Phospholipids and protein adaptation of Emphasis

Authors: Przemysław Bernat Paulina Siewiera Adrian Soboń Jerzy Długoński

Publish Date: 2014/05/03

Volume: 30, Issue: 9, Pages: 2343-2350

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Abstract

A tributyltin TBTresistant strain of Pseudomonas sp isolated from an overworked car filter was tested for its adaptation to TBT The isolate was checked for organotin degradation ability as well as membrane lipid and cellular protein composition in the presence of TBT The phospholipid profiles of bacteria grown with and without increased amounts of TBT were characterized using liquid chromatography/electrospray ionization/mass spectrometry The strain reacted to the biocide by changing the composition of its phospholipids TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid by 58  and phosphatidylethanolamine by 70  An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp was observed These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic In the presence of TBT the abundances of 16 proteins including TonBdependent receptors porins and peroxidases were modified which could indicate a contribution of some enzymes to TBT resistanceTributyltin TBT is a xenoestrogen which disrupts the normal function of human and animal hormonal systems by modelling their activity Endocrine disruption and disruption of lipid homeostasis are the main toxic effects Pagliarani et al 2013 Among organisms attacked by TBT bacterial cells are particularly useful to study the molecular toxicity of membraneactive compounds offering advantages over eukaryotic cells owing to the simple membrane organization Martins et al 2005 TBT has a negative impact on bacterial growth chemotaxis respiration and membrane physical properties in microbial strains Martins et al 2005 Therefore in order to cope with this threat organotinresistant bacteria have developed adaptive responses to the toxicity of TBT Though many authors have reviewed the proposed hypothesis little is known about the mechanism of resistance of microorganisms to organotins Dubey and Roy 2003 Fukushima et al 2012 Among TBTtolerant microorganisms strains from the genus Pseudomonas are often mentioned Roy and Nair 2007 It is also suggested that gene PA0320 in Pseudomonas aeruginosa 25 W as well as other genes such as a transglycosylase homologue in Alteromonas sp and a multidrug efflux transporter in Pseudomonas stutzeri could be involved in stress tolerance against TBT Fukushima et al 2012 However studies on the adaptive stress responses of TBTresistant bacteria including changes in membrane lipid and protein composition are still missingIn this study a systematic analysis of growth rates degradation efficiency regulation of cellular protein and membrane lipid composition of Pseudomonas cultivated with TBT was carried out in order to elucidate cell adaptation to the organotin substrate This knowledge will give some insights into the understanding of the parameters involved in adaptive resistance acquisition by Pseudomonas speciesOrganotins were purchased from Sigma–Aldrich 12Dimyristoylsnglycero3phosphorac1glycerol sodium salt 140/140 PG 12dilauroylsnglycero3phosphoethanolamine 120/120 PE and 12dimyristoylsnglycero3phosphate sodium salt 140/140 PA were purchased from Avanti Polar Lipids All of these compounds were added to a solution of mixed internal standards at the concentration of 01 mg ml−1 IS solution The other chemicals were from J T Baker Fluka and POCh Gliwice Poland All the chemicals were high purity grade reagents Stock solutions of TBT were prepared at 5 mg ml−1 ethanolFor this study Pseudomonas strains isolated from polluted environments were used Based on the results of research concerning Pseudomonas tolerance to TBT Fukushima et al 2012 it was expected that our study would allow us to find a microorganism with high organotinresistanceFivedayold bacterial cultures on malt extract agar slants were used to inoculate 20 ml universal medium per liter 13 g yeast extract 15 g peptone 5 g glucose in 100ml Erlenmeyer flasks The cultivation was carried out on a rotary shaker 140 rev min−1 for 48 h at 28 °C Two ml of the homogenous preculture was introduced into 18 ml of medium with TBT at the concentrations of 10 20 30 and 40 mg l−1 or without the organotin in the control cultures in 100 ml flasks The cultures were incubated for 7 days at 28 °C on a rotary shaker 140 rev min−1 Although Pseudomonas sp B219 is a psychrophilic strain the temperature of 28 °C was recommended by DSMZGerman Collection of Microorganisms for incubation of P proteolytica http//wwwdsmzde Moreover the growth inhibition was not observed at 28 °C Bacterial biomass was separated from culture media by filtering through a Sartorius filter 022 µm and then dried at 105 °C to reach a constant weight The specific growth rate µ was calculated by the leastsquares fitting to the linear part of the semilogarithmic plot of bacterial biomass versus timeBacterial biomass from the stationary phase of growth the samples were withdrawn from 120hold cultures was separated from culture media by centrifugation at 4000×g It was homogenized Misonix Sonicator with 10 ml of a CHCl3–MeOH mixture 21 v/v A total of 30 µl of IS solution was poured into each sample before extraction To the crushed cells 2 ml of 08  NaCl was added the vials were vortexed for 1 min and centrifuged The lower organic phase was collected treated with anhydrous sodium sulphate and evaporated under reduced pressure The residue was then redissolved in 2 ml of methanol/CHCl3 41 v/v and stored at −20 °C pending analysis

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