FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli

The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits. We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB. In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory. This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer. The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.     

Characterization of the endoevaporitic microbial communities in a hypersaline gypsum crust by fatty acid analysis

We have used fatty acid analyses to study the community structure of a layered endoevaporitic microbial community within a gypsum crust that covers the bottom of a saltern evaporation pond in Eilat, Israel. This community, living at a salinity of 218–238 g l⁻¹ total dissolved salts, consists of an upper brown layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, a red-purple layer with both Chromatium and Ectothiorhodospira/Halorhodospira type of purple sulfur bacteria, and a black layer in which dissimilatory sulfate reduction occurs. An olive-green layer is sometimes present below the red-purple layer. Analysis by gas chromatography/mass spectrometry of the fatty acid methyl esters prepared from the different fractions showed characteristic patterns in each layer, and these could be related to fatty acid composition data from the literature and to fatty acid analyses of representative halophilic microorganisms isolated from the site. The nature of the fatty acids in the green layer suggests that the cyanobacteria present there use the oxygen-independent pathway for production of unsaturated fatty acids, a pathway only occasionally encountered in filamentous cyanobacteria. The facultative anaerobic nature of the cyanobacteria in the green layer was confirmed by their ability to perform anoxygenic photosynthesis with sulfide as electron donor. Specific signature fatty acids identified for each layer corresponded well with the microscopic and functional analysis of the biota present. Guest Editor: John M. Melack Saline Waters and their Biota     

Validated Postbiotic Screening Confirms Presence of Physiologically-Active Metabolites,Such as Short-Chain Fatty Acids,Amino Acids and Vitamins in Hylak® Forte

Probiotics and Antimicrobial Proteins - Hylak® forte is a postbiotic that inhibits the growth of pathogenic bacteria by reducing intestinal pH. It is assumed the potential presence of...     

F0 part of the ATP synthase from Escherichia coli. Influence of subunits a, and b, on the structure of subunit c

Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b. Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding [Schneider, E. and Altendorf, K. (1985) EMBO J. 4, 515-518]. The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence. In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID-labelled membrane vesicles of Escherichia coli [Hoppe et al. (1984) Biochemistry 23, 5610-5616]. In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified. The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits.     

Microbial composition of biofilms in a brewery investigated by fatty acid analysis,fluorescence in situ hybridisation and isolation techniques

Biofilms associated with brewery plants can harbour spoiling microorganisms that potentially damage the final product. Most beer-spoiling microorganisms are thought to depend on numerous interactions with the accompanying microbiota. However, there is no information on the microbial community structure of biofilms from bottling plants. The conveyors that transport the bottles to and from the plant are known as potential sources of microbial contamination of beer. Consequently, the material buildup from two conveyors was analysed using a cultivation/isolation approach, and the culture-independent techniques of whole cell fatty acid analysis and fluorescence in situ hybridisation (FISH). Heterogeneous communities were present at both conveyors. Although characteristic fatty acids for Eukarya were present, FISH-signals for Eukarya were extremely low. The Proteobacteria, in particular the Gammaproteobacteria, were abundant at both sample sites. Bacterial isolates were obtained for every dominating group detected by FISH: the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, the Xanthomonadaceae, the Actinobacteria, the Bacteroidetes and the Firmicutes.     

Cutaneous sensitivity to touch and low-frequency vibration in selachians

In 46 isolated and 4in situ preparations of perioral skin, afferent impulse patterns, leading from cutaneous mechanoreceptors and responding to orthogonal square as well as sine-wave stimuli, were investigated in selachians (Scyliorhinus canicula).

Community analysis of biofilters using fluorescence in situ hybridization including a new probe for the Xanthomonas branch of the class Proteobacteria.

Domain-, class-, and subclass-specific rRNA-targeted probes were applied to investigate the microbial communities of three industrial and three laboratory-scale biofilters. The set of probes also included a new probe (named XAN818) specific for the Xanthomonas branch of the class Proteobacteria; this probe is described in this study. The members of the Xanthomonas branch do not hybridize with previously developed rRNA-targeted oligonucleotide probes for the alpha-, beta-, and gamma-Proteobacteria. Bacteria of the Xanthomonas branch accounted for up to 4.5% of total direct counts obtained with 4',6-diamidino-2-phenylindole. In biofilter samples, the relative abundance of these bacteria was similar to that of the gamma-Proteobacteria. Actinobacteria (gram-positive bacteria with a high G+C DNA content) and alpha-Proteobacteria were the most dominant groups. Detection rates obtained with probe EUB338 varied between about 40 and 70%. For samples with high contents of gram-positive bacteria, these percentages were substantially improved when the calculations were corrected for the reduced permeability of gram-positive bacteria when formaldehyde was used as a fixative. The set of applied bacterial class- and subclass-specific probes yielded, on average, 58.5% (+/- a standard deviation of 23.0%) of the corrected eubacterial detection rates, thus indicating the necessity of additional probes for studies of biofilter communities. The Xanthomonas-specific probe presented here may serve as an efficient tool for identifying potential phytopathogens. In situ hybridization proved to be a practical tool for microbiological studies of biofiltration systems.     

Cross-Sectional Comparison of Small Animal [18F]-Florbetaben Amyloid-PET between Transgenic AD Mouse Models

We aimed to compare [¹⁸F]-florbetaben PET imaging in four transgenic mouse strains modelling Alzheimer’s disease (AD), with the main focus on APPswe/PS2 mice and C57Bl/6 mice serving as controls (WT). A consistent PET protocol (N = 82 PET scans) was used, with cortical standardized uptake value ratio (SUVR) relative to cerebellum as the endpoint. We correlated methoxy-X04 staining of β-amyloid with PET results, and undertook ex vivo autoradiography for further validation of a partial volume effect correction (PVEC) of PET data. The SUVR in APPswe/PS2 increased from 0.95±0.04 at five months (N = 5) and 1.04±0.03 (p<0.05) at eight months (N = 7) to 1.07±0.04 (p<0.005) at ten months (N = 6), 1.28±0.06 (p<0.001) at 16 months (N = 6) and 1.39±0.09 (p<0.001) at 19 months (N = 6). SUVR was 0.95±0.03 in WT mice of all ages (N = 22). In APPswe/PS1G384A mice, the SUVR was 0.93/0.98 at five months (N = 2) and 1.11 at 16 months (N = 1). In APPswe/PS1dE9 mice, the SUVR declined from 0.96/0.96 at 12 months (N = 2) to 0.91/0.92 at 24 months (N = 2), due to β-amyloid plaques in cerebellum. PVEC reduced the discrepancy between SUVR-PET and autoradiography from −22% to +2% and increased the differences between young and aged transgenic animals. SUVR and plaque load correlated highly between strains for uncorrected (R = 0.94, p<0.001) and PVE-corrected (R = 0.95, p<0.001) data. We find that APPswe/PS2 mice may be optimal for longitudinal amyloid-PET monitoring in planned interventions studies.