Research paperEffects of elevated growth temperature and heat shock on the lipid composition of the inner and outer membranes of Yersinia pseudotuberculosis
Graphical abstract
Introduction
The lipid matrix of bacterial membranes is the primary target for different environmental factors, which trigger adaptation mechanisms that result in changes in the polar head groups and fatty acid (FA) components of the phospholipids. In particular, alterations in the environmental temperature affect membrane fluidity by regulating the ratio of saturated to unsaturated fatty acids through a bacterial response called “homeoviscous adaptation” [1]. However, the effect of abiotic factors on the phospholipid and fatty acid compositions of bacterial species inhabiting different ecological niches has not been investigated systematically [2].
The cell envelope of gram-negative bacteria consists of inner (IM) and outer membranes (OM) separated by a periplasmic space. Although data are available on the arrangement of individual phospholipids in the envelope of the mesophilic gram-negative bacteria [3], [4], [5], [6], [7], [8], no such data have been reported for psychrotrophic bacteria, including the enteropathogen Yersinia pseudotuberculosis, which is characterized by high ecological plasticity [9]. Despite the important role of membrane lipids in the function and adaptation of bacteria, the available information about the lipid distribution between the IM and OM is mostly limited to the model organism Escherichia coli.
However, recent lipidomic approaches have surprisingly demonstrated that there is little overlap between the Escherichia model lipid profiles and those of specialized pathogens, emphasizing the need for constructing organism-specific lipidomic databases [10], [11]. Moreover, the differences in both the protein and lipid compositions of the IM and OM that were isolated from different gram-negative bacteria were documented [11], [12]. Therefore, data on the distribution of phospholipids between the IM and OM are required to properly interpret the conformational and functional changes of the membrane proteins as the bacteria adapt to the various growth conditions. To our knowledge, there are no reports on the effect of either high growth temperatures or heat shock on the membrane phospholipid and fatty acid compositions in the Yersinia genus. Heat stress can be classified either as a heat adaptation, which assumes that a cell is exposed to a temperature above its optimal growth for long periods, or heat shock, which assumes that a cell is exposed to temperatures above its normal growth maximum for a very short period. Therefore, to understand the response of Y. pseudotuberculosis to both types of heat stress, the changes in the membrane lipid and fatty acid compositions in the heat-adapted and heat-shocked cells were thoroughly investigated by comparing the cells that were either growing at temperatures that correspond to the saprophytic and parasitic phases of the life (at 8 °C and 37 °C, respectively) or subjected to heat shock by a sharp increase in the temperature from 8 °C to 45 °C. Quantitative differences in the phospholipid composition of the IM and OM of the heat-adapted and heat-shocked psychrotrophic Y. pseudotuberculosis cells were observed, which reciprocally (PE and PG) or differently (LPE) change their phospholipid compositions within the envelope compared with mesophilic E. coli.
Section snippets
Strain and growth conditions
Strain 488 of Y. pseudotuberculosis was cultivated in Lysogeny Broth (LB) (Becton, Dickinson and Company). The bacterial cells were grown to log phase (OD600 = 1) at 8 °C and 37 °C under aerobic conditions with shaking at 180 rpm. One part of bacterial cells grown at 8 °C were heat-shocked at 45 °C for 30 min by transferring flask to a water bath shaker (RSB - 12, Remi Elektrotechnik Limited). The bacteria were separated from culture medium by centrifugation at 1200 g for 20 min and then washed
Identification of the inner and outer membranes from the Y. pseudotuberculosis cells
Equilibrium sucrose density gradient centrifugation of the total membrane fraction from Y. pseudotuberculosis resulted in the separation of the crude membrane fraction into three easily visualized, discrete membrane bands. A spectrophotometric analysis of the membrane fractions obtained after centrifugation (Fig. 1A) revealed three distinct peaks at 280 nm absorbance. Peaks I–III were designated in order of decreasing apparent buoyant density (1.25, 1.19 and 1.16 g/cc, respectively). The
Discussion
The cell envelope of all gram-negative bacteria, including enteropathogenic Y. pseudotuberculosis, is composed of OM and IM. Studies on E. coli and some other species of gram-negative bacteria show that the protein and lipid compositions of their OM and IM are essentially different [11], [12]. Therefore, a question arises about the validity of the application of the data on the adaptive changes in the lipids of whole cells of gram-negative bacteria to interpret their relationship with different
Conclusions
The cell envelope of heat-adapted and heat-shocked Y. pseudotuberculosis cells was separated into fractions that were primarily composed of the IM and OM. The results indicate that the adaptive changes in the phospholipid and fatty acid compositions appears to be quantitatively different in the IM and OM of psychotrophic Y. pseudotuberculosis and mesophilic E. coli and E. carotovora. The ability of psychrotrophic Y. pseudotuberculosis to synthesize and maintain a high amount of LPE,
Conflict of interest
None declared.
Author contributions
Contributions of the first two co-authors LD and SB were equal. NS and SB conceived the experiments; SB, LD, MBa, PV performed experimental work; NS, LD, SB, MBc, PV analyzed and wrote the manuscript.
Author agreement
Material submitted in MS “Effect of elevated growth temperature and heat shock on lipid composition of inner and outer membranes of Yersinia pseudotuberculosis” is original; all authors (Ludmila Davydova, Svetlana Bakholdina, Maria Barkina, Peter Velansky, Mikhail Bogdanov, Nina Sanina) are in agreement to have the article published.
Acknowledgements
This work was supported by Russian Science Foundation Grant (project 15-15-00035). Authors thank Khomenko V.A. for the protein standard of OmpF-like porin from Y. pseudotuberculosis.
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