Inner membrane proteins were solubilized with 2% triton X-‐100 or Nonidet 40, and the outer membrane fraction was pelleted by centrifugation at 16.000 g for 30 min at 4ºC, and solubilized in 30 µl of 100 mM Tris–HCl pH 8, 2% SDS buffer, while supernatants were recovered in a new tube for inner membrane protein extraction (see bellow). Outer membrane protein extracts were boiled during 5 min and maintained at -‐20ºC until use.
6.2. Inner membrane protein extraction
Supernatants recovered from outer membrane proteins protocol (Bucarey et al., 2006; Lobos & Mora, 1991), containing the proteins associated with the inner membrane were precipitated by the addition of 2 volumes of cold acetone and centrifuged at 16.000 g for 10 min at 4ºC. Pellets were air dried and resuspended in 30 µl of Tris–HCl 100 mM, pH 8 buffer, 2% SDS, then boiled during 5 min and maintained at -‐20ºC until use.
6.3. Protein quantification
Based on the method described by Bradford (Bradford, 1976), outer and inner proteins were quantified by using Bio-‐Rad Protein Assay (cat no. 500-‐0006). Dye reagent was diluted 1:4 times in distilled water and filtered through Whatman filter (celullose, no.1, grade >11 µm) to remove all possible reagent particles.
Five dilutions of Bovine Serum Albumin (BSA) (0.5 mg ml-‐1, Sigma) were prepared to elaborate the calibration curve, whose concentrations ranging from 0 to 75 µg ml-‐1 (Table 6). Samples and standards were prepared in 0.15M of NaCl (final volume of 200 µl). After the addition of 2ml of diluted dye reagent, samples were vigorously mixed and incubated at room temperature for at least 5 min.
Standards were measured in duplicate at 595 nm in a Hitachi U 2900 spectrophotometer.
Proteins samples were prepared by mixing 8 µl of the extract with 192 µl of 0.15 M NaCl and treated in the same way as standards. A linear regression absorbance/ concentration was calculated and the exact concentrations of the unknown samples were determined by interpolation considering the dilution factor of the samples. Five microliters of loading buffer were added to aliquots containing 20 µg of protein (30 µl as final volume) and, as indicated above, boiled during 5 min and maintained at -‐20ºC until use.
Materials and Methods
6.4. Matrix Assisted Laser Desorption/Ionization-‐ time of flight Mass Spectrometry (MALDI-‐TOF MS) Cultures (1.5 ml) were pelleted at 16.000 g for 3 min and resuspended in 500μl 70 % ethanol. The
Materials and Methods
-‐58-‐
6.4.1. Mass spectra analyses
All mass spectra profiles were grouped in a matrix and evaluated by Hierarchical clustering using the software PRIMER-‐E ® (Plymouth). Based on the presence and absent of signals, a dendrogram of the mass spectra was obtained by single linkage agglomerate similarity calculations. In addition, the data were analyzed in SIMCA-‐P 11.5 (Umetrics, Umea, Sweden). Supervised partial least square discriminative analysis (PLS-‐DA) applying the orthogonal signal correction (OSC) was used in order to evaluate the dependence of signals with the time and stress conditions (Sjöström et al., 1986; Stahle & Wold, 1987).
7. Lipopolysaccharide (LPS) analysis
7.1. LPS extraction
LPS was prepared by following two different methods: the first is a modification of the method described by Hitchcock (Hitchcock & Brown, 1983), in which exponential cultures of M8 and M31 strains of S. ruber were harvested by centrifugation and pellets were resuspended in water until reaching a
DO420 nm= 0.4. Suspensions (1.5 ml) were centrifuged at 16.000 g for 10 minutes, supernatants were
discarded and pellets resuspended in 50 µl of lysis buffer (2% SDS, 4% β-‐mercaptoethanol, 10% glycerol, 1M Tris pH 6.8, 0.02% bromophenol-‐blue) and boiled for 5 min. After adding 5 µl of proteinase K (5 mg ml-‐1 in water solution, Roche), samples were incubated for 1 hr at 60 ºC. Finally, 20 µl of solution were loaded in a 12% polyacrylamide gel (see below).
In the second method, proposed by Busse (Busse et al., 1989), 2 ml of exponential cultures of M8 and M31 strains were harvested by centrifugation at 16.000 g for 10 min at 4°C and bacterial pellet was frozen and lyophilized. Freeze-‐dried cultures were resuspended in 700 µl of lysis buffer (0.5M Tris pH 6.8, 2%SDS, 10% glycerol, 0.02% bromophenol-‐blue). Samples were mixed and boiled for 5 min. When samples were cold, 10 µl of proteinase K (5 mg ml-‐1 in water solution, Roche) were added and incubated for 1 h at 65 ºC. Finally samples were boiled for 5 min to inactivate proteinase K. Samples were centrifuged at 9.300 g for 10 min, and 20 µl of supernatant-‐solution were loaded in a 12%
polyacrylamide gel (see below).
Materials and Methods
-‐59-‐
7.2. Detection of LPS by silver staining
To check the LPS composition, extracts were subjected to the polyacrylamide gel electrophoresis (Laemmli 1970). It included a 5% stacking gel and a 12% separating gel. Tris-‐glycine buffer (pH 8.3) was used as electrode buffer. The electrophoresis was carried out as indicated in seccion 8.2.
Lipopolysaccharides extracts (20 µl) were loaded into the gel and the electrophoresis was performed at 70 V during 1 hr and then 100 V, until the dye front reached the bottom of the gel (about 2 h). After electrophoresis, silver staining was carried out according to Tsai and Frasch (Tsai & Frasch, 1982). The gel was kept overnight in a fixative solution containing 40% ethanol and 5% acetic acid in a clean plastic box. Next, the fixative solution was replaced by 0.7% periodic acid in 40% ethanol and 5% acetic acid to oxidize the LPS during 5 min. Subsequently, three washes of 15 min were performed using double distilled water. Finally, the gel was immersed in fresh staining reagent (150 ml) for 30 min. The staining reagent was prepared as follows: 4 ml of concentrated ammonium and 28 ml of 0.1 N sodium hydroxide were added to 115 ml of double distilled water and 5 ml silver nitrate (1g dissolved in 5 ml of MiliQ water). The concentrated ammonium was added drop by drop with magnetic stirring. Transient brown precipitate was formed when each drop of silver nitrate solution was added, but it disappeared within seconds. After all silver nitrate solution was added, the staining reagent was added to the gel and incubated under agitation at room temperature during 30 min. After staining, three 10 min washes in water were performed. Later, the gel was immersed in 300 ml of formaldehyde developer solution (containing 75 mg of citric acid and 0.75 ml of 37% formaldehyde) and incubated with slight agitation at room temperature until the visualitzation of the LPS bands. To prevent the saturation of color in the gel, development was stopped by adding 1% acetic acid solution and incubation at room temperature during 2 to 5 min. Finally, gels were dried by immersion in a solution containing 25% ethanol and 2% glycerol for 20 min.
Materials and Methods
-‐60-‐
8. Electrophoresis
8.1. Agarose gel electrophoresis
Nucleic acids and amplification products were resolved in 1.5% agarose gels (EEO, Prodanisa) or 1.5%
agarose MS 8 (Prodanisa) in 1X TAE buffer (40mM Tris, 20 Mm acetic acid, 1 mM EDTA) at 5 V/cm. Five microliters of each sample was mixed with 10X loading buffer (0.1% bromophenol blue, 0.1M EDTA, 0.1% SDS, and 50% glycerol). Markers 1kb (from 250 to 10.000 bp, Promega), 100-‐1000 bp (mbl), λ DNA/Hind III-‐EcoRI (from 564 to 21.226 bp, REAL) and λ DNA/PstI (from 247 to 11501 bp, Fermentas) were used as molecular weight references.
Gels were stained during 10 min in fluorescent dye ethidium bromide solution (1µg ml-‐1), washed in distilled water for 20 min and visualized in an UV transiluminator (Syngene) at 312 nm. Pictures of gels were taken by G-‐BOX system (Syngene) or Gel printer plus system (TDI) and printed in Sony digital graphic printer UP-‐D897.
8.2. Polyacrylamide gel electrophoresis (SDS-‐PAGE)
Sodium dodecyl sulfate polyacrylamide electrophoresis was performed by the method of Laemmli (Laemmli, 1970), with 5% (w/v) of 30% acrylamide mix (acrylamide/bis 29:1 (Biorad)) in the stacking gel and 12% or 12.5% of 30% acrylamide mix (Biorad) in the resolving gel. Compositions of stacking and resolving gels are shown in table 7. Only outer membrane proteins were separated in 12.5% resolving gel that also contained NaCl for a better visualization as recommended by Lobos and Mora (Lobos &
Mora, 1991). Extract proteins samples (20 µg in a maximum volume of 30 µl) were mixed with 5 µl of loading buffer (65.5 mM Tris-‐HCl pH 6.75, 20% glycerol, 4% SDS, 0.05% β-‐mercaptoethanol, 0.002%
bromophenol-‐blue), and boiled during 5 min before loading onto the gel.
Electrophoresis was performed in a Protean dual-‐vertical-‐slab-‐gel apparatus (Bio-‐Rad) with 1X running Tris-‐Glycine buffer (3.4% Tris, 14% glycine, and 1% SDS) at a constant voltage of 70V for the stacking gel (1 hour) and 100 V for the resolving gel during 4 h. Gels were rinsed in distilled water for few minutes, fixed for 20-‐30 min in the fixing solution (10% trichloroacetic acid, 50% isopropanol alcohol) and stained over night with Coomasie blue solution (BioRad). Gels were unstained in 10% acetic acid, 15%
isopropanol solution or in distilled water. Protein-‐patterns of samples were analyzed two times and in some cases four times.
Materials and Methods
8.3. Denaturing Gradient Gel Electrophoresis (DGGE)
One milimeter thick 6% (w/v) polyacrilamide gels (40% acrylamide/bis 37.5 :1 (Biorad)) with 40-‐80%
Materials and Methods
-‐62-‐
9. Physiological and biochemical tests
Salt requirement for growth was determined by growing the isolates in liquid SW medium with different concentrations of NaCl (5, 10, 15, 20, 25 and 30%). Similarly, the requirement for Mg+2 was tested in medium containing MgCl2 at the following concentrations: 0, 5, 10, 50, 170 and 400 mM. The experiments were performed in duplicate in 96-‐well microtiter plates. Two hundred microliters of each medium were inoculated with 5 µl of a well grown suspension of each strain (OD600nm = 1.0). Growth was monitored spectrophotometrically at 600 nm after the incubation period (7–14 days at 37ºC without shaking). The pH range for growth was determined in liquid 25% SW medium adjusted to the desired pH values (5.0, 5.2, 5.8, 6.2, 7.2, 7.4, 8.2 and 8.5). The microtiter plates were inoculated and incubated for 4 weeks as described above. Catalase production was performed by suspending single colonies in 3% (v/v) hydrogen peroxide; production of gas was checked for up to 5 min post-‐hydrogen peroxide addition, while cytochrome oxidase production was evaluated by moisting a piece of filter paper with N,N,N',N'-‐
tetramethyl-‐p-‐phenylenediamine solution (1%) and spreading a colony on the moisten filter paper (Smibert & Krieg, 1994; Stan-‐Lotter et al., 2002). The analytical systems API ZYM and API 20NE (BioMerieux) were used for the analysis of additional enzyme activities and for assimilation tests respectively (Stan-‐Lotter et al., 2002).
Strips were inoculated with a cell culture grown in 4 M NaCl and brought to a suspension density recommended by the manufacturers, incubated for up to 24 h (API ZYM) or 3 weeks (API 20NE). Tests were performed by triplicate for each strain.
10. Metabolomics
10.1. Metabolite extracts preparation
A total of 3ml of cell suspension grown on liquid media were collected by centrifugation (16,000 g, 2 min at 4 ºC). Cell-‐free supernatant (2 ml) was stored for further chromatographic extraction. Supernatant was acidified by the addition of 50 ml of 98–100% formic acid (Merck KGaA). Pelleted biomass was then resuspended in 1ml of bidistilled water, and sonicated to obtain a clear lysate extract. This lysate was then acidified by the addition of 50 ml of 98–100% formic acid. After the acidification, the clear lysate formed insoluble aggregates that could be separated from the soluble fraction by centrifugation.
Materials and Methods
-‐63-‐
The clear supernatant was stored for further fractionation, and the insoluble pellet was resuspended in 500 ml of methanol. Sample preparation resulted in three complementary fractions: the extracellular (E), cellular soluble (CS) and cellular insoluble (CI) fractions.
10.2. Solid-‐phase extraction
Both acidified extracellular and cellular soluble fractions were solid phase extracted using Bond Elut C18 columns (Varian Inc). This chromatography enables the isolation of the organic molecules on the basis of their nonspecific interaction and retention to the C18 material. Columns were activated by washing the column with 2ml of methanol, followed by 2ml MilliQ water and finally 2ml 1% formic acid in MilliQ water. Fractions were mixed with 3 ml of 1% formic acid and placed within the columns to bind organics compounds. This purification procedure removes the high-‐salt charge of the media and extracts, which may interfere during the electrospray procedure by ion suppression (Li et al., 2006). Columns were finally washed with 2ml of 1% formic acid and the retained fraction was recovered with 2ml of methanol.
10.3. ICR-‐FT/MS procedure
Broad band mass spectra were acquired on a Bruker (Bremen, Germany) APEX Qe ICR-‐FT/MS with 12 T superconducting magnet and an Apollo I electrospray (ESI) source, whereas high-‐resolution spectra were acquired with an Apollo II ESI source in positive and negative modes. The samples were infused in methanol with a microelectrospray source at a flow rate of 120 ml h-‐1 with a nebulizer gas pressure of 20 p.s.i. and a drying gas pressure of 15 p.s.i. (200 ºC). Spectra were externally calibrated on clusters of arginine (10 mg L-‐1 in methanol), and calibration errors in the relevant mass ranges were always below 100 p.p.b., which is the pre-‐requisite for an adequate elementary composition assignment.
Relative standard deviation in the intensity values of the peaks was routinely lower than 5% in these analysis conditions. The spectra were acquired with a time domain of 1 megaword (where 1 data word corresponds to 32 bits) with a mass range of 150– 2000m/z. The spectra were zero filled to a processing size of 2 megawords. A sine apodization (technical term for changing the shape of an electrical signal) was performed before Fourier transformation of the time domain transient. The ion accumulation time in the ion source was set to 0.2 s and 1024 scans were accumulated for one spectrum. ICR-‐FT/MS spectra were exported to peak lists at a signal-‐to-‐noise ratio (S/N)=1. From these lists, possible elemental formulas were calculated for each peak in batch mode by a software tool written in Helmholtz Zentrum München (Formulae®).
Materials and Methods
-‐64-‐
The generated formulas were validated by setting sensible chemical constraints (nitrogen rule, atomic oxygen to carbon ratio O/C ≤ 1, element counts: hydrogen H≤ (2+C2), carbon C≤ 100, oxygen O≤80, nitrogen N≤ 5 and sulfur S≤ 1) and only the masses in conjunction with their automated generated theoretical isotope pattern (existence of the 13C isotope) were taken into consideration (Hertkorn et al., 2007). The obtained reduced peak lists were compared in m/z at 5 p.p.m. and the corresponding intensity matrices were generated for further statistical analysis.
10.4. Statistical analysis
The data were imported and analyzed in SIMCA-‐P 11.5 (Umetrics, Umea, Sweden) and SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). Different multivariate techniques from unsupervised principal component analysis (PCA) to supervised partial least square discriminative analysis (PLS-‐DA) were used in order to reduce the different datasets and extrapolate informative masses from the different experimental conditions (Sjöström et al., 1986; Stahle & Wold, 1987) (Kemsley, 1996; Vong et al., 1988).
PCA is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of uncorrelated variables called principal components. Partial least squares (PLS) projections to latent structures are a regression extension of PCA (Wold et al., 1987). It uses the X variables (matrix of masses) as predictors, and dummy variables (belonging or not belonging to a given class coded as 1/0; that is, origin of isolation, stage of growth, and stress condition) as response variables (Y variables). Besides, the PLS-‐DA and PLS (applying the orthogonal signal correction (OSC)) modelling could be used to determine the relative concentration of the metabolites of interest. PLS-‐DA are the modelling often used in metabolomic field for classification of the samples (Barker et al. 2003; Bylesjö et al. 2006; Trygg and Wold et al. 2002a, b).
In all cases, three modalities (extracellular, cellular soluble and cellular insoluble) were calculated independently but in each analyzed dataset only a fraction was chosen as the descriptive power of the model. The descriptive power can be defined by several terms, most directly the fraction of the sum of squares (SS) of all the Y explained by the current component (R2Y(cum)) and Q2(cum). R2Y provides an estimate of how well the model fits the Y data, and Q2 provides an estimate of how well the model predicts the Y data. Pareto scaling of the intensity values with a logarithmic transformation of the data was chosen in some cases to consider all masses equally, including those with medium-‐ and low-‐
intensity values (van den Berg et al., 2006).
Materials and Methods
-‐65-‐
Score scatter plots and loading plots were generated. The score scatter plots present a view of how well the classes are separated on the basis of their x variables. In the loading plots, the different masses characteristic for each of the classes are differentiated. From these analyses, a list of discriminative masses (m/z) for the different geographical area, growth stage or stress conditions were chosen according to their correlation coefficient value. Those having the highest coefficients were considered to be relevant (that is, variables (m/z) with a correlation value higher than |0.002|).
Interpretation of the regression coefficients provides information pertaining to the metabolic explanation of class differences (Holmes and Antti, 2002) based on the fact that each coefficient is related to a specific elemental composition. Thus, masses associated with the highest correlation coefficient were represented in the van Krevelen projection in order to visualize their differences in chemical composition (H/C versus O/C) (Holmes & Antti, 2002; Wu et al., 2004).
10.5. Metabolites identification
Lists of discriminative masses were evaluated and assigned with the use of bioinformatics tools as MassTRIX (www.masstrix.org) (Suhre & Schmitt-‐Kopplin, 2008) and the Japanese metabolome (www.metabolome.jp) databases. The masses that were found to be discriminative for each of the different stress responses were crosschecked using the MassTRIX database and the annotation was done using the published genome of the M31 strain as a reference (Mongodin et al. 2005). Additionally MassTRIX gave valuable information through the identification of certain metabolites; in the meanwhile it was possible to characterize the experiments in relation with its biological context.
11. Microscopy techniques
Cell numbers and morphology of the organisms were observed under an optic microscope Zeiss Axio Imager A1.
11.1. Sample fixation
Culture samples (900 µl) were fixed at 4ºC during 16 h, with 37% formaldehyde (Sigma) in order to obtain a final concentration of 4% formaldehyde (Antón et al., 1999). Samples for fluorescence in situ hybridization analyses were pelleted at 16.000 g for 10 min and resuspended in cold ice 50% PBS 4X-‐
ethanol solution and storaged at -‐20ºC until use.
Materials and Methods
-‐66-‐
Before staining, all samples were diluted (10-‐1 and 10-‐2) in sterile 4X PBS, and 100 µl of suspensions were filtered thought GTTP Isopore filters of 0.22 µm pore size and 16 mm diameter (Milipore). Filters were stored at -‐20ºC until use.
11.2. 4’-‐6-‐diamidino-‐2-‐phenylindole stain (DAPI)
In order to determine the number of cells present in each culture, a piece of filter was stained with 25-‐
30 µl of 4'-‐6-‐diamidino-‐2-‐phenylindole solution (1 µg ml-‐1)(Porter & Feig, 1980) during 1.5 min at room temperature. Then, washed with sterile MiliQ water and absolute ethanol and dried at room temperature and darkness. Finally, filter was mounted with a drop of Citiflour AF1 (Citifluor ltd) and covered in a microscope slide. Cells were quantified using a fluorescence microscope (Axio imager.A1, Zeiss) with filter set 49 (G 365, FT 395, BP 445/50, Zeiss). Counts are reported as means calculated from 15 randomly chosen microscope fields. Fifteen microscope fields of 1200 µm2 were the optimum number of fields with the lowest standard deviation. A number of fields > 15 did not produced significant modifications in the means and the standard deviations.
11.3. Fluorescence in situ hybridization (FISH)
To evaluate the integrity and ribosome containing of Salinibacter cells, a piece of filter was hybridized with EHB-‐412 monolabel probe (Antón et al., 1999; Antón et al., 2000). EHB-‐412 probe (5’-‐
TACGCCCCATAGGGGTGT-‐3’; 50 µg ml-‐1) was diluted in sterile MiliQ water to a final concentration of 1 µg ml-‐1. The hybridization was performed at 45% formamide and the hybridization buffer was prepared as follows: 360 µl 5M NaCl, 40 µl 1M Tris-‐HCl pH 8.0, 904. 5 µl formamide, 695.5 µl Mili Q water, and 2 µl of 10% SDS. Filters were placed on a clean slide and each was hybridized with 20 µl of hybridization mix (4 µl probe, 16 µl hybridization buffer). Slides were placed into a hybridization chamber and incubated at 46ºC during 2 h. Then, to eliminate the unspecific hybridizations, filters were immersed in a washing buffer (300 µl 5M NaCl, 1 ml 1M Tris-‐HCl pH 8.0, 500 µl 0.5 M EDTA, MiliQ water to complete a final volume of 50 ml, and 50 µl of 10% SDS) and incubated at 48ºC during 15 min. After washing, filters were dried at room temperature and darkness and then, stained with DAPI. Finally, hibridized cells were quantified using a fluorescence microscope (Axio imager.A1, Zeiss) with filter set 49 (G 365, FT 395, BP 445/50, Zeiss) for DAPI, and the HQ: Cy3 filter set (AF analysentechnik; HQ 545/30, Q 570 lp, HQ610/75).
Counts were reported as means calculated from 15 randomly chosen microscope fields and the percentage of hibridized cells was calculated based on the total of DAPI counts of each sample.
IV. RESULTS AND DISCUSSION
Results and Discussion: Chapter 1
-‐69-‐
CHAPTER 1: Intraspecific diversity and biogeography of S.ruber strains
1.1.Background
Growth of the extremely halophilic bacterium S. ruber (Antón et al., 2000) is constrained to relatively small water bodies in restricted areas on Earth. S. ruber has been isolated from different areas of the world, and in sites as diverse as Mediterranean coastal solar salterns (Peña et al., 2005) or the remote Andean Peruvian salterns of Maras at 3,380 m above sea level (Maturrano et al., 2006a). The extreme conditions and geographical isolation of its environments are optimal circumstances for observing allopatric speciation (Coyne & Orr, 2004; Whitaker, 2006). Preliminary analyses based on fingerprinting genomic traits, such as PFGE or RAPD, although indicating a certain incipient trend, did not render a clear cut geographical discrimination among isolates (Peña et al., 2005). In order to discern biogeographical patterns in S. ruber, ten strains from five different locations were selected to study, through MLSA, the intraspecific diversity within the same group. For this, twelve protein-‐coding genes, which had been observed as phylogenetically informative, were selected (Sória-‐Carrasco et al., 2007). In addition, a metabolomic approach by Ion Cyclotron Resonance Fourier Transform Mass Spectrometry (ICR-‐FT/MS) was performed to evaluate some phenotypic evidence for allopatric segregation of members of S ruber, by identification of phenotypic patterns of the chemical extracts of this strain collection (as detailed in Materials and Methods section).
1.2.Multilocus sequence analysis (MLSA)
Multilocus sequence analysis was applied to study the intraspecific diversity of S. ruber strains isolated from different geographic localizations. The obtained sequences of specific coding-‐protein genes from each strain were concatenated and analyzed to calculate the total number of synonymous or nonsynonymous substitutions. Finally, different phylogenetic reconstructions were applied in order to evaluate stability of the genealogies by including and excluding the 16S rRNA gene sequences.
1.2.1.Amplification of protein-‐coding genes
A total of ten S. ruber strains (Table 3) were selected in order to study their intraspecific diversity by MLSA. The selected strains were representative of three main geographic areas: Mediterranean (M8,
A total of ten S. ruber strains (Table 3) were selected in order to study their intraspecific diversity by MLSA. The selected strains were representative of three main geographic areas: Mediterranean (M8,