Rhizosphere microflora of plants used for the phytoremediation of bitumen-contaminated soil

The microbial communities and their degradative potential in rhizospheres of alfalfa (Medicago sativa) and reed (Phragmites australis) and in unplanted soil in response to bitumen contamination of soil were studied in pot experiments. According to the results of fluorescence microscopy, over a period of 27 months, bitumen contamination of soil reduced the total number of microorganisms more significantly (by 75%) in unplanted than in rhizosphere soil (by 42% and 7% for reed and alfalfa, respectively) and had various effects on some important physiological groups of microorganisms such as actinomycetes as well as nitrogen-fixing, nitrifying, denitrifying, ammonifying, phosphate-solubilizing, sulphur-oxidizing, cellulolytic and hydrocarbon-degrading microorganisms. The changes in the physiological structure of the microbial community under bitumen contamination were found to hinge on not merely the presence of plants but also their type. It was noted that the rhizosphere microflora of alfalfa was less inhibited by hydrocarbon pollution and had a higher degradative potential than the rhizosphere microflora of reed.     

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.     

Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.     

Implementation of the main chain directed assignment strategy. Computer assisted approach.

A computer-assisted procedure has been developed to apply the main chain directed (MCD) assignment strategy to the analysis of 1H NMR spectra of proteins. The underlying mathematical foundation of this procedure, termed MCDPAT, is presented. MCDPAT is based upon the expanded library of MCD patterns defined previously (A.J. Wand and S.J. Nelson. 1991. Biophys. J. 59:1101-1112), and has been evaluated with both simulated and experimental data from the protein ubiquitin. The influence of the precision, spectral variation, and inherent degeneracy upon the design of the procedure is explored. Several approaches have been taken to overcome the uncertainty introduced by these variables. These include a hierarchical approach to both primary pattern recognition and subsequent construction of MCD-defined units of secondary structure. It is shown that the MCDPAT procedure, in conjunction with automated statistically based spectral analysis, leads to the successful MCD assignment of the protein ubiquitin. The implications and limitations of this approach are discussed.     

Proton resonance assignments of horse ferricytochrome c

Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) was used to obtain extensive resonance assignments in the 1H NMR spectrum of horse ferricytochrome c. Assignments were made for the main-chain and C beta protons of 102 residues (all except Pro-44 and Gly-84) and the majority of side-chain protons. As starting points for the assignment of the oxidized protein, a limited set of protons was initially assigned by use of 2D NMR magnetization transfer methods to correlate resonances in the oxidized form with assigned resonances in the reduced form [Wand, A. J., Di Stefano, D. L., Feng, Y., Roder, H., & Englander, S. W. (1989) Biochemistry (preceding paper in this issue)]. Given the complexity of the spectrum due to the size of this protein (104 residues) and its paramagnetic center, the initial search for side-chain spin systems in J-correlated spectra was successful only for the simplest side chains, but the majority of NH-C alpha H-C beta H subspin systems (NAB sets) could be identified at this stage. The subsequent search for sequential NOE connectivities focused on NAB sets, with use of previously assigned residues to place NOE-connected segments within the amino acid sequence. Selective proton labeling of either the slowly or the rapidly exchanging amide sites was used to simplify the spectra, and systematic work at two temperatures was used to resolve ambiguities in the 2D NMR spectra. These approaches, together with the use of magnetization transfer methods to correlate reduced and oxidized cytochrome c spectra, provide multiple cross-checks to verify assignments.     

Determinants of protein hydrogen exchange studied in equine cytochrome c.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.     

移植的5—羟色胺能神经元跨软脊膜迁入大鼠脊髓的研究

本文对移植的5-HT神经元从蛛网膜下腔跨软脊膜迁移进入脊髓作了初步研究。将含有5-HT细胞的胚胎中缝核组织小块或神经细胞悬浮液作为移植物,以5-HT免疫组织化学方法跟踪移植细胞,结果如下:(1)在低胸水平横切脊髓,10d后,横断脊髓内的5-HT纤维消失。(2)横切脊髓(方法同上)后,立即将中缝核组织小块移植在胸腰段脊髓的蛛网膜下腔,一月后.在横断脊髓内出现5-HT阳性神经元和纤维。5-HT纤维能在灰白质内延伸。(3)脊髓横断后,若以中缝核的细胞悬浮液代替组织小块,作上述移植,则在移植区附近的灰质内出现大量的5-HT阳性神经元。这些神经元在灰质内的分布范围与神经细胞悬浮液在蛛网膜下腔的移植范围相一致。迁入神经元能在灰质内重新形成5-HT阳性纤维网。(4)经上述移植后,灰质内出现的5-HT阳性纤维随远离细胞体而变得稀疏。白质内的5-HT阳性纤维远比灰质内稀少。本实验结果表明:移植在脊髓蛛网膜下腔的脑干5-HT细胞能跨软脊膜迁移进入脊髓。     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.