Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.

Proteorhodopsin (PR) is an outward light-driven proton pump observed in marine eubacteria. Despite many structural and functional similarities to bacteriorhodopsin (BR) in archaea, which also acts as an outward proton pump, the mechanism of the photoinduced proton release and uptake is different between two H(+)-pumps. In this study, we investigated the pH dependence of the photocycle and proton transfer in PR reconstituted with the phospholipid membrane under alkaline conditions. Under these conditions, as the medium pH increased, a blue-shifted photoproduct (defined as Ma), which is different from M, with a pKa of ca. 9.2 was produced. The sequence of the photoinduced proton uptake and release during the photocycle was inverted with the increase in pH. A pKa value of ca. 9.5 was estimated for this inversion and was in good agreement with the pKa value of the formation of Ma (∼ 9.2). In addition, we measured the photoelectric current generated by PRs attached to a thin polymer film at varying pH. Interestingly, increases in the medium pH evoked bidirectional photocurrents, which may imply a possible reversal of the direction of the proton movement at alkaline pH. On the basis of these findings, a putative photocycle and proton transfer scheme in PR under alkaline pH conditions was proposed.

[Diversity and Physiological and Biochemical Properties of Heterotrophic Bacteria. Isolated from Lake Baikal Neuston.]

For heterotrophic microorganisms (44 strains) isolated-from the surface film of Lake Baikal, iden- tification was carried out and their. physiological and biochemical characteristics were determined. Com- pared to the water column, diversity of cultured heterotrophs was low, indicating formation of stable micro- bial communities at the air-water interphase interface. Heterotrophic bacteria isolated from the surface mi- crolayer exhibited the enzymatic activity comparable to that for strains form other biofilm associations. Deinococcusfi6us strain NA202 'vas the most active component of the community, capable of utilization of the broadest spectrum of mono- and disaccharides,'sugars, and amino acids. This strain possessed the highest diversity of extracellular enzymes and was the most resistant to UV radiation. The physiological and bio- chemical properties of this strain may-be responsible for its adaptation to survival in extreme conditions of the surface microlayer. Our results improve our understanding of occurrence of UV-resistant strains in freshwater ecosystems.

Growth of Dunaliella tertiolecta and associated bacteria in photobioreactors.

The aim of this study was to test three flat-plate photobioreactor configurations for cultivation of marine green alga Dunaliella tertiolecta under non-axenic growth conditions and to characterize and quantify the associated bacteria. The photobioreactor cultivations were conducted using tap water-based media. Static mixers intended to enhance mixing and light utilization did not generally increase algal growth at the low light intensities used. The maximum biomass concentration (measured as volatile suspended solids) and maximum specific growth rate achieved in the flat plate with no mixer were 2.9 g l⁻¹ and 1.3 day⁻¹, respectively. Based on quantitative polymerase chain reaction, bacterial growth followed the growth of D. tertiolecta. Based on 16S rDNA amplification and denaturing gradient gel electrophoresis profiling, heterotrophic bacteria in the D. tertiolecta cultures mainly originated from the non-axenic algal inocula, and tap water heterotrophs were not enriched in high chloride media (3 % salinity). Bacterial communities were relatively stable and reproducible in all flat-plate cultivations and were dominated by Gammaproteobacteria, Flavobacteria, and Alphaproteobacteria.

Occurrence of resistance to antibiotics, UV-B, and arsenic in bacteria isolated from extreme environments in high-altitude (above 4400 m) Andean wetlands.

High-altitude Andean wetlands are pristine environments with extreme conditions such as high UV radiation, high heavy metal content (mainly arsenic), high salinity, and oligotrophy. In this paper, the UV-B resistance and tolerance to arsenic of phylogenetically characterized bacteria (Actinobacteria [six isolates], Firmicutes [four isolates], and gamma-Proteobacteria [three isolates]) isolated from Laguna Vilama (4400-m altitude) and Laguna Azul (4560 m) were determined. In addition, given that multiple antibiotic resistances were also determined, a relationship between antibiotic resistances as a consequence of mutagenic ability or in relation to metal resistance is proposed. High UV-B resistances were found, since after 30 min (0.7 KJ m(-2)) and 60 min (1.4 KJ m(-2)) of irradiation, most of the studied bacteria did not show a decreased survival; what is more, many of them had an improved survival with the increased doses. Augmentations in mutagenesis rates were observed after UV-B irradiation in only 4 of the 13 tested isolates. Arsenite tolerance was also established in 8 of the 13 tested strains: Staphylococcus saprophyticus A3 and Micrococcus sp. A7, which were able to grow in media containing up to 10 mM As(III). Finally, predominance of antibiotic resistances (azithromycin, erythromycin, clarithromycin, roxithromycin, streptomycin, chloramphenicol, gentamycin, kanamycin, tetracycline, and ampicillin) was found, in all the isolated strains from both wetlands, with unexpectedly high minimal inhibitory concentrations (MICs; >2 mg mL(-1)) for macrolides. These results demonstrate that in extreme environments like high-altitude wetlands there is a correlation of multiresistances to UV-B radiation and arsenic, and that antibiotic resistances are also widespread in these pristine environments, where antibiotic selective pressure is supposed to be absent.

Microbial oxidation of CH(4) at different temperatures in landfill cover soils.

Biological oxidation of CH(4) is an important constraint on the emission of this gas from areas, such as landfills to the atmosphere. We studied the effect of temperature on methanotrophic bacteria in three different landfill cover soils, incubated in the laboratory. In samples of a young cover, consisting of wood chips and sewage sludge, the phospholipid fatty acids (PLFAs), regarded as biomarkers for type I methanotrophs (16:1omega5t, 16:1omega6c, 16:1omega8c), primarily increased at low temperatures (5-10 degrees C). On the other hand, the PLFA marker for type II methanotrophs (18:1omega8c) was highly elevated only at 20 degrees C. These results suggest that temperature can determine the selection of methanotroph populations.

Engineering photocycle dynamics. Crystal structures and kinetics of three photoactive yellow protein hinge-bending mutants.

Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues.