Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain,Ochrobactrum sp. Strain SJY1

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.     

Trace detection of picloram using an electrochemical immunosensor based on three-dimensional gold nanoclusters

Picloram, a herbicide widely used for broadleaf weed control, is persistent and mobile in soil and water with adverse health and environmental effects. It is important to develop a sensitive method for accurate detection of trace picloram in the environment. In this article, a type of ordered three-dimensional (3D) gold (Au) nanoclusters obtained by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane is reported. Bovine serum albumin (BSA)-picloram was immobilized on the 3D Au nanoclusters by self-assembly, and then competitive immunoreaction with picloram antibody and target picloram was executed. The horseradish peroxidase (HRP)-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The electrodeposited Au nanoclusters built direct electrical contact and immobilization interface with protein molecules without postmodification and positioning. Under the optimal conditions, the linear range for picloram determination was 0.001-10 μg/ml with a correlation coefficient of 0.996. The detection and quantification limits were 5.0 × 10⁻⁴ and 0.0021 μg/ml, respectively. Picloram concentrations in peach and excess sludge supernatant extracts were tested by the proposed immunosensor, which exhibited good precision, sensitivity, selectivity, and storage stability.     

Microsatellite DNA markers for population-genetic studies of blue mackerel (Scomber australasicus) and cross-specific amplification in S. japonicus

Blue mackerel (Scomber australasicus) is targeted by large-scale purse-seiners in the western North Pacific, and its stock structure is still contentious. Herein, we described 10 polymorphic microsatellite loci for blue mackerel. The number of alleles among 32 individuals surveyed ranged from five to 27 (average of 16.2 alleles per locus). Departures from Hardy-Weinberg expectation were observed at two loci. Cross-specific amplification in the congener, S. japonicus, was successful, except for one locus, revealed to be diagnostic for these congeners. These microsatellite loci will be useful tools to address queries in population genetic structure, fishery management unit and taxonomic species status in the genus Scomber.     

<Emphasis Type="Italic">Rubellimicrobium roseum</Emphasis> sp. nov., a Gram-negative bacterium isolated from the forest soil sample

A novel pink-coloured, non-spore-forming, non-motile, Gram-negative bacterium, designated YIM 48858^T, is described by using a polyphasic approach. The strain can grow at pH 6.5–9 (optimum at pH 7) and 25–30°C (optimum at 28°C). NaCl is not required for its growth. Positive for oxidase and catalase. Urease activity, nitrate reduction, starch and Tween 80 tests are negative reaction. 16S rRNA gene sequence similarity studies showed that strain YIM 48858^T is a member of the genus Rubellimicrobium, with similarities of 96.3, 95.7 and 95.5% to Rubellimicrobium mesophilum MSL-20^T, Rubellimicrobium aerolatum 5715S-9^T and Rubellimicrobium thermophilum DSM 16684^T, respectively. Q-10 was the predominant respiratory ubiquinone as in the other members of the genus Rubellimicrobium. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphoglycolipid, glycolipid and the major fatty acids were C18:1 ω7c, C16:0 and C10:0 3-OH, which are very different from the valid published species. The DNA G + C content was 67.7 mol%. Both phylogenetic and chemotaxonomic evidence supports that YIM 48858^T is a novel species of the genus Rubellimicrobium, for which the name Rubellimicrobium roseum sp. nov. is proposed. The type strain is YIM 48858^T (=CCTCC AA 208029^T =KCTC 23202^T).     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.