Ribonucleotides differentially modulate oral glutamate detection thresholds.

The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3 mM) of 5 different 5'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08 mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046 mM. In contrast to the purines, the pyrimidines uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.

Role of the Tumor Microenvironment in Regulating Pancreatic Cancer Therapy Resistance.

Pancreatic cancer has a notoriously poor prognosis, exhibits persistent drug resistance, and lacks a cure. Unique features of the pancreatic tumor microenvironment exacerbate tumorigenesis, metastasis, and therapy resistance. Recent studies emphasize the importance of exploiting cells in the tumor microenvironment to thwart cancers. In this review, we summarize the hallmarks of the multifaceted pancreatic tumor microenvironment, notably pancreatic stellate cells, tumor-associated fibroblasts, macrophages, and neutrophils, in the regulation of chemo-, radio-, immuno-, and targeted therapy resistance in pancreatic cancer. The molecular insight will facilitate the development of novel therapeutics against pancreatic cancer.

Bitter Taste Receptors (T2Rs) are Sentinels that Coordinate Metabolic and Immunological Defense Responses.

In addition to being responsible for bitter taste, type 2 taste receptors (T2Rs) regulate endocrine, behavioral, and immunological responses. T2R agonists include indicators of incoming threats to metabolic homeostasis, pathogens, and irritants. This review will provide an overview of T2R-regulated processes throughout the body that function defensively. We propose a broader definition of T2Rs as chemosensory sentinels that monitor toxic, metabolic, and infectious threats and initiate defensive responses.

Sequential anaerobic and aerobic bioaugmentation for commingled groundwater contamination of trichloroethene and 1,4-dioxane.

Chlorinated solvents, notably trichloroethene (TCE), and the cyclic ether stabilizer, 1,4-dioxane (dioxane), have been frequently detected commingling in contaminated aquifers. Here we developed a sequential anaerobic and aerobic treatment strategy effective to mitigate the co-contamination of TCE and dioxane, particularly when dioxane is present at ppb levels relevant to many impacted sites. After the primary anaerobic treatment by a halorespiring consortium SDC-9, TCE was effectively removed, though lingering less-chlorinated metabolites, vinyl chloride (VC) and cis-dichloroethene (cDCE). Subsequent aerobic bioaugmentation with Azoarcus sp. DD4, a cometabolic dioxane degrader, demonstrated the ability of DD4 to degrade dioxane at an initial concentration of 20 µg/L to below 0.4 µg/L and its dominance (~7%) in microcosms fed with propane. Even better, DD4 can also transform VC and cDCE in tandem, though cDCE and VC at relatively high concentrations (e.g., 1 mg/L) posed inhibition to propane assimilation and cell growth of DD4. Mutagenesis of DD4 revealed group-2 toluene monooxygenase and group-5 propane monooxygenase are responsible for cDCE and VC co-oxidation, respectively. Overall, we demonstrated the feasibility of a treatment train combining reductive dehalogenation and aerobic co-oxidation processes in tandem to not only effectively clean up prevalent co-contamination of TCE and dioxane at trace levels but also mitigate persistent products (e.g., cDCE and VC) when complete reductive dehalogenation of less-chlorinated ethenes occurs slowly in the field.

Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4.

Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multicomponent monooxygenases in Azoarcus sp. strain DD4, a Gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved to be the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active cooxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility can lead to broad applications, spanning from environmental remediation and water treatment to biocatalysis in green chemistry.IMPORTANCE Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective cooxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with nontoxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implications in the field of environmental microbiology and beyond.

Distinct Catalytic Behaviors between Two 1,4-Dioxane-Degrading Monooxygenases: Kinetics, Inhibition, and Substrate Range.

Monitored natural attenuation (MNA) and engineered bioremediation have been recognized as effective and cost-efficient in situ treatments to mitigate 1,4-dioxane (dioxane) contamination. Dioxane metabolism can be initiated by two catabolic enzymes, propane monooxygenase (PRM) and tetrahydrofuran monooxygenase (THM), belonging to the group-6 and 5 of soluble di-iron monooxygenase family, respectively. In this study, we comprehensively compared catalytic behaviors of PRM and THM when individually expressed in the heterologous host, Mycobacterium smegmatis mc2-155. Kinetic results revealed a half-saturation coefficient (Km) of 53.0 ± 13.1 mg/L for PRM, nearly 4 times lower than that of THM (235.8 ± 61.6 mg/L), suggesting that PRM has a higher affinity to dioxane. Exposure with three common co-contaminants (1,1-dichloroethene, trichloroethene, and 1,1,1-trichloroethane) demonstrated that PRM was also more resistant to their inhibition than THM. Thus, dioxane degraders expressing PRM may be more physiologically and ecologically advantageous than those with THM at impacted sites, where dioxane concentration is relatively low (e.g., 250 to 1000 µg/L) with co-occurrence of chlorinated solvents (e.g., 0.5 to 8 mg/L), underscoring the need of surveying both PRM and THM-encoding genes for MNA potential assessment. PRM is also highly versatile, which breaks down cyclic molecules (dioxane, tetrahydrofuran, and cyclohexane), as well as chlorinated and aromatic pollutants, including vinyl chloride, 1,2-dichloroethane, benzene, and toluene. This is the first report regarding the ability of PRM to degrade a variety of short-chain alkanes and ethene in addition to dioxane, unraveling its pivotal role in aerobic biostimulation that utilizes propane, isobutane, or other gaseous alkanes/alkenes (e.g., ethane, butane, and ethene) to select and fuel indigenous microorganisms to tackle the commingled contamination of dioxane and chlorinated compounds.

Complete Genome Sequence of Azoarcus sp. Strain DD4, a Gram-Negative Propanotroph That Degrades 1,4-Dioxane and 1,1-Dichloroethylene.

Azoarcus sp. strain DD4 can cometabolically degrade 1,4-dioxane and 1,1-dichloroethylene (1,1-DCE) when grown with propane and other substrates. The complete genome sequence of strain DD4 reveals a diverse collection of bacterial monooxygenase genes that may contribute to its versatility in degrading commingled groundwater pollutants.

Membrane-Disrupting Nanofibrous Peptide Hydrogels.

Self-assembled peptide nanofibers can form biomimetic hydrogels at physiological pH and ionic strength through noncovalent and reversible interactions. Inspired by natural antimicrobial peptides, we designed a class of cationic amphiphilic self-assembled peptides (CASPs) that self-assemble into thixotropic nanofibrous hydrogels. These constructs employ amphiphilicity and high terminal charge density to disrupt bacterial membranes. Here, we focus on three aspects of the self-assembly of these hydrogels: (a) the material properties of the individual self-assembled nanofibers, (b) emergence of bulk-scale elasticity in the nanofibrous hydrogel, and (c) trade-off between the desirable material properties and antimicrobial efficacy. The design of the supramolecular nanofibers allows for higher-order noncovalent ionic cross-linking of the nanofibers into a viscoelastic network. We determine the stiffness of the self-assembled nanofibers via the peak force quantitative nanomechanical atomic force microscopy and the bulk-scale rheometry. The storage moduli depend on peptide concentration, ionic strength, and concentration of multivalent ionic cross-linker. CASP nanofibers are demonstrated to be effective against Pseudomonas aeruginosa colonies. We use nanomechanical analysis and microsecond-time scale coarse-grained simulation to elucidate the interaction between the peptides and bacterial membranes. We demonstrate that the membranes stiffen, contract, and buckle after binding to peptide nanofibers, allowing disruption of osmotic equilibrium between the intracellular and extracellular matrix. This is further associated with dramatic changes in cell morphology. Our studies suggest that self-assembled peptide nanofibrils can potentially acts as membrane-disrupting antimicrobial agents, which can be formulated as injectable hydrogels with tunable material properties.

Potential application of an Aspergillus strain in a pilot biofilter for benzene biodegradation.

A biofilter with fungus was developed for efficient degradation of benzene, which can overcome the potential risk of leakage commonly found in such services. Results indicated that the optimum parameter values were temperature 40 °C, pH 6, and 500 mg L-1 of the initial benzene concentration. Besides, the empty bed residence time and inlet load range of biofilter were set to 20 s and 21.23-169.84 g m-3 h-1 respectively. Under these conditions, this biofilter can obtain the maximum removal efficiency of more than 90%, the eliminating capacity could be up to 151.67 g m-3 h-1. Furthermore, scanning electron microscopy was used to investigate three filler materials for packing fungus biofilm. This is the first study introducing an Aspergillus strain for benzene removal and these results highlight that the development of this biofilter has the potential scaling-up application as gas-processing of industrial wastes.

Uptake, translocation and metabolism of decabromodiphenyl ether (BDE-209) in seven aquatic plants.

Terrestrial plant uptake of PBDEs from contaminated soils has been widely reported recently. In this study the fate of deca-BDE within a plant/PBDEs/aquatic environment system was investigated through simulated pot experiments. Accumulations of the total PBDEs and deca-BDE were observed in tissues of seven test aquatic plant species, namely Phragmites australis, Cyperus papyrus, Alternanthera philoxeroides, Colocasia esculenta, Scirpus validus, Acorus calamus and Oryza sativa. In all seven plants, O. sativa leads the uptake and accumulation both in the total PBDEs (444.8 ng g(-1)) and deca-BDE (368.0 ng g(-1)) in roots. Among the six common phytoremediation aquatic plants, A. calamus leads the uptake (236.2 ng g(-1)), and P. australis leads the translocation (Cshoot/Croot = 0.35), while A. philoxeroides (43.4%) and P. australis (80.0%) lead in the metabolism efficiencies in the root and shoot, respectively. The detection of seventeen lesser brominated PBDE congeners provided the debromination evidence, and the specific PBDEs profiles in test plant species indicated there is no common metabolic pattern. Furthermore, a relative high proportion of lesser brominated PBDE congeners in shoots suggested the possible metabolic difference between roots and shoots. Finally, a noticeable percentage of penta- and octa-BDE derived from deca-BDE also hint the ecological risk in deca-BDE use. This comparative research on the aquatic plants provide a broad vision on the understanding of plant/PBDEs/aquatic environment interaction system, and may be applied to remediate PBDEs in contaminated waters and sediments.

Polymorphism of the glucosyltransferase gene (ycjM) in Escherichia coli and its use for tracking human fecal pollution in water.

This study examined polymorphism of the glucosyltransferase gene (ycjM) in fecal Escherichia coli isolates and evaluated the use of the sequence polymorphism for measuring human fecal pollution in water. Significant nucleotide variations were observed through comparative analysis of the ycjM sequences of 70 E. coli strains isolated from the feces of humans, domestic livestock, and wild animals. Three distinct types of ycjM sequences were found: universal-ycjM, human/chicken-ycjM, and human-ycjM. Using the human-ycjM sequences, both a polymerase chain reaction (PCR), Hycj-PCR and a quantitative PCR, Hycj-qPCR, were developed. As shown by the Hycj-PCR amplification, the human-ycjM marker appeared to be highly associated with the E. coli strains isolated from human feces, based on the analysis of 370 E. coli strains isolated from humans and seven other animal species. Similarly, the human-ycjM marker was highly linked with human feces, as demonstrated by the Hycj-PCR assay, when using 337 fecal DNA samples from 16 host animal sources, including both domestic and wild animals. Overall, the specificity and sensitivity of the human-ycjM marker for differentiating between the feces of humans and those of nonhuman groups were 99.7% and 100%, respectively; the prevalence of the marker appeared to be greater than 50% in the human-feces-associated E. coli population. In addition, our study showed that the quantification of human E. coli by the Hycj-qPCR was linearly correlated with the anthropogenic activity within a watershed. Our study suggests that this novel human-ycjM marker and the resulting PCR-based methods developed should be useful for measuring human-associated E. coli and human fecal pollution in water.

Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM).

This study is to tackle the challenge posed by the "naturalized" Escherichia coli population against the worldwide practice of E. coli-based water quality monitoring. In the literature, the putative glucosyltransferase gene (ycjM) of E. coli has been identified in silico to be one of the 114 genes specific to enteric E. coli. Based on the sequence of E. coli K-12 MG1655, a PCR assay (ycjPCR) targeting ycjM was developed in this study. As demonstrated by the ycjPCR assay using 367 E. coli strains isolated from animal feces, 97.2% of the isolates carried the ycjM with variations from 93.9% to 100% among nine different host sources, but none of the 17 strains of non-E. coli bacteria and only 23.0% of the environment-isolated cryptic Escherichia strains contained the ycjM. These data experimentally confirmed ycjM to be enteric specific. Our study also showed that the ycjPCR assay was superior to the commonly used tuf- or uidA-based PCR methods in differentiating enteric E. coli from ß-D-glucuronidase-positive environmental bacteria. Furthermore, study on 190 E. coli isolates from water samples, using EPA Method 1603 followed by bacterial identification with Biolog MicroStation™ and ycjPCR assay, indicated that the prevalence of ycjM in the E. coli water isolates had a significant (p < 0.05, odds ratio ) spatial variation from 69.6% to 93.8%. These data suggest that E. coli profile using EPA Method 1603 or other ß-D-glucuronidase-activity-based methods may need further analysis using the ycjM profile to accurately determinate fecal pollution in water.

[Effects of nitrate on organic removal and microbial community structure in the sediments].

The strategy promoted pollutant degradation and transformation under the anaerobic circumstance by adding nitrate as an electron acceptor has been widely used in sediment bioremediation. However, few literature reports on organic removal characteristics and microbial community responses in the contaminated river sediment under the nitrate reduction condition. Methods including the polar and non-polar chemical fractionation, relative abundance detection of organic matters by GC-MS were combined and applied to investigate organic removals and PCR-DGGE analysis was used for microbial community structures in sediment incubation systems with or without calcium nitrate addition. The results indicated that the addition of calcium nitrate could significantly enhance removal efficiencies of organic pollutants. The removal efficiency of total organic carbon (TOC) and the total peak area of organic matters in GC-MS were 47.25% and 29.55% which were higher than those of the control. The effect descending order of organic pollutants was: silicon materials > alkanes > polycyclic aromatic hydrocarbons > heterocyclic compounds > olefins > benzene homologues > polar compounds > phthalates > aldehydes and ketones > alkyl esters. And removal rates of silicon materials, the persistent organic pollutants, benzene homologues and heterocyclic compounds were 46.73%, 36.25%, 23.19% and 35.92% which were higher than those of the control. The PCR-DGGE profile of bacterial 16S rDNA V3 fragments showed obviously different microbial community structures between the treatment and the control systems. Blastn analysis revealed that sequences of 10 predominant bands from DGGE profile were closely related to Proteobacteria, Actinobacteria, Clostridia, Chloroflexi, Caldiserica and uncultured bacterium. The research findings provide some helpful scientific information for promoting organic pollutant removal of river sediment by nitrate reduction.

[Characteristics of organic pollutants in the sediments from a typical electronics industrial zone].

In order to investigate the contamination status of organic pollutants in a river of a typical electrical equipment industrial area, Ronggui, Foshan, the sediments were sampled for the composition, concentration and occurrence analysis of organic pollutants. The polar and non-polar fractionation methods were employed for the fingerprint establishment of organic pollutants. One hundred and seventy-one of organic chemicals including ten categories of alkanes, alkenes, polycyclic aromatic hydrocarbons, benzene, heterocyclic compounds, phthalate esters, aldehydes, ketones, polar compounds, silicon-containing material as well as alkyl esters were examined. The number of different categories of the detected organic pollutants in a descending order was: alkanes > polar compounds > polycyclic aromatic hydrocarbons > aldehydes and ketones > heterocyclic compounds > benzene homologues, phthalate ester > alkyl esters > silicon material > olefins. The abundance of detected organic pollutants in a descending order was: alkanes > polar compounds > alkyl esters > olefins > polycyclic aromatic hydrocarbons > phthalates > silicon material > aldehydes and ketones > heterocyclic compounds > benzene homologues. Among the 51 kinds of alkanes detected, nonadecane accounted for 14.83%, and the persistent organic pollutants accounted for 2.33% of the total organic matter. Compared to similar studies, there were 51 kinds of alkanes and they accounted for 55.5% of the total organic chemicals, showing high diversity and abundance. In addition, some electronics industry-related organic pollutants such as silicone materials were also detected in high frequency.

[Distribution and potential ecological risk of polycyclic aromatic hydrocarbons in the sediments from typical electronics industrial zone].

In order to investigate the polycyclic aromatic hydrocarbons (PAHs) contamination status in a river of a typical electrical equipment industrial area, Ronggui, Foshan, 8 sediments simples were collected. The results showed that 12 components of PAHs were detected in all sediment samples, and the other 4 components were also detected in different degree. The total 16 EPA priority PAHs contents in surface sediments varied from 343.5 microg x kg(-1) to 2 099 microg x kg(-1) (dry wt), with an average of 1215.9 microg x kg(-1), and no significant variation was found in vertical distribution. The 2 to 3 aromatic rings were the dominant PAHs, and they accounted for 50.69% to 82.5% of the total PAHs. Diagnostic ratios were used to identify the possible sources of PAHs, and the results indicated that PAHs were mainly pyrogenic origin. Sediments were polluted by PAHs heavily according to Maliszewska-Kordybach's standard, the very high risk level was also confirmed by the contamination factors assessment which was investigated by comparison with the background area (SO). The assessment by the method of sediment quality guidelines (SQGs) and relative contamination factor (RCF) demonstrated that adverse biological toxicity effect might occasionally happen in a few sampling sites, and the ecological risk components included acenaphthene (Ace), fluorine (Flu), and phenanthrene (Phe).

[Horizontal and vertical distribution of polybrominated diphenyl ethers (PBDEs) in river sediment from a typical electrical equipment industrial area].

Polybrominated diphenyl ethers (PBDEs) were determined in sediment collected from a river in a typical electrical equipment industrial area, Ronggui, Foshan. Eight samples were collected from river sediment. The results indicated that PBDEs were presented in all samples. Spatial trends showed that the concentrations of PBDEs in all sediment samples were ranged from 62 ng x g(-1) to 349 ng x g(-1), with an average of 178 ng x g(-1). The predominant congener was BDE-209 (90% - 99%), which ranged from 56-337 ng x g(-1), with an average of 171 ng x g(-1). Some of congeners such as BDE-196, 197 and 203 may be were the degradation product of BDE-209. Vertically, the concentrations of PBDEs were increased with the depth, whereas the concentrations were 147 ng x g(-1) in layer 0-10 cm and 260 ng x g(-1) in layer 30-40 cm, respectively. Distribution profile of PBDEs in vertical direction was similar in different depths. The commercial deca-BDE (94%), which contained BDE-209, 208, 207 and 206, was the dominate pollutant with minor contributions from penta-and octa-BDEs. The results suggested that this area was polluted by PBDEs and BDE-209 was the most dominate congener, which is related with the manufacturing activities for electrical equipment.

Bar-coded pyrosequencing reveals the responses of PBDE-degrading microbial communities to electron donor amendments.

Polybrominated diphenyl ethers (PBDEs) can be reductively degraded by microorganisms under anaerobic conditions. However, little is known about the effect of electron donors on microbial communities involved in PBDEs degradation. Here we employed 454 Titanium pyrosequencing to examine the phylogenetic diversity, composition, structure and dynamics of microbial communities from microcosms under the conditions of different electron donor amendments. The community structures in each of the five alternate electron donor enrichments were significantly shifted in comparison with those of the control microcosm. Commonly existing OTUs between the treatment and control consortia increased from 5 to 17 and more than 50% of OTUs increased around 13.7 to 186 times at least in one of the microcosms after 90-days enrichment. Although the microbial communities at different taxonomic levels were significantly changed by different environmental variable groups in redundancy analysis, significant correlations were observed between the microbial communities and PBDE congener profiles. The lesser-brominated PBDE congeners, tri-BDE congener (BDE-32) and hexa-BDE, were identified as the key factors shaping the microbial community structures at OTU level. Some rare populations, including the known dechlorinating bacterium, Dehalobacter, showed significant positive-correlation with the amounts of PBDE congeners in the consortia. The same results were also observed on some unclassified bacteria. These results suggest that PBDEs-degrading microbial communities can be successfully enriched, and their structures and compositions can be manipulated through adjusting the environmental parameters.

Effects of electron donors on anaerobic microbial debromination of polybrominated diphenyl ethers (PBDEs).

Polybrominated diphenyl ethers (PBDEs) are a class of widely used flame retardants that have been highly accumulated in sediments. It is reported that microorganisms play an important role in the reductive debromination of PBDEs in anaerobic sediments. However, little is known about the effects of electron donors on the microbial community structure and their debromination capacity in PBDE transformation. In this study, alternate carbon substrates were used as electron donors to enrich the PBDE-debrominating microbial consortia to evaluate the effects of electron donors on PBDE microbial debromination. Decabromodiphenyl ether (BDE-209) was found to be the dominant (more than 50%) PBDEs congener in all consortia, and the percentage of BDE-209 was deceased by 12% (methanol), 11% (ethanol), 8% (acetate), 9% (lactate), 5% (pyruvate), and 11% (no electron donors), while the relative abundances of most lesser-brominated PBDEs increased after 90-day incubation compared to the initial profile of PBDEs. Substantial shifts in the microbial community structure among different amendments were observed based on denaturing gradient gel electrophoresis results. Pseudomonas spp. were identified to be the predominant organisms and the abundances of Band R, which was associated with Pseudomonas sp. SCSWA09, was well correlated with the biodegradation rate of BDE-209. Finally, the microbial community structure was highly correlated with the concentration of deca-BDE, octa-BDE and total nitrogen. These results provide insights into in situ bioremediation of environments contaminated by PBDEs and our understanding of microbial ecology associated with PBDE-debromination.

Characterization of two components of the 2-naphthoate monooxygenase system from Burkholderia sp. strain JT1500.

2-Naphthoate monooxygenase, a two-protein system, encoded by the nmoA and nmoB genes, was heterologously overexpressed in Escherichia coli. The proteins used for functional characterization were purified to over 90% homogeneity by affinity chromatography. The oxidative component EnmoA (47.9 kDa) lacked substrate catalysis capability on its own, and the reductive component EnmoB (33.4 kDa) and its truncated derivate EnmoB(T) (25 kDa) possessed nearly identical independent flavin reductase activities, c. 130 micromol min(-1) mg(-1) of protein. The inframe fusioned protein EnmoB(T)A (65.2 kDa), containing NmoB(T) and NmoA peptides showed a stable 2-naphthoate monooxygenase activity of 1.2 micromol min(-1) mg(-1) of protein. This is the first report on the purification of a fused form of a two-component flavoprotein monooxygenase. In the specificity experiment, FAD and NADH were shown to be preferred cosubstrates for EnmoB and EnmoB(T). All these data suggest that NmoB(T)A is a two-component flavoprotein monooxygenase, consisting of an oxygenase and a reductase component. NmoA is a member of the class D flavoprotein monooxygenase, and NmoB is an independent NADH:Flavin oxidoreductase.

lambdaN gene expression regulated by translation termination in ribosome L24 mutant.

Besides transcription regulation, gene expression is also regulated at translation level. Although translation regulation is mainly mediated by translation initiation, an abundance of evidence shows that the termination phase of translation is also important for gene expression. The expression of lambdaN gene is down regulated at translation level in L24 mutant, however the precise mechanism still remains unknown. We report here that in an L24 mutant strain, the expression of lac-lambdaN and GST-lambdaN is decreased to 25% and 50% of that in wild type T83 strain respectively. Strikingly, the yield of GST-lambdaN fusion protein in L24 mutant can be restored to the level as in T83 wild type strain by changing the two codons upstream lambdaN stop codon. These findings imply that the stop codon and its context are involved in the translation regulation. The possible reason is that the translation termination complex containing L24 mutant ribosome may not dissociate properly in stop code region. This failure of disengagement from mRNA will slow down the process of following ribosomes, and consequently decrease the efficiency of lambdaN gene expression.