RESUMEN
The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other ß-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.
Asunto(s)
Modelos Moleculares , beta-Lactamasas , beta-Lactamasas/química , beta-Lactamasas/metabolismo , beta-Lactamasas/ultraestructura , beta-Lactamasas/genética , Alcaligenes faecalis/enzimología , Alcaligenes faecalis/química , Conformación Proteica , Zinc/química , Zinc/metabolismo , Cristalografía por Rayos X , Dominio Catalítico , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Secuencia de Aminoácidos , Sitios de UniónRESUMEN
Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 âNH2 OHâN2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.
Asunto(s)
Alcaligenes faecalis , Alcaligenes faecalis/química , Alcaligenes faecalis/genética , Alcaligenes faecalis/metabolismo , Amoníaco/metabolismo , Sitios de Unión , Factores de Transcripción/genética , Transcripción Genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Picolinic acid (PA) is a natural toxic pyridine derivative as well as an important intermediate used in the chemical industry. In a previous study, we identified a gene cluster, pic, that responsible for the catabolism of PA in Alcaligenes faecalis JQ135. However, the transcriptional regulation of the pic cluster remains known. This study showed that the entire pic cluster was composed of 17 genes and transcribed as four operons: picR, picCDEF, picB4B3B2B1, and picT1A1A2A3T2T3MN. Deletion of picR, encoding a putative MarR-type regulator, greatly shortened the lag phase of PA degradation. An electrophoretic mobility shift assay and DNase I footprinting showed that PicR has one binding site in the picR-picC intergenic region and two binding sites in the picB-picT1 intergenic region. The DNA sequences of the three binding sites have the palindromic characteristics of TCAG-N4-CTNN: the space consists of four nonspecific bases, and the four palindromic bases on the left and the first two palindromic bases on the right are strictly conserved, while the last two bases on the right vary among the three binding sites. An in vivo ß-galactosidase activity reporter assay indicated that 6-hydroxypicolinic acid but not PA acted as a ligand of PicR, preventing PicR from binding to promoter regions and thus derepressing the transcription of the pic cluster. This study revealed the negative transcriptional regulation mechanism of PA degradation by PicR in A. faecalis JQ135 and provides new insights into the structure and function of the MarR-type regulator. IMPORTANCE The pic gene cluster was found to be responsible for PA degradation and widely distributed in Alpha-, Beta-, and Gammaproteobacteria. Thus, it is very necessary to understand the regulation mechanism of the pic cluster in these strains. This study revealed that PicR binds to three sites of the promoter regions of the pic cluster to multiply regulate the transcription of the pic cluster, which enables A. faecalis JQ135 to efficiently utilize PA. Furthermore, the study also found a unique palindrome sequence for binding of the MarR-type regulator. This study enhanced our understanding of microbial catabolism of environmental toxic pyridine derivatives.
Asunto(s)
Alcaligenes faecalis , Alcaligenes faecalis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Intergénico , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Ácidos Picolínicos , Unión Proteica , Piridinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.
Asunto(s)
Alcaligenes faecalis , Bacillus , Alcaligenes faecalis/química , Bacillus/genética , Bacillus/metabolismo , Carbono/metabolismo , Ferredoxinas/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Familia de Multigenes , Oxigenasas/metabolismo , Ácidos Picolínicos/metabolismo , Piridinas/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu2+ , Zn2+ , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.
Asunto(s)
Alcaligenes faecalis , Alcaligenes faecalis/química , Alcaligenes faecalis/genética , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Ácidos Mandélicos/química , Ácidos Mandélicos/metabolismo , Pichia/genética , Pichia/metabolismo , SaccharomycetalesRESUMEN
AfIP-1A/1B is a two-component insecticidal protein identified from the soil bacterium Alcaligenes faecalis that has high activity against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). Previous results revealed that AfIP-1A/1B is cross-resistant to the binary protein from Bacillus thuringiensis (Bt), Cry34Ab1/Cry35Ab1 (also known as Gpp34Ab1/Tpp35Ab1; Crickmore et al., 2020), which was attributed to shared binding sites in WCR gut tissue (Yalpani et al., 2017). To better understand the interaction of AfIP-1A/1B with its receptor, we have systematically evaluated the binding of these proteins with WCR brush border membrane vesicles (BBMVs). Our findings show that AfIP-1A binds directly to BBMVs, while AfIP-1B does not; AfIP-1B binding only occurred in the presence of AfIP-1A which was accompanied by the presence of stable, high molecular weight oligomers of AfIP-1B observed on denaturing protein gels. Additionally, we show that AfIP-1A/1B forms pores in artificial lipid membranes. Finally, binding of AfIP-1A/1B was found to be reduced in BBMVs from Cry34Ab1/Cry35Ab1-resistant WCR where Cry34Ab1/Cry35Ab1 binding was also reduced. The reduced binding of both proteins is consistent with recognition of a shared receptor that has been altered in the resistant strain. The coordination of AfIP-1B binding by AfIP-1A, the similar structures between AfIP-1A and Cry34Ab1, along with their shared binding sites and cross-resistance, suggest a similar role for AfIP1A and Cry34Ab1 in receptor recognition and docking site for their cognate partners, AfIP-1B and Cry35Ab1, respectively.
Asunto(s)
Alcaligenes faecalis/genética , Proteínas Bacterianas/genética , Insecticidas/farmacología , Mariposas Nocturnas/genética , Alcaligenes faecalis/química , Alcaligenes faecalis/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Agentes de Control Biológico/química , Agentes de Control Biológico/metabolismo , Tracto Gastrointestinal/microbiología , Control de Insectos , Insecticidas/química , Larva/genética , Larva/crecimiento & desarrollo , Larva/microbiología , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/microbiología , Control Biológico de VectoresRESUMEN
Alcaligenes faecalis is the predominant Gram-negative bacterium inhabiting gut-associated lymphoid tissues, Peyer's patches. We previously reported that an A.â faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll-like receptorâ 4 (TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A.â faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A.â faecalis. We synthesized three lipidâ A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1- and 4'-phosphates. Hexaacylated A.â faecalis lipidâ A showed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit a discrete interleukin-6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate.
Asunto(s)
Alcaligenes faecalis/química , Lípido A/química , Lipopolisacáridos/química , Animales , Conformación de Carbohidratos , Línea Celular , Humanos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/metabolismo , Lípido A/farmacología , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ratones , Receptor Toll-Like 4/agonistasRESUMEN
This study aimed at evaluating the antagonistic activity of 16 bacterial strains for the control of brown rot disease caused by Monilinia fructigena, and M. laxa under in vitro and a semi-commercial large-scale trial. These bacterial antagonists' belonging to the genera Alcaligenes, Bacillus, Brevibacterium, Pantoea, Pseudomonas, and Serratia were previously proven effective for control of fire blight of apple. The in vitro dual culture bioassay showed the highest inhibition rates of mycelial growth ranging from 55 to 95% and from 43 to 94% for M. fructigena and M. laxa, respectively. The in vivo bioassay showed moderate and strong inhibition for M. fructigena and M. laxa, respectively. The inhibition rates were dependent on incubation time as well as pathogen virulence. The free-cell bacterial filtrate revealed substantial mycelial growth inhibition ranging from 66 to 86%. The inhibition of conidial germination was from 32 to 78%, suggesting the involvement of metabolites in their biocontrol activity. The antifungal effect of the volatile compounds (VCOs) was observed for all bacteria with mycelial inhibition varying from 12 to 70%. Overall, their efficacy was substantially affected by the nature of the bacterial strains and the modes of action. Taken together, these results underscore that ACBC1 and SF14 for M. fructigena and SP10 and ACBP1 for M. laxa were the most effective bacterial strains. These strains were confirmed effective in a semi-commercial large-scale trial. Interestingly, their efficacies were found to be comparable to those of both commercial BCAs (B. subtilis Y1336 and P. agglomerans P10c), but slightly lower than thiophanate-methyl fungicide. The ability of most bacterial strains to produce lytic enzymes (Amylase, Protease or Cellulase) and lipopeptides (bacillomycin, fengycin, iturin and surfactin) was demonstrated by biochemical and molecular analyzes. Therefore, our findings suggest that the bacterial antagonists ACBC1, SF14, SP10 and ACBP1, have the potential to prevent brown rot disease.
Asunto(s)
Alcaligenes faecalis/química , Ascomicetos/fisiología , Bacillus amyloliquefaciens/química , Fungicidas Industriales/farmacología , Pantoea/química , Enfermedades de las Plantas/microbiología , Alcaligenes faecalis/metabolismo , Bacillus amyloliquefaciens/metabolismo , Frutas/microbiología , Fungicidas Industriales/química , Fungicidas Industriales/metabolismo , Malus/microbiología , Pantoea/metabolismoRESUMEN
Picolinic acid (PA) is a natural toxic pyridine derivative. Microorganisms can degrade and utilize PA for growth. However, the full catabolic pathway of PA and its physiological and genetic foundation remain unknown. In this study, we identified a gene cluster, designated picRCEDFB4B3B2B1A1A2A3, responsible for the degradation of PA from Alcaligenes faecalis JQ135. Our results suggest that PA degradation pathway occurs as follows: PA was initially 6-hydroxylated to 6-hydroxypicolinic acid (6HPA) by PicA (a PA dehydrogenase). 6HPA was then 3-hydroxylated by PicB, a four-component 6HPA monooxygenase, to form 3,6-dihydroxypicolinic acid (3,6DHPA), which was then converted into 2,5-dihydroxypyridine (2,5DHP) by the decarboxylase PicC. 2,5DHP was further degraded to fumaric acid through PicD (2,5DHP 5,6-dioxygenase), PicE (N-formylmaleamic acid deformylase), PicF (maleamic acid amidohydrolase), and PicG (maleic acid isomerase). Homologous pic gene clusters with diverse organizations were found to be widely distributed in Alpha-, Beta-, and Gammaproteobacteria Our findings provide new insights into the microbial catabolism of environmental toxic pyridine derivatives.IMPORTANCE Picolinic acid is a common metabolite of l-tryptophan and some aromatic compounds and is an important intermediate in organic chemical synthesis. Although the microbial degradation/detoxification of picolinic acid has been studied for over 50 years, the underlying molecular mechanisms are still unknown. Here, we show that the pic gene cluster is responsible for the complete degradation of picolinic acid. The pic gene cluster was found to be widespread in other Alpha-, Beta-, and Gammaproteobacteria These findings provide a new perspective for understanding the catabolic mechanisms of picolinic acid in bacteria.
Asunto(s)
Alcaligenes faecalis/genética , Alcaligenes faecalis/metabolismo , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Ácidos Picolínicos/metabolismo , Alcaligenes faecalis/química , Alcaligenes faecalis/enzimología , Proteínas Bacterianas/genética , Biodegradación Ambiental , Redes y Vías Metabólicas , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Ácidos Picolínicos/químicaRESUMEN
5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned from Alcaligenes faecalis JQ135. HpaM was a monocomponent flavin adenine dinucleotide (FAD)-dependent monooxygenase and shared low identity (only 28 to 31%) with reported monooxygenases. HpaM catalyzed the ortho decarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH dependent. The apparent Km values of HpaM for 5HPA and NADH were 45.4 µM and 37.8 µM, respectively. The genes hpaX, hpaD, and hpaF were found to encode 2,5DHP dioxygenase, N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation in A. faecalis JQ135. Furthermore, the gene maiA, which encodes a maleic acid cis-trans isomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.IMPORTANCE Unlike the benzene ring, the uneven distribution of the electron density of the pyridine ring influences the positional reactivity and interaction with enzymes; e.g., the ortho and para oxidations are more difficult than the meta oxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, the ortho hydroxylations of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while the meta hydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed the ortho decarboxylative hydroxylation of 5HPA. In addition, we found that the maiA gene coding for maleic acid cis-trans isomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives.
Asunto(s)
Alcaligenes faecalis/metabolismo , Piridinas/metabolismo , Alcaligenes faecalis/química , Alcaligenes faecalis/enzimología , Alcaligenes faecalis/genética , Amidohidrolasas/química , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Flavina-Adenina Dinucleótido/metabolismo , Hidroxilación , Cinética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Operón , Filogenia , Piridinas/químicaRESUMEN
Measuring solid-state electron transport (ETp) across proteins allows studying electron transfer (ET) mechanism(s), while minimizing solvation effects on the process. ETp is, however, sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurements allow extending ETp studies to low temperatures, with the concomitant resolution of lower current densities, because of the larger electrode contact areas. Thus, earlier we reported temperature-independent ETp via the copper protein azurin (Az), from 80 K until denaturation, whereas for apo-Az ETp was temperature dependent above 180 K. Deuteration (H/D substitution) may provide mechanistic information on the question of whether the ETp involves H-bonds in the solid state. Here we report results of kinetic deuterium isotope effect (KIE) measurements on ETp through holo-Az as a function of temperature (30-340 K). Strikingly, deuteration changed ETp from temperature independent to temperature dependent above 180 K. This H/D effect is expressed in KIE values between 1.8 (340 K) and 9.1 (≤ 180 K). These values are remarkable in light of the previously reported inverse KIE on ET in Az in solution. We ascribe the difference between our KIE results and those observed in solution to the dominance of solvent effects in the latter (larger thermal expansion in H(2)O than in D(2)O), whereas in our case the KIE is primarily due to intramolecular changes, mainly in the low-frequency structural modes of the protein caused by H/D exchange. The observed high KIE values are consistent with a transport mechanism that involves through-H-bonds of the ß-sheet structure of Az, likely also those in the Cu coordination sphere.
Asunto(s)
Alcaligenes faecalis/química , Azurina/metabolismo , Transporte de Electrón/fisiología , Conformación Proteica , Azurina/química , Dicroismo Circular , Medición de Intercambio de Deuterio , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , TemperaturaRESUMEN
Mimicking a concept of nature for the hydrolysis of biopolymers, the Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was fused to a polymer binding module (PBM) to enhance the hydrolysis of the polyester poly(1,4-butylene adipate) (PBA). Namely, the binding module of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (Thc_Cut1_PBM) was attached to the cutinase via two different linker sequences varying in length. In order to investigate the adsorption behavior, catalytically inactive mutants both of Thc_Cut1 and Thc_Cut1_PBM were successfully constructed by site-directed mutagenesis of serine 131 to alanine. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis revealed that the initial mass increase during enzyme adsorption was larger for the inactive enzymes linked with the PBM as compared to the enzyme without the PBM. The hydrolysis rates of PBA were significantly enhanced when incubated with the active, engineered Thc_Cut1_PBM as compared to the native Thc_Cut1. Thc_Cut1_PBM completely hydrolyzed PBA thin films on QCM-D sensors within approximately 40 min, whereas twice as much time was required for the complete hydrolysis by the native Thc_Cut1.
Asunto(s)
Proteínas Bacterianas/química , Biomimética/métodos , Butileno Glicoles/química , Hidrolasas de Éster Carboxílico/química , Polímeros/química , Actinobacteria/química , Actinobacteria/enzimología , Alanina/química , Alanina/genética , Alcaligenes faecalis/química , Alcaligenes faecalis/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Butileno Glicoles/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hidrólisis , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Polímeros/metabolismo , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/genética , Relación Estructura-ActividadRESUMEN
A powerful strategy for long-term and diffusional-resistance-minimized whole-cell biocatalysis in biphasic systems is reported where individually encapsulated bacteria are employed as robust and recyclable Pickering interfacial biocatalysts. By individually immobilizing bacterial cells and optimizing the hydrophobic/hydrophilic balance of the encapsulating magnetic mineral shells, the encased bacteria became interfacially active and locate at the Pickering emulsion interfaces, leading to dramatically enhanced bioconversion performances by minimizing internal and external diffusional resistances. Moreover, inâ situ product separation and biocatalyst recovery was readily achieved using a remote magnetic field. Importantly, the mineral shell effectively protected the entire cell from long-term organic-solvent stress, as shown by the reusability of the biocatalysts for up to 30â cycles, while retaining high stereoselective catalytic activities, cell viabilities, and proliferative abilities.
Asunto(s)
Bacterias/metabolismo , Alcaligenes faecalis/química , Alcaligenes faecalis/efectos de los fármacos , Alcaligenes faecalis/metabolismo , Bacterias/química , Bacterias/efectos de los fármacos , Biocatálisis , Células Inmovilizadas/química , Células Inmovilizadas/metabolismo , Emulsiones/química , Fluoresceínas/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Magnetismo , Microscopía Confocal , Nanoestructuras/químicaRESUMEN
Although at low concentrations, arsenic commonly occurs naturally as a local geological constituent. Whereas both arsenate and arsenite are strongly toxic to life, a number of prokaryotes use these compounds as electron acceptors or donors, respectively, for bioenergetic purposes via respiratory arsenate reductase, arsenite oxidase and alternative arsenite oxidase. The recent burst in discovered arsenite oxidizing and arsenate respiring microbes suggests the arsenic bioenergetic metabolisms to be anything but exotic. The first goal of the present review is to bring to light the widespread distribution and diversity of these metabolizing pathways. The second goal is to present an evolutionary analysis of these diverse energetic pathways. Taking into account not only the available data on the arsenic metabolizing enzymes and their phylogenetical relatives but also the palaeogeochemical records, we propose a crucial role of arsenite oxidation via arsenite oxidase in primordial life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.
Asunto(s)
Arsénico/metabolismo , Metabolismo Energético , Alcaligenes faecalis/química , Alcaligenes faecalis/enzimología , Arseniato Reductasas/química , Arseniato Reductasas/metabolismo , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Conformación ProteicaRESUMEN
The rate and kinetic isotope effect (KIE) on proton transfer during the aromatic amine dehydrogenase-catalyzed reaction with phenylethylamine shows complex pressure and temperature dependences. We are able to rationalize these effects within an environmentally coupled tunneling model based on constant pressure molecular dynamics (MD) simulations. As pressure appears to act anisotropically on the enzyme, perturbation of the reaction coordinate (donor-acceptor compression) is, in this case, marginal. Therefore, while we have previously demonstrated that pressure and temperature dependences can be used to infer H-tunneling and the involvement of promoting vibrations, these effects should not be used in the absence of atomistic insight, as they can vary greatly for different enzymes. We show that a pressure-dependent KIE is not a definitive hallmark of quantum mechanical H-tunneling during an enzyme-catalyzed reaction and that pressure-independent KIEs cannot be used to exclude tunneling contributions or a role for promoting vibrations in the enzyme-catalyzed reaction. We conclude that coupling of MD calculations with experimental rate and KIE studies is required to provide atomistic understanding of pressure effects in enzyme-catalyzed reactions.
Asunto(s)
Alcaligenes faecalis/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Fenetilaminas/metabolismo , Alcaligenes faecalis/química , Alcaligenes faecalis/metabolismo , Cinética , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Presión , Conformación Proteica , Protones , TermodinámicaRESUMEN
The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.
Asunto(s)
Acetonitrilos/metabolismo , Alcaligenes faecalis/metabolismo , Ácidos Mandélicos/metabolismo , Acetonitrilos/química , Alcaligenes faecalis/química , Biocatálisis , Células Inmovilizadas/química , Células Inmovilizadas/metabolismo , Cetrimonio , Compuestos de Cetrimonio/química , Ácidos Mandélicos/química , EstereoisomerismoRESUMEN
Two bacterial stains were isolated from the activated sludge and identified as Leucobacter sp. and Alcaligenesfaecalis by 16S rDNA sequencing. Pure cultures of these two strains, representing well or poorly settled bacteria, were used to investigate the mechanism of bioflocculation in activated sludge. Based on the analyses of the characteristics of cells hydrophobicity, zeta-potential, flocculation ability and extracellular polymeric substance (EPS) composition under different growth stages, it was found that the ratio of cell EPS protein had the highly influence on zeta-potential and hydrophobicity, which were important factors to bioflocculation. Cellulase and Proteinase K could destroy the extracellular biopolymer and resulted in a decrease in the hydrophobicity and zeta-potential. However, in our study, the flocculation characteristics exhibited differently in relation to cellulase and Proteinase K. Flocculation of cells treated with cellulase and Proteinase K decreased sharply, and then recovered quickly in cellulase treatment, while cells treated with Proteinase K showed no sign of recovery. This reveals that the presence of protein in extracellular biopolymer plays an important role to the bioflocculation of cells.
Asunto(s)
Alcaligenes faecalis/química , Aguas del Alcantarillado/microbiología , Alcaligenes faecalis/fisiología , Alcaligenes faecalis/ultraestructura , Proteínas Bacterianas/química , Carbohidratos/química , Celulasa , Endopeptidasa K , Floculación , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
The dynamics of the reduced form of the blue copper protein pseudoazurin from Alcaligenes faecalis S-6 was investigated using (15)N relaxation measurements with a focus on the dynamics of the micro- to millisecond time scale. Different types of conformational exchange processes are observed in the protein on this time scale. At low pH, the protonation of the C-terminal copper-ligated histidine, His81, is observed. A comparison of the exchange rates in the presence and absence of added buffers shows that the protonation is the rate-limiting step at low buffer concentrations. This finding agrees with previous observations for other blue copper proteins, e.g., amicyanin and plastocyanin. However, in contrast to plastocyanin but similar to amicyanin, a second conformational exchange between different conformations of the protonated copper site is observed at low pH, most likely triggered by the protonation of His81. This process has been further characterized using CPMG dispersion methods and is found to occur with a rate of a few thousands per second. Finally, micro- to millisecond motions are observed in one of the loop regions and in the alpha-helical regions. These motions are unaffected by pH and are unrelated to the conformational changes in the active site of pseudoazurin.
Asunto(s)
Alcaligenes faecalis/química , Azurina/química , Cobre , Metaloproteínas/química , Tampones (Química) , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Conformación ProteicaRESUMEN
Bioelectrochemical systems use microbes as catalysts for current production or consumption. Up to now only a few microbes have been demonstrated to be capable of both outward and inward extracellular electron transfer (EET) (i.e. bidirectional electron transfer). However, the mechanisms of electron exchange between microbes and extracellular solids remain uncertain. Here, we showed that Alcaligenes faecalis catalyzed an outward EET and generated electricity at a poised potential of +0.3V vs. SHE, whereas it conducted an inward EET for autotrophic denitrification at -0.5V vs. SHE. Both cyclic voltammetry and in situ electrochemical FTIR spectroscopy revealed that different redox components were utilized during the outward and inward EET. Electron transport inhibitor experiments indicated for the first time that complex I, II, III, and the quinone pool on the plasma membrane were involved in the bidirectional EET. Comparative proteomics showed that the protein expression profile of outward-EET biofilms differed greatly from those of inward-EET biofilms, implying that the pili and outer membrane proteins might be responsible for the interfacial outward and inward EET, respectively. These results suggest different electron transport conduits of A. faecalis biofilms could be used for bidirectional EET.
Asunto(s)
Alcaligenes faecalis/aislamiento & purificación , Técnicas Biosensibles , Proteómica , Alcaligenes faecalis/química , Fuentes de Energía Bioeléctrica , Biopelículas/crecimiento & desarrollo , Transporte de Electrón , Electrones , Quinonas/químicaRESUMEN
La contaminación por plásticos petroquímicos es una grave amenaza para el medio ambiente que requiere im-plementar alternativas como los bioplásticos para lograr un desarrollo sostenible. Los polihidroxialcanoatos (PHA) son polímeros utilizados para la producción de plásticos biodegradables y que han llamado la atención como sustitutos de los plásticos de base fósil. Sin embargo, el costo de producción de los PHA constituye una barrera para su producción industrial a gran escala. Las de bacterias de hábitats salinos son microorganismos prometedores para la síntesis de PHA debido a sus características tales como altos requisitos de salinidad que previenen la contaminación microbiana, la alta presión osmótica intracelular que permite una fácil lisis celular para purificar los PHA y la capacidad para usar un amplio espectro de sustratos. La presente investigación planteó determinar las cepas nativas de bacterias halófilas y halotolerantes de la Laguna de Ayarza capaces de producir PHA, establecer la capacidad que tienen de utilizar residuos agrícolas para la producción de PHA y determinar su eficiencia. Esto se logró a través de la inoculación de las cepas productoras de PHA en medios de fermentación con pulpa de café, cáscaras de plátanos y salvado de trigo lo que permitió determinar las cepas más eficientes. Se encontró que las bacterias productoras de PHA pertenecen a las especies: Alcaligenes faecalis, Bacillus idriensis, Bacillus megaterium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis y Sta-phylococcus capitis y que las cepas AP21-14, AP21-10 y AP21-03 mostraron los mejores resultados que podrían ser prometedores para la producción a nivel industrial.
Pollution by petrochemical plastics is a serious threat to the environment that requires the implementation of al-ternatives such as bioplastics to achieve sustainable development. Polyhydroxyalkanoates (PHAs) are polymers used for the production of biodegradable plastics and have drawn attention as substitutes for fossil-based plastics. However, the cost of producing PHAs constitutes a barrier to their large-scale industrial production. Bacteria from saline environments bacteria are promising microorganisms for PHA synthesis due to their characteristics such as high salinity requirements that prevent microbial contamination, high intracellular osmotic pressure that allows easy cell lysis to purify PHAs, and the ability to use a broad spectrum of substrates. This research project aimed to determine the native strains of halophilic and halotolerant bacteria from Laguna de Ayarza capable of producing PHA, establish their ability to use agricultural residues for the production of PHA, and determine their efficiency. This was achieved through the inoculation of the PHA-producing strains in fermentation media with coffee pulp, banana peels and wheat bran, which allowed determining the most efficient strains. It was found that the PHA-producing bacteria belong to the species: Alcaligenes faecalis, Bacillus idriensis, Bacillus mega-terium, Exiguobacterium acetylicum, E. aurantiacum, Pseudomonas cuatrocienegasensis and Staphylococcus capitis and that the strains AP21-14, AP21-10 and AP21-03 showed the best results that could be promising for production at an industrial level.