Directed Evolution and In Silico Analysis of Reaction Centre Proteins Reveal Molecular Signatures of Photosynthesis Adaptation to Radiation Pressure

Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one

Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3).

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity

Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of <i>Chlamydomonas reinhardtii</i> under Extreme Environment in Space

<div><p>Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, <i>Chlamydomonas reinhardtii,</i> which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the Q_B binding pocket and in the environment surrounding Tyr-161 (Y_Z) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the Q_A⁻ state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.</p></div

Amino acid substitutions and their localization in the D1 protein of the analysed <i>C. reinhardtii</i> random mutants survived after neutrons and protons exposures.

<p>Amino acid substitutions and their localization in the D1 protein of the analysed <i>C. reinhardtii</i> random mutants survived after neutrons and protons exposures.</p

Polyphasic chlorophyll fluorescence transient under different atrazine concentrations.

<p>The fluorescence transients of reference strain IL and P162S/F211S and L200I mutants are presented as curves of the relative variable fluorescence, <i>V_t</i> = (F_t-F₀)/(F_m-F₀). Typical <i>V_t</i> curves for each strain and herbicide treatment are presented; the characteristic points <i>O-J-I-P</i> are also indicated.</p

Comparison of the <i>C. reinhardtii</i> strains culture chlorophyll and optical density during growth.

<p>The development of the reference strain, IL, and D1 random mutants is presented as a ratio of the total chlorophyll mL⁻¹ and the corresponding OD₇₅₀ mL⁻¹. The cell cultures were grown for a period of 88 h in TAP medium under continues illumination of 50 µmol m⁻² s⁻¹ at 24°C and 150 rpm agitation. Average values from four experiments are presented, ±SE, n = 4. For the sake of clarity the standard error bars of the mutants values are omitted.</p

Physiological parameters of the reference strain IL and D1 random mutants of <i>C. reinhardtii</i> selected for their PSII long-term stability under oxidative stress-related conditions.

<p>The total chlorophyll content, the maximum quantum yield of PSII photochemical reaction (F_v/F_m), the efficiency of the electron transport through PSII primary and secondary quinones (<i>1-V_J</i>) and the oxygen evolution rate (measured at 350 µmol m⁻² s⁻¹ light intensity) were determined in cell cultures in exponential growth phase (<i>OD₇₅₀</i> = 0.45±0.02). Average values are presented, ±SE, 4<<i>n</i><12; the asterisks indicate values that are not significantly different from the IL strain at p≤0.05 (Mann-Whitney U Test).</p

Limits of detection of IL and selected D1 random mutants to three different herbicides, representing the lower herbicide molar concentration inducing detectable inhibition of strains PSII electron transport efficiency.

<p>Limits of detection of IL and selected D1 random mutants to three different herbicides, representing the lower herbicide molar concentration inducing detectable inhibition of strains PSII electron transport efficiency.</p