analyzing the PCR products in an ethidium bromide-stained agarose or polyacrylamide gel. PASA is based on oligonucleotide primers containing one or more 3' mismatch with the target DNA making it refractory to primer extension by Thermus aquaticus DNA polymerase lacking the 3' to 5' exonuclease proofreading activity because of which it is also called amplification refractory mutation system-PCR (ARMS-PCR). This technique has found application in detection of allele, mutation, single-nucleotide polymorphisms (SNPs) causing genetic and infectious diseases. This chapter describes an approach of cohort PASA in context of genotyping single and double mutant worms generated to study the process of cell migration and axon outgrowth in C. elegans. Single worm-based cohort PASA allows genotyping for identification of single base mutations; particularly it is convenient method to detect mutations without a visible phenotype.


Assuntos
Caenorhabditis elegans , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Caenorhabditis elegans/genética , Genótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Taq Polimerase , Thermus
2.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33883282

RESUMO

To initiate transcription, the holoenzyme (RNA polymerase [RNAP] in complex with σ factor) loads the promoter DNA via the flexible loading gate created by the clamp and ß-lobe, yet their roles in DNA loading have not been characterized. We used a quasi-Markov State Model (qMSM) built from extensive molecular dynamics simulations to elucidate the dynamics of Thermus aquaticus holoenzyme's gate opening. We showed that during gate opening, ß-lobe oscillates four orders of magnitude faster than the clamp, whose opening depends on the Switch 2's structure. Myxopyronin, an antibiotic that binds to Switch 2, was shown to undergo a conformational selection mechanism to inhibit clamp opening. Importantly, we reveal a critical but undiscovered role of ß-lobe, whose opening is sufficient for DNA loading even when the clamp is partially closed. These findings open the opportunity for the development of antibiotics targeting ß-lobe of RNAP. Finally, we have shown that our qMSMs, which encode non-Markovian dynamics based on the generalized master equation formalism, hold great potential to be widely applied to study biomolecular dynamics.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Simulação de Dinâmica Molecular , Thermus/enzimologia , Cadeias de Markov
3.
Curr Microbiol ; 78(5): 1903-1913, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33786643

RESUMO

In this study, using a metagenomic approach, we explore the bacterial diversity of compost sites categorized based on their ambient temperatures. The two sites were Reckong Peo in the lower Himalayas and Tambaram in the southern region of the country, namely, CPR and CT. Following assembly of the raw reads from shotgun metagenomics, similarity hits were generated using NCBI BLAST + and SILVA database. A total of 1463 and 1483 species were annotated from CPR and CT. A species-level annotation was performed using a python-based literature search pipeline revealing their growth characteristics. Thermophiles Thermomonospora curvata and Thermus scotoductus were among the prominent species in CT. CPR too was seen abundant with Acidothermus cellulolyticus and Moorella thermoacetica, constituting 10% of the population. Nearly 3% of the identified species in the site CPR were psychrophilic. Although found higher in CPR, psychrophilic species were identified in CT too. Flavobacterium and Psychrobacter spp. were present in both sites without any significant changes in their relative distribution contrary to the thermophilic species abundance (z = - 4.3). Akin to the sequenced samples, database-derived metagenomes also showed similar distribution of thermophiles and psychrophiles. Identifying such peculiar prevalence of extremophiles can be central to understanding extended growth temperatures.


Assuntos
Compostagem , Metagenômica , Actinobacteria , Moorella , Temperatura , Thermus
4.
Nucleic Acids Res ; 49(7): 4129-4143, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33784404

RESUMO

Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.


Assuntos
Proteínas de Bactérias , DNA Helicases , DNA de Cadeia Simples/metabolismo , Thermus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica
5.
Appl Environ Microbiol ; 87(11)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33741620

RESUMO

A novel gene cluster involved in the degradation of lignin-derived monoaromatics such as p-hydroxybenzoate, vanillate, and ferulate has been identified in the thermophilic nitrate reducer Thermus oshimai JL-2. Based on conserved domain analyses and metabolic pathway mapping, the cluster was classified into upper- and peripheral-pathway operons. The upper-pathway genes, responsible for the degradation of p-hydroxybenzoate and vanillate, are located on a 0.27-Mb plasmid, whereas the peripheral-pathway genes, responsible for the transformation of ferulate, are spread throughout the plasmid and the chromosome. In addition, a lower-pathway operon was also identified in the plasmid that corresponds to the meta-cleavage pathway of catechol. Spectrophotometric and gene induction data suggest that the upper and lower operons are induced by p-hydroxybenzoate, which the strain can degrade completely within 4 days of incubation, whereas the peripheral genes are expressed constitutively. The upper degradation pathway follows a less common route, proceeding via the decarboxylation of protocatechuate to form catechol, and involves a novel thermostable γ-carboxymuconolactone decarboxylase homolog, identified as protocatechuate decarboxylase based on gene deletion experiments. This gene cluster is conserved in only a few members of the Thermales and shows traces of vertical expansion of catabolic pathways in these organisms toward lignoaromatics.IMPORTANCE High-temperature steam treatment of lignocellulosic biomass during the extraction of cellulose and hemicellulose fractions leads to the release of a wide array of lignin-derived aromatics into the natural ecosystem, some of which can have detrimental effects on the environment. Not only will identifying organisms capable of using such aromatics aid in environmental cleanup, but thermostable enzymes, if characterized, can also be used for efficient lignin valorization. However, no thermophilic lignin degraders have been reported thus far. The present study reports T. oshimai JL-2 as a thermophilic bacterium with the potential to use lignin-derived aromatics. The identification of a novel thermostable protocatechuate decarboxylase gene in the strain further adds to its significance, as such an enzyme can be efficiently used in the biosynthesis of cis,cis-muconate, an important intermediate in the commercial production of plastics.


Assuntos
Ácidos Cumáricos/metabolismo , Lignina/metabolismo , Parabenos/metabolismo , Thermus/metabolismo , Ácido Vanílico/metabolismo , Genes Bacterianos , Família Multigênica , Thermus/genética
6.
Microbiologyopen ; 10(1): e1149, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33415847

RESUMO

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.


Assuntos
DNA Polimerase I/genética , DNA Polimerase I/metabolismo , Thermus/enzimologia , Thermus/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Polimerase I/química , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Taq Polimerase/genética , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
7.
J Microbiol Biotechnol ; 31(1): 43-50, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33046683

RESUMO

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Sequência de Aminoácidos , Amilose , Carboidratos , Catálise , Ciclização , Ciclodextrinas/metabolismo , Deinococcus/enzimologia , Escherichia coli/enzimologia , Sistema da Enzima Desramificadora do Glicogênio/classificação , Sistema da Enzima Desramificadora do Glicogênio/genética , Cinética , Filogenia , Thermus/enzimologia
8.
Microbiol Res ; 243: 126655, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33279728

RESUMO

How cell morphology is maintained in thermophilic bacteria is unknown. In this study, the functions and mechanisms of the potential cell shape determinants (e.g. MreB, MreC, MreD and RodA homologues) of the model extremely thermophilic bacterium Thermus thermophilus were initially analyzed. Deletion of mreC, mreD or rodA only resulted in heterozygous mutants indicating that these genes are all essential. In the MreB-inhibited (by A22) strain and the heterozygous mreC, mreD or rodA mutant, cell morphologies were drastically changed, and enlarged spherical cells were eventually dead indicating that they are vital for cell shape maintenance. When fused to sGFP, MreB, MreC, MreD, RodA, and the enzymes involved in peptidoglycan synthesis (e.g. PBP2 and MurG) exhibited similar subcellular localization pattern, appearing as patches, or bands slightly angled to the cell length. The localizations and functions of all the 6 proteins required a natural peptidoglycan synthesis pattern, additionally those of MreD, RodA and MurG were dependent on MreB polymerization. Consistently, through comprehensive bacterial two-hybrid analyses, it was revealed that MreB could interact with itself, MreC, MreD, RodA and MurG, and MreC could associate with PBP2. In conclusion, in T. thermophilus, MreB, MreC, MreD, RodA and the peptidoglycan synthesis enzymes probably form a network of interactions centered with MreB and bridged with MreC, thereby maintaining cell morphology.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Peptidoglicano/biossíntese , Thermus/citologia , Thermus/metabolismo , Proteínas de Bactérias/genética , Parede Celular/genética , Regulação Bacteriana da Expressão Gênica , Thermus/genética
9.
J Dairy Sci ; 104(3): 2632-2640, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33358792

RESUMO

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.


Assuntos
Bacteriocinas , Lactococcus lactis , Nisina , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Thermus
10.
Nat Commun ; 11(1): 6033, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247115

RESUMO

MicroRNAs (miRNAs) are short (19-24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3'-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.


Assuntos
MicroRNAs/análise , MicroRNAs/isolamento & purificação , Sequência de Bases , Linhagem Celular , Humanos , Thermus/genética
11.
Chem Commun (Camb) ; 56(68): 9850-9853, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32716419

RESUMO

By combining X-ray crystallography, electron paramagnetic resonance techniques and density functional theory-based modelling, we provide evidence for a direct coordination of the product analogue, phosphate, to the molybdenum active site of a sulfite dehydrogenase. This interaction is mimicking the still experimentally uncharacterized reaction intermediate proposed to arise during the catalytic cycle of this class of enzymes. This work opens new perspectives for further deciphering the reaction mechanism of this nearly ubiquitous class of oxidoreductases.


Assuntos
Molibdênio/química , Fosfatos/química , Sulfito Desidrogenase/química , Domínio Catalítico , Cristalografia por Raios X , Teoria da Densidade Funcional , Espectroscopia de Ressonância de Spin Eletrônica , Ligação de Hidrogênio , Sulfito Desidrogenase/metabolismo , Thermus/enzimologia
12.
Microb Ecol ; 80(3): 614-626, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32474659

RESUMO

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2-) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Óxido Nítrico/metabolismo , Nitrito Redutases/genética , Thermus/genética , Proteínas de Bactérias/metabolismo , Nitrito Redutases/metabolismo , Thermus/enzimologia
13.
Funct Integr Genomics ; 20(5): 621-631, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32377887

RESUMO

Chaperones are important molecular machinery that assists proteins to attain their native three-dimensional structure crucial for function. Earlier studies using experimental evolution showed that chaperones impose a relaxation of sequence constraints on their "client" proteins, which may lead to the fixation of slightly deleterious mutations on the latter. However, we hypothesized that such a phenomenon might be harmful to the organism in a natural physiological condition. In this study, we investigated the evolutionary rates of chaperone client and non-client proteins in five model organisms from both prokaryotic and eukaryotic lineages. Our study reveals a slower evolutionary rate of chaperone client proteins in all five organisms. Additionally, the slower folding rate and lower aggregation propensity of chaperone client proteins reveal that the chaperone may play an essential role in rescuing the slightly disadvantageous effects due to random mutations and subsequent protein misfolding. However, the fixation of such mutations is less likely to be selected in the natural population.


Assuntos
Evolução Molecular , Chaperonas Moleculares , Proteínas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Mutação , Agregados Proteicos , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Thermus/genética
14.
Proc Natl Acad Sci U S A ; 117(20): 10856-10864, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371489

RESUMO

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.


Assuntos
DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Thermus/genética
15.
Protein Expr Purif ; 174: 105676, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32442498

RESUMO

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.


Assuntos
Anti-Infecciosos , Bacteriófagos , Endopeptidases , Siphoviridae , Thermus/virologia , Proteínas Virais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bacteriófagos/enzimologia , Bacteriófagos/genética , Endopeptidases/biossíntese , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Siphoviridae/enzimologia , Siphoviridae/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/farmacologia
16.
Appl Microbiol Biotechnol ; 104(10): 4407-4415, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32232528

RESUMO

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted ß-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards ß-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M-1 s-1) and HorGH1-M229R (13,260 M-1 s-1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce ß-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of ß-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted ß-glycosidases. •ß-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.


Assuntos
Proteínas de Bactérias/genética , Firmicutes/genética , Glicosídeo Hidrolases/genética , Thermus/genética , beta-Glucosidase/genética , Sequência de Aminoácidos , Arginina/genética , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Firmicutes/enzimologia , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/metabolismo , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Especificidade por Substrato , Thermus/enzimologia , beta-Glucosidase/metabolismo
17.
J Appl Microbiol ; 129(5): 1287-1296, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32330366

RESUMO

AIM: To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. METHODS AND RESULTS: TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5 U TfAM g-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. CONCLUSIONS: TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Saccharomyces cerevisiae/genética , Amido/metabolismo , Thermus/enzimologia , Amilose/metabolismo , Proteínas de Bactérias/genética , Ciclodextrinas/biossíntese , Ciclodextrinas/química , Sistema da Enzima Desramificadora do Glicogênio/genética , Manihot/química , Ervilhas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Temperatura , Thermus/genética
18.
Nucleic Acids Res ; 48(5): 2762-2776, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32009148

RESUMO

OLD family nucleases contain an N-terminal ATPase domain and a C-terminal Toprim domain. Homologs segregate into two classes based on primary sequence length and the presence/absence of a unique UvrD/PcrA/Rep-like helicase gene immediately downstream in the genome. Although we previously defined the catalytic machinery controlling Class 2 nuclease cleavage, degenerate conservation of the C-termini between classes precludes pinpointing the analogous residues in Class 1 enzymes by sequence alignment alone. Our Class 2 structures also provide no information on ATPase domain architecture and ATP hydrolysis. Here we present the full-length structure of the Class 1 OLD nuclease from Thermus scotoductus (Ts) at 2.20 Å resolution, which reveals a dimerization domain inserted into an N-terminal ABC ATPase fold and a C-terminal Toprim domain. Structural homology with genome maintenance proteins identifies conserved residues responsible for Ts OLD ATPase activity. Ts OLD lacks the C-terminal helical domain present in Class 2 OLD homologs yet preserves the spatial organization of the nuclease active site, arguing that OLD proteins use a conserved catalytic mechanism for DNA cleavage. We also demonstrate that mutants perturbing ATP hydrolysis or DNA cleavage in vitro impair P2 OLD-mediated killing of recBC-Escherichia coli hosts, indicating that both the ATPase and nuclease activities are required for OLD function in vivo.


Assuntos
Trifosfato de Adenosina/metabolismo , Biocatálise , Endonucleases/química , Endonucleases/metabolismo , Thermus/enzimologia , Adenosina Trifosfatases/química , Sequência Conservada , Hidrólise , Metais/metabolismo , Modelos Moleculares , Mutação/genética , Domínios Proteicos
19.
Nat Commun ; 11(1): 610, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001694

RESUMO

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L.


Assuntos
Complexo I de Transporte de Elétrons/química , NADPH Desidrogenase/química , Fotossíntese , Thermus/enzimologia , Sítios de Ligação , Carotenoides/química , Membrana Celular/química , Transporte de Elétrons , Complexo I de Transporte de Elétrons/ultraestrutura , Ferredoxinas/química , Ferredoxinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Modelos Moleculares , NADPH Desidrogenase/ultraestrutura , Plastoquinona/química , Plastoquinona/metabolismo , Domínios Proteicos , Subunidades Proteicas/química , Homologia Estrutural de Proteína
20.
Viruses ; 12(2)2020 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-32050494

RESUMO

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 µg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.


Assuntos
Anti-Infecciosos/farmacologia , Endopeptidases/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Proteínas Virais/farmacologia , Animais , Bacteriófagos/enzimologia , Bacteriófagos/genética , Feminino , Genoma Viral , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Pele/efeitos dos fármacos , Pele/microbiologia , Pele/patologia , Thermus/virologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia
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