Explore the vast range of titles available.

Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. Diaphorobacter sp. strain JS3051 utilizes 2,3-dichloronitrobenzene (23DCNB), a toxic anthropogenic compound, as the sole carbon, nitrogen, and energy source for growth, but the metabolic pathway and its origins are unknown. Here, we establish that a gene cluster ( dcb ), encoding a Nag-like dioxygenase, is responsible for the initial oxidation of the 23DCNB molecule. The 2,3-dichloronitrobenzene dioxygenase system (DcbAaAbAcAd) catalyzes conversion of 23DCNB to 3,4-dichlorocatechol (34DCC). Site-directed mutagenesis studies indicated that residue 204 of DcbAc is crucial for the substrate specificity of 23DCNB dioxygenase. The presence of glutamic acid at position 204 of 23DCNB dioxygenase is unique among Nag-like dioxygenases. Genetic, biochemical, and structural evidence indicate that the 23DCNB dioxygenase is more closely related to 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 than to the 34DCNB dioxygenase from Diaphorobacter sp. strain JS3050, which was isolated from the same site as strain JS3051. A gene cluster ( dcc ) encoding the enzymes for 34DCC catabolism, homologous to a clc operon in Pseudomonas knackmussii strain B13, is also on the chromosome at a distance of 2.5 Mb from the dcb genes. Heterologously expressed DccA catalyzed ring cleavage of 34DCC with high affinity and catalytic efficiency. This work not only establishes the molecular mechanism for 23DCNB mineralization, but also enhances the understanding of the recent evolution of the catabolic pathways for nitroarenes. IMPORTANCE Because anthropogenic nitroaromatic compounds have entered the biosphere relatively recently, exploration of the recently evolved catabolic pathways can provide clues for adaptive evolutionary mechanisms in bacteria. The concept that nitroarene dioxygenases shared a common ancestor with naphthalene dioxygenase is well established. But their phylogeny and how they evolved in response to novel nitroaromatic compounds are largely unknown. Elucidation of the molecular basis for 23DCNB degradation revealed that the catabolic pathways of two DCNB isomers in different isolates from the same site were derived from different recent origins. Integrating structural models of catalytic subunits and enzymatic activities data provided new insight about how recently modified enzymes were selected depending on the structure of new substrates. This study enhances understanding and prediction of adaptive evolution of catabolic pathways in bacteria in response to new chemicals.

To improve biofilter performance, the microbial community of a biofilter must be clearly defined. In this study, the performance of a lab-scale polyurethane biofilter for treating waste gas with low loads of nitrobenzene (NB) (< 20 g m-3 h-1) was investigated when using different empty bed residence times (EBRT) (64, 55.4 and 34 s, respectively). In addition, the variations of the bacterial community in the biofilm on the longitudinal distribution of the biofilters were analysed by using Illumina MiSeq high-throughput sequencing. The results showed that NB waste gas was successfully degraded in the biofilter. High-throughput sequencing data suggested that the phylum Actinobacteria and genus Rhodococcus played important roles in the degradation of NB. The variations of the microbial community were attributed to the different intermediate degradation products of NB in each layer. The strains identified in this study were potential candidates for purifying waste gas effluents containing NB.

We explored the feasibility of using urinary metabolites of p-chloronitrobenzene (p-CNB) as exposure biomarkers of occupational p-CNB exposure. Forty-two workers exposed to p-CNB during their jobs at a chemical enterprise in Shaoxing, Zhejiang Province, China were included in the exposure group, while administrative personnel who do not come into contact with p-CNB at work were included in the control group. Each worker in the exposure group was equipped with a personal air sampler to collect airborne p-CNB samples, and urine samples were collected at the conclusion of each shift. After sample collection, the airborne p-CNB concentrations and urinary metabolite concentrations were detected by gas chromatography-tandem mass spectrometry and ultra-performance liquid chromatography - quadrupole - orbitrap high resolution mass spectrometry, respectively. The urinary metabolite concentrations were corrected by the content of urinary creatinine. Subsequently, the correlations between the urinary metabolite concentrations and the p-CNB time-weighted average (TWA) concentrations were assessed using correlation analysis. All p-CNB TWA concentrations measured in this study were below the occupational exposure limit in the Chinese national standards. In the exposure group, N-acetyl-S-(4-nitrophenyl)-L-cysteine (NANPC), 2-chloro-5-nitrophenol (2C5NP), p-chloroacetanilide (p-CAA), p-chlorooxanilic acid (p-COA), 2-amino-5-chlorophenol (2A5CP), and p-chloroaniline (p-CA) were detected at varying levels. The percentages of NANPC, 2C5NP, p - CAA, p - COA, 2A5CP, and p - CA were 64.1%, 5.1%, 0.3%, 15.1%, 5.1%, and 10.3%, respectively. We found extremely significant positive relationships ( p  < 0.01) between the urinary metabolite concentrations (p-CA, 2C5NP, 2A5CP, NANPC, and p-COA) and the p-CNB TWA concentrations, with respective correlation coefficients of 0.827, 0.673, 0.790, 0.714, and 0.741. Thus, these five metabolites may be used as exposure biomarkers of occupational p-CNB exposure. Moreover, among these metabolites, NANPC was identified as the most suitable exposure biomarker because it had the highest correlation coefficient and the highest content in urine.

We genetically encoded the photocaged amino acid 4,5-dimethoxy-2-nitrobenzylserine (DMNB-Ser, 1 ) in Saccharomyces cerevisiae in response to the amber nonsense codon TAG. This amino acid was converted to serine in living cells by irradiation with relatively low-energy blue light and was used to noninvasively photoactivate phosphorylation of the transcription factor Pho4, which controls the cellular response to inorganic phosphate 1 . When substituted at phosphoserine sites that control nuclear export of Pho4, 1 blocks phosphorylation and subsequent export by the receptor Msn5 (ref. 2 ). We triggered phosphorylation of individual serine residues with a visible laser pulse and monitored nuclear export of Pho4-GFP fusion constructs in real time. We observed distinct export kinetics for differentially phosphorylated Pho4 mutants, which demonstrates dynamic regulation of Pho4 function. This methodology should also facilitate the analysis of other cellular processes involving free serine residues, including catalysis, biomolecular recognition and ion transport.

Background/aimsLeukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6–13 months, leukodystrophy and peripheral neuropathy.MethodsExome analysis, determination of alkaline ceramidase activity catalysing the conversion of C18:1-ceramide to sphingosine and D-ribo-C12-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phytoceramide to NBD-C12-fatty acid using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and thin layer chromatography, respectively, and sphingolipid analysis in patients’ blood by LC-MS/MS.ResultsThe patients were homozygous for p.E33G in the ACER3, which encodes a C18:1-alkaline ceramidase and C20:1-alkaline ceramidase. The mutation abolished ACER3 catalytic activity in the patients’ cells and failed to restore alkaline ceramidase activity in yeast mutant strain. The levels of ACER3 substrates, C18:1-ceramides and dihydroceramides and C20:1-ceramides and dihydroceramides and other long-chain ceramides and dihydroceramides were markedly increased in the patients’ plasma, along with that of complex sphingolipids, including monohexosylceramides and lactosylceramides.ConclusionsHomozygosity for the p.E33G mutation in the ACER3 gene results in inactivation of ACER3, leading to the accumulation of various sphingolipids in blood and probably in brain, likely accounting for this new form of childhood leukodystrophy.

Protein-lipid interactions in cellular membranes modulate central cellular functions, are often transient in character, but occur too intermittently to be readily observable. We introduce transient state imaging (TRAST), combining sensitive fluorescence detection of fluorophore markers with monitoring of their dark triplet state transitions, allowing imaging of such protein-lipid interactions. We first determined the dark state kinetics of the biomembrane fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) in lipid vesicles, and how its triplet state is quenched by spin-labels in the same membranes. We then monitored collisional quenching of NBD-lipid derivatives by spin-labelled stearic acids in live cell plasma membranes, and of NBD-lipid derivatives by spin-labelled G-Protein Coupled Receptors (GPCRs). We could then resolve transient interactions between the GPCRs and different lipids, how these interactions changed upon GPCR activation, thereby demonstrating a widely applicable means to image and characterize transient molecular interactions in live cell membranes in general, not within reach via traditional fluorescence readouts.

Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-alpha, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D(2) (PGD(2)) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD(3). This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD(2) receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD(2) signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs.

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

Nitrobenzene dioxygenase (NBDO) is known to add both atoms of molecular oxygen to the aromatic ring of nitrobenzene to form catechol. It is assembled by four subunits of which the alpha subunit is responsible for catalysis. As an oxidizing enzyme, it has a potential use in the detoxification of industrial waste and the synthesis of pharmaceuticals and food ingredients; however, not much work has been done studying its structure-function correlations. We used several protein engineering approaches (neutral drift libraries, random libraries, two types of focused libraries, and family shuffling) to engineer NBDO for the production of the highly potent antioxidant, hydroxytyrosol (HTyr), from the substrate 3-nitrophenethyl alcohol (3NPA). We obtained a triple mutant, F222C/F251L/G253D, which is able to oxidize 3NPA 375-fold better than wild type with a very high regioselectivity. In total, we identified four positions which are important for acquisition of new specificities, of which only one is well-known and studied. Based on homology modeling, it is suggested that these mutations increase activity by vacating extra space within the active site for the larger substrate and also by hydrogen bonding to the substrate. The best variant had acquired a stabilizing mutation which was beneficial only in this mutant. Thus, we have achieved two goals, the first is the enzymatic production of HTyr, and the second is valuable information regarding the structure-function correlations of NBDO.

The zero-valent iron (ZVI) corrosion products and their functions were investigated in the combined ZVI and anaerobic sludge system. Results showed that ZVI corrosion occurred, and the reductive transformation and dechlorination of p -chloronitrobenzene ( p -ClNB) by the anaerobic sludge were enhanced. In the combined systems with different types of ZVIs and mass ratios of anaerobic sludge to ZVI, a considerable amount of suspended iron compounds was produced and coated onto the microbial cells. However, the microbial cellular structure was damaged, and the p -ClNB reductive transformation was affected adversely after the long-term presence of nanoscale ZVI (NZVI) or reduced ZVI (RZVI) with a high concentration of 5 g L −1 . The oxidized products of FeOOH and Fe 3 O 4 were found on the surface of ZVI, which are speculated to act as electron mediators and consequently facilitate the utilization of electron donors by the anaerobic microbes.