Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.     

Hyperhydration induced by glycerol ingestion: hormonal and renal responses

The simultaneous time courses of hydromineral hormones (renin-aldosterone system, arginine vasopressin, and atrial natriuretic peptide) and renal responses were examined during and after the completion of hyperhydration induced by glycerol and fluid ingestion. Eight healthy young male Caucasian subjects participated in two separate trials, each including three consecutive phases in a thermoneutral environment. Phases 1 and 3 involved a 90-min period at rest, while phase 2 involved a 120-min period at rest designed to provide either (i) euhydration (control trial) or (ii) hyperhydration induced by ingestion of glycerol (1.1 g/kg body mass) and fluid (21.4 mL/kg body mass). During the 2-h time period of glycerol and fluid ingestion, urine flow, urine osmolality, and plasma levels of hydromineral hormones remained at basal values. In contrast, after hyperhydration completion during phase 3, the diuresis increased markedly together with a dilution of the urine (p < 0.05) while hormonal responses did not change. These results indicate significant differences in renal responses during and after hyperhydration completion and suggest that these changes are independent of fluid-regulating hormonal responses.     

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.     

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates

Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed. A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.     

Linear dichroism of membrane-bound reaction centers from Rhodobacter sphaeroides: Alterations of the B band induced by site-specific mutations

Low temperature absorption and linear dichroism (LD) measurements were performed on oriented membranes containing wild type Rhodobacter sphaeroides reaction centers, a mutant reaction center with the change Phe M197 to Arg (FM197R), and a double mutant reaction center where, in addition, Gly M203 was replaced by Asp (FM197R/GM203D). The monomeric bacteriochlorophyll band (B), which is highly congested in the wild type reaction center, was separated into two bands in the mutant reaction centers peaking 10 nm (single mutant) or 15 nm (double mutant) apart. This separation arose principally from changes in the interaction of the protein with the L-side monomer bacteriochlorophyll B_L.The ability to separate the B bands is extremely useful in spectroscopic studies. The orientations of the two monomer-type transitions contributing to the B band were similar in all three reaction centres studied, and were asymmetric with respect to the orientation axis, with the transition mostly associated with B_L making a smaller angle with the C₂ axis. Differences in the LD observed in wild type membrane-bound or isolated reaction centers can be ascribed either to differences in shifts of the B transitions or to differences in the orientation axis.     

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.     

Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.     

Effects of habitat fragmentation and road density on the distribution pattern of the moor frog Rana arvalis

1. The effects of habitat fragmentation on the distribution pattern of the moor frog Rana arvalis were investigated. Also, the possible isolation effects of the road network were taken into account.
2. Indications were found that habitat fragmentation partly explains the distribution pattern of the moor frog. The statistical models showed a positive effect of pond size (or marsh area) and a negative effect of road density on the probability of occupation of a moorland pond.
3. Because of the strong correlation between habitat quality variables and isolation variables, no unambiguous effects of isolation, described as the amount of suitable terrestrial habitat (moorland) in the surroundings of a moorland pond in a radius of 100–2000 m, could be demonstrated.
4. Spatial differences in road density can play a role in the selection of optimal locations for nature protection areas. The regression model used in this study predicts a reduced occupation probability in 55% of the study area. In the part of the study area adjacent to a motorway, occupation probability is lowered to less than 30%.
5. European studies of habitat fragmentation on amphibian species revealed a mean distance between occupied ponds of <1 km in all studies. This could be a general rule of thumb for persistent amphibian populations. Effects of pond size on the probability of occupation were more variable.
6. When discussing the effects of habitat fragmentation on amphibians and other ground dwelling species, the negative effects of roads are often underestimated.     

Response of selected antioxidants and pigments in tissues of Rosa hybrida and Fuchsia hybrida to supplemental UV-A exposure

The effect of supplemental UV-A (320–400 nm) radiation on tissue absorption at 355 nm, levels of various antioxidants (ascorbate, glutathione, carotenoids and flavonoids) and of antioxidant scavenging capacity were investigated with leaves and petals of Rosa hybrida , cv. Honesty and with leaves, petals and sepals of Fuchsia hybrida , cv. Dollarprinzessin. Supplemental UV-A did not result in visible changes in plant morphology of either species. In leaves it induced small increases in levels of chlorophylls a and b , the carotenoids antheraxanthin, lutein and β-carotene, and high increases in the flavonols quercetin and kaempferol. Petals hardly responded, while the coloured sepals of fuchsia showed an increase in quercetin derivatives. HPLC of unhydrolysed flavonoids showed that individual quercetin derivatives in leaves of both species and kaempferol derivatives in rose leaves increased 2-fold. Some kaempferol derivatives in fuchsia leaves were more than 2-fold enhanced or were newly induced by supplemental UV-A. Increases in l-ascorbic acid levels in fuchsia leaves, and decreases in rose leaves as result of supplemental UV-A were observed, but differences appeared statistically not significant, while l-ascorbate levels remained unchanged in the other tissues investigated. Anthocyanins and reduced glutathione levels were unaffected in all tissues. The combined UV-A induced increases in concentrations of these antioxidant species, did not lead to significant increases in antioxidant capacity of tissues, measured as Trolox equivalents in 50%-ethanol extracts. Light absorption at 355 nm of leaf extracts was significantly increased upon UV-A exposure. Our results indicate that the major protection towards UV-A exposure, in particular in the leaves, will originate from absorption of irradiation, and not from scavenging reactive oxygen species.