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1.
Biochem J ; 478(15): 3047-3062, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34338286

RESUMO

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.


Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Cátions Monovalentes/metabolismo , Ativação Enzimática , Potássio/metabolismo , Zoogloea/isolamento & purificação , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetil-CoA C-Acetiltransferase/química , Acetil-CoA C-Acetiltransferase/genética , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Cátions Monovalentes/química , Cristalografia por Raios X , Cinética , Modelos Moleculares , Mutação , Potássio/química , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Especificidade por Substrato , Zoogloea/enzimologia , Zoogloea/genética
2.
Bioresour Technol ; 340: 125690, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34352640

RESUMO

A Mn(II) oxidizing-denitrifying and tetracycline (TC) removal bacterium Zoogloea sp. MFQ7 was isolated in this study. Nitrogen removal was 83.49% by nitrogen balance experiment. The maximum removal efficiencies of nitrate, Mn(II), and TC by strain MFQ7 within 96 h was 100.00, 74.56, and 63.59% at C/N of 2.0, pH of 7.0, Mn(II) of 20 mg L-1, temperature of 30.0 °C, and TC of 0.2 mg L-1. SEM illustrated that biogenic manganese oxides (BMO) was petal-like, XRD and XPS analyses confirmed that MnO2 was the main component of BMO. Besides, the maximum adsorption capacity of BMO for TC was 52.21 mg g-1. FTIR detected the changes in TC adsorption by BMO. Pseudo-second-order model (R2 = 0.994) explained the adsorption kinetics of TC on BMO and Langmuir isotherm model (R2 = 0.983) suggested that it was homogeneous adsorption, thermodynamics data (ΔG < 0, ΔH = 18.31 kJ mol-1, ΔS = 72.8 J (mol*K)-1) confirmed that adsorption was endothermic and spontaneous.


Assuntos
Poluentes Químicos da Água , Zoogloea , Adsorção , Concentração de Íons de Hidrogênio , Cinética , Manganês , Compostos de Manganês , Nitratos , Óxidos , Tetraciclina , Termodinâmica , Poluentes Químicos da Água/análise
3.
Bioresour Technol ; 336: 125326, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34052544

RESUMO

A kind of gel beads loaded with iron-carbon powder (Fe-C), Zoogloea sp. L2, and riboflavin (VB2) were prepared through cross-linking of sodium alginate (SA) to establish an immobilized bioreactor. The optimal ratio of SA beads was adjusted by orthogonal experiment. The change of oxidation-reduction potential (ORP) and the concentration of Fe2+ and Fe3+ showed that the addition of VB2 as a redox mediator can promote denitrification. Under the optimal conditions (carbon to nitrogen (C/N) ratio = 2.0, pH = 7.0, and hydraulic retention time (HRT) = 8 h), the nitrate removal efficiency (NRE) of bioreactor reached 98.48% (1.99 mg L-1h-1). Furthermore, Fourier transform infrared spectrometer (FTIR), Fluorescence excitation-emission matrix (EEM), X-ray diffraction (XRD), and gas chromatography (GC) analysis revealed that the immobilization and denitrification of the immobilized bioreactor were excellent. High throughput sequencing also showed that Zoogloea played a vital role in nitrate removal.


Assuntos
Zoogloea , Alginatos , Reatores Biológicos , Carbono , Desnitrificação , Ferro , Nitratos , Nitrogênio , Riboflavina
4.
Artigo em Inglês | MEDLINE | ID: mdl-33054534

RESUMO

This study investigates the stability of a pure oxygen aeration-activated sludge system for petrochemical wastewater treatment under high organic concentration and non-steady food-to-microorganism (F/M) ratio conditions. Sludge settling characteristics maintained relatively stable conditions with an F/M ratio variation from 0.15 ± 0.04 to 0.33 ± 0.07 kg COD/kg MLSS⋅d, while the excess F/M ratio (0.44 ± 0.16 kg COD/kg MLSS⋅d) resulted in deterioration of the organic removal and sludge-water separation performances. Loosely bound extracellular polymeric substances (EPS) showed more significant effect on sludge settleability than the tightly bound EPS. The genus Hydrogenophaga was related to organic removal performance, while Zoogloea and Chitinophaga were related to the effluent quality of suspended solids. The excess F/M ratio also caused an increase in Zoogloea and Chitinophaga, whereas the toxicity of petrochemical wastewater resulted in decreased abundance of Hydrogenophaga. These changes caused deterioration of the organic removal and sludge-water separation performances.


Assuntos
Reatores Biológicos/microbiologia , Oxigênio/metabolismo , Petróleo/análise , Esgotos/microbiologia , Águas Residuárias/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Aerobiose , Bacteroidetes/metabolismo , Comamonadaceae/metabolismo , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Zoogloea/metabolismo
5.
Int J Syst Evol Microbiol ; 70(10): 5312-5318, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32841112

RESUMO

A motile, Gram-stain-negative, rod-shaped bacterium, designated G-4-1-14T, was obtained from forest soil sampled at Gwanggyo mountain, Gyeonggi-do, Republic of Korea. Cells were colourless, aerobic, grew optimally at 28-35 °C and hydrolysed DNA and casein. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain G-4-1-14T formed a lineage within the genus Zoogloea. The closest members were Zoogloea resiniphila ATCC 70068T (98.6 % sequence similarity), Zoogloea caeni EMB43T (98.2 %), Zoogloea oryzae A-7T (97.7 %), Zoogloea ramigera IAM 12136T (96.9 %) and Zoogloea oleivorans BucT (96.2 %). The major respiratory quinone was ubiquinone-8 and the principal polar lipids were phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The predominant cellular fatty acids were summed feature 3 (iso-C15 :0 2-OH/C16  : 1 ω7c) and C16 : 0. The DNA G+C content was 65.9 mol%. The average nucleotide identity and digital DNA-DNA hybridization relatedness values between strain G-4-1-14T and other type strains were ≤81.6 and ≤24.9 %, respectively, which are below the species demarcation thresholds. Based on the results of phenotypic, phylogenetic and genomic analyses, strain G-4-1-14T represents a novel species in the genus Zoogloea, for which the name Zoogloea dura sp. nov. is proposed. The type strain is G-4-1-14T (=KACC 21618T=NBRC 114358T). In addition, we propose emendation of the genus Zoogloea and the species Zoogloea oryzae and Zoogloea ramigera.


Assuntos
Florestas , Filogenia , Microbiologia do Solo , Zoogloea/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fixação de Nitrogênio , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Ubiquinona/química , Zoogloea/isolamento & purificação
6.
Bioresour Technol ; 311: 123533, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32460131

RESUMO

Denitrification in mixed culture system has been extensively researched to date, but few studies have focused on accelerating the process using redox mediators to promote electron transfer. Strain L2, an iron-reducing bacteria, can remove 75.44% of nitrate under temperature of 30.60 °C, pH of 6.75 and Fe2+ concentration of 27.86 mg·L-1. Additionally, the removal rate of nitrate reached 1.516 mg·L-1·h-1 in 8 h with the addition of 0.030 mmol·L-1 2-hydroxy-1,4-naphthoquinone (HNQ), which increased by 1.38 times than control group. Furthermore, analysis by fluorescence spectroscopy, flow cytometer and gas chromatography demonstrated that HNQ positively stimulated denitrification. This study provides a reference for enhancing denitrification in mixed culture and lays the foundation for the practical application of redox mediators in groundwater treatment.


Assuntos
Naftoquinonas , Zoogloea , Desnitrificação , Nitratos , Oxirredução
7.
Arch Microbiol ; 202(2): 421-426, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31659381

RESUMO

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.


Assuntos
Biodegradação Ambiental , Oxigenases/metabolismo , Tolueno/metabolismo , Zoogloea/metabolismo , Composição de Bases/genética , Catecóis , Metabolismo Energético/fisiologia , Genoma Bacteriano/genética , Água Subterrânea/microbiologia , Redes e Vias Metabólicas , Zoogloea/genética
8.
Environ Microbiol ; 20(5): 1677-1692, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29473278

RESUMO

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP-CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK-PrsR two-component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc-forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post-translational modifications, could not rescue the long-rod morphology of the rpoN mutant. Consistently, PEP-CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc-forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.


Assuntos
Esgotos/microbiologia , Águas Residuárias/microbiologia , Zoogloea/fisiologia , Proteínas de Bactérias/metabolismo , Floculação , Fator sigma/metabolismo , Eliminação de Resíduos Líquidos , Águas Residuárias/química
9.
Huan Jing Ke Xue ; 38(2): 665-671, 2017 Feb 08.
Artigo em Chinês | MEDLINE | ID: mdl-29964524

RESUMO

A completely mixed aeration system was introduced in domestic sewage treatment, and the simultaneous nitrification and denitrification process and microbial community structure were further investigated. The pilot-scale experimental results showed that the operation of the system was stable and the average chemical oxygen demand (COD), NH4+-N and total nitrogen (TN) removal were 93.2%, 96.9% and 75.2%, respectively. The effluent COD, NH4+-N and TN were better than the standard of level A stipulated by Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant (GB 18918-2002) in the absence of external carbon source. The sludge derived from the system showed a significant denitrification capacity and the denitrification rate was 2.86 times higher than that in the wastewater treatment plant. In addition, the existence of aerobic denitrifying bacteria in the system was confirmed by the PCR amplification of genes encoding perplasmic nitrate reductases. The high-throughput sequencing results revealed that Zoogloea, Thauera and Dechloromonas were dominant in the completely mixed aeration system.


Assuntos
Reatores Biológicos/microbiologia , Desnitrificação , Nitrificação , Nitrogênio/isolamento & purificação , Betaproteobacteria , Esgotos , Thauera , Águas Residuárias , Zoogloea
10.
Water Res ; 102: 494-504, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27403872

RESUMO

Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea. Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes.


Assuntos
Esgotos/química , Zoogloea , Bactérias , Floculação , Genômica , Eliminação de Resíduos Líquidos
11.
Bioresour Technol ; 214: 645-652, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27187569

RESUMO

In this paper, we employ atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP) technique, phospholipid fatty acids (PLFA) and MiSeq analysis to study the effects of traditional dissolved oxygen (DO) levels (0.71-1.32mg/L, 2.13-3.02mg/L and 4.31-5.16mg/L) on surface force, cell membrane properties and microbial community dynamics of activated sludge. Results showed that low DO level enhanced the surface force and roughness of activated sludge; the medium DO level decreased cell membrane fluidity by reducing the synthesis of branched fatty acids in the cell membrane; high DO level resulted in the highest protein content in the effluent by EEM scanning. Abundance of Micropruina, Zoogloea and Nakamurella increased and Paracoccus and Rudaea decreased with the increase of DO levels. RDA analysis suggested that saturated fatty acids (SFA), anteiso-fatty acids (AFA) and iso-fatty acids (IFA) were closely related to effluent quality as well as some genera.


Assuntos
Consórcios Microbianos/fisiologia , Oxigênio/metabolismo , Esgotos/química , Esgotos/microbiologia , Actinomycetales/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Cocos Gram-Positivos/metabolismo , Microscopia de Força Atômica , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Eliminação de Resíduos Líquidos/métodos , Zoogloea/metabolismo
12.
Bioresour Technol ; 201: 1-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26615496

RESUMO

Conductive carbon felts (Cf) were used as biofilm carriers in bioelectrochemical reactors to enhance the electrical stimulation on treatment of phenol-containing synthetic wastewater. In batch test, phenol biodegradation was accelerated under an optimum direct current (DC), which was 2mA for Cf biofilm carriers, lower than that for non-conductive white foam carriers. The stimulation effect was consistent with Adenosine Triphosphate contents in biofilms. The long-term operation further demonstrated that a high and stable phenol removal efficiency could be achieved with applied DC of 2mA, and intermittent DC application was better than continuous one, with phenol removal efficiency of over 97%. Although the quantities of whole microbial communities kept at a high level under all conditions, special microorganisms related with genera of Zoogloea and Desulfovibrio were distinctively enriched under intermittent applied DC pattern. This study shows that the electrical stimulation is potentially effective for biofilm reactors treating phenol-containing wastewater.


Assuntos
Biofilmes , Reatores Biológicos , Consórcios Microbianos/fisiologia , Fenóis/química , Águas Residuárias/química , Purificação da Água/métodos , Trifosfato de Adenosina/química , Biodegradação Ambiental , Carbono/química , Fibra de Carbono , Desulfovibrio/fisiologia , Estimulação Elétrica , Zoogloea/fisiologia
13.
Int J Mol Sci ; 16(5): 10038-60, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25946341

RESUMO

Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h-1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53%±1.69% and 58.65%±0.61%, respectively. The ammonia removal rate reached 44.12%±1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5-9 mg/L, pH 8-9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41%±3.17% (sterilized) and 44.88%±4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p<0.05). High C/N was beneficial for nitrate reduction (p<0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p>0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.


Assuntos
Desnitrificação , Água Potável/química , Zoogloea/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Água Potável/microbiologia , Água Subterrânea/microbiologia , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , RNA Ribossômico/genética , Purificação da Água/métodos , Zoogloea/genética , Zoogloea/isolamento & purificação
14.
Int J Syst Evol Microbiol ; 65(Pt 1): 274-279, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25342113

RESUMO

A floc-forming, Gram-stain-negative, petroleum hydrocarbon-degrading bacterial strain, designated Buc(T), was isolated from a petroleum hydrocarbon-contaminated site in Hungary. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Buc(T) formed a distinct phyletic lineage within the genus Zoogloea. Its closest relative was found to be Zoogloea caeni EMB43(T) (97.2% 16S rRNA gene sequence similarity) followed by Zoogloea oryzae A-7(T) (95.9%), Zoogloea ramigera ATCC 19544(T) (95.5%) and Zoogloea resiniphila DhA-35(T) (95.4%). The level of DNA-DNA relatedness between strain Buc(T) and Z. caeni EMB43(T) was 31.6%. Cells of strain Buc(T) are facultatively aerobic, rod-shaped, and motile by means of a polar flagellum. The strain grew at temperatures of 5-35 °C (optimum 25-28 °C), and at pH 6.0-9.0 (optimum 6.5-7.5). The predominant fatty acids were C16:0, C10 : 0 3-OH, C12:0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major respiratory quinone was ubiquinone-8 (Q-8) and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 63.2 mol%. On the basis of the chemotaxonomic, molecular and phenotypic data, isolate Buc(T) is considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea oleivorans sp. nov. is proposed. The type strain is Buc(T) ( =DSM 28387(T) =NCAIM B 02570(T)).


Assuntos
Biofilmes , Petróleo/microbiologia , Filogenia , Zoogloea/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , Biodegradação Ambiental , DNA Bacteriano/genética , Ácidos Graxos/química , Hungria , Hidrocarbonetos/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fosfatidiletanolaminas/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/química , Zoogloea/genética , Zoogloea/isolamento & purificação
15.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 12): 3212-25, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25478839

RESUMO

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nß2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.


Assuntos
Acetil-CoA C-Aciltransferase/química , Mitocôndrias/enzimologia , Acetil-CoA C-Aciltransferase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Coenzima A/química , Coenzima A/metabolismo , Cristalografia por Raios X , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Zoogloea/enzimologia
16.
Water Res ; 47(2): 503-16, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23182667

RESUMO

Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this might have on drinking water quality.


Assuntos
Água Potável/microbiologia , Hidrologia/métodos , Methylophilus/crescimento & desenvolvimento , Pseudomonas/crescimento & desenvolvimento , Sphingomonadaceae/crescimento & desenvolvimento , Abastecimento de Água , Biofilmes/crescimento & desenvolvimento , Água Potável/química , Sequenciamento de Nucleotídeos em Larga Escala , Hidrologia/instrumentação , Methylophilus/classificação , Methylophilus/isolamento & purificação , Methylophilus/fisiologia , Viabilidade Microbiana , Tipagem Molecular , Oxalobacteraceae/classificação , Oxalobacteraceae/crescimento & desenvolvimento , Oxalobacteraceae/isolamento & purificação , Oxalobacteraceae/fisiologia , Análise de Componente Principal , Pseudomonas/classificação , Pseudomonas/isolamento & purificação , Pseudomonas/fisiologia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , Reologia/métodos , Estações do Ano , Sphingomonadaceae/classificação , Sphingomonadaceae/isolamento & purificação , Sphingomonadaceae/fisiologia , Fatores de Tempo , Reino Unido , Qualidade da Água , Zoogloea/classificação , Zoogloea/crescimento & desenvolvimento , Zoogloea/isolamento & purificação , Zoogloea/fisiologia
17.
Environ Sci Technol ; 45(20): 8794-803, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21875070

RESUMO

On-site aerobic sequencing batch reactor (SBR) treatment plants are implemented in many Australian wineries to treat the large volumes of associated wastewater they generate. Yet very little is known about their microbiology. This paper represents the first attempt to analyze the communities of three such systems sampled during both vintage and nonvintage operational periods using molecular methods. Alphaproteobacterial tetrad forming organisms (TFO) related to members of the genus Defluviicoccus and Amaricoccus dominated all three systems in both operational periods. Candidatus 'Alysiosphaera europaea' and Zoogloea were codominant in two communities. Production of high levels of exocellular capsular material by Zoogloea and Amaricoccus is thought to explain the poor settleability of solids in one of these plants. The dominance of these organisms is thought to result from the high COD to N/P ratios that characterize winery wastes, and it is suggested that manipulating this ratio with nutrient dosing may help control the problems they cause.


Assuntos
Reatores Biológicos/microbiologia , Eliminação de Resíduos Líquidos/métodos , Vinho , Aerobiose , Alphaproteobacteria/metabolismo , Austrália , Zoogloea/metabolismo
18.
ISME J ; 5(5): 896-907, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21107441

RESUMO

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1-18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.


Assuntos
Reatores Biológicos/microbiologia , Hidroxibutiratos/metabolismo , Microbiologia Industrial , Temperatura , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Biomassa , Zoogloea/crescimento & desenvolvimento , Zoogloea/metabolismo
19.
Lett Appl Microbiol ; 51(4): 428-35, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20809924

RESUMO

AIMS: Single-walled carbon nanotubes (SWNTs) are likely to become increasingly widespread and yet their environmental impact is not well understood. The purpose of the current study was to evaluate the impact of SWNTs on microbial communities in a 'sentinel' environmental system, activated sludge batch-scale reactors. METHODS AND RESULTS: Triplicate batch reactors were exposed to SWNTs and compared to control reactors exposed to impurities associated with SWNTs. Automated ribosomal intergenic spacer analysis (ARISA) was used to assess bacterial community structure in each reactor. SWNT exposure was found to impact microbial community structure, while SWNT-associated impurities had no effect, compared to controls. 16S rRNA gene sequence analysis indicated that dominant phylotypes detected by ARISA included members of the families Sphingomonadaceae and Cytophagacaceae and the genus Zoogloea. ARISA results indicated an adverse impact of SWNTs on the sphingomonad relative to other community members. Changes in community structure also occurred in both SWNT-exposed and control reactors over the experimental time period and with the date on which activated sludge was obtained from a wastewater treatment facility. CONCLUSIONS: These results indicate that SWNTs differentially impact members of the activated sludge reactor bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that community structure was affected by SWNTs indicates that this emerging contaminant differentially impacted members of the activated sludge bacterial community and raises the concern that SWNTs may also affect the services it provides.


Assuntos
Ecossistema , Nanotubos de Carbono , Esgotos/microbiologia , Eliminação de Resíduos Líquidos/métodos , Reatores Biológicos , Cytophagaceae/genética , Cytophagaceae/crescimento & desenvolvimento , DNA Bacteriano/genética , DNA Espaçador Ribossômico/análise , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Sphingomonadaceae/genética , Sphingomonadaceae/crescimento & desenvolvimento , Zoogloea/genética , Zoogloea/crescimento & desenvolvimento
20.
Biochemistry ; 48(46): 11011-25, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19842716

RESUMO

The biosynthetic thiolase catalyzes a Claisen condensation reaction between acetyl-CoA and the enzyme acetylated at Cys89. Two oxyanion holes facilitate this catalysis: oxyanion hole I stabilizes the enolate intermediate generated from acetyl-CoA, whereas oxyanion hole II stabilizes the tetrahedral intermediate of the acetylated enzyme. The latter intermediate is formed when the alpha-carbanion of acetyl-CoA enolate reacts with the carbonyl carbon of acetyl-Cys89, after which C-C bond formation is completed. Oxyanion hole II is made of two main chain peptide NH groups, whereas oxyanion hole I is formed by a water molecule (Wat82) and NE2(His348). Wat82 is anchored in the active site by an optimal set of hydrogen bonding interactions, including a hydrogen bond to ND2(Asn316). Here, the importance of Asn316 and His348 for catalysis has been studied; in particular, the properties of the N316D, N316A, N316H, H348A, and H348N variants have been determined. For the N316D variant, no activity could be detected. For each of the remaining variants, the k(cat)/K(m) value for the Claisen condensation catalysis is reduced by a factor of several hundred, whereas the thiolytic degradation catalysis is much less affected. The crystal structures of the variants show that the structural changes in the active site are minimal. Our studies confirm that oxyanion hole I is critically important for the condensation catalysis. Removing either one of the hydrogen bond donors causes the loss of at least 3.4 kcal/mol of transition state stabilization. It appears that in the thiolytic degradation direction, oxyanion hole I is not involved in stabilizing the transition state of its rate limiting step. However, His348 has a dual role in the catalytic cycle, contributing to oxyanion hole I and activating Cys89. The analysis of the hydrogen bonding interactions in the very polar catalytic cavity shows the importance of two conserved water molecules, Wat82 and Wat49, for the formation of oxyanion hole I and for influencing the reactivity of the catalytic base, Cys378, respectively. Cys89, Asn316, and His348 form the CNH-catalytic triad of the thiolase superfamily. Our findings are also discussed in the context of the importance of this triad for the catalytic mechanism of other enzymes of the thiolase superfamily.


Assuntos
Acetil-CoA C-Acetiltransferase/química , Acetil-CoA C-Acetiltransferase/metabolismo , Asparagina/química , Histidina/química , Zoogloea/enzimologia , Acetilcoenzima A/química , Acetil-CoA C-Acetiltransferase/genética , Substituição de Aminoácidos/genética , Biocatálise , Calorimetria , Domínio Catalítico/genética , Dicroísmo Circular , Coenzima A/química , Cristalografia por Raios X , Estabilidade Enzimática/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática , Termodinâmica , Temperatura de Transição , Água/química
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