Disentangling mechanisms involved in the adaptation of the cyanobacterium Microcystis aeruginosa to the extreme sulphureous water from Los Baños de la Hedionda (S Spain)

Ma1Vc Set 1 Set 2 No. of replicate cultures: 90 40 No. of cultures with cells in the rank: 0 73 0 1 - 104 1 0 104-105 1 0 105-106 6 0 > 106 9 40 CV of the no. of LHWresistant cells per culture 2.351 0.214 One-tailed Z-test for comparison of CVs 10.891, p<0.0001 (significant) Adaptation process Genetic μ (mutants cell-1 generation-1) 2.1 × 10-7 Mutation rate (μ) and mutationselection equilibrium (q): The mutation rate, μ was calculated as: μ = (-loge Po)/(Nt-No); where N0 and Nt are the number of cells at the start and at the end of the propagation period (i.e. before the addition LHW), respectively (see Fig. 1). The parameter P0 was computed as the proportion of Set 1 cultures showing no mutant cells after LHW exposure. On the other hand, q, the mutationselection equilibrium; q= μ/(μ-s) is the frequency of the LHW-resistant allele, and s is the coefficient of selection against this resistant allele, calculated as follows: s= 1- (mr / ms), where mr and ms are the growth rates on non-selective medium of resistant and sensible strains, respectively. As resistant strain, a 106 cells culture from Set 1 was isolated and maintained in the culture collection. Culture maintenance and sulphide management: Ma1Vc strain was grown in 100-mL cell culture flasks, with 20 mL of BG-11 medium, under continuous irradiance of 50 μmol m−2 s−1, at 20 °C. To measure the Lethal Dosis (LD), sulphur was added every 24 hours from a Na2S-aqueous NaOH master stock solution(pH ~13, with concentration between 19-23 mM). . HEPES 20 mM was used to maintain the culture at pH 7. At this pH, sulfur (S) are in the forms SH- (50%) and H2S (50%) . Fig. 1 Growing rate (m) of wild-type LHW-sensitive and LHW-resistant strains under non selective (BG11 medium) and selective (100 µM S) Data are mean ± SD (n= 4). Table 1: FLUCTUATION ANALYSIS of LHW-sensitivity to LHW-resistance transformation in the strain Ma1Vc of the cyanobacteria M. aeruginosa Fig. 2 Growing rate (m) as a function of S concentration on LHW-sensitive and LHW-resistant strain of M. aeruginosa Ma1Vc. Data are mean ± SD (n= 4). LD is indicated on the graph. Fluctuation analysis (FA): Two sets were used under the FA (Fig. 1). The fist one (Set 1) started with 90 flasks, inoculated with 103 cells. When the cell concentration was above 106, the culture medium was changed by LHW (selective conditions) every three days, (150-200 µM). A control (Set 2) was used to measure the experimental error. At the end of the experiment, cell number in each flask was measured using a hematocytometer. Los Baños de la Hedionda (Málaga, S Spain) is a natural sulphureous spa, where sulphide can reach a concentration of 150-200 µM. Although this ion has biocide properties (1) ,a rich flora can be found in this extreme environment (2). Thus, the adaptation mechanisms allowing resistance of photosynthetic microorganisms to these sulphureous waters were studied using a modified Luria–Delbrück (3) fluctuation analysis. For this purpose, the adaptation of the cyanobacteria Microcystis aeruginosa (Kützing) Kützing Ma1Vc strain (isolated from a non-sulphureous freshwater reservoir) to La Hedionda waters (LHW) was analyzed, in order to find out if it was achieved by a physiological adaptation process (acclimation) or by the selection of rare spontaneous mutations (genetic adaptation). On this last case, the frequency of the LHW-resistant genetic variant can be calculated. Introduction Material and Methods Results and Discussion Conclusions Disentangling mechanisms involved in the adaptation of the cyanobacterium Microcystis aeruginosa to the extreme sulphureous water from Los Baños de la Hedionda (S Spain) •Genetic adaptation was the phenomenon that allowed Ma1Vc strain to survive on sulphide mediums, with a mutation rate 2.1 × 10 -7 cell division. The mutationselection equilibrium was 9.4 × 10 -7 cells . •It could be hypothesized that this cyanobacterium could adapt to sulphureous environment by the selection of favored mutants. References Acknowledgments Elena Martín-Clemente1, Ignacio José Melero-Jiménez1, Andreas Reul2, Miguel Hernández-López3, Enrique Salvo1, Elena Bañares-España1, María Jesús García-Sánchez1, Antonio Flores-Moya1 1 Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain; 2Departamento de Ecología y Geología, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain; 3 Departamento de Química Analítica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain 1. Czyzewski, B. K., & Wang, D. N. (2012). Identification and characterization of a bacterial hydrosulphide ion channel. Nature, 483(7390), 494-497. 2. Flores‐Moya, A., Costas, E., Bañares‐España, E., García‐Villada, L., Altamirano, M., & López‐Rodas, V. (2005). Adaptation of Spirogyra insignis (Chlorophyta) to an extreme natural environment (sulphureous waters) through preselective mutations. New Phytologist, 166(2), 655-661. 3. Luria, S. E., & Delbrück, M. (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics, 28(6), 491. The CV of the number of cells between the Set 1 and the Set 2 was significantly different (p<0.0001), so the adaptation process that allows resistance was GENETIC. On absence of selective agent (BG11 medium), the ms was higher than mr (Fig. 1). That is because the physiological mutation cost, that decreases the wild-type growing rate. Nevertheless, the mr was significantly higher on presence of sulfur (100 µM) than the ms. Indeed, the resistant LD was almost three times higher than the sensible LD (Fig 2). Using both ms and mr growing rates on BG11, a coefficient of selection (s) of 0.222 was computed. The estimation of the frequency (q) of LHW-resistant allelles in wild-type populations of Ma1Vc, was calculated to be 9.4 × 10 -7 cells. Do you want to know more about this experiment? Get in contact with us. Sensitive-cells LD= 122 µM •This work has been financially supported by the Spanish Ministry of Science and Innovation through CGL2014-53682-P project. •Besides, the Predoctoral State Grant from Scientific and Technical Research and Innovation Plan, Spanish Ministry of Economy, Industry and Competitiveness I+D+i ECC/1402/2013, 2015 0 0,1 0,2 0,3 0,4 0,5 0,6 BG11 100 µM S m (doublings day -1) Sensitive-cells Resistant-cells 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 50 100 150 200 250 300 m (doublings day -1) [S] µM Sensitive-cells Resistant-cells Resistant-cells LD= 294 µM