Membrane Fluidity during cold adaption

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Temperature is main cause for the changes in physical behaviour and properties biological membranes. Lipid behaviour is extremely sensitive to temperature. Plasma membrane is important part of a cell and it separates living cell from their respective surroundings. The function of this plasma membrane mainly depends on the lipid bilayer conditions and its state. The state of plasma membrane makes it vulnerable to changes in external environment changes such as temperature. Most organisms have lipid bilayers which are entirely/mostly fluid at normal temperature moreover they have disordered arrangement of fatty acyl. As the temperature reduces, bilayers undergo a reversible changes from a state of disordered to more ordered or non-fluid arrangement of fatty acyl. The temperature at the middle of this entire process is called as transition temperature. Unsaturated fatty acids helps to keep any membrane more fluid at lower temperature with the help double bonds from the kinks in the fatty acid tails which prevents adjacent lipids from packing tightly [1].

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This paper discusses the research by Jana Beranová et al [2] and MichaelH.W.Weber et al [3] in membrane fluidity during cold adaption. Jana Beranová et al mainly describe that the membrane fluidity adapts itself with respect to changes in composition of medium.B. subtilis fatty acid desaturase is an important factor in membrane adaption for different temperature conditions and this procedure has been explained by MichaelH.W.Weber et al.

The living cells can be classified into two classes namely: eukaryotes and prokaryotes. Eukaryotes can be as a cell which has nucleus and prokaryotes can be defined as a cell which has not nucleus. Cells are made of many crucial parts that work together and perform specific functions.Basically, phospholipid bilayer forms a membrane and works as a separator between two compartments which are nothing but inside and outside part of cell. It regulates what enters and exits through the channels. Phospholipid bilayer has proteins which regulates specific function associated with plasma membrane [4]. There are four categories of phospholipids which are present in plasma membrane.First is phosphatidylcholine which has choline as a main group and these are available in egg yolk and soybeans. Second category is phosphatidylethanolamine which generally found in nervous tissue and composes around 20% of lipids. Third category is phosphatidylserine which is important because of its cell signalling function. Last category is sphingomyelin which has a polar head group. The almost half of the lipid content is made by these four phospholipids. The other part of the membrane may contain glycolipids and cholesterol [5].

The study of membrane fluidity is very useful for encompassing different classes of phospholipids and the its components like fatty acids [6]. It also provides the important information about fluid matrix for protein organization. Cell membrane fluidity mainly depends upon the nature of fatty acid found in phospholipids of the cell membrane. The degree of unsaturation of array of fatty acids decides membrane fluidity. Different biological systems use different mechanism to compensate with environment changes and maintain membrane fluidity. The most used mechanism is feedback mechanism which changes degree of unsaturation with respect to environment changes to keep membrane fluidity constant [7]. Basically, cellular processes are functions of membrane lipid fluidity. It is possible to change lipid fluidity by changing factors on which it depend e.g. temperature compensation, genetic manipulations etc. [8]. The subsequent part of this paper, the effect of ambient temperature on membrane fluidity has been discussed with reference to research done by Jana Beranová et al and MichaelH.W.Weber et al.

Prokaryotic cell adapts itself with a change in external temperature of their environment. These changes are carried out mainly in accordance with membrane fluidity.Membrane fluidity adaption in bacillus subtilis is carried out by long term adaption and desaturation of fatty acid chains after cold shock. It is well proved that the main factor that decides membrane fluidity is anteiso fatty acid precursor isoleucine [9]. Also, many studies have revealed nature of fatty acids in relation with lower growth temperature [9].The long term adaption is carried out by changing the ratio of anteiso to isobranched fatty acids. Generally, the increased ratio has used in long term adaption. The fatty acid structure is dependent on precursor availability. But at lower temperature, there is increase in antiso which would increase the overall ratio of anteiso to iso [9].The another mechanism involves use of fatty acids desaturase. It desaturates the fatty acid chains in phospholipids. Membrane fluidity phase is important because it regulates the different functions of membrane. In order to do this, bacteria changes the component composition of fatty acid chains.

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The change in composition of fatty acid is important to keep adequate lipid fluidity in low growth temperature. The long term adaption technique has not been well studied. However, different mechanisms for short term adaption of membrane fluidity have been proposed.Desaturation of fatty acid is catalyse by the induction of fatty acid in membrane. This entire process is regulated by kinase DesK which works as a sensor and DesR which regulates the response [10]. Here, the membrane fluidity is mainly controlled by contents of DesK which detects the changes in lipid fluidity. The increase in anteisobranched fatty acid precursors like C15:0 andC17:0 restrain the fatty acid desaturase path [9].

The cold adaption behaviour of the cytoplasmic membrane in bacillus subtilis in different media has been explained. The different media used are mineral medium with glucose, mineral medium with kycerol and complex medium. As explained earlier, the main factor that affects the cold adaption behaviour is the composition of fatty acid because it regulates the fluidity of membrane at low temperature. The changes in transition temperature of gel and crystalline phase of lipids have been demonstrated in subsequent part of this research. DPH fluorescence anisotropy is used to measure the membrane fluidity which essentially measures the temperature profiles.

The response of basillussubtilis during membrane adaption to low temperature has been studied in second paper. The basillussubtilis fatty acid desaturase des has important role during cold shock. Many investigations have shown that plasma membranes are complex structures and its physical statesdepend upon the different content and their respective amount in membrane lipid as well as temperature. So, along with the other organism it is important for bacteria to compensate their composition of lipids in membrane. This is important to keep membrane fluid and functioning properly. Generally, control of membrane fluidity is carried out in three different ways. First is homeoviscous adaption. Second is induction of cis configured double bonded carbon. Third mechanism involves a protein that works as a sensor which detects the physical states with respect to membrane fluidity. The homeostatic regulation of bilayer order is a property of functional importance [11]. Third mechanism is carried out by protein class known as protein kinase so fatty acid desaturases enzymes can work as sensors for measurement or control of membrane fluidity. This paper describes that basillussubtilis fatty acid desaturase has a key role in cold adaption because it works as a back up source if source of exogenous isoleucine is not available.

Methods

Jana Beranová et alhas used bacillus subtilis 168 to develop a bacterial strain. Cultivation procedure is carried out in different medium which are explained above. This cultivation is performed at temperature 40 °C and 20 °C. For the study of behaviour in cold shock, temperature reduced suddenly from 40 °C to 20 °C. The membrane isolation is carried out during this cold shock and the incubation is performed for 30 minute at 40 °C and for 60 minute for 20 °C. The measurement for anisotropy is taken by stopping frozen situation only once. Protein detection is carried out by using Pierce BCA protein assay. The next step is lipid isolation and mixture of chloroform methanol is used for it. The cultured cells in 60mM phosphate buffer which has a pH of 7.4 mingled with chloroform methanol. This experiment is performed for 2 hour at normal room temperature. After centrifugation of mixture extraction of chloroform and methanol is carried out and the isolated lipid stored at -20 ËšC. For fatty acid analysis, 0.3mg isolated lipid is used. The reaction of lipids with methanol at 37 ËšC for 15 min gives fatty acid methyl ester as a product. Gas chromatography is used to separate out fatty acids and automatic identification system is used for detecting peaks due to fatty acids.

MichaelH.W.Weber et al. has used E. coli XL1-Blue as a host for cell culture and medium is designed by using B. subtilis JH642 and B. subtilisMW_Ddes. These are used for fatty acid analysis. Cells are harvested by using centrifugation for 5 minute at 4 ËšC.K3PO4, KCl, MgSO4 buffer is used to wash out all the sediments. Again, gas chromatography is used to analyse and separate fatty acid methyl ester.

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Results

The experiments carriedout by Jana Beranová et alfor the determination of growth of bacillus subtilis in different media at cultivation temperatures Tc40°C and Tc20°C. Also, they have taken results after sudden decrease in temperature from Tc40°C to Tc20°C. Results are taken in three different mediums explained above. The following figure shows the growth of bacillus subtilis in three mediums for cultivation temperature and during cold shock. Here, abbreviations are CM stands for complex medium with glucose, MMGlu stands for mineral medium with glucose and MMGlyc stands for mineral medium with glycerol.

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Figure 1.growth of bacillus subtilis in different media [2].

The fatty acid synthesis is performed by comparative study of fatty acid analysis of bacillus subtilis which are cultured in different mediums for different cultivation temperature. Figure 2 shows the graphical interpretation of composition of bacillus subtilis fatty acids at different growth conditions. The recorded fatty acid data shows the percentage of fatty acid in figure A. Figure B shows percentage for each structural type of fatty acid.

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Figure 2.the graphical interpretation of composition of bacillus subtilis fatty acids at different growth conditions

The important finding is that, the amount of fatty acid desaturase induced after the cold shock remains same for different growth mediums.As shown in figure 3, the effect of growth condition on expression in bacillus subtilis after cold shock is almost same.the amount of fatty acid desaturase inducedis expressed by term β-galactosidase activity.All the results are the mean values of the entire set of results for each individual media.

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Figure 3. Effect of growth condition on expression in bacillus subtilis after cold shock

MichaelH.W.Weber et al has demonstrated the importance of bacillus subtilis fatty acid desaturase by developing a model which has erythromycin resistance mls cassette instead of des gene. The analysis of des deletion mutant is carried out at standard (37 degree centigrade) and at cold shock (15 degree centigrade) and results are compared with analysis of parental strain bacillussubtilis.All the measurements taken for growth shows almost same results for mutant and parental strains for different temperature.The optical density for bacillus subtilis des deletion mutant is depend upon amount of isoleucine and if it is not available the optical density goes on decreasing. Figure 4 growth of mutant in different media with absence and presence of exogenous isoleucine sources. The triangles in figure shows the cultures grown without induced expression of des and with induced expression of des in the absence of isoleucine.

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Figure 4.Growth of bacillus subtilis, fatty acid desaturase deletion mutant at different conditions.

The analysis of unsaturated fatty acids synthesization by bacillus subtilis is carried out by comparative study of bacillus subtilis JH642 and bacillus subtilis MW mutant des.The comparison between the results in accordance with availability of isoleucine shows that unsaturated fatty acid products are synthesized under all conditions in the absence of isoleucine.Whereas, unsaturated fatty acid species are only generated with presence of isoleucine only at 15 ËšC during growth condition. In addition to this, the absence of isoleucine produces complex fatty acids with parental and mutant strains. Figure 5 shows the comparative analysis of unsaturated fatty acid species from bacillus subtilis JH642 in proportional to different growth conditions. The following graph shows amount of UFA with respect to total cultured content.

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Figure 5.comparative analysis of unsaturated fatty acid species from bacillus subtilis JH642 in proportional to different growth conditions.

Both Jana Beranová et al and MichaelH.W.Weber et al have demonstrated the same work related to fatty acid functional behaviour in bacillus subtilis with respect to different medium conditions. The presence of isoleucine as precursor for antiso-branched fatty acid affect the level of this fatty acid which would further affect the survival of cell during cold shock.The sufficient data is provided by Jana Beranová et al for demonstration as compare to second paper. The concept of homeoviscous adaption proposed by Sinensky 1974 [11] which include the shortening of fatty acid acyl chains, differentiation of cis configured carbon double bonds has been explained by both researchers. As per MichaelH.W.Weber et al, the concept of homeoviscous adaption is achievable by creating double bonds into acyl chains. This can be carried out by anaerobically while carrying out fatty acid synthesis. Also, this can be done by aerobically by changing fatty acids compositions with the help of fatty acid desaturase.However, research by Jana Beranová et al demonstrated that homeoviscous adaptation is not validating for common behaviour of membrane during cold adaption.Here, they introduce a concept of homeoviscous efficacy which can be explained as the degree of changes due to temperature in membrane physical state which is compensated by modifications in composition of membrane lipid.

Effect of different media and different cultivation temperature with respect to membrane properties has been studied in both papers to determine the advantage for cold adaption after cold shock. Overall, both the papers explained the concept of membrane fluidity and the factors that affect it which is necessary to determine membrane behaviour at low temperature conditions.