Cryotolerance strategies of Pseudomonads isolated from the rhizosphere of Himalayan plants

Microorganisms have a range of evolutionary adaptations and physiological acclimation mechanisms that allow them to survive and remain active in the conditions of environmental stress. Adaptation towards stress condition is indispensable for survival, mainly when it causes alterations to the cell metabolism. Sudden decrease in temperature has severe effects on microbial cells, like, reduction of membrane fluidity, decrease in ribosome efficiency, and increased stabilization of secondary structures of nucleic acids, which may affect transcription, translation and DNA replication (Phadtare et al. 2000). In order to survive under freezing conditions, bacteria have developed various strategies for their endurance, such as, maintenance of membrane fluidity, constant metabolic activities etc. (Ramos et al. 2001). Additionally, it has been suggested that trehalose, glycerol and sorbitol are the major cryoprotectants for prokaryotic cells to response the freezing damage, thereby causing the maintenance of some enzymatic functions in-vivo (Yamashita et al. 2002). However, a limited information is available about the cryoprotectants that are responsible for the freezing resistance mechanisms of bacteria. Bacteria often encounter freezing conditions and can survive in extremely cold environments, like, the high altitude regions of Himalaya. In frozen environments, bacteria are exposed to conditions that necessitate the removal of water to maintain the structure and function of the bacterial cell. As water contributes to the stabilization of various macromolecular structures, any significant deviation from the accessibility of water due to dehydration, desiccation or an alteration of its physical state from aqueous phase to an ice crystal form poses a severe threat to the normal cell functions and survival of organism (Beall 1983; Crowe et al. 1984).

In this regard, regulatory proteins and key metabolic enzymes require adjustments to cope with the temperature shifts in order to maintain a balanced microbial growth at the new environmental temperature. Under such conditions, the synthesis of specific cryoprotectant molecules might be enhanced that act as chemical chaperons and protect the cellular proteins from freezing temperature. Scanty reports are available on psychrotrophic bacterial cryotolerance strategies and related responsible molecules except cold shock (Csps) and cold acclimation (Caps)s proteins. Although, bacterial cryotolerance has been investigated in relevance with role of trehalose and glycine betine in Escherichia coli and Bacillus subtilis, respectively (Jones et al. 1987; Willimsky et al. 1992). But, very little is known about the possibility of other molecules responsible for the survival of bacteria subjected to freezing challenge by an adaptation of the microbial cells to low temperatures particularly in psychrotolerant/psychrophilic bacteria (Margesin and Schinner 1999; Mishra et al. 2010).

In the upper parts of north west (NW) Himalaya, winter is mostly characterized by intermittent snow cover (November to March) and fluctuating subfreezing temperatures, while summer displays intense desiccating sunshine punctuated by infrequent rains (Mishra et al. 2008; 2011; Bisht et al. 2013). These conditions pose additional challenges to microbial species that may endure summer temperatures as high as 30°C and winter temperatures that can dip to -10°C, as well as alternating freezing and thawing periods during the cold season. At these temperatures, microorganisms might be injured or killed as a result of cold shock, freezing, prolonged exposure to subzero temperatures, and subsequent warming, and injury or death is often due to damage to membranes or cell walls that results in changes in permeability, as well as damage to DNA. Given these challenges, the fact that soil bacteria thrive in NW Himalayan regions is a testimony either to environmental heterogeneity or to the remarkable adaptive abilities of these psychrotrophic microbes (Srinivas et al. 2011; Bisht et al. 2013). The precise mechanisms or molecular strategies underlying the cellular adaptations of psychrotrophic bacterial cells in cold conditions are not clear and needs to be addressed, particularly for varying genus of Pseudomonas. In this context, the present study was undertaken to investigate the freezing survival strategies operated in six psychrotrophic Pseudomonas strains (P. lurida NPRs3, P. lurida NPRp15, P. sp. PPERs23, P. putida PGRs4, P. sp. PGERs17 and P. fluorescens PPRs4) isolated previously from rhizosphere of NW Himalayan plants (Mishra et al. 2011; Bisht et al. 2013).

The Himalayan region provides an opportunity to obtain microbes that have experienced extended exposure to cold temperatures, reduced water activities, radiation and low nutrient accessibility. The cold adaptation related properties of psychrotrophic Pseudomonas cells showed high cellular metabolism activities in cold conditions (Mishra et al. 2008; 2009; 2011). It has been well established that the cold-active enzymes and efficient growth rates are used to facilitate and maintain the adequate metabolic fluxes at near freezing temperature for cold-adaptation (Shivaji and Prakash 2010). The great metabolic flexibility of Pseudomonas species allows them to inhabit diverse environments and capable of a high level environmental ubiquity even in cold/freezing habitats (Timmis 2002; Remold et al. 2011; Cray et al. 2013b). This study clearly demonstrates that cold acclimatized cells (grown at 4°C) had higher freezing-thawing survival over non-acclimatized cells (grown at 28°C), and linear modeling analysis clearly proved that the incubation temperature was the main factor for Pseudomonads’ freezing survival. The overall freezing-thawing survival of Pseudomonas strains at -10°C was found be 98% (Additional file 3: Table S1). This feature of Pseudomonads suggests their survival persistence in Himalayan extreme freezing-thawing conditions during winter season. One of the major freezing survival strategy might be the evolution of strains that are capable of utilizing a large number of carbon sources (Ponder et al. 2005). This might be more relevant because the alpine environment is highly heterogeneous with pockets of specific carbon compounds, a large number of bacterial strains that have recently gained or lost the ability to grow on a particular source of carbon may exist and supported the adaptability, versatility and environmental ubiquity, and prevalence of Pseudomonas genus in the Himalayan region (Mishra et al. 2010; Remold et al. 2011; Cray et al. 2013b).

Ice crystal formation is the primary risk associated with the freezing-thawing of microbial cells and leads to membrane damage, and parallels the situation of dehydration/desiccation of the cells. Crowe et al. (1984) reported that the rehydration condition causes most damage to the cells. At low temperatures (both above and below 0°C) the intracellular environment usually becomes dehydrated and this increases solute concentration as well as free radical formation. As a result cold and solute-stress are to a large extent inseparable (Chin et al. 2010). A key component of cryotolerance in bacterial cells is tolerance to desiccation, solute-induced stress, and oxidative stress (Hallsworth et al. 2003; Bhaganna et al. 2010; Gülez et al. 2012; Pablo et al. 2013; Jonathan et al. 2013). Lowering of ice nucleation temperature and controlling the freezing temperature and shape of the ice crystal have been identified as two possible strategies for microbial cells to avoid freezing conditions (Kawahara et al. 1991). Whereas development of freezing tolerance by producing cryoprotectant compounds or adaptation of cytoplasmic enzymes to cold conditions for protecting cytoplasmic components is the third strategy used by microbial cells to survive in freezing conditions as these molecules depress freezing point for the protection of cells (Yamashita et al. 2002). In the present study, all Pseudomonas strains didn’t display the presence of type-I and/or type-II ice nuclei as found in ‘ice plus’ bacteria. Thus, all the collected strains were considered as ‘ice minus’ bacteria because they lack Ina proteins present on bacterial cell wall that act as a nucleation centre for ice crystals, which are mostly active in between -2 to -7°C. All Pseudomonas strains demonstrated very low level of type-III ice nuclei which is typically active between -7 to -10°C. Presentation of less ice nuclei by the Pseudomonads indicates their freeze survival strategy by lowering the ice nucleating temperature. Therefore, it can be suggested that low ice nucleation activity of Pseudomonads makes them capable of inhibiting the ice formation, which might required for freezing survival.

Microbes produce EPS, which is stored as a thick gel surrounding the cell, the major ecological characteristic of EPS is that it can form and maintain protective microhabitats around microorganism in aquatic and frozen atmosphere (Stoderegger and Herndl 1998; Decho 1990; Tamaru et al. 2005). We found that EPS production by the Pseudomonas strains was higher at lower temperatures (4 or 15°C) in comparison to their optimal growth temperature (28°C). Enhanced EPS production by the Pseudomonads at low temperature suggested that EPS plays an important role in desiccation protection or prevention of drying of bacterial cells from freezing temperature (Figures 2 and 4; Roberson and Firestone 1992). The production of EPS is associated with the biofilm formation. The fact that the Himalayan strains overproduce EPS at low temperature might suggests that under these conditions, the Pseudomonas strains show higher biofilm formation, and even the process of root colonization enhances at low temperature (Mishra et al. 2011).

Studies of low temperature tolerance in microbial cells demonstrated that the flexibility of cellular macromolecules can be the limiting factor/failure-point for growth windows at low temperatures, and showed that a chaperonin (chao- and kosmotrop) can extend the biotic window for growth down to lower temperatures (Margesin and Schinner 1999; Ferrer et al. 2003). Hence, it can be assumed that the collected Pseudomonads were also following the third type cold evading strategy to thrive in freezing conditions by synthesizing various chaperonin/cryoprotectants, i.e., sugars, polyols and amino acids, in order to protect their cytoplasmic components. These cryoprotectants are known to depress freezing point to evade crystallization (Chattopadhyay 2002).

Raffinose, like other sugars plays a cryoprotective role by interacting with membrane lipids and proteins and decreases the risk of intracellular ice-crystal formation that causes cellular osmotic dehydration during cryopreservation (Agca et al. 2002; Tuncer et al. 2010). The effect of raffinose related to oxidative stress has been considered as an indirect effect of sugar signaling and triggers the production of specific reactive oxygen species (ROS), such as, hydroxyl radicals’ scavengers (Van-den Ende and Valluro 2009). Though, the role of raffinose has been defined in recent years in alleviation of oxidative stress and as a cryoprotectant (Van-den Ende and Valluro 2009; Tuncer et al. 2010), but, still no reports are available related to the accumulation of raffinose in bacterial cells in response to cold stress conditions. Here, we found high accumulation of intracellular raffinose content in all the tested Pseudomonads in response to their growth at 4°C prior to freezing at -10 and/or -40°C, and it was further confirmed through linear modeling analysis and supported its role in bacterial freeze survival (Figure 4). Likewise trehalose, raffinose is a kosmotrophic substance that has a stabilizing effect on macromolecular structure (Cray et al. 2013a). Therefore, it may possible or it can be hypothesized that the protective effect of raffinose as observed in current study of Himalayan Pseudomonads utilizes a different mechenism from that of glycerol and fructose (Chin et al. 2010).

The significance of chao- and kosmotropicity for the maintenance of structure and activities of macromolecular systems have been well characterized in-vitro, whereas, the degree to which they facilitate and/or limit the activities of cellular macromolecules in-vivo remains relatively unclear (Duda et al. 2004; Chin et al. 2010; Cray et al. 2013a). Nevertheless, it has been established that chaotropicity-mediated stresses elicit specific stress responses in microbial cells (Bhaganna et al. 2010). Chaotropicity has been shown to not only limit life processes but can render potential environmental habitats (Hallsworth et al. 2007; Cray et al. 2013a). The kosmotropicity nature of trehalose (non-reducing disaccharide) plays an important role in developing the ability of organisms to resist against adverse environmental conditions (Kandror et al. 2002; Cray et al. 2013a). Trehalose stabilizes the membrane and proteins by replacing water and preserves the intracellular water structure (Sano et al. 1999). High intracellular trehalose accumulation was found in all Pseudomonas strains except P. fluorescence PPRs4 cells when they grown at 4°C prior to freezing and the same was supported by linear modeling analysis (Figure 4). Our findings were in congruence with earlier studies where accumulation of higher trehalose was related with its cryoprotectant function (Kaasen et al. 1992; Mitta et al. 1997). Regarding P. fluorescens PPRs4, we can speculate that trehalose might replaced by sucrose and plays similar role of cryoprotectant, as higher sucrose accumulation was noticed in the said strain (Cray et al. 2013b).

The cryoprotectant property of glucose has been previously documented by Koda et al. (2002). On the similar lines, we found that P. lurida NPRp15 cells accumulated higher glucose content when grown at 28°C and also demonstrated freezing survival at -10°C. Hence, it can be suggested that glucose plays a significant role in cryoprotection of microbial cells. Two more kosmotropic solutes mannitol and sorbitol act as stress protectants and has been previously investigated (Chatuverdi et al. 1997; Kets et al. 1996; Bhaganna et al. 2010). The principle role of mannitol for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic P. putida has been discovered recently (Bhaganna et al. 2010). Earlier studies reported that mannitol accumulation increases in microbial cells under various stress treatments, like, heat, salt and/or their combination (Managbanag and Torzilli 2002; Chatuverdi et al. 1997). Analogous to above, we also noticed enhanced accumulation of intracellular mannitol and sorbitol in all the Pseudomonas strains except PPRs4 strain grown at 4 and 28°C, and it can be assumed that glycerol might replaced by mannitol/sorbitol in these Pseudomonas strains as we failed to detect glycerol in bacterial cells.

Moreover, the collected cold tolerant Pseudomonas strains were found to protect cytoplasmic components by synthesizing specific free amino acids needed for freezing survival and cold adaptation of the microbial cells. These amino acids act as chemical chaperones which prevent the aggregation of cellular proteins during stress conditions and their possible function is to regulate the fluidity of membrane at lower temperatures (Chattopadhyay and Jagannadham 2001; Chattopadhyay 2002; Ferrer et al. 2003; Bhaganna et al. 2010; Jonathan et al. 2013). Enhanced production of intracellular proline has been reported in microorganisms in order to improve their freeze tolerance and osmotic stress (Morita et al. 2003; Jonathan et al. 2013; Kempf and Bremer 1998). This indicates that proline accumulation might be a general protective strategy against freeze stress evasion. Additionally, the intracellular accumulation of charged amino acids, viz., arginine, aspartic acid and glutamate also seems to enhance microbial freeze tolerance and act as cryoprotectants (Shima et al. 2003; Jenkelunas 2013). These amino acids thought to play important roles as general acids in enzyme active centers, as well as in maintaining the solubility and ionic character of proteins (Shima et al. 2003; Jenkelunas 2013). In view of previous reports, high accumulation of intracellular proline, arginine and glutamate in collected Pseudomonads suggests their cryoprotective role for freezing survival. Cysteine and methionine are sulphur-containing amino acids. Cysteine is a powerful antioxidant and can react with itself to form an oxidized dimer by forming a disulfide bond. The environment within a cell is too reducing for disulfides to form, but in the extracellular environment, disulfides can form and play a key role in stabilizing many proteins (Sen 2005; Ladenstein and Ren 2008). Disulfide bonds are important for protection of bacteria as a reversible switch that turns a protein on or off when bacterial cells are exposed to oxidation reactions. Hydrogen peroxide (H₂O₂) in particular could severely damage DNA and kill the bacterium at low concentrations if not for the protective action of the SS-bond (Ladenstein and Ren 2008). Likewise, methionine mostly acts as a precursor amino acid for glutathione. It plays an important role in the antioxidant defense mechanism by reacting readily with oxidants to form methionine sulfoxide (Livine et al. 1999). The present study suggests that high intracellular cysteine and methionine synthesis in collected Pseudomonads grown at 4°C might be an integral part of cold survival strategy to avoid damages from oxidative stress during cold conditions.

In conclusion, it is the physicochemical diversity of stress protectants produced by Pseudomonads that confer their remarkable tenacity and stress tolerance. Each type of compatible solute/cryoprotectant has protective effects via different mechanisms. Accumulation of diverse amino acids, sugars and polyols (including EPS) under cold stress are important characteristics of Himalayan psychrotrophic Pseudomonas strains. The most novel and intriguing finding of this study was, intrecellular accumulation of raffinose, cysteine and aspartic acid in bacterial cells as a key metabolites at low temperature. The characterization of these traits are potentially important for beginning to understand these adaptations in microbial community present in Himalayan region. The present findings are part of unfolded field of stress biology, and it will surely have implications for the studies related to microbial diversity present in extreme conditions of high altitudes.