Complete genome sequence of the naphthalene-degrading Pseudomonas putida strain ND6.

Pseudomonas putida strain ND6 is an efficient naphthalene-degrading bacterium. The complete genome of strain ND6 was sequenced and annotated. The genes encoding the enzymes involved in catechol degradation by the ortho-cleavage pathway were found in the chromosomal sequence, which indicated that strain ND6 is able to metabolize naphthalene by the catechol meta- and ortho-cleavage pathways.

Predicting in vitro lung deposition behavior of combined dry powder inhaler via rheological properties.

Dry powder inhaler (DPI) for pulmonary delivery is currently the primary treatment for asthma and COPD (chronic obstructive pulmonary disease), an increasing number of combined DPIs (containing two or more drugs in one inhaler) have been developed to complement the effect of single DPIs. Based on our previous studies, the rheological properties can be a potential tool used to predict the in vitro lung deposition behavior of DPI formulations. However, it is unknown whether such a prediction model is suitable for combination systems. Therefore, this study aimed to verify the applicability of using powder rheological properties to predict in vitro drug deposition behavior in combined DPI formulations. Two drugs (fluticasone propionate and salmeterol xinafoate) and their combination of DPI formulations were prepared using fine lactose content (in the range of 1%-20%) as a variable. The physicochemical properties of the powder mixtures such as particle size and content uniformity were characterized. The rheological properties of the powder mixtures were measured by FT4 rheometer, the aerodynamic behavior of the DPI formulations was evaluated by a new generation impactor (NGI), and the effect of flowability and adhesion on the deposition of the fine particle fraction (FPF) was investigated by principal component analysis (PCA). The results showed that the combined DPI formulations with larger particle interaction forces have certain differences from the aerodynamic behavior of the single DPI formulations. The regularity of rheological properties affecting FPF revealed in single DPI is still applicable to combined DPI, the parameters basic flowability energy (BFE), representing flowability, and flow factor (ff), Cohesion representing adhesion, can be well linearly related to the FPF. The results of the principal component analysis showed that better flowability and suitable adhesion contributed to higher in vitro deposition of the drug in the formulation, and the contribution of adhesion (75.42%) was greater than that of flowability (24.58%). In conclusion, rheological properties is an effective tool for predicting the deposition behavior of DPI not only in single but also in combined DPIs.

Composition and variability of biofouling organisms in seawater reverse osmosis desalination plants.

Seawater reverse osmosis (SWRO) membrane biofouling remains a common challenge in the desalination industry, but the marine bacterial community that causes membrane fouling is poorly understood. Microbial communities at different stages of treatment processes (intake, cartridge filtration, and SWRO) of a desalination pilot plant were examined by both culture-based and culture-independent approaches. Bacterial isolates were identified to match the genera Shewanella, Alteromonas, Vibrio, and Cellulophaga based on 16S rRNA gene sequencing analysis. The 16S rRNA gene clone library of the SWRO membrane biofilm showed that a filamentous bacterium, Leucothrix mucor, which belongs to the gammaproteobacteria, accounted for nearly 30% of the clone library, while the rest of the microorganisms (61.2% of the total clones) were related to the alphaproteobacteria. 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that bacteria colonizing the SWRO membrane represented a subportion of microbes in the source seawater; however, they were quite different from those colonizing the cartridge filter. The examination of five SWRO membranes from desalination plants located in different parts of the world showed that although the bacterial communities from the membranes were not identical to each other, some dominant bacteria were commonly observed. In contrast, bacterial communities in source seawater were significantly different based on location and season. Microbial profiles from 14 cartridge filters collected from different plants also revealed spatial trends.

Detection and quantification of enteroviruses in coastal seawaters from Bohai Bay, Tianjin, China.

An 8-month survey was conducted to detect and quantify enteroviruses in Tianjin coastal seawaters of Bohai Bay to assess coastal water quality. Ten water samples were collected from Bohai Bay for the detection and quantification of enteroviruses by conventional reverse transcription polymerase chain reaction (RT-PCR) and SYBR Green real-time quantitative RT-PCR (qRT-PCR). Total viral nucleic acid was extracted from 500 mL of seawater samples concentrated by Centricon plus-70 centrifugal filter devices. The viral recovery rate was 29.1% based on viral seeding study. The centrifugal ultrafiltration method applied is effective for viral recovery from small volume of polluted water, which may have broader applications to monitoring human virus in aquatic environment. Our results indicated that there was a severe viral contamination in seawater of Bohai Bay. Enteroviruses were detected at concentrations ranging from 1.7 x 10(6) to 6.3 x 10(7) copies/L by qRT-PCR. Sequencing analyses identified that all of the twenty clones as poliovirus type 2. This is the first quantitative report of human viruses in coastal waters of a metropolitan city in China. This study emphasized the importance for the local and central governments to monitor and assess the water quality.

Synthesis, herbicidal activities, and 3D-QSAR of 2-cyanoacrylates containing aromatic methylamine moieties.

A series of novel 2-cyanoacrylates containing different aromatic rings were synthesized, and their structures were characterized by (1)H NMR, elemental analysis, and single-crystal X-ray diffraction analysis. Their herbicidal activities against four weeds and inhibition of photosynthetic electron transport against isolated chloroplasts (the Hill reaction) were evaluated. Both in vivo and in vitro data showed that the compounds containing benzene, pyridine, and thiazole moieties gave higher activities than those containing pyrimidine, pyridazine, furan, and tetrahedronfuran moieties. To further explore the comprehensive structure-activity relationship on the basis of in vitro data, comparative molecular field analysis (CoMFA) was performed, and the results showed that a bulky and electronegative group around the para-position of the aromatic rings would have the potential for higher activity, which offered important structural insights into designing highly active compounds prior to the next synthesis.

Isolation and characterization of atrazine-degrading Arthrobacter sp. AD26 and use of this strain in bioremediation of contaminated soil.

A bacterial strain (AD26) capable of utilizing atrazine as a sole nitrogen source for growth was isolated from an industrial wastewater sample by enrichment culture. The 16S rRNA gene sequencing identified AD26 as an Arthrobacter sp. PCR assays indicated that AD26 contained atrazine-degrading genes trzN and atzBC. The trzN gene of AD26 only differs from the trzN of Arthrobacter aurescens TC1 by one base (A-->T at 907) and one amino acid (Met-->Leu at 303). The specific activity of trzN of AD26 in crude atrazine-containing minimal media than two well characterized atrazine-degrading bacteria, Pseudomonas sp. ADP and Arthrobacter aurescens TC1. After incubating for 48 h at 30 degrees C, the OD(600) of AD26 reached 2.6 compared with 1.33 of ADP. AD26 was capable of degrading 500 mg/L of atrazine in minimal medium at 95% in 72 h, while the degradative rates by TC1 and ADP were only 90% and 86%, respectively. A bioremediation trial of contaminated soil has indicated that AD26 can degrade as high as 98% of atrazine contained in soil (300 mg/kg) after incubating for 20 d at 26 degrees C, nominating this strain as a good candidate for use in bioremediation programs.

Synthesis, crystal structure, and biological activities of 2-cyanoacrylates containing furan or tetrahydrofuran moieties.

A series of novel cyanoacrylates containing furan or tetrahydrofuran moieties were synthesized, and their structures were characterized by 1H NMR, elemental analysis, and single-crystal X-ray diffraction analysis. The herbicidal, plant growth regulatory, fungicidal, and antiviral activities of these cyanoacrylates were evaluated. The results of herbicidal activities showed that most of these cyanoacrylates exhibited higher herbicidal activities against dicotyledonous weeds than monocotyl-edonous weeds, and the compounds containing the tetrahydrofuran moiety gave higher herbicidal activities than corresponding furan analogues; (Z)-ethoxyethyl 2-cyano-3-isopropyl-3-(tetrahydrofuran-3-yl)methaneaminoacrylate showed excellent herbicidal activities against amaranth pigweed in postemergence treatment at a dose of 375 g/ha. At the same time, these cyanoacrylates had interesting plant growth regulatory activities, and some compounds stimulated radicle growth of cucumber, whereas some compounds had an inhibitory effect. These cyanoacrylates showed fungicidal activities as well.

Biodegradation of phenol by free and immobilized Acinetobacter sp. strain PD12.

A new phenol-degrading bacterium with high biodegradation activity and high tolerance of phenol, strain PD12, was isolated from the activated sludge of Tianjin Jizhuangzi Wastewater Treatment Facility in China. This strain was capable of removing 500 mg phenol/L in liquid minimal medium by 99.6% within 9 h and metabolizing phenol at concentrations up to 1100 mg/L. DNA sequencing and homologous analysis of 16S rRNA gene identified PD12 to be an Acinetobacter sp. Polyvinyl alcohol (PVA) was used as a gel matrix to immobilize Acinetobacter sp. strain PD12 by repeated freezing and thawing. The factors affecting phenol degradation of immobilized cells were investigated, and the results showed that the immobilized cells could tolerate a high phenol level and protected the bacteria against changes in temperature and pH. Storage stability and reusability tests revealed that the phenol degradation functions of immobilized cells were stable after reuse for 50 times or storing at 4 degrees C for 50 d. These results indicate that immobilized Acinetobacter sp. strain PD12 possesses a good application potential in the treatment of phenol-containing wastewater.

[Cloning and expression of catA gene from Pseudomonas putida ND6 and study on the catechol cleavage pathway].

Catechol 1,2-dioxygenase gene, calA, from naphthalene-degrading plasmid pND6-1 of Pseudomonas putida ND6, was cloned and expressed in Escherichia coli. Enzymic properties of the expressed product were investigated. The results indicated that the Km and Vmax of the enzyme are 0.019 mol/L and 1.434 mol/(min x mg), respectively. The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50 degrees C for 45 min. Fe2+ could enhance the enzyme activity by 292%. The enzyme displayed a lower activity against 4-chlorocatechol and belongs to group I of catechol 1,2-dioxygenases. When naphthalene was used as a substrate for growth of strain ND6, catechol 1, 2-dioxygenase and catechol 2,3-dioxygenase activities were both detected in their crude extract. However, when strain ND6 was grown on benzoate, rho-hydroxybenzoic acid or phenylacetic acid as a sole source of carbon the activity of catechol 1,2-dioxygenase was much higher than that of catechol 2,3-dioxygenase. These indicated that strain ND6 is able to metabolize naphthalene by catechol meta- and ortho-cleavage pathways. When benzoate, rho-hydroxybenzoic acid and phenylacetic acid were used as growth substrates, strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.

Overexpression, purification and characterization of a new salicylate hydroxylase from naphthalene-degrading Pseudomonas sp. strain ND6.

A new salicylate hydroxylase from naphthalene-degrading Pseudomonas sp. strain ND6, NahU, has been identified. The nahU is an isofunctional gene of the classic salicylate hydroxylase gene, nahG, and situated outside the transcriptional unit forming the naphthalene degradation lower pathway. Both genes, nahU and nahG of Pseudomonas sp. ND6, have been cloned and overexpressed in Escherichia coli BL21 (DE3). NahU contains 429 amino acid residues and NahG contains 434 amino acid residues. SDS-PAGE analysis showed that both NahG and NahU are about 47 kDa. Both enzymes exhibit broad substrate specificities and metabolize salicylate, sulfosalicylate, aspirin, methylsalicylate, chlorosalicylate and 3,5-dinitrosalicylate. The comparison of the Km and Vmax values for NahG and NahU demonstrated that NahU possesses a higher binding ability to salicylate and cofactors and catalytic efficiency.

Complete nucleotide sequence and organization of the naphthalene catabolic plasmid pND6-1 from Pseudomonas sp. strain ND6.

Pseudomonas sp. strain ND6, which was isolated from industrial wastewater in Tianjin, China, was capable of dissimilating naphthalene as sole carbon and energy sources. We identified one plasmid, pND6-1, which was associated with the metabolism of naphthalene and determined the complete nucleotide sequence of pND6-1 (101,858 bp) using a whole-genome-shotgun approach. Computational analyses indicated that the naphthalene metabolism of the strain ND6 is associated with this plasmid. This is the first report of a complete sequence of naphthalene catabolic plasmid. pND6-1 encodes 102 putative coding sequences (CDSs). Among them, 23 CDSs were predicted to be involved in naphthalene catabolism, 14 were predicted to be involved in transposition and integration, 2 encoded putative transporters, 3 were putative transcriptional regulators, and 9 were proteins necessary for plasmid replication and partitioning. Most of the naphthalene catabolic genes of pND6-1 have 99-100% identity in amino acid sequences homologous to their nearest counterparts found in plasmid pDTG1, NAH7 and in a chromosome region in Pseudomonas stutzeri AN10 except for two duplicated genes (ND013 and ND016). Results of this study indicated that globally distributed naphthalene catabolic genes are highly conserved among different bacterial species.

Complete nucleotide sequence of plasmid pND6-2 from Pseudomonas putida ND6 and characterization of conjugative genes.

Pseudomonas putida ND6 was shown to be capable of growth using naphthalene as sole carbon and energy sources. One plasmid from this strain, pND6-1, which was associated with the metabolism of naphthalene, was characterized; and the complete nucleotide sequence (101,858bp) and annotation of the plasmid were reported previously. In this paper, another plasmid (117,003bp) from P. putida ND6, pND6-2, was sequenced and 136 putative coding sequences (CDSs) were annotated. Among them, 9 CDSs were predicted to be involved in replication and partition of the plasmid, 32 CDSs encoded proteins associated with plasmid conjugative transfer, 2 CDSs encoded transcriptional regulators, 9 CDSs were necessary for DNA-associated function (including metabolism, recombination and repair), and 5 CDSs encoded proteins associated with other functions. The filter matting experiment indicated that pND6-2 is a helper plasmid, which could assist the naphthalene catabolic plasmid pND6-1 without any conjugative genes being transferred from P. putida ND6 to Escherichia coli AD256.

Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6.

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.

Design, synthesis, and herbicidal activities of novel 2-cyanoacrylates containing isoxazole moieties.

A series of novel 2-cyanoacrylates containing an isoxazole moiety were designed and synthesized. Their structures were characterized by (1)H NMR and elemental analysis (or high-resolution mass spectrometry). Their herbicidal activities against four species were evaluated, and the results indicated that some of the title compounds showed excellent herbicidal activities against rape and amaranth pigweed in postemergence treatment even at a dose of 75 g/ha.

[Detection and quantification of hepatitis A virus in Tianjin coastal seawater of Bohai Bay].

Hepatitis A virus (HAV) is single-strainded RNA virus that causes infectious hepatitis A. Detection and quantification of hepatitis A virus in Tianjin coastal seawater of Bohai Bay were carried out by conventional RT-PCR and SYBR Green real-time quantitative RT-PCR using the primers based on the conserved sequence at the VP1-VP2 genes of HAV. The nine samples were taken at Tianjin coastal seawater of Bohai Bay locating in the south of Tanggu, in summer, autumn and winter of 2007 and spring of 2008. For viral detection, seawater samples were concentrated either using a small ultrafiltration system (Millipore Pellicon Mini TFF) or a Centriprep-100 centrifugal ultrafiltration device (Millipore Centricon Plus-70). RT-PCR analysis showed that a 192 bp HAV cDNA was amplified from all nine seawater samples and the sequence identities of these cDNAs to the homologous sequence in the GenBank were between 95% and 100%. SYBR Green real-time quantitative RT-PCR analysis indicated that HAV concentration in these samples ranged from 5.35 x 10(6) to 4.51 x 10(7) virus particles/L.

[Isolation and identification of phenol-degrading strains and the application in biotreatment of phenol-containing wastewater].

Ten phenol-degrading bacterial strains were isolated from mixture of activated sludge and wastewater of a petroleum chemical plant. The five isolates (PD1, PD2, PD6, PD7 and PD39) were identified as Pseudomonas sp.,the four (PD4, PD5, PD8 and PD9) as Acinetobacter sp.,and the one (PD3) as Comamonas sp.by 16S rDNA sequence. Biodegradation characteristics of phenol, optimal conditions for growth, substrate range, activities of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, and biotreatment of phenol-containing wastewater of Pseudomonas sp.PD39 were investigated in detail. The results indicated that the optimal conditions for growth and degradation of strain PD39 are beginning pH of medium 7.0, growth temperature 30 degrees C, concentration of phenol 800 mg/L. PD39 was capable of metabolizing phenol at concentrations up to 1200 mg/L and removing 637 mg/L in industrial wastewater by 99.96% in 72 h. This strain possesses a good application potential as a bioaugmentation strain in the activated sludge system for treatment of phenol-containing wastewater.

Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization.

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.