Elsevier

Biochimie

Volume 123, April 2016, Pages 103-109
Biochimie

Research paper
Effects of elevated growth temperature and heat shock on the lipid composition of the inner and outer membranes of Yersinia pseudotuberculosis

https://doi.org/10.1016/j.biochi.2016.02.004Get rights and content

Highlights

  • Elevated growth temperature and heat shock increase LPE content predominantly in OM.

  • The content of anionic PG and neutral PE are regulated within OM reciprocally.

  • The ratio between unsaturated and saturated FAs remains higher in IM than that in OM.

  • Heat shock unlike elevated growth temperature does not change FA composition.

Abstract

Differences in the distribution of individual phospholipids between the inner (IM) and outer membranes (OM) of gram-negative bacteria have been detected in mesophilic Escherichia, Erwinia and Salmonella species but have never been investigated in the psychrotrophic Yersinia genus. Therefore, the influence of an elevated growth temperature and heat shock on the phospholipid and fatty acid (FA) compositions of the fractionated Yersinia pseudotuberculosis envelope was investigated. The shift of the growth temperature from 8 °C to 37 °C to mimic the switch from saprophytic to parasitic growth of this bacteria and the exposure of the cells to heat shock, which was induced by a sharp increase in the temperature from 8 °C to 45 °C, increased the lysophosphatidylethanolamine content from zero and 1% to 6% and 10% in the IM and OM, respectively. These changes were accompanied by a decrease in the phosphatidylethanolamine (PE) content and a drastic increase (up to 3-fold higher) in the phosphatidylglycerol (PG) level in the OM of the bacteria, which increases the net negative charge of the cell envelope. The levels of the predominant saturated palmitic (16:0) and cyclopropane FAs were approximately 1.5- and 7.5-fold higher, respectively, but the content of the predominant unsaturated palmitoleic (16:1n-7) and cis-vaccenic (18:1n-7) FAs was approximately 10–30-fold lower in both membranes that were isolated from the cells grown at elevated temperatures. Due to these changes, reflecting the process of “homeoviscous adaptation”, the ratio between the unsaturated and saturated FAs decreased but remained higher in the IM than that in the OM. Simultaneously, no significant changes were observed in the FA composition of cells subjected to heat shock, demonstrating a difference between the responses of the heat-shocked and heat-adapted Y. pseudotuberculosis. The unique ability of Y. pseudotuberculosis to reciprocally regulate the ratio of anionic PG and net neutral PE and therefore adjust the negative charge of the OM may be a common strategy used by pathogenic bacteria to promote the barrier function of the OM.

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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