IV. RESULTS AND DISCUSSION
2.2.1. Growth kinetics
CHAPTER 2: Survival and response to adverse conditions in S. ruber M8 and M31 strains
2.1. Background
As described in the previous chapter, the use of the metabolomic approach by high-‐field ion cyclotron Fourier transform mass spectrometry (ICR-‐FT/MS) allowed to discern biogeographical patterns in S. ruber populations worldwide distributed. This permitted a non-‐targeted search for special metabolic traits considered as relevant in the organism’s phenotype, showing a higher resolution power than the molecular techniques previously applied in S. ruber population studies (Antón et al., 2008; Rosselló-‐Mora et al., 2008).
In this chapter, a combination of conventional cultivation methods, molecular techniques and a metabolomic approach were applied to study the main growth features of the closest S. ruber strains (M8 and M31) hitherto isolated and sequenced (Peña et al., 2010). Special attention has been focused on the changes occurring in the transition from the exponential growth to the stationary phase, and the response to three major different environmental stresses in which S. ruber may be exposed in its natural hypersaline habitat : i) oxygen depletion, ii) dilution of salts present in the culture medium, and iii) decrease in the incubation temperature.
2.2. Growth curves
Growth curves are widely used in Microbiology to study the increase in population size or biomass of a given microorganism. In liquid culture, most of bacterial growth displays a characteristic four-‐phase pattern of growth. The initial lag phase comprises a period of slow growth in which the bacteria are adapting to the new incubation conditions. This phase is followed by a logarithmic phase in which the growth is optimum and the cell numbers increase at ever increasing rates with time. After, a stationary phase occurs, when culture enters in a steady-‐state equilibrium in which the rate of cell growth is balanced by the rate of cell death. The death phase occurs because of a loss of limiting nutrients (due to their incorporation into cells during log-‐
phase growth) or a build-‐up of toxins (due to their release during log-‐phase growth, e.g., fermentative products).
2.2.1. Growth kinetics
Kinetic of growth was followed through optical density measurements (OD600) that were plotted versus time (expressed in hours), as shown in Fig. 18. Both strains behaved differently in their OD changes. The exponential phase for both organisms started 72 hours after inoculation and occurred during approximately 144 h (M8 strain) to 168 h (M31 strain).
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In addition, during the exponential phase, both strains showed different generation times of 9.3 h for M8 and 10.1 hours for M31. These values differ from previous observations in which generation times ranged from 14 to 18 hours (Antón et al., 2002), but the differences may be related to the higher yeast extract concentration used in our study (0.2%) in comparison to the previous one (0.1%).
The higher OD reached during the exponential growth was at 168 h and 216 h for M8 and M31, respectively.
Besides, OD measures decreased when the cells entered into the stationary phase, which occurred at 216 h (M8 strain) and 240 h (M31 strain) after inoculation (Fig. 18).
Figure 18: Growth curves of Salinibacter ruber M8 (squares) and M31 (circles). Growth was monitored at OD600 (empty symbols) and by DAPI counts (filled symbols). Data are represented as log OD and log (cells mL-‐1) with time. Plotted points are an average of two independent measurements.
2.2.2. Variations of cell numbers and FISH-‐detectable cells along the growth curve
In parallel to OD measures, the increase in absolute cell counts during the growth was followed by DAPI staining and expressed as log cell mL-‐1 versus time (Fig. 18). Although both cultures were inoculated approximately with the same amount of cells (109 mL-‐1), the highest OD reached by M8 corresponded to 5.5 x
1010 cells mL-‐1, whereas the maximum value of M31 corresponded to 5 ± 0.05 x 1010 cells mL-‐1 (Fig. 18 and
Table 11). However, the lower OD value together with higher cell counts observed in M31 strain can be explained by the formation of cell aggregates that bias the OD measures, a phenomenon that was not observed for M8 (data not shown). In the stationary phase, the cell numbers decreased to 2.1 ± 0.19 x 1010 and 3.1 ± 0.04 x 1010 cells mL-‐1 in M8 and M31, respectively (Fig. 18 and Table 11).
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In addition, FISH analyses were performed by using the EHB-‐412 probe in order to evaluate the fraction of cells that may show a decrease in the intensity signal as a response to a reduction in their ribosome’ s content along the time (Amann et al., 1995). As expected, during exponential phase both strains showed values that were close to 100% of the total cell counts (Table 11) whereas during stationary phase the FISH-‐
detectable cells decreased to about 87-‐91% respect to the total cells counts obtained by DAPI (Table 11).
Differences among these two strains regarding growth kinetics have been already reported by competition experiments, suggesting direct competition between the two closely related populations (Peña et al., 2010).
Under standard conditions, M8 cells outcompeted M31 cells since in salt-‐saturated medium the density of M31 cells in mixed cultures was roughly up to 30-‐fold higher than M8 cells, while in pure cultures these difference was only two fold (Peña et al., 2010).
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2.2.3. Culturability along the growth curve
The culturable fraction (CFUs counts) at the highest development of the exponential phase was equivalent to 44% and 53% of the corresponding DAPI counts in M8 and M31, respectively (Table 11). This is a known phenomenon that has been previously reported in other sort of samples (e.g. Amann et al., 1995; Hoefel et al., 2003; Marques et al., 2005). As mentioned before, during exponential phase about 98% of cells may be all metabolically active as indicated by the high rate of hybridization with the specific FISH probe. Thus, the observed discrepancy in counts may be related to the culturing procedure itself. For example, studies of resuscitation of Vibrio vulnificus showed that the plating medium itself could be a factor of nonculturability.
Besides, the elevated nutrient content may be toxic in some manner and might prevent colony development onto solid media (Whitesides & Oliver, 1997). Also factors as aeration, salinity and cell washing have been reported to influence the culturability (Oliver et al., 1991).
As expected, the differences between CFUs and total counts were more dramatic in the stationary phase, where just 9% of the DAPI stained cells grew onto plate, whereas the FISH counts just dropped to 90% (Table 11). The decreased in CFU counts in the stationary phase can be explained by cell death (Nyström, 2004).
However, due to the still high number of FISH detectable cells, it cannot be excluded that an important proportion of them remained under a viable but non culturable state (Oliver, 2005), still with an active metabolism (Marques et al., 2005), or even under a dormant phenotype (Bloomfield et al., 1998). Given the high number of FISH detectable cells, a live/dead test commercial kit was not used because they relay on the cell membrane integrity (Boulos et al., 1999), and viability or death may not be always related to compromised membranes (Kramer et al., 2009).
2.3. Stress dynamics
Changes in environmental conditions impose a variety of stress for microorganisms, which in turn have developed different strategies to survive. Bacteria must sense the changes and then respond with appropriate alterations in gene expression and protein activity, which also involve changes in the characteristics of cells populations. To study the response of S. ruber to different stress conditions, M8 and M31 strains were grown under optimal conditions until reach the exponential phase. At this point, cultures were divided into four aliquots, one was kept as control and the remaining three were submitted to differents stresses as mentioned in Materials and Methods section.
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In this regard, it should be noted that due to the low buffering capacity of the used medium, the effect of bubbling with N2:CO2 in the cultures in order to achieve the oxygen depletion was also accompanied by a pH drop from 7.2 to 5.8. After different times under stress incubation, the dynamics of each strain under different stresses was followed by quantification of cells numbers, quantification of FISH-‐hybridized cells, and changes in the culturability.
2.3.1. Cells abundances and FISH-‐detectable cells numbers under stress conditions
As it can be seen in Fig. 19, both strains responded similarly and all stresses promoted a continuous decrease in the total cell numbers observed by DAPI, in some cases to nearly the half of the initial value. Oxygen depletion and dilution promoted a similar effect on the decrease of cell counts, whereas during temperature stress, this effect was less pronounced and cells did not always show an abrupt drop. As example, during the first two hours of treatment, the most remarkable changes were on M31 cells, which showed a much higher sensitivity to both stresses than M8. This decrease in the number of cells respect to the control condition was about 20% (M8) and 40% (M31) for oxygen depletion, and 40% (M8) and 60% (M31) for osmotic stress. At this point, it is important to note that the changes observed under anoxia may not be only due to the oxygen depletion, but also to the decrease of the pH value from 7.2 to 5.8 since in previous experiments S. ruber showed a decrease in the growth yields when the pH was reduced to 6.0 (Antón et al., 2002). On the other hand, during the first two hours of temperature stress, M8 cells showed a lower sensitivity than M31 cells (about 10% versus 30%), a trend that remained during all tested stress conditions (Fig. 19).
Although the number of cells decreased in all sampled points and stress conditions studied, the fraction of FISH detectable cells did not decrease so strongly, and in the worst case, the detection rates dropped about 13% with respect to the control condition (Fig. 19).
2.3.2. Culturability changes in stress conditions
The reduction in cell abundances observed by DAPI were mirrored by the culturable numbers (CFU), the last showing more abrupt changes. The differences between total cell counts and CFU were already apparent after 2 h under every stress conditions with respect to the control, with a significant loss of culturability.
During this period, the culturability of M8 cells decreased in each stress condition. M31 seemed to be mostly influenced by the dilution stress, being less susceptible to the oxygen and temperature conditions during the same period of incubation (Fig. 19).
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Actually, this strain has been shown to outcompete M8 in high salt concentrations, but less fit in lower ionic strength (Peña et al., 2010), a fact that would be in accordance with its higher sensitivity to the dilution of the culture media.
Dilution and oxygen promoted an abrupt drop in the culturability in both strains, which reached the minimal values after 40 h, but they retained similar FISH detection rates, as occurred in the exponential–stationary phase transition. However, the temperature effect showed a different dynamics. Both strains strongly reduced their culturability, reaching the minimal values just after 16 h, but this culturability reduction was recovered when the incubation time was prolonged for 40 h under low temperature, at which point the initial rates were not only recovered, but also increased to 100% of the DAPI counts (Fig. 18). Besides, this phenomenon was observed even when the incubation at low temperature was prolonged up to 30 days (see below).
Figure 19: Culturability and cell counts of M8 and M31 strains under different stress conditions. All points correspond to the average of two independent measurements. In the graph it is represented the number of total cells determined by DAPI staining and the FISH hybridized cells (expressed as cells mL-‐1 with time), and the number of cells grown on agar plates expressed as CFU mL-‐1 with time.
2.3.4. Cells numbers, FISH counts, and culturability changes during prolonged temperature stress conditions Both strains responded similarly to the prolonged temperature stress, promoting a slight decrease of total cell numbers observed by DAPI which was not accompanied by a decrease in the fraction of FISH-‐detectable cells (Fig. 20).
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Contrary to previous observations, the prolonged incubation at 4ºC promoted an increase in the number of FISH hybridized cells respect to control condition (Fig. 20). On the other hand, the culturability under prolonged low temperature showed the same behavior observed at 40 h of stress, increasing the culturability to 100% (Fig. 20). The minor changes in the number of cells, accompanied with an increase in the ribosome content as well as the recovery of culturability under low temperature, supports the previous hypothesis that under low temperature incubation, S. ruber may generate a survival strategy to re-‐adapt to low temperatures probably by adopting a dormant state.
As mentioned before, cells could display increased stability after extended cold incubation (Weichart &
Kjelleberg, 1996), which could promote the change to cultivable phenotype as observed in Figs 19 and 20. A recovery of the culturable fraction is a phenomenon that has been observed in species such as Vibrio parahaemolyticus (Coutard et al. 2007) in which a proportion of viable but non-‐culturable cells (VBNC) subjected to low temperatures remained viable after a temperature upshift, suggesting the re-‐growth of these cells rather than resuscitation of all bacteria of the initial inoculum (Coutard et al. 2007). In addition, the use of exponential cultures to study the stress response could have effects in the culturability, since the growth phase prior to cold incubation has been identified as the major determinant for maintenance the culturability at low temperatures in species as Vibrio vulnificus (Oliver et al., 1991). Thus, S. ruber may generate a survival strategy to re-‐adapt to the new environmental conditions, which would promote a readjustment of their cellular machinery to shift the non-‐culturable state to a culturable state.
Figure 20: Culturability and cell counts of M8 and M31 strains under prolonged temperature decrease (30 days). All points correspond to the average of two independent measurements, and represent the number of total cells determined by DAPI staining and FISH (expressed as cells mL-‐1 vs time), and the number of cells grown on agar plates (expressed as CFU mL-‐1 vs time).
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2.4. Metabolome comparisons
As in the previous chapter, in order to study the changes during the different phases of growth and the response to adverse conditions, standard microbiological techniques were supplemented with a metabolomic study of chemical extracts of M8 and M31 strains by using high-‐field ICR-‐FT/MS. Different metabolomic comparisons were performed in order to characterize the metabolic response in both strains.
2.4.1. Common metabolome composition in both strains along the different phases of the growth curve The complete set of metabolomes during the growth curve measurements of both strains rendered a total of 18,054 unique masses signals at S/N=1, from which 7,174 were attributed to distinct elementary compositions containing the elements C, H, O, N and S (Table 12). These 7,174 masses corresponded to the total masses used for the statistical analyses, which provide all information about the significant differences between different growth phases that allowed the metabolomic discrimination of exponential and stationary phases of growth (Barker et al. 2003; Bylesjö et al. 2006; Trygg and Wold et al. 2002a, b).
Besides, in order to study the changes during the stationary phase, the common metabolome composition during this phase was also analyzed, rendering a total of 11,138 unique masses, from which 4,167 were attributed to distinct elementary compositions (C, H, O, N,S) (Table 13). Lastly, the common stationary metabolome was also considered to analyze the masses that increased or decreased in intensity during this phase of growth (see below).
2.4.1.1. Statistical analysis and models
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 (Barker et al. 2003; Bylesjö et al.
2006; Trygg and Wold et al. 2002a, b). A multiple regression analysis on the different phases of the growth curve revealed a clear separation among the growth states. PLS-‐DA models with Orthogonal Signal Correction (OSC) were first applied to the three cellular fractions analysed (see M y M). The inspection of the models created for E, CS, and CI fractions showed differences between different states of growth (Fig. S1, S2).
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The number of common masses considered for growth phase discrimination after statistical analysis is also specified.
Results and Discussion: Chapter 2 three dependent variables (Y1=initial phase, Y2=exponential phase and Y3=stationary phase) was always highly significant (p<0.001 for all) showing three different groups corresponding to the initial, exponential and stationary growth phases, all of them analyzed in positive mode. The model was validated with 200 permutations, revealing the absence of over fitting.
2.4.1.2. Analysis of discriminative masses
The numbers of discriminative masses in each cellular fraction were between 800 and 990, where the
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However, the most relevant markers for the common discriminative masses of the stationary phase metabolomes were mainly CHO and CHON compounds, with both high (H:C) and low (O:C) ratios (Figure 22b). Probably these compounds are aliphatic in structure and associated to some peptides and carbohydrates and mainly saturated and unsaturated lipids, which could be involved in the composition of cell membrane (Hertkorn et al., 2007). Also during stationary phase, CHO and CHON compounds that contain sulphur (S) were poorly represented and could be related to a decrease of biosynthetic processes requiringing sulphur, such as amino acids, proteins and vitamins (Nyström, 2004; Sekowska et al., 2000).
Some studies in Campilobacter jejuni and marine bacterial strains have showed changes in membrane fatty acid composition when the cells enter in the stationary phase, thereby increasing the surface hydrophobicity (Martínez-‐Rodriguez & Mackey, 2005; Syakti et al., 2006). These changes appear to represent a restricted physiological response to the conditions existing in halophilic stationary phase cultures, due to the hydrophilic cell surface that makes the cell more attractive to water molecules in a water-‐poor environment.
Also, the hydrated cell surface may help the cell to obtain cytoplasmic water and thereby prevent desiccation (Martínez-‐Rodriguez & Mackey, 2005; Ventosa, 2006).
Finally, we retrieved all the m/z values that showed intensities with increasing or decreasing values during the stationary phase (and with respect to the exponential phase). In this regard, the analysis of the stationary phase metabolome showed about 230 masses with increased intensities and 325 with decreased values, which were concentrated mainly in the insoluble cellular fraction and extracellular fraction respectively (Table 13). Those masses were considered as the most discriminative for the assay and were used for the metabolite annotation, because it would explain the most abrupt changes respect to the exponential phase (see below).
Results and Discussion: Chapter 2 metabolites of general metabolome databases (www.metabolome.jp, www.genome.jp/kegg) shown in grey in the figure.
(b) All discriminating m/z values of the stationary phase of the M8 and M31 strains coloured as a function of their elementary composition, where most discriminative metabolites contain CHO (only a few metabolites contain sulphur or nitrogen).
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For the transition to the stationary phase, only 14% of the total discriminative masses could be annotated, with the majority of them remaining unidentified. Of the annotated masses, approximately 34% could be associated to the aminosugar and aminoacids metabolisms, whereas 29% were involved in the glycerolipid and glycerophospholipid metabolism pathways, being the most important those shown in Table 14. Although the rest of the identified metabolites were associated to cyano-‐aminoacids (17%) and nitrogen (10%) metabolisms, and 10% were distributed in different metabolic pathways (Table S1), these metabolites not allowed us to speculate about a clear physiological response of S. ruber during the stationary phase.
Altogether, the results suggested that the most representative changes were related to modifications on the composition of the cell envelope. For example, during exponential phase we could identify compounds involved in the synthesis of peptidoglycan (N-‐Acetyl-‐D-‐glucosamine, N-‐Acetyl-‐D-‐mannosamine and N-‐Acetyl-‐
D-‐galactosamine), that remained undetected in the extracellular fraction during the stationary phase (Table 14). The decrease of these compounds during stationary phase could be related to the size of most bacteria that is considerably reduced upon entry into stationary phase, being the peptidoglycan a polymeric structure responsible for maintaining cell shape, as well as counteracting the osmotic pressure of the cytoplasm (Nyström, 2004). Also, it can affect the cell wall turnover and recycling of Gram-‐negative bacteria such as Escherichia coli, in which all of the amino acids and amino sugars of peptidoglycan are recycled, making them available for the cell to resynthesize more peptidoglycan or to use as an energy source during stationary phase (Park & Uehara, 2008).
Moreover during stationary phase, changes in the saturation/unsaturation ratio and in the length of the acyl-‐
chain of main membrane fatty acids, such as glycerolipids and glycerophospholipids were recognized (Table 14). Studies in marine bacterial pure cultures strains show that the composition of the phospholipid fatty acid could be strongly influenced by both, the carbon source and the growth phase (Syakti et al., 2006). In some halophilic bacteria, these changes may include the increase in the proportion of charged phospholipids at
chain of main membrane fatty acids, such as glycerolipids and glycerophospholipids were recognized (Table 14). Studies in marine bacterial pure cultures strains show that the composition of the phospholipid fatty acid could be strongly influenced by both, the carbon source and the growth phase (Syakti et al., 2006). In some halophilic bacteria, these changes may include the increase in the proportion of charged phospholipids at