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Cryotolerance strategies of Pseudomonads isolated from the rhizosphere of Himalayan plants

Cryotolerance strategies of Pseudomonads isolated from the rhizosphere of Himalayan plants The cold stress biology of psychrotrophic Pseudomonas strains isolated from the rhizosphere of Himalayan plants have been explored to evaluate their cryotolerance characteristcs. Pseudomonas strains were examined for stress metabolites, viz., exopolysaccharide (EPS) production, intracellular sugar, polyols and amino acid content, ice nucleation activity, and their freezing survival at −10 and −40°C, respectively. High freezing survival was observed for the Pseudomonas strains that were grown at 4°C prior to their freezing at −10 or −40°C. Increased EPS production was noticed when Pseudomonas strains were grown at lower temperatures, i.e., 4 and 15°C, in comparison with their optimal growth temperature of 28°C. All Pseudomonas strains showed low level of type-III class ice nucleation activity at −10°C after 96 h. Considerable differences were noticed in accumulated contents of various intracellular sugars, polyols, amino acids for all Pseudomonas strains when they grown at two different temperatures, i.e., 4 and 28°C, respectively. The unusual complement of stress protectants especially, raffinose, cysteine and aspartic acid that accumulated in the bacterial cells at low temperature was novel and intriguing finding of this study. The finding that raffinose is a key metabolite accumulated at low temperature is an exciting discovery, and to the best of our information this is first report ever signifying its role in bacterial cold tolerance. Keywords: Psychrotrophic; Pseudomonas; Cold tolerance; Raffinose; Exopolysaccharide; Free amino acids Background causing the maintenance of some enzymatic functions in- Microorganisms have a range of evolutionary adaptations vivo (Yamashita et al. 2002). However, a limited information and physiological acclimation mechanisms that allow them is available about the cryoprotectants that are responsible to survive and remain active in the conditions of environ- for the freezing resistance mechanisms of bacteria. Bacteria mental stress. Adaptation towards stress condition is indis- often encounter freezing conditions and can survive in ex- pensable for survival, mainly when it causes alterations to tremely cold environments, like, the high altitude regions of the cell metabolism. Sudden decrease in temperature has Himalaya. In frozen environments, bacteria are exposed to severe effects on microbial cells, like, reduction of mem- conditions that necessitate the removal of water to maintain brane fluidity, decrease in ribosome efficiency, and in- the structure and function of the bacterial cell. As water creased stabilization of secondary structures of nucleic contributes to the stabilization of various macromolecular acids, which may affect transcription, translation and DNA structures, any significant deviation from the accessibility of replication (Phadtare et al. 2000). In order to survive under water due to dehydration, desiccation or an alteration of its freezing conditions, bacteria have developed various stra- physical state from aqueous phase to an ice crystal form tegies for their endurance, such as, maintenance of mem- poses a severe threat to the normal cell functions and sur- brane fluidity, constant metabolic activities etc. (Ramos vival of organism (Beall 1983; Crowe et al. 1984). et al. 2001). Additionally, it has been suggested that treha- In this regard, regulatory proteins and key metabolic lose, glycerol and sorbitol are the major cryoprotectants for enzymes require adjustments to cope with the temperature prokaryotic cells to response the freezing damage, thereby shifts in order to maintain a balanced microbial growth at the new environmental temperature. Under such condi- tions, the synthesis of specific cryoprotectant molecules * Correspondence: shekhar.bisht@hotmail.com might be enhanced that act as chemical chaperons and pro- Department of Biotechnology, HNB Garhwal University (A Central University), Srinagar 246174, Uttarakhand, India tect the cellular proteins from freezing temperature. Scanty Full list of author information is available at the end of the article © 2013 Bisht et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bisht et al. SpringerPlus 2013, 2:667 Page 2 of 13 http://www.springerplus.com/content/2/1/667 reports are available on psychrotrophic bacterial cryotoler- EPS production ance strategies and related responsible molecules except EPS production was found to be higher at lower incubation cold shock (Csps) and cold acclimation (Caps)s proteins. temperatures (4 or 15°C) in comparison to the optimal Although, bacterial cryotolerance has been investigated in growth temperature (28°C) in all the Pseudomonas strains relevance with role of trehalose and glycine betine in (Figure 2). At 4°C, P. lurida NPRs3 produced 2.75 and 8.8 Escherichia coli and Bacillus subtilis, respectively (Jones folds higher EPS in comparison to EPS produced at 15 and et al. 1987; Willimsky et al. 1992). But, very little is known 28°C, respectively. At 15°C, P. lurida NPRs3 showed 3.2 about the possibility of other molecules responsible for folds higher EPS production in comparison with the same the survival of bacteria subjected to freezing challenge by cells grown at 28°C. Similarly, the P. lurida NPRp15 cells an adaptation of the microbial cells to low temperatures demonstrated 1.38 and 7.0 folds higher EPS production at particularly in psychrotolerant/psychrophilic bacteria (Margesin 4°C compared to the cells grown at 15 and 28°C, respect- and Schinner 1999; Mishra et al. 2010). ively. At 15°C, the P. lurida NPRp15 culture produced 5.07 In the upper parts of north west (NW) Himalaya, winter folds greater EPS as comparedtoculture grownat 28°C. is mostly characterized by intermittent snow cover Likewise, the cells of Pseudomonas sp. PPERs23 grown at (November to March) and fluctuating subfreezing tempera- 4°C showed 23.33 and 21.31% higher EPS accumulation as tures, while summer displays intense desiccating sunshine compared to the cells cultivated at 15 and 28°C, respect- punctuated by infrequent rains (Mishra et al. 2008; 2011; ively. However, the Pseudomonas sp. PPERs23 culture Bisht et al. 2013). These conditions pose additional chal- grown at 15 and 28°C showed almost similar EPS accumu- lenges to microbial species that may endure summer tem- lation (Figure 2). Similarly, the P. putida PGRs4 cells peratures as high as 30°C and winter temperatures that can showed 23.14% greater EPS accumulation at 4°C in com- dip to −10°C, as well as alternating freezing and thawing parison to the P. putida PGRs4 cells grown at 28°C. At periods during the cold season. At these temperatures, mi- 15°C, the P. putida PGRs4 culture showed 17.6% enhanced croorganisms might be injured or killed as a result of cold EPS production than the P. putida PGRs4 cells cultivated shock, freezing, prolonged exposure to subzero tempera- at 28°C. Likewise, the cells of Pseudomonas sp. PGERs17 tures, and subsequent warming, and injury or death is often cultivated at 4°C showed 66.0% increased EPS production due to damage to membranes or cell walls that results in in comparison to the cells grown at 28°C, whereas, the changes in permeability, as well as damage to DNA. Given Pseudomonas sp. PGERs17 cells separately grown at 4 and these challenges, the fact that soil bacteria thrive in NW 15°C failed to show significant difference in EPS accumula- Himalayan regions is a testimony either to environmental tion. P. fluorescens PPRs4 demonstrated almost double heterogeneity or to the remarkable adaptive abilities of amount of EPS production at 4°C growth temperature in these psychrotrophic microbes (Srinivas et al. 2011; Bisht comparison to cells grown at 15 or 28°C. et al. 2013). The precise mechanisms or molecular stra- tegies underlying the cellular adaptations of psychrotrophic INA of psychrotolerant Pseudomonas strains bacterial cells in cold conditions are not clear and needs to The INA of Pseudomonads had been measured to deter- be addressed, particularly for varying genus of Pseudo- mine the catalytic sites present in the bacterial cells respon- monas. In this context, the present study was undertaken sible for ice formation. None of the collected strains to investigate the freezing survival strategies operated demonstrated type-I or type-II category INA measured in six psychrotrophic Pseudomonas strains (P. lurida at −5°C. All Pseudomonads showed low level of type-III NPRs3, P. lurida NPRp15, P. sp. PPERs23, P. putida INA (active between −7to −10°C) measured at −10°C after PGRs4, P. sp. PGERs17 and P. fluorescens PPRs4) isolated 24, 72 and 96 h of bacterial growth (Table 1). The type-III previously from rhizosphere of NW Himalayan plants INA of all Pseudomonas strains was found to be low at long (Mishra et al. 2011; Bisht et al. 2013). (96 h) incubation of the culture. Whereas, the same was noted high for cultures incubated for short (24 h) time Results period. The highest INA was found in P. fluorescens PPRs4 Bacterial growth and freeze survival andthe lowest INAwas observedin Pseudomonas sp. Freezing survival studies of Pseudomonas strains re- PGERs17 after 24 h incubation period. The mean INA (log -1 vealed that strains which were grown at 4°C prior to ice nuclei CFU )of P. lurida NPRs3, P. lurida NPRp15, freezing separately at −10 and −40°C demonstrated sig- and P. fluorescens PPRs4 was noticed to be higher at 4°C nificantly higher freezing survival rather than cultures after 24 h growth incubation in comparison to 96 h of bac- which were grown at 28°C prior to freezing (Figure 1). It terial growth incubation at the same temperature. Whereas, was observed that Pseudomonas strains grown at low after 24 h growth incubation at 4°C, Pseudomonas sp. temperature (e.g., 4°C) have a survival advantage upon PPERs23, P. putida PGRs4, and Pseudomonas sp. PGERs17 freezing tolerance compared to their optimal growth showed difference of 0.85, 0.97 and 0.77 log ice nuclei -1 temperature (28°C). CFU , respectively, as compared to 96 h growth incubation Bisht et al. SpringerPlus 2013, 2:667 Page 3 of 13 http://www.springerplus.com/content/2/1/667 Figure 1 Percentage survival of Pseudomonads subjected to freezing temperature [−10 and −40°C] shifted from two different incubation temperatures (4 and 28°C). Note: All values are mean of three independent replicates and bar represents the standard error of mean. at 4°C. These differences indicated lowering in INA of all significant increase in intracellular raffinose was no- Pseudomonas strains with long incubation time (96 h) in ticed during cold condition (at 4°C) in all the Pseudo- cold conditions (e.g., 4°C). monas isolates (Table 2, Figure 3). Accumulation of sucrose molecule at 4°C was found in P. fluorescens Accumulation of intracellular sugars and polyols PPRs4, while,at28°Citwas notdetected. Likewise, Remarkable variations in terms of accumulation of glucose molecule was not detected in bacterial growth various intracellular sugars and polyols were noticed at 4°C for all the isolates, while P. lurida NPRp15 through HPLC chromatogram (Additional file 1: showed noticeable amount of glucose at 28°C. Trehal- Figure S1), when all the Pseudomonas cells were ose was accumulated in high amount in all the iso- grown at 4 and 28°C (Table 2). The bacterial intracel- lates at 4°C. Pseudomonas sp. PPERs23 accumulated lular sugar content was expressed in terms of μgper significant amount of trehalose, mannitol and sorbitol, -1 -1 mg of cell dry weight (μgmg CDW ). It was found when grown at 28°C (Table 2). Accumulation of man- that the accumulation of intracellular sugars varied in nitol was found only in four isolates, interestingly different psychrotrophic Pseudomonas strains at 4 and three out of four isolates showed higher accumulation 28°C. Such changes were observed in the amount of of mannitol at 28°C, P. putida PGRs4 showed higher glucose, trehalose, sucrose, mannitol and sorbitol. accumulation of mannitol at 4°C (Table 2). Greater Figure 3 shows the grouping of stress metabolites accumulation of sorbitol was noticed for NPRs3, based on their accumulation at two different tempera- PGRs4 and PGERs17 strains, when their cells were tures for individual strain and indicates variation grown at 4°C in comparison to the cells grown at in accumulation of stress metabolites for each iso- 28°C. Whereas, Pseudomonas sp. PPERs23 accumu- late. Most importantly, a prominent and statistically lated significant amount of sorbitol at 28°C. Bisht et al. SpringerPlus 2013, 2:667 Page 4 of 13 http://www.springerplus.com/content/2/1/667 Figure 2 EPS accumulation by Pseudomonas strains at three different incubation temperatures (4, 15 and 28°C). Note: All values are mean of three independent replicates and bar represents the standard error of mean. The alphabet letters (a, b, c) in the column for individual Pseudomonas strain indicate significant differences at 4, 15 and 28°C incubation temperature. Accumulation of intracellular amino acids phenylalanine at 4°C was found among all the isolates, The intracellular free amino acids’ concentrations in on the other hand reduction in concentrations of gly- Pseudomonas strains were investigated under optimal cine, glutamic acid, lysine and methionine at 28°C was (28°C) and low (4°C) temperature conditions. HPLC observed for most of the isolates. The P. lurida NPRs3 chromatograms of the Pseudomonas cells for 17 amino cells showed highest increase in concentration of aspar- acids tested from their intracellular extract demonstrated tic acid, serine, glycine, arginine, and proline at 4°C, significant variations at 4 and 28°C growth temperatures while no change was observed in lysine concentration. (Additional file 2: Figure S2 and Table 3). The intracellu- Substantial increase in accumulation of isoleucine (50.4 lar amino acids’ expression pattern varied from strain to folds) and cysteine (5.6 folds) was noticed, whereas no strain at 4 and 28°C, and the most prominent increase changes were observed for glutamic acid and methionine was observed in the concentrations of aspartic acid, pro- for P. lurida NPRp15 cells grown at 4°C. Prominent in- line and cysteine at 4°C (Table 3 and Figure 3). All crease in phenylalanine (24.2 folds) followed by histidine Pseudomonas strains exhibited statistically significant in- (4.6 folds), threonine (3.6 folds) and leucine (3.5 folds), crease in their aspartic acid (1.3 to 3.0 folds) and proline and minor increase in tyrosine concentrations were (1.1 to 2.8 folds) contents at 4°C. Significant accumula- observed at 4°C in P. putida PGRs4 cells (Table 3). For tion of serine, arginine, threonine, cysteine, leucine and Pseudomonas sp. PGERs17 cells, enhanced concentrations Table 1 Ice nucleation activity of cold tolerant Pseudomonas strains at −10°C temperature Cold tolerant Culture incubation at 4°C Pseudomonas 24 h 72 h 96 h strains -1 Ice nucleation activity of bacterial culture at −10°C (log ice nuclei cfu ) After After After After Mean After After After After Mean After After After After Mean 5 min 10 min 15 min 20 min 5 min 10 min 15 min 20 min 5 min 10 min 15 min 20 min P. lurida −8.47 −8.47 −8.47 −7.36 −7.15 −9.19 −9.19 −8.89 −8.49 −8.11 −9.17 −9.17 −8.8 −8.75 −8.47 NPRs3 P. lurida −7.43 −7.43 −7.33 −7.24 −7.17 −8.49 −8.49 −8.14 −8.08 −8.01 −8.96 −8.96 −8.78 −8.62 −8.56 NPRp15 Pseudomonas −6.68 −6.68 −6.68 −6.68 −6.38 −7.9 −7.9 −7.9 −7.56 −7.26 −7.74 −7.74 −7.74 −7.43 −7.23 sp. PPERs23 P. putida −7.58 −7.40 −6.82 −6.65 −6.18 −7.33 −7.24 −7.17 −7.17 −7.36 −8.49 −8.14 −8.08 −7.15 −7.15 PGRs4 Pseudomonas −7.92 −7.82 −7.78 −7.62 −7.52 −8.47 −8.37 −8.27 −8.24 −8.07 −8.89 −8.59 −8.69 −8.49 −8.29 sp. PGERs17 P. fluorescens −6.82 −6.65 −5.86 −5.80 −5.88 −8.49 −7.92 −6.82 −6.65 −6.65 −8.58 −8.19 −7.9 −7.56 −7.26 PPRs4 Bisht et al. SpringerPlus 2013, 2:667 Page 5 of 13 http://www.springerplus.com/content/2/1/667 Table 2 Quantitative analysis of intracellular sugars and polyols content of Pseudomonas strains by HPLC -1 -1 Pseudomonas strains Temperature Content (μgmg cell dry weight )* Sugar’ content Polyols content Raffinose Sucrose Trehalose Glucose Mannitol Sorbitol P. lurida NPRs3 28°C 8.92 ± 1.52a - - - 2.73 ± 0.52a - 4°C 19.79 ± 2.26b - 1.89 ± 0.21 - 2.23 ± 0.34a 9.36 ± 1.61 P. lurida NPRp15 28°C - - - 105.4 ± 3.9 43.23 ± 3.63 - 4°C 41.69 ± 3.39b - 3.19 ± 0.81 - - - Pseudomonas sp. PPERs23 28°C 3.18 ± 1.28a - 16.46 ± 1.62b - 7.12 ± 1.07b 22.06 ± 1.96b 4°C 13.41 ± 1.76b - 2.49 ± 0.48a - 0.55 ± 0.01a 3.80 ± 1.56a P. putida PGRs4 28°C 4.38 ± 0.97a - 0.44 ± 0.09a - - 3.17 ± 1.01a 4°C 11.25 ± 1.13b - 3.14 ± 0.84b - 3.92 ± 0.66 50.49 ± 3.68b Pseudomonas sp. PGERs17 28°C 1.56 ± 0.54a - - - - 0.91 ± 0.03a 4°C 9.90b - 3.02 ± 1.6 - - 4.32 ± 0.98b P. fluorescens PPRs4 28°C 6.76 ± 1.38 - - - - - 4°C 14.22 ± 1.74 3.33 ± 1.25 - - - - *(−): Not detected. Note: All values are mean of three (n = 3) experiments, followed by ± Standard deviation. Letters (a,b) in the same column for each Pseudomonas strain indicate significant difference at 4°C and 28°C incubation temperatures. were found for tyrosine (21.3 folds), threonine (4.2 folds), optimum growth temperature of 28°C. The parameters glutamic acid (3.9 folds), and proline (2.7 folds) at 4°C, placed at the negative side of the factor F2 showed less whilenochanges were noticed forarginine(Table3). Max- accumulation and less activity at 28°C, and high correl- imum increase was found for cysteine (46.5 folds) followed ation with cold temperature. Only freezing survival par- by leucine (7.4 fold), isoleucine (6.3 folds) and serine (4.5 ameter was found on the negative side of the factor F2 folds) in P. fluorescens PPRs4 cells at 4°C (Table 3). How- for five Pseudomonas strains with exception of NPRs3 ever, low concentrations of glutamic acid, tyrosine, and ly- cells (Figure 3[a]). The parameters which were grouped sine were found in the same cells at 4°C (Table 3). together in the PCA plot showed high correlation, whereas parameters which were grouped in opposite dir- Stastistical analysis ection indicated the negative correlation. The parame- Correlation analysis proved existence of significant rela- ters placed at the middle of the PCA model reflected no tionship between the measured cold stress parameters correlation with the factors (i.e., growth temperatures) and the bacterial growth conditions (i.e., temperature: and showed either equal or nil accumulation. These coordinate). The PCA for correlation of individual iso- include no accumulation of glucose and sucrose late has been shown in Figure 3. The first two factorial for five strains except NPRp and PPRs4, respectively axes represent 94.7 to 97.14% variance in the data. Ex- (Figure 3[b,f]). cept Pseudomonas NPRs3, factor F1 represented the The relationship/effect of measured cold stress metab- grouping of stress metabolites and reflected their sub- olites on bacterial freezing survival at −10 and −40°C stantial accumulation at 4°C. Factor F2 represented the was evaluated using automated linear modeling (ALM) grouping of stress metabolites accumulated maximally at analysis (Figure 4). The model contained only important 28°C. Maximum stress metabolites were accumulated at predictor(s) (i.e., stress metabolites) for freezing survival. 4°C for two Pseudomonas isolates, i.e., NPRp15 and Both the models were found statistically significant PPRs4 (Figure 3[b,f]). The parameters placed at a strong (p < 0.05) with 70 to 75% accuracy. Six cold stress pa- positive side of the factor F1 were highly correlated with rameters were identified as important predictors for the cold temperature and indicated higher accumulation freeze survival at −10°C (Figure 4[a]). The incubation at cold temperature. However, the parameters which temperature showed significant correlation (p = 0.000) were placed at negative side of the factor F1 showed with freeze survival at −10°C and demonstrated import- negative correlation with the cold temperature and indi- ant role (42%) in bacterial freeze survival. Bacterial sur- cated less or nil production at cold temperature. Simi- vival to freezing conditions was paralleled by an increase larly, the parameters which were placed at the strong in the intracellular raffinose level and showed significant positive side of the axis of factor F2 indicated minor role association (p = 0.000) with freezing survival at −10°C, in cold adaptation and showed higher accumulation at and also suggested that raffinose contribute in bacterial Bisht et al. SpringerPlus 2013, 2:667 Page 6 of 13 http://www.springerplus.com/content/2/1/667 Figure 3 Principal component analysis (PCA) of stress metabolites profile of Pseudomonas strains grown at 4 and 28°C. [a] P. lurida NPRs3 [b] P. lurida NPRp15 [c] Pseudomonas sp. PPERs23 [d] P. putida PGRs4 [e] Pseudomonas sp. PGERs17 [f] P. fluorescens PPRs4. (Factor map of rows (metabolite); stress metabolite with similar distributions of appearance with increases in cold condition occur in similar positions on the map). freeze survival. Likewise, high accumulation of cysteine, level environmental ubiquity even in cold/freezing habi- trehalose, aspartic acid and proline showed positive rela- tats (Timmis 2002; Remold et al. 2011; Cray et al. tionship with bacterial high freezing survival at −10°C 2013b). This study clearly demonstrates that cold accli- with 15.6, 9.1, 6.5 and 3.2% contribution, respectively. matized cells (grown at 4°C) had higher freezing- EPS and sorbitol were identified as most important thawing survival over non-acclimatized cells (grown at stress metabolites (predictors) for bacterial freeze sur- 28°C), and linear modeling analysis clearly proved that vival at −40°C (Figure 4[b]). the incubation temperature was the main factor for Pseudomonads’ freezing survival. The overall freezing- Discussion thawing survival of Pseudomonas strains at −10°C was The Himalayan region provides an opportunity to obtain found be 98% (Additional file 3: Table S1). This feature microbes that have experienced extended exposure to of Pseudomonads suggests their survival persistence in cold temperatures, reduced water activities, radiation Himalayan extreme freezing-thawing conditions during and low nutrient accessibility. The cold adaptation re- winter season. One of the major freezing survival strat- lated properties of psychrotrophic Pseudomonas cells egy might be the evolution of strains that are capable of showed high cellular metabolism activities in cold condi- utilizing a large number of carbon sources (Ponder et al. tions (Mishra et al. 2008; 2009; 2011). It has been well 2005). This might be more relevant because the alpine established that the cold-active enzymes and efficient environment is highly heterogeneous with pockets of growth rates are used to facilitate and maintain the ad- specific carbon compounds, a large number of bacterial equate metabolic fluxes at near freezing temperature for strains that have recently gained or lost the ability to cold-adaptation (Shivaji and Prakash 2010). The great grow on a particular source of carbon may exist and metabolic flexibility of Pseudomonas species allows them supported the adaptability, versatility and environmental to inhabit diverse environments and capable of a high ubiquity, and prevalence of Pseudomonas genus in the Bisht et al. SpringerPlus 2013, 2:667 Page 7 of 13 http://www.springerplus.com/content/2/1/667 Table 3 Quantitative analysis (HPLC) of intracellular amino acids’ content of Pseudomonas strains at 4°C and 28°C growth temperatures Amino acid (pico mole Pseudomonas strains -1 -1 mg cell dry wt. )* P. lurida NPRs3 P. lurida NPRp15 Pseudomonas sp. PPERs23 P. putida PGRs4 Pseudomonas sp. P. fluorescens PPRs4 PGERs17 At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C Aspartic acid 65.8 ± 1.5b 39.9 ± 0.9a 33.1 ± 0.7b 20.3 ± 0.5a 14.1 ± 0.3b 11.1 ± 0.2a 333.9 ± 7.5b 166.5 ± 3.7a 51.7 ± 1.2b 27.5 ± 0.6a 18.0 ± 0.5b 5.9 ± 0.3a Proline 4355 ± 98b 1997 ± 45a 7317 ± 164b 3751.2 ± 1880 ± 42b 1229.7 ± 27a 1467 ± 33b 1352.2 ± 30a 4733.3 ± 106b 1738.2 ± 1613 ± 26b 586.5 ± 13a 84a 39a Cysteine 32.8 ± 0.7a 89.7 ± 2b 439.4 ± 9.9b 78.2 ± 1.8a 67.8 ± 1.5 - 89.9 ± 2b 52.5 ± 1.2a 30.8 ± 0.7b 17.4 ± 0.4a 71.8 ± 1.8b 1.5 ± 0.1a Serine 485.0 ± 57.9 ± 1.3a 28.1 ± 0.6b 23.0 ± 0.5a 12.0 ± 0.4a 21.6 ± 0.5b 14.2 ± 0.3b 7.7 ± 0.2a 13.6 ± 0.3a 19.0 ± 0.4b 13.1 ± 0.6b 2.9 ± 0.1a 10.9b Glutamic acid 11.8 ± 0.3a 26.2 ± 0.6b 14.8 ± 0.3a 16.1 ± 0.4a 8.8 ± 0.2b 3.9 ± 0.1a 26.1 ± 0.6a 46.5 ± 1.1b 28.7 ± 0.6b 7.4 ± 0.2a 5.9 ± 0.3a 8.0 ± 0.4b Glycine 662.0 ± 347.5 ± 7.8a 274.5 ± 6.2a 370.7 ± 8.3b 350.8 ± 7.9a 184.7 ± 4.2b 137.7 ± 3.1b 121.5 ± 2.7a 351.2 ± 7.9a 533.3 ± 12b 288.8 ± 6.2b 272.9 ± 6.1a 14.9b Histidine 97.6 ± 2.2a 192.6 ± 4.3b 148.2 ± 3.3b 134.4 ± 3a 50.8 ± 1.1a 63.0 ± 1.4b 20.9 ± 0.5b 4.6 ± 0.1a 103.9 ± 2.3b 55.6 ± 1.3a 73.9 ± 1.8b 65.9 ± 1.5a Arginine 158.1 ± 3.6b 27.6 ± 0.6a 12.9 ± 0.3b 9.8 ± 0.2a 4.5 ± 0.1b 1.2 ± 0.1a 58.2 ± 1.3a 81.1 ± 1.8b 8.2 ± 0.2a 9.4 ± 0.2a 7.7 ± 0.3b 4.4 ± 0.1a Threonine 190.5 ± 4.3a 1084.3 ± 9194.7 ± 20b 3757.1 ± 5143.0 ± 6722.1 ± 6510.4 ± 1795 ± 40.4a 3003.8 ± 714.0 ± 16a 6479.2 ± 3890.4 ± 24b 84a 115a 151b 146b 67.6b 57b 87a Alanine 57.5 ± 1.3a 167.73.8b 107.9 ± 2.4b 94.1 ± 2.1a 38.3 ± 0.9a 39.7 ± 0.9a 104.6 ± 2.4a 890.9 ± 20b 91.0 ± 2b 66.3 ± 1.5b 46.5 ± 1.3a 50.1 ± 1.1b Tyrosine 30.2 ± 0.7a 46.5 ± 1.2b 39.2 ± 0.9b 19.6 ± 0.4a 10.7 ± 0.3a 9.5 ± 0.4a 6.5 ± 0.1a 3874.7 ± 16.8 ± 0.4b 0.8a 14.0 ± 0.5a 166.8 ± 3.8b 87b Valine 27.1 ± 0.6a 64.9 ± 1.5b 40.0 ± 1.9b 23.2 ± 0.5a 9.5 ± 0.2a 8.3 ± 0.3a 3.1 ± 0.1a 26.4 ± 0.6b 17.0 ± 0.4b 6.5 ± 0.1a 14.1 ± 0.4b 7.0 ± 0.2a Methionine 11.6 ± 0.3a 37.50.8b 18.5 ± 0.4a 18.9 ± 0.4a 5.5 ± 0.1a 7.3 ± 0.2b 13.2 ± 0.3b 10.0 ± 0.2a 12.8 ± 0.3a 11.4 ± 0.3a 10.3 ± 0.3a 10.2 ± 0.2a Lysine 40.0 ± 0.9a 41.6 ± 0.9a 58.5 ± 1.3a 153.7 ± 3.5b 9.8 ± 0.5a 20.2 ± 0.5b 303.5 ± 6.8a 301.2 ± 6.8a 69.1 ± 1.6a 120.9 ± 2.7b 5.1 ± 0.2a 11.3 ± 0.3b Isoleucine 16.3 ± 0.4a 54.4 ± 1.2b 778.3 ± 15.4 ± 0.3a 0.2a 0.4a 49.8 ± 1.1b 27.9 ± 0.6a - - 34.5 ± 0.7b 5.5 ± 0.2a 17.5b Leucine 9.7 ± 0.2a 18.5 ± 0.4b 103.6 ± 2.3b 12.3 ± 0.4a 6.1 ± 0.2a 5.1 ± 0.1a 78.9 ± 1.8b 22.6 ± 0.5a 9.1 ± 0.3b 6.7 ± 0.2a 73.1 ± 1.3b 9.9 ± 0.3a Phenylalanine 21.2 ± 0.5a 81.6 ± 1.8b 38.6 ± 0.9b 23.9 ± 0.5a 6.6 ± 0.3a 5.4 ± 0.2a 118.9 ± 2.7b 4.9 ± 0.1a 8.3 ± 0.2b 4.9 ± 0.1a 130.1 ± 3.3b 5.4 ± 0.1a *(−): Not detected. Note: All values are mean of three (n = 3) experiments, followed by ± Standard deviation. Letters (a,b) in the same column for each Pseudomonas strain indicate significant difference at 4°C and 28°C incubation temperatures. Bisht et al. SpringerPlus 2013, 2:667 Page 8 of 13 http://www.springerplus.com/content/2/1/667 Figure 4 The combined analysis of relationship/effect of stress metabolite with/on freezing survival [(4a) -10 degree C; (4b) -40 degree C] of Pseudomonas strains [automated linear model (95% CI)]. Himalayan region (Mishra et al. 2010; Remold et al. between −2to −7°C. All Pseudomonas strains demon- 2011; Cray et al. 2013b). strated very low level of type-III ice nuclei which is typ- Ice crystal formation is the primary risk associated ically active between −7to −10°C. Presentation of less with the freezing-thawing of microbial cells and leads to ice nuclei by the Pseudomonads indicates their freeze membrane damage, and parallels the situation of dehy- survival strategy by lowering the ice nucleating dration/desiccation of the cells. Crowe et al. (1984) re- temperature. Therefore, it can be suggested that low ice ported that the rehydration condition causes most nucleation activity of Pseudomonads makes them cap- damage to the cells. At low temperatures (both above able of inhibiting the ice formation, which might re- and below 0°C) the intracellular environment usually be- quired for freezing survival. comes dehydrated and this increases solute concentra- Microbes produce EPS, which is stored as a thick gel tion as well as free radical formation. As a result cold surrounding the cell, the major ecological characteristic and solute-stress are to a large extent inseparable (Chin of EPS is that it can form and maintain protective mi- et al. 2010). A key component of cryotolerance in bac- crohabitats around microorganism in aquatic and frozen terial cells is tolerance to desiccation, solute-induced atmosphere (Stoderegger and Herndl 1998; Decho 1990; stress, and oxidative stress (Hallsworth et al. 2003; Tamaru et al. 2005). We found that EPS production by Bhaganna et al. 2010; Gülez et al. 2012; Pablo et al. the Pseudomonas strains was higher at lower tempera- 2013; Jonathan et al. 2013). Lowering of ice nucleation tures (4 or 15°C) in comparison to their optimal growth temperature and controlling the freezing temperature temperature (28°C). Enhanced EPS production by the and shape of the ice crystal have been identified as two Pseudomonads at low temperature suggested that EPS possible strategies for microbial cells to avoid freezing plays an important role in desiccation protection or pre- conditions (Kawahara et al. 1991). Whereas development vention of drying of bacterial cells from freezing of freezing tolerance by producing cryoprotectant com- temperature (Figures 2 and 4; Roberson and Firestone pounds or adaptation of cytoplasmic enzymes to cold 1992). The production of EPS is associated with the bio- conditions for protecting cytoplasmic components is the film formation. The fact that the Himalayan strains over- produce EPS at low temperature might suggests that third strategy used by microbial cells to survive in freez- ing conditions as these molecules depress freezing point under these conditions, the Pseudomonas strains show for the protection of cells (Yamashita et al. 2002). In the higher biofilm formation, and even the process of root colonization enhances at low temperature (Mishra et al. present study, all Pseudomonas strains didn’t display the presence of type-I and/or type-II ice nuclei as found in 2011). ‘ice plus’ bacteria. Thus, all the collected strains were Studies of low temperature tolerance in microbial cells demonstrated that the flexibility of cellular macromole- considered as ‘ice minus’ bacteria because they lack Ina proteins present on bacterial cell wall that act as a nucle- cules can be the limiting factor/failure-point for growth ation centre for ice crystals, which are mostly active in windows at low temperatures, and showed that a Bisht et al. SpringerPlus 2013, 2:667 Page 9 of 13 http://www.springerplus.com/content/2/1/667 chaperonin (chao- and kosmotrop) can extend the biotic et al. 1999). High intracellular trehalose accumulation window for growth down to lower temperatures (Marge- was found in all Pseudomonas strains except P. fluores- sin and Schinner 1999; Ferrer et al. 2003). Hence, it can cence PPRs4 cells when they grown at 4°C prior to freez- be assumed that the collected Pseudomonads were also ing and the same was supported by linear modeling following the third type cold evading strategy to thrive analysis (Figure 4). Our findings were in congruence in freezing conditions by synthesizing various chaper- with earlier studies where accumulation of higher trehal- onin/cryoprotectants, i.e., sugars, polyols and amino ose was related with its cryoprotectant function (Kaasen acids, in order to protect their cytoplasmic components. et al. 1992; Mitta et al. 1997). Regarding P. fluorescens These cryoprotectants are known to depress freezing PPRs4, we can speculate that trehalose might replaced point to evade crystallization (Chattopadhyay 2002). by sucrose and plays similar role of cryoprotectant, as Raffinose, like other sugars plays a cryoprotective role by higher sucrose accumulation was noticed in the said interacting with membrane lipids and proteins and de- strain (Cray et al. 2013b). creases the risk of intracellular ice-crystal formation that The cryoprotectant property of glucose has been previ- causes cellular osmotic dehydration during cryopreserva- ously documented by Koda et al. (2002). On the similar tion (Agca et al. 2002; Tuncer et al. 2010). The effect of raf- lines, we found that P. lurida NPRp15 cells accumulated finose related to oxidative stress has been considered as an higher glucose content when grown at 28°C and also dem- indirect effect of sugar signaling and triggers the production onstrated freezing survival at −10°C. Hence, it can be sug- of specific reactive oxygen species (ROS), such as, hydroxyl gested that glucose plays a significant role in cryoprotection radicals’ scavengers (Van-den Ende and Valluro 2009). of microbial cells. Two more kosmotropic solutes mannitol Though, the role of raffinose has been defined in recent and sorbitol act as stress protectants and has been previ- years in alleviation of oxidative stress and as a cryoprotec- ously investigated (Chatuverdi et al. 1997; Kets et al. 1996; tant (Van-den Ende and Valluro 2009; Tuncer et al. 2010), Bhaganna et al. 2010). The principleroleofmannitolfor but, still no reports are available related to the accumula- the de novo-synthesized polyol mannitol in osmoadaptation tion of raffinose in bacterial cells in response to cold stress of a heterotrophic P. putida has been discovered recently conditions. Here, we found high accumulation of intracellu- (Bhaganna et al. 2010). Earlier studies reported that manni- lar raffinose content in all the tested Pseudomonads in re- tol accumulation increases in microbial cells under various sponse to theirgrowthat4°C priortofreezing at −10 and/ stress treatments, like, heat, salt and/or their combination or −40°C, and it was further confirmed through linear (Managbanag and Torzilli 2002; Chatuverdi et al. 1997). modeling analysis and supported its role in bacterial Analogous to above, we also noticed enhanced accumula- freeze survival (Figure 4). Likewise trehalose, raffinose is tion of intracellular mannitol and sorbitol in all the Pseudo- a kosmotrophic substance that has a stabilizing effect on monas strains except PPRs4 strain grown at 4 and 28°C, macromolecular structure (Cray et al. 2013a). Therefore, and it can be assumed that glycerol might replaced by man- it may possible or it can be hypothesized that the pro- nitol/sorbitol in these Pseudomonas strains as we failed to tective effect of raffinose as observed in current study of detect glycerol in bacterial cells. Himalayan Pseudomonads utilizes a different mechenism Moreover, the collected cold tolerant Pseudomonas from that of glycerol and fructose (Chin et al. 2010). strains were found to protect cytoplasmic components The significance of chao- and kosmotropicity for the by synthesizing specific free amino acids needed for maintenance of structure and activities of macromolecu- freezing survival and cold adaptation of the microbial lar systems have been well characterized in-vitro, cells. These amino acids act as chemical chaperones whereas, the degree to which they facilitate and/or limit which prevent the aggregation of cellular proteins during the activities of cellular macromolecules in-vivo remains stress conditions and their possible function is to regu- relatively unclear (Duda et al. 2004; Chin et al. 2010; late the fluidity of membrane at lower temperatures Cray et al. 2013a). Nevertheless, it has been established (Chattopadhyay and Jagannadham 2001; Chattopadhyay that chaotropicity-mediated stresses elicit specific stress 2002; Ferrer et al. 2003; Bhaganna et al. 2010; Jonathan responses in microbial cells (Bhaganna et al. 2010). et al. 2013). Enhanced production of intracellular proline Chaotropicity has been shown to not only limit life pro- has been reported in microorganisms in order to im- cesses but can render potential environmental habitats prove their freeze tolerance and osmotic stress (Morita (Hallsworth et al. 2007; Cray et al. 2013a). The kosmo- et al. 2003; Jonathan et al. 2013; Kempf and Bremer tropicity nature of trehalose (non-reducing disaccharide) 1998). This indicates that proline accumulation might be plays an important role in developing the ability of or- a general protective strategy against freeze stress evasion. ganisms to resist against adverse environmental condi- Additionally, the intracellular accumulation of charged tions (Kandror et al. 2002; Cray et al. 2013a). Trehalose amino acids, viz., arginine, aspartic acid and glutamate stabilizes the membrane and proteins by replacing water also seems to enhance microbial freeze tolerance and act and preserves the intracellular water structure (Sano as cryoprotectants (Shima et al. 2003; Jenkelunas 2013). Bisht et al. SpringerPlus 2013, 2:667 Page 10 of 13 http://www.springerplus.com/content/2/1/667 These amino acids thought to play important roles as regions of NW Himalaya (Mishra et al. 2011; Bisht et al. general acids in enzyme active centers, as well as in 2013). Bacterial cultures were maintained on Nutrient Agar maintaining the solubility and ionic character of proteins (NA) and Kings B slants, respectively, and preserved in 60% (Shima et al. 2003; Jenkelunas 2013). In view of previous glycerol at −80°C. The submission details of all the six reports, high accumulation of intracellular proline, ar- Pseudomonas strains and their growth curve studies (at ginine and glutamate in collected Pseudomonads sug- three different temperatures, i.e., 4°C, 15°C and 28°C) have gests their cryoprotective role for freezing survival. been published earlier (Mishra et al. 2008; 2009; 2011). All Cysteine and methionine are sulphur-containing amino reagents were of analytical grade and procured from Merck, acids. Cysteine is a powerful antioxidant and can react Sigma Aldrich, HiMedia Laboratories. with itself to form an oxidized dimer by forming a disul- fide bond. The environment within a cell is too reducing Assessment of survival after freezing-thawing for disulfides to form, but in the extracellular environ- Six bacterial cell samples (6 strains × 3 replicates = 18) ment, disulfides can form and play a key role in stabiliz- from each Pseudomonas strain were prepared and each ing many proteins (Sen 2005; Ladenstein and Ren 2008). set was grown separately into 5 ml LB medium at two Disulfide bonds are important for protection of bacteria different temperatures 4 and 28°C (48 h incubation as a reversible switch that turns a protein on or off when period for 4°C culture and 24 h for 28°C culture). Both bacterial cells are exposed to oxidation reactions. Hydro- cultures (4 and 28°C) were kept separately at preset gen peroxide (H O ) in particular could severely damage temperature of −10 and −40°C temperature for 48, 72 2 2 DNA and kill the bacterium at low concentrations if not and 96 h. Bacterial cultures were thawed at room for the protective action of the SS-bond (Ladenstein and temperature, appropriate dilutions were plated and incu- Ren 2008). Likewise, methionine mostly acts as a precur- bated at 28°C for 48 h and CFU counts were measured sor amino acid for glutathione. It plays an important role for 48, 72 and 96 h of freeze shifted bacterial cultures. in the antioxidant defense mechanism by reacting readily Freeze-thaw survival of Pseudomonas spp. was deter- with oxidants to form methionine sulfoxide (Livine et al. mined at −10 and −40°C by comparing the log CFU 1999). The present study suggests that high intracellular counts before and after the freezing treatment. All ex- cysteine and methionine synthesis in collected Pseudo- periments were performed in duplicates. monads grown at 4°C might be an integral part of cold survival strategy to avoid damages from oxidative stress Quantification of exopolysaccharide (EPS) production during cold conditions. Bacterial cells were grown in 100 ml Kings B and nutri- ent broth at three different temperatures, i.e., 4, 15 and Conclusions 28°C for 48 h. Following the incubation, bacterial cells In conclusion, it is the physicochemical diversity of stress were harvested and EPS was extracted following the protectants produced by Pseudomonads that confer their method of Underwood et al. (1995). Precisely, the bac- remarkable tenacity and stress tolerance. Each type of com- terial cells were centrifuged at 10,000 rpm (Sigma Model patible solute/cryoprotectant has protective effects via dif- 2 K15, Rotor No. 12132) at 4°C for 15 min. The cell pel- ferent mechanisms. Accumulation of diverse amino acids, let was washed twice with sterile distilled water, treated sugars and polyols (including EPS) under cold stress are with 10 mM EDTA (w/v), vortexed for 15 min, and fi- important characteristics of Himalayan psychrotrophic nally recentrifuged at 10,000 rpm for 20 min at 4°C to Pseudomonas strains. The most novel and intriguing find- extract the cell-bound EPS. Extraction process was re- ing of this study was, intrecellular accumulation of raffi- peated and EPS samples were pooled and precipitated nose, cysteine and aspartic acid in bacterial cells as a key using chilled acetone and centrifuged at 10,000 rpm for metabolites at low temperature. The characterization of 10 min. The cell pellet was collected and their dry these traits are potentially important for beginning to weight was measured. understand these adaptations in microbial community present in Himalayan region. The present findings are part Determination of ice nucleation activity of unfolded field of stress biology, and it will surely have Ice nucleation activities (INA) [ice nuclei per colony- implications for the studies related to microbial diversity forming-unit (CFU)] of potential cold tolerant Pseudo- present in extreme conditions of high altitudes. monas strains were measured by freeze-drop method (Vali 1971; Lindow 1990). Bacterial cultures were grown Materials and methods into 100 ml Luria broth (LB) medium at 4°C for 24 to Bacterial culture conditions and chemicals 96 h. One ml of culture was centrifuged at 6,000 rpm The psychrotrophic Pseudomonas strains used in the for 5 min at 4°C and collected pellet was washed twice present study were previously isolated from different plant with 0.85% phosphate buffer saline (PBS; pH 7.2; w/v). root zones that were collected from the high altitude The cell pellet was resuspended into 1.0 ml of phosphate Bisht et al. SpringerPlus 2013, 2:667 Page 11 of 13 http://www.springerplus.com/content/2/1/667 buffer (pH 7.2) and vortexed vigorously. Nearly 10 μl were procured from Waters Corporation, USA. Acetonitrile (equivalent to 30 drops) of the cell suspension was and deionised water were used as solvent-B. Samples were placed on a parafilm coated aluminum boat floating on prepared according to the manufacturer’s instruction. The an ethanol bath at preset temperature of −5 and −10°C. chromatographic analysis was performed on HPLC system The number of frozen droplets were counted after 2, 5, as mentioned in the upper section using multi ƛ fluores- 15 and 20 min, respectively and bacterial concentration cence detector (Waters 2475) attached with photodiode was measured by plating of serial dilution of bacterial array detector (Waters 2996). Ten μl sample was injected cells on Kings B medium followed by incubation for in a Waters AccQ� Tag™ (Waters, Ireland) column and sep- th 48 h. The ice nucleation activity was calculated and rated by mobile phase (60% acetonitrile and 1/10 concen- -1 expressed in log ice nuclei CFU (Vali 1971; Lindow tration of Waters AccQ� Tag™ buffer)atgradient flowrate -1 1990). of 1 ml min . The chromatographic parameters, like, de- tector gain value, column temperature, and run time were Preparation of intracellular cell extract for detection of maintained as 10, 25°C and 65 min, respectively. Intracellu- cryoprotectants lar free amino acids were quantified by measuring peak area Two sets (6 × 2 = 12) of each bacterial isolate was prepared using the external standard method and samples were ana- and grown separately into 100 ml LB broth medium at 4 lyzed in triplicate. and 28°C for 36 h. Afterwards, the cultures were centri- fuged at 5°C for 10 min at 8,000 rpm and resultant cell pel- Stastistical analysis lets were washed three times with 0.85% PBS (pH 7.2). The Descriptive statistics was employed to represent the means cell pellets were resuspended into 1.0 ml of phosphate buf- andstandarddeviations. Student’s t-test was used to com- fer (pH 7.2) and disrupted by sonication (Soni Prep 150, pare the mean values at 4 and 28°C for stress metabolites. Sanyo) in3cyclesat8 μm (amplitude) for 2 min with In order to investigate the expression pattern of stress me- 45 sec cooling interval. The cell debris of each culture was tabolites for individual isolate and its correlation with removed by centrifugation at 10,000 for 15 min at 4°C. The growth temperatures (4 and 28°C), Principle component supernatant was filtered (0.22 μm) and samples were stored analysis (PCA) was performed for all cold stress related at −20°C. Standard sugar solutions of specific concentration measured parameters along with the growth temperature. -1 (100, 200, 300, 400 and 500 mg l )werepreparedinphos- PCA analysis was carried out using the XLSTAT (version phate buffer (pH 7.0). This experimental part was per- 2013) program. Additionally, to predict the relationship and formed in duplicate. effect of cold stress metabolites on bacterial freezing sur- vival at −10 and −40°C, an automated linear modeling Analysis of intracellular sugars (ALM), using forward stepwise with information criterion High performance liquid chromatography (HPLC) (Waters (standard model) was performed. For the modeling, data Corporation, USA) analysis was employed to analyze the gathered during bacterial growthat boththe temperatures intracellular sugars. The HPLC system consisted of an iso- (4 and 28°C) were combined and analyzed using SPSS pro- cratic pump (Waters, 600 Delta), DES-1008D interface (D- gram. The linear modeling was performed with the goal of Link, China), Waters temperature controller model TC2 selection of the most explanatory model that can explain and evaporative light scattering detector (Waters 2424 the relationship and effect between independent (stress me- ELSD) controlled by the ‘Empower’ program. Waters tabolite) and dependent (freezing survival) variables. Spherisorb 5 μmNH (250 × 4.6 mm) chromatographic column was used during the analysis. Ten μlofthe sample Additional files was injected into the Spherisorb column and separated with the mobile phase (67% acetonitrile and 33% water) at a flow Additional file 1: Figure S1a. HPLC chromatogram of intracellular -1 sugars’ and polyols’ contents of Pseudomonas strains grown at 4 and rate of 1 ml min . The chromatographic parameters, like, 28°C. Figure S1b. HPLC chromatogram of intracellular sugars and gas pressure, detector gain value, column temperature, run polyols’ contents of Pseudomonas lurida NPRp15 Grown at 4°C and time and nebulizer tube temperature were 50 psi, 10, 25°C, 28°C. Figure S1c. HPLC chromatogram of intracellular sugars and polyols’ contents of Pseudomonas sp. PPERs23 grown at 4°C and 28°C. 30 min and 60°C, respectively. Various sugars, like, Figure S1d. HPLC chromatogram of intracellular sugars and polyols’ D-Xylose, D-Glucose, D-Sorbitol, Trehalose and Raffinose contents of Pseudomonas putida PGRs4 grown at 4°C and 28°C. were quantified using external standard method, and Figure S1e. HPLC chromatogram of intracellular sugars and polyols’ contents of Pseudomonas sp. PGERs17 grown at 4°C and 28°C. samples were analyzed in triplicate. Additional file 2: Figure S2a. HPLC chromatogram of intracellular amino acids’ contents of Pseudomonas strains grown at 4 and 28°C. Analysis of intracellular free amino acids Figure S2b. HPLC chromatogram of intracellular amino acid contents of Amino acid standard H-kit, amino acid solvent-A (Aqueous Pseudomonas lurida NPRp15 grown at 4°C and 28°C. Figure S2c. HPLC chromatogram of intracellular amino acid contents of Pseudomonas buffer, Waters AccQ� Tag™), derivatization regent that con- sp. PPERs23 grown at 4°C and 28°C. Figure S2d. HPLC chromatogram of tain AccQ� Fluro Borate buffer and AccQ� Fluro reagent Bisht et al. SpringerPlus 2013, 2:667 Page 12 of 13 http://www.springerplus.com/content/2/1/667 Duda VI, Danilevich VN, Suzina NF, Shorokhova AP, Dmitriev VV, Mokhova intracellular amino acid contents of Pseudomonas putida PGRs4 grown at ON, Akimov VN (2004) Changes in the fine structure of microbial cells 4°C and 28°C. Figure S2e. HPLC chromatogram of intracellular amino induced by chaotropic salts. Microbiology 73:341–349 acid contents of Pseudomonas sp. PGERs17 grown at 4°C and 28°C. Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN (2003) Figure S2f. HPLC chromatogram of intracellular amino acid contents of Chaperonins govern growth of Escherichia coli at low temperatures. Pseudomonas fluorescens PPRs4 grown at 4°C and 28°C. Nat Biotechnol 21(11):1266–1267 Additional file 3: Table S1. Comparative analysis of stress metabolites Gülez G, Dechesne A, Workman CT, Smets BF (2012) Transcriptome dynamics accumulation/production at cold (4°C) and optimum growth temperature of Pseudomonas putida KT2440 under water stress. Appl Environ (28°C) for all six Pseudomonas strains (combined average of all strains). Microbiol 78(3):676–683 Hallsworth JE, Heim S, Timmis KN (2003) Chaotropic solutes cause water stress in Pseudomonas putida. Environ Microbiol 12:1270–1280 Competing interests Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JLM, D’Auria G, Alves FDL et al The authors report no conflict of interests. (2007) Limits of life in MgCl -containing environments: chaotropicity defines the window. Environ Microbiol 9:801–813 Authors’ contributions Jenkelunas P (2013) Production and Assessment of Pacific Hake Hydrolysates as a SCB conducted the study and prepared the manuscript. SH helped in Cryoprotectant. M.Sc thesis The University of British Columbia, Canada, https:// manuscript preparation which includes manuscript writing, reviewing and circle.ubc.ca/bitstream/handle/2429/43921/ubc_2013_spring_jenkelunas_peter. editing. GKJ provided technical advice and supervised research work. PKM pdf?sequence=3 (last accesed 7 May 2013) provided research project management role and supervised research work. Jonathan A, Cray A, Bell NW, Bhaganna P, Allen YM, Timson DJ et al (2013) The biology All the authors read and approved the final manuscript. of habitat dominance; can microbes behave as weeds? Microb Biotechnol, doi:10.1111/1751-7915.12027 Jones PG, Van Bogelen RA, Neidhardt FC (1987) Induction of proteins in response Acknowledgements to low temperature in Eschericha coli. J Bacteriol 169:2092–2095 The research leading to these results has funding from the Indian Council Kaasen I, Falkenberg P, Styrvold OB, Stroem AR (1992) Molecular cloning and of Agricultural Research (ICAR), New Delhi, India, under grant agreement physical mapping of the otsBA genes, which encode the osmoregulatory entitled ‘Development of bacterial consortium to alleviate cold stress’ a part of national level mega project ‘Application of microorganisms in agriculture trehalose pathway of Escherichia coli: evidence that transcription is and allied sectors (AMAAS)’. Special thanks, to Dr. J.C Bhatt, Director, Dr. J.K. activated by Kat F (AppR). J Bacteriol 174:889–898 Kandror O, DeLeon A, Goldberg AL (2002) Trehalose synthesis is induced upon Bisht, Head, CPD & Technical cell, V.P.K.A.S, Almora-263601, Uttarakhand, India exposure of Escherichia coli to cold and is essential for viability at low for their support and encouragement during study. temperatures. 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Limnol Oceanogr 40:1243–1253 7 Convenient online submission Vali G (1971) Quantitative evolution of experimental results on the 7 Rigorous peer review heterogeneous freezing nucleation of super cooled liquids. J Atmos Sci 7 Immediate publication on acceptance 28:402–409 7 Open access: articles freely available online Van-den Ende W, Valluro R (2009) Sucrose, sucrosyl oligosaccharides, and 7 High visibility within the fi eld oxidative stress: scavenging and salvaging. J Exp Bot 60:9–18 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png SpringerPlus Springer Journals

Cryotolerance strategies of Pseudomonads isolated from the rhizosphere of Himalayan plants

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Abstract

The cold stress biology of psychrotrophic Pseudomonas strains isolated from the rhizosphere of Himalayan plants have been explored to evaluate their cryotolerance characteristcs. Pseudomonas strains were examined for stress metabolites, viz., exopolysaccharide (EPS) production, intracellular sugar, polyols and amino acid content, ice nucleation activity, and their freezing survival at −10 and −40°C, respectively. High freezing survival was observed for the Pseudomonas strains that were grown at 4°C prior to their freezing at −10 or −40°C. Increased EPS production was noticed when Pseudomonas strains were grown at lower temperatures, i.e., 4 and 15°C, in comparison with their optimal growth temperature of 28°C. All Pseudomonas strains showed low level of type-III class ice nucleation activity at −10°C after 96 h. Considerable differences were noticed in accumulated contents of various intracellular sugars, polyols, amino acids for all Pseudomonas strains when they grown at two different temperatures, i.e., 4 and 28°C, respectively. The unusual complement of stress protectants especially, raffinose, cysteine and aspartic acid that accumulated in the bacterial cells at low temperature was novel and intriguing finding of this study. The finding that raffinose is a key metabolite accumulated at low temperature is an exciting discovery, and to the best of our information this is first report ever signifying its role in bacterial cold tolerance. Keywords: Psychrotrophic; Pseudomonas; Cold tolerance; Raffinose; Exopolysaccharide; Free amino acids Background causing the maintenance of some enzymatic functions in- Microorganisms have a range of evolutionary adaptations vivo (Yamashita et al. 2002). However, a limited information and physiological acclimation mechanisms that allow them is available about the cryoprotectants that are responsible to survive and remain active in the conditions of environ- for the freezing resistance mechanisms of bacteria. Bacteria mental stress. Adaptation towards stress condition is indis- often encounter freezing conditions and can survive in ex- pensable for survival, mainly when it causes alterations to tremely cold environments, like, the high altitude regions of the cell metabolism. Sudden decrease in temperature has Himalaya. In frozen environments, bacteria are exposed to severe effects on microbial cells, like, reduction of mem- conditions that necessitate the removal of water to maintain brane fluidity, decrease in ribosome efficiency, and in- the structure and function of the bacterial cell. As water creased stabilization of secondary structures of nucleic contributes to the stabilization of various macromolecular acids, which may affect transcription, translation and DNA structures, any significant deviation from the accessibility of replication (Phadtare et al. 2000). In order to survive under water due to dehydration, desiccation or an alteration of its freezing conditions, bacteria have developed various stra- physical state from aqueous phase to an ice crystal form tegies for their endurance, such as, maintenance of mem- poses a severe threat to the normal cell functions and sur- brane fluidity, constant metabolic activities etc. (Ramos vival of organism (Beall 1983; Crowe et al. 1984). et al. 2001). Additionally, it has been suggested that treha- In this regard, regulatory proteins and key metabolic lose, glycerol and sorbitol are the major cryoprotectants for enzymes require adjustments to cope with the temperature prokaryotic cells to response the freezing damage, thereby shifts in order to maintain a balanced microbial growth at the new environmental temperature. Under such condi- tions, the synthesis of specific cryoprotectant molecules * Correspondence: shekhar.bisht@hotmail.com might be enhanced that act as chemical chaperons and pro- Department of Biotechnology, HNB Garhwal University (A Central University), Srinagar 246174, Uttarakhand, India tect the cellular proteins from freezing temperature. Scanty Full list of author information is available at the end of the article © 2013 Bisht et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Bisht et al. SpringerPlus 2013, 2:667 Page 2 of 13 http://www.springerplus.com/content/2/1/667 reports are available on psychrotrophic bacterial cryotoler- EPS production ance strategies and related responsible molecules except EPS production was found to be higher at lower incubation cold shock (Csps) and cold acclimation (Caps)s proteins. temperatures (4 or 15°C) in comparison to the optimal Although, bacterial cryotolerance has been investigated in growth temperature (28°C) in all the Pseudomonas strains relevance with role of trehalose and glycine betine in (Figure 2). At 4°C, P. lurida NPRs3 produced 2.75 and 8.8 Escherichia coli and Bacillus subtilis, respectively (Jones folds higher EPS in comparison to EPS produced at 15 and et al. 1987; Willimsky et al. 1992). But, very little is known 28°C, respectively. At 15°C, P. lurida NPRs3 showed 3.2 about the possibility of other molecules responsible for folds higher EPS production in comparison with the same the survival of bacteria subjected to freezing challenge by cells grown at 28°C. Similarly, the P. lurida NPRp15 cells an adaptation of the microbial cells to low temperatures demonstrated 1.38 and 7.0 folds higher EPS production at particularly in psychrotolerant/psychrophilic bacteria (Margesin 4°C compared to the cells grown at 15 and 28°C, respect- and Schinner 1999; Mishra et al. 2010). ively. At 15°C, the P. lurida NPRp15 culture produced 5.07 In the upper parts of north west (NW) Himalaya, winter folds greater EPS as comparedtoculture grownat 28°C. is mostly characterized by intermittent snow cover Likewise, the cells of Pseudomonas sp. PPERs23 grown at (November to March) and fluctuating subfreezing tempera- 4°C showed 23.33 and 21.31% higher EPS accumulation as tures, while summer displays intense desiccating sunshine compared to the cells cultivated at 15 and 28°C, respect- punctuated by infrequent rains (Mishra et al. 2008; 2011; ively. However, the Pseudomonas sp. PPERs23 culture Bisht et al. 2013). These conditions pose additional chal- grown at 15 and 28°C showed almost similar EPS accumu- lenges to microbial species that may endure summer tem- lation (Figure 2). Similarly, the P. putida PGRs4 cells peratures as high as 30°C and winter temperatures that can showed 23.14% greater EPS accumulation at 4°C in com- dip to −10°C, as well as alternating freezing and thawing parison to the P. putida PGRs4 cells grown at 28°C. At periods during the cold season. At these temperatures, mi- 15°C, the P. putida PGRs4 culture showed 17.6% enhanced croorganisms might be injured or killed as a result of cold EPS production than the P. putida PGRs4 cells cultivated shock, freezing, prolonged exposure to subzero tempera- at 28°C. Likewise, the cells of Pseudomonas sp. PGERs17 tures, and subsequent warming, and injury or death is often cultivated at 4°C showed 66.0% increased EPS production due to damage to membranes or cell walls that results in in comparison to the cells grown at 28°C, whereas, the changes in permeability, as well as damage to DNA. Given Pseudomonas sp. PGERs17 cells separately grown at 4 and these challenges, the fact that soil bacteria thrive in NW 15°C failed to show significant difference in EPS accumula- Himalayan regions is a testimony either to environmental tion. P. fluorescens PPRs4 demonstrated almost double heterogeneity or to the remarkable adaptive abilities of amount of EPS production at 4°C growth temperature in these psychrotrophic microbes (Srinivas et al. 2011; Bisht comparison to cells grown at 15 or 28°C. et al. 2013). The precise mechanisms or molecular stra- tegies underlying the cellular adaptations of psychrotrophic INA of psychrotolerant Pseudomonas strains bacterial cells in cold conditions are not clear and needs to The INA of Pseudomonads had been measured to deter- be addressed, particularly for varying genus of Pseudo- mine the catalytic sites present in the bacterial cells respon- monas. In this context, the present study was undertaken sible for ice formation. None of the collected strains to investigate the freezing survival strategies operated demonstrated type-I or type-II category INA measured in six psychrotrophic Pseudomonas strains (P. lurida at −5°C. All Pseudomonads showed low level of type-III NPRs3, P. lurida NPRp15, P. sp. PPERs23, P. putida INA (active between −7to −10°C) measured at −10°C after PGRs4, P. sp. PGERs17 and P. fluorescens PPRs4) isolated 24, 72 and 96 h of bacterial growth (Table 1). The type-III previously from rhizosphere of NW Himalayan plants INA of all Pseudomonas strains was found to be low at long (Mishra et al. 2011; Bisht et al. 2013). (96 h) incubation of the culture. Whereas, the same was noted high for cultures incubated for short (24 h) time Results period. The highest INA was found in P. fluorescens PPRs4 Bacterial growth and freeze survival andthe lowest INAwas observedin Pseudomonas sp. Freezing survival studies of Pseudomonas strains re- PGERs17 after 24 h incubation period. The mean INA (log -1 vealed that strains which were grown at 4°C prior to ice nuclei CFU )of P. lurida NPRs3, P. lurida NPRp15, freezing separately at −10 and −40°C demonstrated sig- and P. fluorescens PPRs4 was noticed to be higher at 4°C nificantly higher freezing survival rather than cultures after 24 h growth incubation in comparison to 96 h of bac- which were grown at 28°C prior to freezing (Figure 1). It terial growth incubation at the same temperature. Whereas, was observed that Pseudomonas strains grown at low after 24 h growth incubation at 4°C, Pseudomonas sp. temperature (e.g., 4°C) have a survival advantage upon PPERs23, P. putida PGRs4, and Pseudomonas sp. PGERs17 freezing tolerance compared to their optimal growth showed difference of 0.85, 0.97 and 0.77 log ice nuclei -1 temperature (28°C). CFU , respectively, as compared to 96 h growth incubation Bisht et al. SpringerPlus 2013, 2:667 Page 3 of 13 http://www.springerplus.com/content/2/1/667 Figure 1 Percentage survival of Pseudomonads subjected to freezing temperature [−10 and −40°C] shifted from two different incubation temperatures (4 and 28°C). Note: All values are mean of three independent replicates and bar represents the standard error of mean. at 4°C. These differences indicated lowering in INA of all significant increase in intracellular raffinose was no- Pseudomonas strains with long incubation time (96 h) in ticed during cold condition (at 4°C) in all the Pseudo- cold conditions (e.g., 4°C). monas isolates (Table 2, Figure 3). Accumulation of sucrose molecule at 4°C was found in P. fluorescens Accumulation of intracellular sugars and polyols PPRs4, while,at28°Citwas notdetected. Likewise, Remarkable variations in terms of accumulation of glucose molecule was not detected in bacterial growth various intracellular sugars and polyols were noticed at 4°C for all the isolates, while P. lurida NPRp15 through HPLC chromatogram (Additional file 1: showed noticeable amount of glucose at 28°C. Trehal- Figure S1), when all the Pseudomonas cells were ose was accumulated in high amount in all the iso- grown at 4 and 28°C (Table 2). The bacterial intracel- lates at 4°C. Pseudomonas sp. PPERs23 accumulated lular sugar content was expressed in terms of μgper significant amount of trehalose, mannitol and sorbitol, -1 -1 mg of cell dry weight (μgmg CDW ). It was found when grown at 28°C (Table 2). Accumulation of man- that the accumulation of intracellular sugars varied in nitol was found only in four isolates, interestingly different psychrotrophic Pseudomonas strains at 4 and three out of four isolates showed higher accumulation 28°C. Such changes were observed in the amount of of mannitol at 28°C, P. putida PGRs4 showed higher glucose, trehalose, sucrose, mannitol and sorbitol. accumulation of mannitol at 4°C (Table 2). Greater Figure 3 shows the grouping of stress metabolites accumulation of sorbitol was noticed for NPRs3, based on their accumulation at two different tempera- PGRs4 and PGERs17 strains, when their cells were tures for individual strain and indicates variation grown at 4°C in comparison to the cells grown at in accumulation of stress metabolites for each iso- 28°C. Whereas, Pseudomonas sp. PPERs23 accumu- late. Most importantly, a prominent and statistically lated significant amount of sorbitol at 28°C. Bisht et al. SpringerPlus 2013, 2:667 Page 4 of 13 http://www.springerplus.com/content/2/1/667 Figure 2 EPS accumulation by Pseudomonas strains at three different incubation temperatures (4, 15 and 28°C). Note: All values are mean of three independent replicates and bar represents the standard error of mean. The alphabet letters (a, b, c) in the column for individual Pseudomonas strain indicate significant differences at 4, 15 and 28°C incubation temperature. Accumulation of intracellular amino acids phenylalanine at 4°C was found among all the isolates, The intracellular free amino acids’ concentrations in on the other hand reduction in concentrations of gly- Pseudomonas strains were investigated under optimal cine, glutamic acid, lysine and methionine at 28°C was (28°C) and low (4°C) temperature conditions. HPLC observed for most of the isolates. The P. lurida NPRs3 chromatograms of the Pseudomonas cells for 17 amino cells showed highest increase in concentration of aspar- acids tested from their intracellular extract demonstrated tic acid, serine, glycine, arginine, and proline at 4°C, significant variations at 4 and 28°C growth temperatures while no change was observed in lysine concentration. (Additional file 2: Figure S2 and Table 3). The intracellu- Substantial increase in accumulation of isoleucine (50.4 lar amino acids’ expression pattern varied from strain to folds) and cysteine (5.6 folds) was noticed, whereas no strain at 4 and 28°C, and the most prominent increase changes were observed for glutamic acid and methionine was observed in the concentrations of aspartic acid, pro- for P. lurida NPRp15 cells grown at 4°C. Prominent in- line and cysteine at 4°C (Table 3 and Figure 3). All crease in phenylalanine (24.2 folds) followed by histidine Pseudomonas strains exhibited statistically significant in- (4.6 folds), threonine (3.6 folds) and leucine (3.5 folds), crease in their aspartic acid (1.3 to 3.0 folds) and proline and minor increase in tyrosine concentrations were (1.1 to 2.8 folds) contents at 4°C. Significant accumula- observed at 4°C in P. putida PGRs4 cells (Table 3). For tion of serine, arginine, threonine, cysteine, leucine and Pseudomonas sp. PGERs17 cells, enhanced concentrations Table 1 Ice nucleation activity of cold tolerant Pseudomonas strains at −10°C temperature Cold tolerant Culture incubation at 4°C Pseudomonas 24 h 72 h 96 h strains -1 Ice nucleation activity of bacterial culture at −10°C (log ice nuclei cfu ) After After After After Mean After After After After Mean After After After After Mean 5 min 10 min 15 min 20 min 5 min 10 min 15 min 20 min 5 min 10 min 15 min 20 min P. lurida −8.47 −8.47 −8.47 −7.36 −7.15 −9.19 −9.19 −8.89 −8.49 −8.11 −9.17 −9.17 −8.8 −8.75 −8.47 NPRs3 P. lurida −7.43 −7.43 −7.33 −7.24 −7.17 −8.49 −8.49 −8.14 −8.08 −8.01 −8.96 −8.96 −8.78 −8.62 −8.56 NPRp15 Pseudomonas −6.68 −6.68 −6.68 −6.68 −6.38 −7.9 −7.9 −7.9 −7.56 −7.26 −7.74 −7.74 −7.74 −7.43 −7.23 sp. PPERs23 P. putida −7.58 −7.40 −6.82 −6.65 −6.18 −7.33 −7.24 −7.17 −7.17 −7.36 −8.49 −8.14 −8.08 −7.15 −7.15 PGRs4 Pseudomonas −7.92 −7.82 −7.78 −7.62 −7.52 −8.47 −8.37 −8.27 −8.24 −8.07 −8.89 −8.59 −8.69 −8.49 −8.29 sp. PGERs17 P. fluorescens −6.82 −6.65 −5.86 −5.80 −5.88 −8.49 −7.92 −6.82 −6.65 −6.65 −8.58 −8.19 −7.9 −7.56 −7.26 PPRs4 Bisht et al. SpringerPlus 2013, 2:667 Page 5 of 13 http://www.springerplus.com/content/2/1/667 Table 2 Quantitative analysis of intracellular sugars and polyols content of Pseudomonas strains by HPLC -1 -1 Pseudomonas strains Temperature Content (μgmg cell dry weight )* Sugar’ content Polyols content Raffinose Sucrose Trehalose Glucose Mannitol Sorbitol P. lurida NPRs3 28°C 8.92 ± 1.52a - - - 2.73 ± 0.52a - 4°C 19.79 ± 2.26b - 1.89 ± 0.21 - 2.23 ± 0.34a 9.36 ± 1.61 P. lurida NPRp15 28°C - - - 105.4 ± 3.9 43.23 ± 3.63 - 4°C 41.69 ± 3.39b - 3.19 ± 0.81 - - - Pseudomonas sp. PPERs23 28°C 3.18 ± 1.28a - 16.46 ± 1.62b - 7.12 ± 1.07b 22.06 ± 1.96b 4°C 13.41 ± 1.76b - 2.49 ± 0.48a - 0.55 ± 0.01a 3.80 ± 1.56a P. putida PGRs4 28°C 4.38 ± 0.97a - 0.44 ± 0.09a - - 3.17 ± 1.01a 4°C 11.25 ± 1.13b - 3.14 ± 0.84b - 3.92 ± 0.66 50.49 ± 3.68b Pseudomonas sp. PGERs17 28°C 1.56 ± 0.54a - - - - 0.91 ± 0.03a 4°C 9.90b - 3.02 ± 1.6 - - 4.32 ± 0.98b P. fluorescens PPRs4 28°C 6.76 ± 1.38 - - - - - 4°C 14.22 ± 1.74 3.33 ± 1.25 - - - - *(−): Not detected. Note: All values are mean of three (n = 3) experiments, followed by ± Standard deviation. Letters (a,b) in the same column for each Pseudomonas strain indicate significant difference at 4°C and 28°C incubation temperatures. were found for tyrosine (21.3 folds), threonine (4.2 folds), optimum growth temperature of 28°C. The parameters glutamic acid (3.9 folds), and proline (2.7 folds) at 4°C, placed at the negative side of the factor F2 showed less whilenochanges were noticed forarginine(Table3). Max- accumulation and less activity at 28°C, and high correl- imum increase was found for cysteine (46.5 folds) followed ation with cold temperature. Only freezing survival par- by leucine (7.4 fold), isoleucine (6.3 folds) and serine (4.5 ameter was found on the negative side of the factor F2 folds) in P. fluorescens PPRs4 cells at 4°C (Table 3). How- for five Pseudomonas strains with exception of NPRs3 ever, low concentrations of glutamic acid, tyrosine, and ly- cells (Figure 3[a]). The parameters which were grouped sine were found in the same cells at 4°C (Table 3). together in the PCA plot showed high correlation, whereas parameters which were grouped in opposite dir- Stastistical analysis ection indicated the negative correlation. The parame- Correlation analysis proved existence of significant rela- ters placed at the middle of the PCA model reflected no tionship between the measured cold stress parameters correlation with the factors (i.e., growth temperatures) and the bacterial growth conditions (i.e., temperature: and showed either equal or nil accumulation. These coordinate). The PCA for correlation of individual iso- include no accumulation of glucose and sucrose late has been shown in Figure 3. The first two factorial for five strains except NPRp and PPRs4, respectively axes represent 94.7 to 97.14% variance in the data. Ex- (Figure 3[b,f]). cept Pseudomonas NPRs3, factor F1 represented the The relationship/effect of measured cold stress metab- grouping of stress metabolites and reflected their sub- olites on bacterial freezing survival at −10 and −40°C stantial accumulation at 4°C. Factor F2 represented the was evaluated using automated linear modeling (ALM) grouping of stress metabolites accumulated maximally at analysis (Figure 4). The model contained only important 28°C. Maximum stress metabolites were accumulated at predictor(s) (i.e., stress metabolites) for freezing survival. 4°C for two Pseudomonas isolates, i.e., NPRp15 and Both the models were found statistically significant PPRs4 (Figure 3[b,f]). The parameters placed at a strong (p < 0.05) with 70 to 75% accuracy. Six cold stress pa- positive side of the factor F1 were highly correlated with rameters were identified as important predictors for the cold temperature and indicated higher accumulation freeze survival at −10°C (Figure 4[a]). The incubation at cold temperature. However, the parameters which temperature showed significant correlation (p = 0.000) were placed at negative side of the factor F1 showed with freeze survival at −10°C and demonstrated import- negative correlation with the cold temperature and indi- ant role (42%) in bacterial freeze survival. Bacterial sur- cated less or nil production at cold temperature. Simi- vival to freezing conditions was paralleled by an increase larly, the parameters which were placed at the strong in the intracellular raffinose level and showed significant positive side of the axis of factor F2 indicated minor role association (p = 0.000) with freezing survival at −10°C, in cold adaptation and showed higher accumulation at and also suggested that raffinose contribute in bacterial Bisht et al. SpringerPlus 2013, 2:667 Page 6 of 13 http://www.springerplus.com/content/2/1/667 Figure 3 Principal component analysis (PCA) of stress metabolites profile of Pseudomonas strains grown at 4 and 28°C. [a] P. lurida NPRs3 [b] P. lurida NPRp15 [c] Pseudomonas sp. PPERs23 [d] P. putida PGRs4 [e] Pseudomonas sp. PGERs17 [f] P. fluorescens PPRs4. (Factor map of rows (metabolite); stress metabolite with similar distributions of appearance with increases in cold condition occur in similar positions on the map). freeze survival. Likewise, high accumulation of cysteine, level environmental ubiquity even in cold/freezing habi- trehalose, aspartic acid and proline showed positive rela- tats (Timmis 2002; Remold et al. 2011; Cray et al. tionship with bacterial high freezing survival at −10°C 2013b). This study clearly demonstrates that cold accli- with 15.6, 9.1, 6.5 and 3.2% contribution, respectively. matized cells (grown at 4°C) had higher freezing- EPS and sorbitol were identified as most important thawing survival over non-acclimatized cells (grown at stress metabolites (predictors) for bacterial freeze sur- 28°C), and linear modeling analysis clearly proved that vival at −40°C (Figure 4[b]). the incubation temperature was the main factor for Pseudomonads’ freezing survival. The overall freezing- Discussion thawing survival of Pseudomonas strains at −10°C was The Himalayan region provides an opportunity to obtain found be 98% (Additional file 3: Table S1). This feature microbes that have experienced extended exposure to of Pseudomonads suggests their survival persistence in cold temperatures, reduced water activities, radiation Himalayan extreme freezing-thawing conditions during and low nutrient accessibility. The cold adaptation re- winter season. One of the major freezing survival strat- lated properties of psychrotrophic Pseudomonas cells egy might be the evolution of strains that are capable of showed high cellular metabolism activities in cold condi- utilizing a large number of carbon sources (Ponder et al. tions (Mishra et al. 2008; 2009; 2011). It has been well 2005). This might be more relevant because the alpine established that the cold-active enzymes and efficient environment is highly heterogeneous with pockets of growth rates are used to facilitate and maintain the ad- specific carbon compounds, a large number of bacterial equate metabolic fluxes at near freezing temperature for strains that have recently gained or lost the ability to cold-adaptation (Shivaji and Prakash 2010). The great grow on a particular source of carbon may exist and metabolic flexibility of Pseudomonas species allows them supported the adaptability, versatility and environmental to inhabit diverse environments and capable of a high ubiquity, and prevalence of Pseudomonas genus in the Bisht et al. SpringerPlus 2013, 2:667 Page 7 of 13 http://www.springerplus.com/content/2/1/667 Table 3 Quantitative analysis (HPLC) of intracellular amino acids’ content of Pseudomonas strains at 4°C and 28°C growth temperatures Amino acid (pico mole Pseudomonas strains -1 -1 mg cell dry wt. )* P. lurida NPRs3 P. lurida NPRp15 Pseudomonas sp. PPERs23 P. putida PGRs4 Pseudomonas sp. P. fluorescens PPRs4 PGERs17 At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C At 4°C At 28°C Aspartic acid 65.8 ± 1.5b 39.9 ± 0.9a 33.1 ± 0.7b 20.3 ± 0.5a 14.1 ± 0.3b 11.1 ± 0.2a 333.9 ± 7.5b 166.5 ± 3.7a 51.7 ± 1.2b 27.5 ± 0.6a 18.0 ± 0.5b 5.9 ± 0.3a Proline 4355 ± 98b 1997 ± 45a 7317 ± 164b 3751.2 ± 1880 ± 42b 1229.7 ± 27a 1467 ± 33b 1352.2 ± 30a 4733.3 ± 106b 1738.2 ± 1613 ± 26b 586.5 ± 13a 84a 39a Cysteine 32.8 ± 0.7a 89.7 ± 2b 439.4 ± 9.9b 78.2 ± 1.8a 67.8 ± 1.5 - 89.9 ± 2b 52.5 ± 1.2a 30.8 ± 0.7b 17.4 ± 0.4a 71.8 ± 1.8b 1.5 ± 0.1a Serine 485.0 ± 57.9 ± 1.3a 28.1 ± 0.6b 23.0 ± 0.5a 12.0 ± 0.4a 21.6 ± 0.5b 14.2 ± 0.3b 7.7 ± 0.2a 13.6 ± 0.3a 19.0 ± 0.4b 13.1 ± 0.6b 2.9 ± 0.1a 10.9b Glutamic acid 11.8 ± 0.3a 26.2 ± 0.6b 14.8 ± 0.3a 16.1 ± 0.4a 8.8 ± 0.2b 3.9 ± 0.1a 26.1 ± 0.6a 46.5 ± 1.1b 28.7 ± 0.6b 7.4 ± 0.2a 5.9 ± 0.3a 8.0 ± 0.4b Glycine 662.0 ± 347.5 ± 7.8a 274.5 ± 6.2a 370.7 ± 8.3b 350.8 ± 7.9a 184.7 ± 4.2b 137.7 ± 3.1b 121.5 ± 2.7a 351.2 ± 7.9a 533.3 ± 12b 288.8 ± 6.2b 272.9 ± 6.1a 14.9b Histidine 97.6 ± 2.2a 192.6 ± 4.3b 148.2 ± 3.3b 134.4 ± 3a 50.8 ± 1.1a 63.0 ± 1.4b 20.9 ± 0.5b 4.6 ± 0.1a 103.9 ± 2.3b 55.6 ± 1.3a 73.9 ± 1.8b 65.9 ± 1.5a Arginine 158.1 ± 3.6b 27.6 ± 0.6a 12.9 ± 0.3b 9.8 ± 0.2a 4.5 ± 0.1b 1.2 ± 0.1a 58.2 ± 1.3a 81.1 ± 1.8b 8.2 ± 0.2a 9.4 ± 0.2a 7.7 ± 0.3b 4.4 ± 0.1a Threonine 190.5 ± 4.3a 1084.3 ± 9194.7 ± 20b 3757.1 ± 5143.0 ± 6722.1 ± 6510.4 ± 1795 ± 40.4a 3003.8 ± 714.0 ± 16a 6479.2 ± 3890.4 ± 24b 84a 115a 151b 146b 67.6b 57b 87a Alanine 57.5 ± 1.3a 167.73.8b 107.9 ± 2.4b 94.1 ± 2.1a 38.3 ± 0.9a 39.7 ± 0.9a 104.6 ± 2.4a 890.9 ± 20b 91.0 ± 2b 66.3 ± 1.5b 46.5 ± 1.3a 50.1 ± 1.1b Tyrosine 30.2 ± 0.7a 46.5 ± 1.2b 39.2 ± 0.9b 19.6 ± 0.4a 10.7 ± 0.3a 9.5 ± 0.4a 6.5 ± 0.1a 3874.7 ± 16.8 ± 0.4b 0.8a 14.0 ± 0.5a 166.8 ± 3.8b 87b Valine 27.1 ± 0.6a 64.9 ± 1.5b 40.0 ± 1.9b 23.2 ± 0.5a 9.5 ± 0.2a 8.3 ± 0.3a 3.1 ± 0.1a 26.4 ± 0.6b 17.0 ± 0.4b 6.5 ± 0.1a 14.1 ± 0.4b 7.0 ± 0.2a Methionine 11.6 ± 0.3a 37.50.8b 18.5 ± 0.4a 18.9 ± 0.4a 5.5 ± 0.1a 7.3 ± 0.2b 13.2 ± 0.3b 10.0 ± 0.2a 12.8 ± 0.3a 11.4 ± 0.3a 10.3 ± 0.3a 10.2 ± 0.2a Lysine 40.0 ± 0.9a 41.6 ± 0.9a 58.5 ± 1.3a 153.7 ± 3.5b 9.8 ± 0.5a 20.2 ± 0.5b 303.5 ± 6.8a 301.2 ± 6.8a 69.1 ± 1.6a 120.9 ± 2.7b 5.1 ± 0.2a 11.3 ± 0.3b Isoleucine 16.3 ± 0.4a 54.4 ± 1.2b 778.3 ± 15.4 ± 0.3a 0.2a 0.4a 49.8 ± 1.1b 27.9 ± 0.6a - - 34.5 ± 0.7b 5.5 ± 0.2a 17.5b Leucine 9.7 ± 0.2a 18.5 ± 0.4b 103.6 ± 2.3b 12.3 ± 0.4a 6.1 ± 0.2a 5.1 ± 0.1a 78.9 ± 1.8b 22.6 ± 0.5a 9.1 ± 0.3b 6.7 ± 0.2a 73.1 ± 1.3b 9.9 ± 0.3a Phenylalanine 21.2 ± 0.5a 81.6 ± 1.8b 38.6 ± 0.9b 23.9 ± 0.5a 6.6 ± 0.3a 5.4 ± 0.2a 118.9 ± 2.7b 4.9 ± 0.1a 8.3 ± 0.2b 4.9 ± 0.1a 130.1 ± 3.3b 5.4 ± 0.1a *(−): Not detected. Note: All values are mean of three (n = 3) experiments, followed by ± Standard deviation. Letters (a,b) in the same column for each Pseudomonas strain indicate significant difference at 4°C and 28°C incubation temperatures. Bisht et al. SpringerPlus 2013, 2:667 Page 8 of 13 http://www.springerplus.com/content/2/1/667 Figure 4 The combined analysis of relationship/effect of stress metabolite with/on freezing survival [(4a) -10 degree C; (4b) -40 degree C] of Pseudomonas strains [automated linear model (95% CI)]. Himalayan region (Mishra et al. 2010; Remold et al. between −2to −7°C. All Pseudomonas strains demon- 2011; Cray et al. 2013b). strated very low level of type-III ice nuclei which is typ- Ice crystal formation is the primary risk associated ically active between −7to −10°C. Presentation of less with the freezing-thawing of microbial cells and leads to ice nuclei by the Pseudomonads indicates their freeze membrane damage, and parallels the situation of dehy- survival strategy by lowering the ice nucleating dration/desiccation of the cells. Crowe et al. (1984) re- temperature. Therefore, it can be suggested that low ice ported that the rehydration condition causes most nucleation activity of Pseudomonads makes them cap- damage to the cells. At low temperatures (both above able of inhibiting the ice formation, which might re- and below 0°C) the intracellular environment usually be- quired for freezing survival. comes dehydrated and this increases solute concentra- Microbes produce EPS, which is stored as a thick gel tion as well as free radical formation. As a result cold surrounding the cell, the major ecological characteristic and solute-stress are to a large extent inseparable (Chin of EPS is that it can form and maintain protective mi- et al. 2010). A key component of cryotolerance in bac- crohabitats around microorganism in aquatic and frozen terial cells is tolerance to desiccation, solute-induced atmosphere (Stoderegger and Herndl 1998; Decho 1990; stress, and oxidative stress (Hallsworth et al. 2003; Tamaru et al. 2005). We found that EPS production by Bhaganna et al. 2010; Gülez et al. 2012; Pablo et al. the Pseudomonas strains was higher at lower tempera- 2013; Jonathan et al. 2013). Lowering of ice nucleation tures (4 or 15°C) in comparison to their optimal growth temperature and controlling the freezing temperature temperature (28°C). Enhanced EPS production by the and shape of the ice crystal have been identified as two Pseudomonads at low temperature suggested that EPS possible strategies for microbial cells to avoid freezing plays an important role in desiccation protection or pre- conditions (Kawahara et al. 1991). Whereas development vention of drying of bacterial cells from freezing of freezing tolerance by producing cryoprotectant com- temperature (Figures 2 and 4; Roberson and Firestone pounds or adaptation of cytoplasmic enzymes to cold 1992). The production of EPS is associated with the bio- conditions for protecting cytoplasmic components is the film formation. The fact that the Himalayan strains over- produce EPS at low temperature might suggests that third strategy used by microbial cells to survive in freez- ing conditions as these molecules depress freezing point under these conditions, the Pseudomonas strains show for the protection of cells (Yamashita et al. 2002). In the higher biofilm formation, and even the process of root colonization enhances at low temperature (Mishra et al. present study, all Pseudomonas strains didn’t display the presence of type-I and/or type-II ice nuclei as found in 2011). ‘ice plus’ bacteria. Thus, all the collected strains were Studies of low temperature tolerance in microbial cells demonstrated that the flexibility of cellular macromole- considered as ‘ice minus’ bacteria because they lack Ina proteins present on bacterial cell wall that act as a nucle- cules can be the limiting factor/failure-point for growth ation centre for ice crystals, which are mostly active in windows at low temperatures, and showed that a Bisht et al. SpringerPlus 2013, 2:667 Page 9 of 13 http://www.springerplus.com/content/2/1/667 chaperonin (chao- and kosmotrop) can extend the biotic et al. 1999). High intracellular trehalose accumulation window for growth down to lower temperatures (Marge- was found in all Pseudomonas strains except P. fluores- sin and Schinner 1999; Ferrer et al. 2003). Hence, it can cence PPRs4 cells when they grown at 4°C prior to freez- be assumed that the collected Pseudomonads were also ing and the same was supported by linear modeling following the third type cold evading strategy to thrive analysis (Figure 4). Our findings were in congruence in freezing conditions by synthesizing various chaper- with earlier studies where accumulation of higher trehal- onin/cryoprotectants, i.e., sugars, polyols and amino ose was related with its cryoprotectant function (Kaasen acids, in order to protect their cytoplasmic components. et al. 1992; Mitta et al. 1997). Regarding P. fluorescens These cryoprotectants are known to depress freezing PPRs4, we can speculate that trehalose might replaced point to evade crystallization (Chattopadhyay 2002). by sucrose and plays similar role of cryoprotectant, as Raffinose, like other sugars plays a cryoprotective role by higher sucrose accumulation was noticed in the said interacting with membrane lipids and proteins and de- strain (Cray et al. 2013b). creases the risk of intracellular ice-crystal formation that The cryoprotectant property of glucose has been previ- causes cellular osmotic dehydration during cryopreserva- ously documented by Koda et al. (2002). On the similar tion (Agca et al. 2002; Tuncer et al. 2010). The effect of raf- lines, we found that P. lurida NPRp15 cells accumulated finose related to oxidative stress has been considered as an higher glucose content when grown at 28°C and also dem- indirect effect of sugar signaling and triggers the production onstrated freezing survival at −10°C. Hence, it can be sug- of specific reactive oxygen species (ROS), such as, hydroxyl gested that glucose plays a significant role in cryoprotection radicals’ scavengers (Van-den Ende and Valluro 2009). of microbial cells. Two more kosmotropic solutes mannitol Though, the role of raffinose has been defined in recent and sorbitol act as stress protectants and has been previ- years in alleviation of oxidative stress and as a cryoprotec- ously investigated (Chatuverdi et al. 1997; Kets et al. 1996; tant (Van-den Ende and Valluro 2009; Tuncer et al. 2010), Bhaganna et al. 2010). The principleroleofmannitolfor but, still no reports are available related to the accumula- the de novo-synthesized polyol mannitol in osmoadaptation tion of raffinose in bacterial cells in response to cold stress of a heterotrophic P. putida has been discovered recently conditions. Here, we found high accumulation of intracellu- (Bhaganna et al. 2010). Earlier studies reported that manni- lar raffinose content in all the tested Pseudomonads in re- tol accumulation increases in microbial cells under various sponse to theirgrowthat4°C priortofreezing at −10 and/ stress treatments, like, heat, salt and/or their combination or −40°C, and it was further confirmed through linear (Managbanag and Torzilli 2002; Chatuverdi et al. 1997). modeling analysis and supported its role in bacterial Analogous to above, we also noticed enhanced accumula- freeze survival (Figure 4). Likewise trehalose, raffinose is tion of intracellular mannitol and sorbitol in all the Pseudo- a kosmotrophic substance that has a stabilizing effect on monas strains except PPRs4 strain grown at 4 and 28°C, macromolecular structure (Cray et al. 2013a). Therefore, and it can be assumed that glycerol might replaced by man- it may possible or it can be hypothesized that the pro- nitol/sorbitol in these Pseudomonas strains as we failed to tective effect of raffinose as observed in current study of detect glycerol in bacterial cells. Himalayan Pseudomonads utilizes a different mechenism Moreover, the collected cold tolerant Pseudomonas from that of glycerol and fructose (Chin et al. 2010). strains were found to protect cytoplasmic components The significance of chao- and kosmotropicity for the by synthesizing specific free amino acids needed for maintenance of structure and activities of macromolecu- freezing survival and cold adaptation of the microbial lar systems have been well characterized in-vitro, cells. These amino acids act as chemical chaperones whereas, the degree to which they facilitate and/or limit which prevent the aggregation of cellular proteins during the activities of cellular macromolecules in-vivo remains stress conditions and their possible function is to regu- relatively unclear (Duda et al. 2004; Chin et al. 2010; late the fluidity of membrane at lower temperatures Cray et al. 2013a). Nevertheless, it has been established (Chattopadhyay and Jagannadham 2001; Chattopadhyay that chaotropicity-mediated stresses elicit specific stress 2002; Ferrer et al. 2003; Bhaganna et al. 2010; Jonathan responses in microbial cells (Bhaganna et al. 2010). et al. 2013). Enhanced production of intracellular proline Chaotropicity has been shown to not only limit life pro- has been reported in microorganisms in order to im- cesses but can render potential environmental habitats prove their freeze tolerance and osmotic stress (Morita (Hallsworth et al. 2007; Cray et al. 2013a). The kosmo- et al. 2003; Jonathan et al. 2013; Kempf and Bremer tropicity nature of trehalose (non-reducing disaccharide) 1998). This indicates that proline accumulation might be plays an important role in developing the ability of or- a general protective strategy against freeze stress evasion. ganisms to resist against adverse environmental condi- Additionally, the intracellular accumulation of charged tions (Kandror et al. 2002; Cray et al. 2013a). Trehalose amino acids, viz., arginine, aspartic acid and glutamate stabilizes the membrane and proteins by replacing water also seems to enhance microbial freeze tolerance and act and preserves the intracellular water structure (Sano as cryoprotectants (Shima et al. 2003; Jenkelunas 2013). Bisht et al. SpringerPlus 2013, 2:667 Page 10 of 13 http://www.springerplus.com/content/2/1/667 These amino acids thought to play important roles as regions of NW Himalaya (Mishra et al. 2011; Bisht et al. general acids in enzyme active centers, as well as in 2013). Bacterial cultures were maintained on Nutrient Agar maintaining the solubility and ionic character of proteins (NA) and Kings B slants, respectively, and preserved in 60% (Shima et al. 2003; Jenkelunas 2013). In view of previous glycerol at −80°C. The submission details of all the six reports, high accumulation of intracellular proline, ar- Pseudomonas strains and their growth curve studies (at ginine and glutamate in collected Pseudomonads sug- three different temperatures, i.e., 4°C, 15°C and 28°C) have gests their cryoprotective role for freezing survival. been published earlier (Mishra et al. 2008; 2009; 2011). All Cysteine and methionine are sulphur-containing amino reagents were of analytical grade and procured from Merck, acids. Cysteine is a powerful antioxidant and can react Sigma Aldrich, HiMedia Laboratories. with itself to form an oxidized dimer by forming a disul- fide bond. The environment within a cell is too reducing Assessment of survival after freezing-thawing for disulfides to form, but in the extracellular environ- Six bacterial cell samples (6 strains × 3 replicates = 18) ment, disulfides can form and play a key role in stabiliz- from each Pseudomonas strain were prepared and each ing many proteins (Sen 2005; Ladenstein and Ren 2008). set was grown separately into 5 ml LB medium at two Disulfide bonds are important for protection of bacteria different temperatures 4 and 28°C (48 h incubation as a reversible switch that turns a protein on or off when period for 4°C culture and 24 h for 28°C culture). Both bacterial cells are exposed to oxidation reactions. Hydro- cultures (4 and 28°C) were kept separately at preset gen peroxide (H O ) in particular could severely damage temperature of −10 and −40°C temperature for 48, 72 2 2 DNA and kill the bacterium at low concentrations if not and 96 h. Bacterial cultures were thawed at room for the protective action of the SS-bond (Ladenstein and temperature, appropriate dilutions were plated and incu- Ren 2008). Likewise, methionine mostly acts as a precur- bated at 28°C for 48 h and CFU counts were measured sor amino acid for glutathione. It plays an important role for 48, 72 and 96 h of freeze shifted bacterial cultures. in the antioxidant defense mechanism by reacting readily Freeze-thaw survival of Pseudomonas spp. was deter- with oxidants to form methionine sulfoxide (Livine et al. mined at −10 and −40°C by comparing the log CFU 1999). The present study suggests that high intracellular counts before and after the freezing treatment. All ex- cysteine and methionine synthesis in collected Pseudo- periments were performed in duplicates. monads grown at 4°C might be an integral part of cold survival strategy to avoid damages from oxidative stress Quantification of exopolysaccharide (EPS) production during cold conditions. Bacterial cells were grown in 100 ml Kings B and nutri- ent broth at three different temperatures, i.e., 4, 15 and Conclusions 28°C for 48 h. Following the incubation, bacterial cells In conclusion, it is the physicochemical diversity of stress were harvested and EPS was extracted following the protectants produced by Pseudomonads that confer their method of Underwood et al. (1995). Precisely, the bac- remarkable tenacity and stress tolerance. Each type of com- terial cells were centrifuged at 10,000 rpm (Sigma Model patible solute/cryoprotectant has protective effects via dif- 2 K15, Rotor No. 12132) at 4°C for 15 min. The cell pel- ferent mechanisms. Accumulation of diverse amino acids, let was washed twice with sterile distilled water, treated sugars and polyols (including EPS) under cold stress are with 10 mM EDTA (w/v), vortexed for 15 min, and fi- important characteristics of Himalayan psychrotrophic nally recentrifuged at 10,000 rpm for 20 min at 4°C to Pseudomonas strains. The most novel and intriguing find- extract the cell-bound EPS. Extraction process was re- ing of this study was, intrecellular accumulation of raffi- peated and EPS samples were pooled and precipitated nose, cysteine and aspartic acid in bacterial cells as a key using chilled acetone and centrifuged at 10,000 rpm for metabolites at low temperature. The characterization of 10 min. The cell pellet was collected and their dry these traits are potentially important for beginning to weight was measured. understand these adaptations in microbial community present in Himalayan region. The present findings are part Determination of ice nucleation activity of unfolded field of stress biology, and it will surely have Ice nucleation activities (INA) [ice nuclei per colony- implications for the studies related to microbial diversity forming-unit (CFU)] of potential cold tolerant Pseudo- present in extreme conditions of high altitudes. monas strains were measured by freeze-drop method (Vali 1971; Lindow 1990). Bacterial cultures were grown Materials and methods into 100 ml Luria broth (LB) medium at 4°C for 24 to Bacterial culture conditions and chemicals 96 h. One ml of culture was centrifuged at 6,000 rpm The psychrotrophic Pseudomonas strains used in the for 5 min at 4°C and collected pellet was washed twice present study were previously isolated from different plant with 0.85% phosphate buffer saline (PBS; pH 7.2; w/v). root zones that were collected from the high altitude The cell pellet was resuspended into 1.0 ml of phosphate Bisht et al. SpringerPlus 2013, 2:667 Page 11 of 13 http://www.springerplus.com/content/2/1/667 buffer (pH 7.2) and vortexed vigorously. Nearly 10 μl were procured from Waters Corporation, USA. Acetonitrile (equivalent to 30 drops) of the cell suspension was and deionised water were used as solvent-B. Samples were placed on a parafilm coated aluminum boat floating on prepared according to the manufacturer’s instruction. The an ethanol bath at preset temperature of −5 and −10°C. chromatographic analysis was performed on HPLC system The number of frozen droplets were counted after 2, 5, as mentioned in the upper section using multi ƛ fluores- 15 and 20 min, respectively and bacterial concentration cence detector (Waters 2475) attached with photodiode was measured by plating of serial dilution of bacterial array detector (Waters 2996). Ten μl sample was injected cells on Kings B medium followed by incubation for in a Waters AccQ� Tag™ (Waters, Ireland) column and sep- th 48 h. The ice nucleation activity was calculated and rated by mobile phase (60% acetonitrile and 1/10 concen- -1 expressed in log ice nuclei CFU (Vali 1971; Lindow tration of Waters AccQ� Tag™ buffer)atgradient flowrate -1 1990). of 1 ml min . The chromatographic parameters, like, de- tector gain value, column temperature, and run time were Preparation of intracellular cell extract for detection of maintained as 10, 25°C and 65 min, respectively. Intracellu- cryoprotectants lar free amino acids were quantified by measuring peak area Two sets (6 × 2 = 12) of each bacterial isolate was prepared using the external standard method and samples were ana- and grown separately into 100 ml LB broth medium at 4 lyzed in triplicate. and 28°C for 36 h. Afterwards, the cultures were centri- fuged at 5°C for 10 min at 8,000 rpm and resultant cell pel- Stastistical analysis lets were washed three times with 0.85% PBS (pH 7.2). The Descriptive statistics was employed to represent the means cell pellets were resuspended into 1.0 ml of phosphate buf- andstandarddeviations. Student’s t-test was used to com- fer (pH 7.2) and disrupted by sonication (Soni Prep 150, pare the mean values at 4 and 28°C for stress metabolites. Sanyo) in3cyclesat8 μm (amplitude) for 2 min with In order to investigate the expression pattern of stress me- 45 sec cooling interval. The cell debris of each culture was tabolites for individual isolate and its correlation with removed by centrifugation at 10,000 for 15 min at 4°C. The growth temperatures (4 and 28°C), Principle component supernatant was filtered (0.22 μm) and samples were stored analysis (PCA) was performed for all cold stress related at −20°C. Standard sugar solutions of specific concentration measured parameters along with the growth temperature. -1 (100, 200, 300, 400 and 500 mg l )werepreparedinphos- PCA analysis was carried out using the XLSTAT (version phate buffer (pH 7.0). This experimental part was per- 2013) program. Additionally, to predict the relationship and formed in duplicate. effect of cold stress metabolites on bacterial freezing sur- vival at −10 and −40°C, an automated linear modeling Analysis of intracellular sugars (ALM), using forward stepwise with information criterion High performance liquid chromatography (HPLC) (Waters (standard model) was performed. For the modeling, data Corporation, USA) analysis was employed to analyze the gathered during bacterial growthat boththe temperatures intracellular sugars. The HPLC system consisted of an iso- (4 and 28°C) were combined and analyzed using SPSS pro- cratic pump (Waters, 600 Delta), DES-1008D interface (D- gram. The linear modeling was performed with the goal of Link, China), Waters temperature controller model TC2 selection of the most explanatory model that can explain and evaporative light scattering detector (Waters 2424 the relationship and effect between independent (stress me- ELSD) controlled by the ‘Empower’ program. Waters tabolite) and dependent (freezing survival) variables. Spherisorb 5 μmNH (250 × 4.6 mm) chromatographic column was used during the analysis. Ten μlofthe sample Additional files was injected into the Spherisorb column and separated with the mobile phase (67% acetonitrile and 33% water) at a flow Additional file 1: Figure S1a. HPLC chromatogram of intracellular -1 sugars’ and polyols’ contents of Pseudomonas strains grown at 4 and rate of 1 ml min . The chromatographic parameters, like, 28°C. Figure S1b. HPLC chromatogram of intracellular sugars and gas pressure, detector gain value, column temperature, run polyols’ contents of Pseudomonas lurida NPRp15 Grown at 4°C and time and nebulizer tube temperature were 50 psi, 10, 25°C, 28°C. Figure S1c. HPLC chromatogram of intracellular sugars and polyols’ contents of Pseudomonas sp. PPERs23 grown at 4°C and 28°C. 30 min and 60°C, respectively. Various sugars, like, Figure S1d. HPLC chromatogram of intracellular sugars and polyols’ D-Xylose, D-Glucose, D-Sorbitol, Trehalose and Raffinose contents of Pseudomonas putida PGRs4 grown at 4°C and 28°C. were quantified using external standard method, and Figure S1e. HPLC chromatogram of intracellular sugars and polyols’ contents of Pseudomonas sp. PGERs17 grown at 4°C and 28°C. samples were analyzed in triplicate. Additional file 2: Figure S2a. HPLC chromatogram of intracellular amino acids’ contents of Pseudomonas strains grown at 4 and 28°C. Analysis of intracellular free amino acids Figure S2b. HPLC chromatogram of intracellular amino acid contents of Amino acid standard H-kit, amino acid solvent-A (Aqueous Pseudomonas lurida NPRp15 grown at 4°C and 28°C. Figure S2c. HPLC chromatogram of intracellular amino acid contents of Pseudomonas buffer, Waters AccQ� Tag™), derivatization regent that con- sp. PPERs23 grown at 4°C and 28°C. Figure S2d. HPLC chromatogram of tain AccQ� Fluro Borate buffer and AccQ� Fluro reagent Bisht et al. SpringerPlus 2013, 2:667 Page 12 of 13 http://www.springerplus.com/content/2/1/667 Duda VI, Danilevich VN, Suzina NF, Shorokhova AP, Dmitriev VV, Mokhova intracellular amino acid contents of Pseudomonas putida PGRs4 grown at ON, Akimov VN (2004) Changes in the fine structure of microbial cells 4°C and 28°C. Figure S2e. HPLC chromatogram of intracellular amino induced by chaotropic salts. Microbiology 73:341–349 acid contents of Pseudomonas sp. PGERs17 grown at 4°C and 28°C. Ferrer M, Chernikova TN, Yakimov MM, Golyshin PN, Timmis KN (2003) Figure S2f. HPLC chromatogram of intracellular amino acid contents of Chaperonins govern growth of Escherichia coli at low temperatures. Pseudomonas fluorescens PPRs4 grown at 4°C and 28°C. Nat Biotechnol 21(11):1266–1267 Additional file 3: Table S1. Comparative analysis of stress metabolites Gülez G, Dechesne A, Workman CT, Smets BF (2012) Transcriptome dynamics accumulation/production at cold (4°C) and optimum growth temperature of Pseudomonas putida KT2440 under water stress. Appl Environ (28°C) for all six Pseudomonas strains (combined average of all strains). Microbiol 78(3):676–683 Hallsworth JE, Heim S, Timmis KN (2003) Chaotropic solutes cause water stress in Pseudomonas putida. Environ Microbiol 12:1270–1280 Competing interests Hallsworth JE, Yakimov MM, Golyshin PN, Gillion JLM, D’Auria G, Alves FDL et al The authors report no conflict of interests. (2007) Limits of life in MgCl -containing environments: chaotropicity defines the window. Environ Microbiol 9:801–813 Authors’ contributions Jenkelunas P (2013) Production and Assessment of Pacific Hake Hydrolysates as a SCB conducted the study and prepared the manuscript. SH helped in Cryoprotectant. M.Sc thesis The University of British Columbia, Canada, https:// manuscript preparation which includes manuscript writing, reviewing and circle.ubc.ca/bitstream/handle/2429/43921/ubc_2013_spring_jenkelunas_peter. editing. GKJ provided technical advice and supervised research work. PKM pdf?sequence=3 (last accesed 7 May 2013) provided research project management role and supervised research work. Jonathan A, Cray A, Bell NW, Bhaganna P, Allen YM, Timson DJ et al (2013) The biology All the authors read and approved the final manuscript. of habitat dominance; can microbes behave as weeds? Microb Biotechnol, doi:10.1111/1751-7915.12027 Jones PG, Van Bogelen RA, Neidhardt FC (1987) Induction of proteins in response Acknowledgements to low temperature in Eschericha coli. 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