Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites.

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M-1 cm-1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light-dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.

Proteomic Signatures of Microbial Adaptation to the Highest Ultraviolet-Irradiation on Earth: Lessons From a Soil Actinobacterium.

In the Central Andean region in South America, high-altitude ecosystems (3500-6000 masl) are distributed across Argentina, Chile, Bolivia, and Peru, in which poly-extremophilic microbes thrive under extreme environmental conditions. In particular, in the Puna region, total solar irradiation and UV incidence are the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. Then, the existence of a network of physiological and molecular mechanisms triggered by ultraviolet light exposure was hypothesized and called "UV-resistome". It includes some or all of the following subsystems: (i) UV sensing and effective response regulators, (ii) UV-avoidance and shielding strategies, (iii) damage tolerance and oxidative stress response, (iv) energy management and metabolic resetting, and (v) DNA damage repair. Genes involved in the described UV-resistome were recently described in the genome of Nesterenkonia sp. Act20, an actinobacterium which showed survival to high UV-B doses as well as efficient photorepairing capability. The aim of this work was to use a proteomic approach together with photoproduct measurements to help dissecting the molecular events involved in the adaptive response of a model High-Altitude Andean Lakes (HAAL) extremophilic actinobacterium, Nesterenkonia sp. Act20, under artificial UV-B radiation. Our results demonstrate that UV-B exposure induced over-abundance of a well-defined set of proteins while recovery treatments restored the proteomic profiles present before the UV-challenge. The proteins involved in this complex molecular network were categorized within the UV-resistome subsystems: damage tolerance and oxidative stress response, energy management and metabolic resetting, and DNA damage repair.

Photolyases and Cryptochromes in UV-resistant Bacteria from High-altitude Andean Lakes.

"High-altitude Andean Lakes" (HAAL) are pristine environments harboring poly-extremophilic microbes that show combined adaptations to physical and chemical stress such as large daily ambient thermal amplitude, extreme solar radiation levels, intense dryness, alkalinity, high concentrations of arsenic (up to 200 ppm) and dissolved salts. In this work, we compared the UV resistance profiles, pigment content and photoreactivation abilities of three UV-resistant bacteria isolated from distinct niches from HAALs, that is Acinetobacter sp. Ver3 (water, Lake Verde; 4400 m), Exiguobacterium sp. S17 (stromatolite, Lake Socompa, 3570 m) and Nesterenkonia sp. Act20 (soil, Lake Socompa, 3570 m). UV resistance ability of HAAL's strains indicate a clear adaptation to high radiation exposure encountered in their original habitat, which can be explained by genetic and physiological mechanisms named as the UV-resistome. Thus, the UV-resistome depends on the expression of a diverse set of genes devoted to evading or repairing the damage it provoked direct or indirectly. As pigment extraction and photoreactive assays indicate the presence of photoactive molecules, we characterized more in detail proteins with homology to photolyases/cryptochromes members (CPF). Phylogenetic analyses, sequence comparison and 3D modeling with bona fide CPF members were used to prove the presence of functional domains and key residues in the novel proteins.

Arsenic metabolism in high altitude modern stromatolites revealed by metagenomic analysis.

Modern stromatolites thrive only in selected locations in the world. Socompa Lake, located in the Andean plateau at 3570 masl, is one of the numerous extreme Andean microbial ecosystems described over recent years. Extreme environmental conditions include hypersalinity, high UV incidence, and high arsenic content, among others. After Socompa's stromatolite microbial communities were analysed by metagenomic DNA sequencing, taxonomic classification showed dominance of Proteobacteria, Bacteroidetes and Firmicutes, and a remarkably high number of unclassified sequences. A functional analysis indicated that carbon fixation might occur not only by the Calvin-Benson cycle, but also through alternative pathways such as the reverse TCA cycle, and the reductive acetyl-CoA pathway. Deltaproteobacteria were involved both in sulfate reduction and nitrogen fixation. Significant differences were found when comparing the Socompa stromatolite metagenome to the Shark Bay (Australia) smooth mat metagenome: namely, those involving stress related processes, particularly, arsenic resistance. An in-depth analysis revealed a surprisingly diverse metabolism comprising all known types of As resistance and energy generating pathways. While the ars operon was the main mechanism, an important abundance of arsM genes was observed in selected phyla. The data resulting from this work will prove a cornerstone for further studies on this rare microbial community.

Functional Green-Tuned Proteorhodopsin from Modern Stromatolites.

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 µs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 µs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.

Forged Under the Sun: Life and Art of Extremophiles from Andean Lakes.

High-altitude Andean lakes (HAAL) are a treasure chest for microbiological research in South America. Their indigenous microbial communities are exposed to extremely high UV irradiation and to multiple chemical extremes (Arsenic, high salt content, alkalinity). Microbes are found both, free-living or associated into microbial mats with different degrees of mineralization and lithification, including unique modern stromatolites located at 3570 m above sea level. Characterization of these polyextremophilic microbes began only recently, employing morphological and phylogenetic methods as well as high-throughput sequencing and proteomics approach. Aside from providing a general overview on microbial communities, special attention is given to various survival strategies; HAAL's microbes present a complex system of shared genetic and physiological mechanisms (UV-resistome) based on UV photoreceptors and stress sensors with their corresponding response regulators, UV avoidance and protection strategies, damage tolerance and UV damage repair. Molecular information will be provided for what is, so far the most studied HAAL molecule, a CPD-Class I photolyase from Acinetobacter Ver3 (Laguna Verde, 4400 m). This work further proposes some strategies that make an appeal for the preservation of HAAL, a highly fragile environment that offers promising and ample research possibilities.

Functional Characterization of a LOV-Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri.

The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time-resolved fluorescence allowed determination of quantum yields for triplet (ΦT  = 0.68 ± 0.03) and photoproduct formation (Φ390  = 0.46 ± 0.05). The lifetime for triplet decay was determined as τT  = 2.4-2.8 µs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light-to-dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven-fold longer lifetime of the triplet state (τT  = 16-18.5 µs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc-LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back-conversion into the dark state was accompanied by a volume expansion. A radioactivity-based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long-lived lit state.

First characterisation of a CPD-class I photolyase from a UV-resistant extremophile isolated from High-Altitude Andean Lakes.

UV-resistant Acinetobacter sp. Ver3 isolated from High-Altitude Andean Lakes (HAAL) in Argentinean Puna, one of the highest UV exposed ecosystems on Earth, showed efficient DNA photorepairing ability, coupled to highly efficient antioxidant enzyme activities in response to UV-B stress. We herein present the cloning, expression, and functional characterization of a cyclobutane pyrimidine dimer (CPD)-class I photolyase (Ver3Phr) from this extremophile to prove its involvement in the previously noted survival capability. Spectroscopy of the overexpressed and purified protein identified flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate (MTHF) as chromophore and antenna molecules, respectively. All functional analyses were performed in parallel with the ortholog E. coli photolyase. Whereas the E. coli enzyme showed the FAD chromophore as a mixture of oxidised and reduced states, the Ver3 chromophore always remained partly (including the semiquinone state) or fully reduced under all experimental conditions tested. Functional complementation of Ver3Phr in Phr(-)-RecA E. coli strains was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) detection. The results identified strong photoreactivation ability in vivo of Ver3Phr while its nonphotoreactivation function, probably related with the stimulation of nucleotide excision repair (NER), was not as manifest as for EcPhr. Whether this is a question of the approach using an exogenous photolyase incorporated in a non-genuine host or a fundamental different behaviour of a novel enzyme from an exotic environment will need further studies.

LOV-domain photoreceptor, encoded in a genomic island, attenuates the virulence of Pseudomonas syringae in light-exposed Arabidopsis leaves.

In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain-regulated two-component system (Pst-Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst-Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst-Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst-Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light-dependent manner. We propose that the function of Pst-Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens.

Extremophilic Acinetobacter strains from high-altitude lakes in Argentinean Puna: remarkable UV-B resistance and efficient DNA damage repair.

High-Altitude Andean Lakes (HAAL) of the South American Andes are almost unexplored ecosystems of shallow lakes. The HAAL are recognized by a remarkably high UV exposure, strong changes in temperature and salinity, and a high content of toxic elements, especially arsenic. Being exposed to remarkably extreme conditions, they have been classified as model systems for the study of life on other planets. Particularly, Acinetobacter strains isolated from the HAAL were studied for their survival competence under strong UV-B irradiation. Clinical isolates, Acinetobacter baumannii and Acinetobacter johnsonii, served as reference material. Whereas the reference strains rapidly lost viability under UV-B irradiation, most HAAL-derived strains readily survived this exposure and showed less change in cell number after the treatment. Controls for DNA repair activity, comparing dark repair (DR) or photo repair (PR), gave evidence for the involvement of photolyases in the DNA repair. Comparative measurements by HPLC-mass spectrometry detected the number of photoproducts: bipyrimidine dimers under both PR and DR treatments were more efficiently repaired in the HAAL strains (up to 85 % PR and 38 % DR) than in the controls (31 % PR and zero DR ability). Analysis of cosmid-cloned total genomic DNA from the most effective DNA-photorepair strain (Ver3) yielded a gene (HQ443199) encoding a protein with clear photolyase signatures belonging to class I CPD-photolyases. Despite the relatively low sequence similarity of 41 % between the enzymes from Ver3 and from E. coli (PDB 1DNPA), a model-building approach revealed a high structural homology to the CPD-photolyase of E. coli.