Characterization of BLUF-photoreceptors present in Acinetobacter nosocomialis.

Acinetobacter nosocomialis is a Gram-negative opportunistic pathogen, whose ability to cause disease in humans is well recognized. Blue light has been shown to modulate important physiological traits related to persistence and virulence in this microorganism. In this work, we characterized the three Blue Light sensing Using FAD (BLUF) domain-containing proteins encoded in the A. nosocomialis genome, which account for the only canonical light sensors present in this microorganism. By focusing on a light-modulated bacterial process such as motility, the temperature dependence of light regulation was studied, as well as the expression pattern and spectroscopic characteristics of the different A. nosocomialis BLUFs. Our results show that the BLUF-containing proteins AnBLUF65 and AnBLUF46 encode active photoreceptors in the light-regulatory temperature range when expressed recombinantly. In fact, AnBLUF65 is an active photoreceptor in the temperature range from 15°C to 37°C, while AnBLUF46 between 15°C to 32°C, in vitro. In vivo, only the Acinetobacter baumannii BlsA's ortholog AnBLUF65 was expressed in A. nosocomialis cells recovered from motility plates. Moreover, complementation assays showed that AnBLUF65 is able to mediate light regulation of motility in A. baumannii ΔblsA strain at 30°C, confirming its role as photoreceptor and in modulation of motility by light. Intra-protein interactions analyzed using 3D models built based on A. baumannii´s BlsA photoreceptor, show that hydrophobic/aromatic intra-protein interactions may contribute to the stability of dark/light- adapted states of the studied proteins, reinforcing the previous notion on the importance of these interactions in BLUF photoreceptors. Overall, the results presented here reveal the presence of BLUF photoreceptors in A. nosocomialis with idiosyncratic characteristics respect to the previously characterized A. baumannii's BlsA, both regarding the photoactivity temperature-dependency as well as expression patterns, contributing thus to broaden our knowledge on the BLUF family.

BlsA Is a Low to Moderate Temperature Blue Light Photoreceptor in the Human Pathogen Acinetobacter baumannii.

Light is an environmental signal that produces extensive effects on the physiology of the human pathogen Acinetobacter baumannii. Many of the bacterial responses to light depend on BlsA, a bluelight using FAD (BLUF)-type photoreceptor, which also integrates temperature signals. In this work, we disclose novel mechanistic aspects of the function of BlsA. First, we show that light modulation of motility occurs only at temperatures lower than 24°C, a phenotype depending on BlsA. Second, blsA transcript levels were significantly reduced at temperatures higher than 25°C, in agreement with BlsA protein levels in the cell which were undetectable at 26°C and higher temperatures. Also, quantum yield of photo-activation of BlsA (lBlsA) between 14 and 37°C, showed that BlsA photoactivity is greatly compromised at 25°C and absent above 28°C. Fluorescence emission and anisotropy of the cofactor together with the intrinsic protein fluorescence studies suggest that the FAD binding site is more susceptible to structural changes caused by increments in temperature than other regions of the protein. Moreover, BlsA itself gains structural instability and strongly aggregates at temperatures above 30°C. Overall, BlsA is a low to moderate temperature photoreceptor, whose functioning is highly regulated in the cell, with control points at expression of the cognate gene as well as photoactivity.

Through the eyes of a pathogen: light perception and signal transduction in Acinetobacter baumannii.

Sunlight is a ubiquitous environmental stimulus for the great majority of living organisms on Earth; therefore it is logical to expect the development of "seeing mechanisms" which lead them to successfully adapt to particular ecological niches. Although these mechanisms were recognized in photosynthetic organisms, it was not until recent years that the scientific community found out about light perception in chemotrophic ones. In this review we summarize the current knowledge about the mechanism of light sensing through the blue light receptor BlsA in Acinetobacter baumannii. We highlight its function as a global regulator that pleiotropically modulates a large number of physiological processes, many of which are linked to the ability of this opportunist pathogen to persist in adverse intrahospital environments. Moreover, we describe with some specific examples the molecular basis of how this photoregulator senses blue light and translates this physical signal by modulating gene expression of target regulons. Finally, we discuss the possible course of these investigations needed to dissect this complex regulatory network, which ultimately will help us better understand the A. baumannii physiology.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor.

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20 ± 0.03 at 15°C < T < 25°C, but is practically inoperative at T > 30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A. baumannii.

Enhancement of photophysical and photosensitizing properties of flavin adenine dinucleotide by mutagenesis of the C-terminal extension of a bacterial flavodoxin reductase.

The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.

Combined mutagenesis and kinetics characterization of the bilin-binding GAF domain of the protein Slr1393 from the Cyanobacterium Synechocystis PCC6803.

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 µs, 390 µs, and 1.5 ms) for the red-to-green conversion, and 1.2 µs, 340 µs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.

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.

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2.

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

Hydrogen-bonding modulation of excited-state properties of flavins in a model of aqueous confined environment.

The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.