A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals.     

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Δasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain χ4072, while in strain χ3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus. Received: 26 August 1998 / Accepted: 9 November 1998     

Expanding the Known Diversity and Environmental Distribution of an Uncultured Phylogenetic Division of Bacteria

Culture-independent molecular phylogenetic methods were used to explore the breadth of diversity and environmental distribution of members of the division-level “candidate” phylogenetic group WS6, recently discovered in a contaminated aquifer and with no cultivated representatives. A broad diversity of WS6-affiliated sequences were cloned from 7 of 12 environments investigated: mainly from anaerobic sediment environments. The number of sequences representing the WS6 candidate division was increased from 3 to 60 in this study. The extent of phylogenetic divergence (sequence difference) in this candidate division was found to be among the largest of any known bacterial division. This indicates that organisms representing the WS6 phylogenetic division offer a broad diversity of undiscovered biochemical and metabolic novelty. These results provide a framework for the further study of these evidently important kinds of organisms and tools, the sequences, with which to do so.     

Mercury resistance in aromatic compound degrading Pseudomonas strains

Abstract: Six of ten Pseudomonas strains selected from environmental samples for their ability to degrade aromatic compounds were found to be mercury resistant. Mercury detoxification proceeded through Hg²⁺ volatilization and the genes involved were chromosomally located. All the mercury resistant strains proved able to degrade aromatic compounds in the presence of Hg²⁺.     

Modeled Effects of Dissolved Organic Carbon and Solar Spectra on Photobleaching in Lake Ecosystems

Dissolved organic matter (DOM) contains molecules that absorb light at various wavelengths. This chromophoric DOM (CDOM) influences the transmission of both visible and ultraviolet energy through water. The absorption of light by CDOM often causes structural changes that reduce its capacity to further absorb light, a process termed ‘photobleaching‘. A model was designed to assess photobleaching through the entire water column of lake ecosystems. The model uses lake morphometry and dissolved organic carbon (DOC) concentration in conjunction with a defined solar spectrum and experimentally measured photobleaching rates to compute the total water columm photobleaching. The model was initially applied to a theoretical ‘average‘ lake using solar spectra for both the north (N) and south (S) temperate western hemispheres and variable DOC from 0.3 to 30 mg L⁻¹. The consequences of varying waveband-specific photobleaching coefficients and lake morphometry were explored in a second set of simulations. Finally, the model was also applied to four temperate northern lakes for which we had prior measurements of CDOM photobleaching rates. The model demonstrates that all three wavebands of solar radiation (UVB, UVA, and PAR) contribute significantly to total water column photobleaching, with UVA being most important. The relative contributions of the three wavebands were invariant for DOC more than 3 mg L⁻¹. Total water column photobleaching at 440 nm was three to five times faster under the UV-enriched solar spectrum of the southern hemisphere. Increasing the lake’s mean depth (from 0.37 to 9.39 m) resulted in five- or 15-fold slower rates of total water column photobleaching for DOC concentrations of 1 or 10 mg L⁻¹, respectively. Varying the waveband-specific photobleaching coefficients by 10-fold resulted in a similar change in total water column photobleaching rates. Applying the model to four specific lakes revealed that photobleaching for the entire water column would reduce CDOM light absorption by 50% in 18–44 days under summer conditions. Received 17 November 1998; accepted 27 June 2000.     

Social isolation-induced increase in the sensitivity of rats to the steroidogenic effect of ethanol

Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3alpha,5alpha-TH PROG, and 3alpha,5alpha-TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group-housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group-housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper-responsiveness of the hypothalamic-pituitary-adrenal (HPA) axis, as was apparent after reduction of GABA-mediated inhibitory tone by isoniazid administration.     

Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg²⁺, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.