2-aminophenol 1,6-dioxygenase: a novel aromatic ring cleavage enzyme purified from Pseudomonas pseudoalcaligenes JS45.

Most bacterial pathways for the degradation of aromatic compounds involve introduction of two hydroxyl groups either ortho or para to each other. Ring fission then occurs at the bond adjacent to one of the hydroxyl groups. In contrast, 2-aminophenol is cleaved to 2-aminomuconic acid semialdehyde in the nitrobenzene-degrading strain Pseudomonas pseudoalcaligenes JS45. To examine the relationship between this enzyme and other dioxygenases, 2-aminophenol 1,6-dioxygenase has been purified by ethanol precipitation, gel filtration, and ion exchange chromatography. The molecular mass determined by gel filtration was 140,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two subunits of 35,000 and 39,000 Da, which suggested an alpha2beta2 subunit structure. Studies with inhibitors indicated that ferrous iron was the sole cofactor. The Km values for 2-aminophenol and oxygen were 4.2 and 710 microM, respectively. The enzyme catalyzed the oxidation of catechol, 6-amino-m-cresol, 2-amino-m-cresol, and 2-amino-4-chlorophenol. 3-Hydroxyanthranilate, protocatechuate, gentisate, and 3- and 4-methylcatechol were not substrates. The substrate range and the subunit structure are unique among those of the known ring cleavage dioxygenases.     

A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation

Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.     

Tuberculosis treatment adherence and fatality in Spain

Background

The adherence to long tuberculosis (TB) treatment is a key factor in TB control programs. Always some patients abandon the treatment or die. The objective of this study is to identify factors associated with defaulting from or dying during antituberculosis treatment.

Methods

Prospective study of a large cohort of TB cases diagnosed during 2006-2007 by 61 members of the Spanish Society of Pneumology and Thoracic Surgery (SEPAR). Predictive factors of completion outcome (cured plus completed treatment vs. defaulters plus lost to follow-up) and fatality (died vs. the rest of patients) were based on logistic regression, calculating odds ratios (OR) and 95% confidence intervals (CI).

Results

Of the 1490 patients included, 29.7% were foreign-born. The treatment outcomes were: cured 792 (53.2%), completed treatment 540 (36.2%), failure 2 (0.1%), transfer-out 33 (2.2%), default 27 (1.8%), death 27 (1.8%), lost to follow-up 65 (4.4%), other 4 (0.3%). Completion outcome reached 93.5% and poor adherence was associated with: being an immigrant (OR = 2.03; CI:1.06-3.88), living alone (OR = 2.35; CI:1.05-5.26), residents of confined institutions (OR = 4.79; CI:1.74-13.14), previous treatment (OR = 2.93; CI:1.44-5.98), being an injecting drug user (IDU) (OR = 9.51; CI:2.70-33.47) and treatment comprehension difficulties (OR = 2.93; CI:1.44-5.98). Case fatality was 1.8% and it was associated with the following variables: age 50 or over (OR = 10.88; CI:1.12-105.01), retired (OR = 12.26;CI:1.74-86.04), HIV-infected (OR = 9.93; CI:1.48-66.34), comprehension difficulties (OR = 4.07; CI:1.24-13.29), IDU (OR = 23.59; CI:2.46-225.99) and Directly Observed Therapy (DOT) (OR = 3.54; CI:1.07-11.77).

Conclusion

Immigrants, those living alone, residents of confined institutions, patients treated previously, those with treatment comprehension difficulties, and IDU patients have poor adherence and should be targeted for DOT. To reduce fatality rates, stricter monitoring is required for patients who are retired, HIV-infected, IDU, and those with treatment comprehension difficulties.     

Aerobic Biodegradation of 2,4-Dinitroanisole by Nocardioides sp. Strain JS1661

2,4-Dinitroanisole (DNAN) is an insensitive munition ingredient used in explosive formulations as a replacement for 2,4,6-trinitrotoluene (TNT). Little is known about the environmental behavior of DNAN. There are reports of microbial transformation to dead-end products, but no bacteria with complete biodegradation capability have been reported. Nocardioides sp. strain JS1661 was isolated from activated sludge based on its ability to grow on DNAN as the sole source of carbon and energy. Enzyme assays indicated that the first reaction involves hydrolytic release of methanol to form 2,4-dinitrophenol (2,4-DNP). Growth yield and enzyme assays indicated that 2,4-DNP underwent subsequent degradation by a previously established pathway involving formation of a hydride-Meisenheimer complex and release of nitrite. Identification of the genes encoding the key enzymes suggested recent evolution of the pathway by recruitment of a novel hydrolase to extend the well-characterized 2,4-DNP pathway.     

The future of ocean governance

Ocean governance is complex and influenced by multiple drivers and actors with different worldviews and goals. While governance encompasses many elements, in this paper we focus on the processes that operate within and between states, civil society and local communities, and the market, including industry. Specifically, in this paper, we address the question of how to move towards more sustainable ocean governance aligning with the sustainable development goals (SDGs) and the UN Ocean Decade. We address three major risks to oceans that arise from governance-related issues: (1) the impacts of the overexploitation of marine resources; (2) inequitable distribution of access to and benefits from marine ecosystem services, and (3) inadequate or inappropriate adaptation to changing ocean conditions. The SDGs have been used as an underlying framework to develop these risks. We identify five drivers that may determine how ocean governance evolves, namely formal rules and institutions, evidence and knowledge-based decision-making, legitimacy of decision-making institutions, stakeholder engagement and participation, and empowering communities. These drivers were used to define two alternative futures by 2030: (a) ‘Business as Usual’—a continuation of current trajectories and (b) ‘More Sustainable Future’—optimistic, transformational, but technically achievable. We then identify what actions, as structured processes, can reduce the three major governance-related risks and lead to the More Sustainable Future. These actions relate to the process of co-creation and implementation of improved, comprehensive, and integrated management plans, enhancement of decision-making processes, and better anticipation and consideration of ambiguity and uncertainty.

     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems

Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation.     

Bacterial degradation of m-nitrobenzoic acid.

Pseudomonas sp. strain JS51 grows on m-nitrobenzoate (m-NBA) with stoichiometric release of nitrite. m-NBA-grown cells oxidized m-NBA and protocatechuate but not 3-hydroxybenzoate, 4-hydroxy-3-nitrobenzoate, 4-nitrocatechol, and 1,2,4-benzenetriol. Protocatechuate accumulated transiently when succinate-grown cells were transferred to media containing m-NBA. Respirometric experiments indicated that the conversion of m-NBA to protocatechuate required 1 mol of oxygen per mol of substrate. Conversions conducted in the presence of 18O2 showed the incorporation of both atoms of molecular oxygen into protocatechuate. Extracts of m-NBA-grown cells cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. These results provide rigorous proof that m-NBA is initially oxidized by a dioxygenase to produce protocatechuate which is further degraded by a 4,5-dioxygenase.     

Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45

Microbial metabolism of nitroarenes via o-aminophenols requires the participation of two key enzymes, a nitroreductase and an hydroxylaminobenzene mutase. The broad substrate ranges of the enzymes suggested that they could be used as biocatalysts for the production of substituted o-aminophenols. We have used enzymes from Pseudomonas pseudoalcaligenes JS45 for the conversion of 4-nitrobiphenyl ether to the corresponding o-aminophenol. Partially purified nitrobenzene nitroreductase reduced 4-nitrobiphenyl ether to the corresponding 4-hydroxylaminobiphenyl ether. Partially purified hydroxylaminobenzene mutase stoichiometrically converted the intermediate to 2-amino-5-phenoxyphenol. The results indicate that the enzyme system can be applied for the production of o-aminophenols useful as intermediates for synthesis of commercially important materials. Journal of Industrial Microbiology & Biotechnology (2000) 24, 301–305. Received 13 October 1999/ Accepted in revised form 31 January 2000     

High sensitivity DNA detection using gold nanoparticle functionalised polyaniline nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF-AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150×10(-12) to 1×10(-6) mol L(-1) and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.