Príloha č. 1. Králová S. (2017) Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp. Syst. Appl. Microbiol., 40(6): 329-333. Doi: 10.1016/j.syapm.2017.06.001 Systematic and Applied Microbiology 40 (2017) 329–333 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ournal homepage: www.elsevier.de/syapm Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp. Stanislava Králová Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic a r t i c l e i n f o a b s t r a c t Article history: Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in Received 9 May 2017 extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the Received in revised form 8 June 2017 membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty Accepted 12 June 2017 acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types Keywords: of major fatty acids than meso- and thermophilic members of this genus, namely C15:1 iso G, C15:0 iso, Flavobacterium C15:0 anteiso, C15:1 ω6c, C15:0 iso 3OH, C17:1 ω6c, C16:0 iso 3OH and C17:0 iso 3OH, summed features 3 Antarctic bacteria ω ω ω Adaptation (C16:1 7cand/or C16:1 6c) and 9 (C16:0 10-methyl and/or C17:1 iso 9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results Fatty acids Cell membrane also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration Cold-shock in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium. © 2017 Elsevier GmbH. All rights reserved. Introduction effort needed to maintain a liquid crystalline state [4,12,25,29,30]. The response of bacterial cells leading to maintenance of a fully Microscopic life in cold regions of the Earth is characterized by functional cell membrane is termed homeoviscous adaptation. a high biodiversity comprised mainly of microorganisms such as Bacteria achieve homeoviscous adaptation to low temperatures bacteria, fungi and microalgae [6]. To survive in conditions typical (cold-shock) by using several mechanisms. These cold-induced of cold regions (cold temperature, a lack of water and/or nutrients, responses are common among different bacterial groups and intense UV irradiation and/or higher salinity) various adaptation include an increase in FA unsaturation, FA methyl branching and/or mechanisms are required [7]. Of these conditions, cold tempera- increasing the iso/anteiso-branched FAs ratio [8,22–24]. Moreover, tures strongly affect microbial cellular functions by influencing cell with a decrease in temperature, the average length of FAs, as well as integrity, water viscosity, diffusion rates, the kinetics of enzyme the sterol/phospholipid ratio, have been shown to decrease [13,25]. reactions and membrane fluidity [7]. These changes in different combinations and quantities allow bac- In recent years, researchers have intensively studied cold- teria to retain membrane function and fluidity [24] and thereby adapted bacteria in order to determine the adaptations related to sustain the survival of these bacterial cells. their cytoplasmic membrane [13]. The composition of the mem- Previously, a number of bacterial species have been studied brane fatty acids (FAs) strongly affects membrane properties by in order to characterize their membranes or the mechanisms influencing fluidity and/or biological functions, such as solute of their membrane temperature adaptation. These studies were uptake, nutrient transport, osmotic pressure and/or energy pro- mainly focused on Gram-positive bacteria, such as members of duction [4]. These functions are directly associated with natural the genera Bacillus, Clostridium, Micrococcus or Listeria monocyto- forms of biomembranes represented by the semi-liquid state of genes [2,8,20,21,31]. Within the Gram-negative bacteria, the main lipid structures. It is well known that environmental conditions and object of chemotaxonomic studies has been the cell wall structure stresses strongly affect cell membrane structure, due to the cellular [10,14,15,19,27,29]. This current study focused on the cold-adapted Flavobacterium spp. isolated from Antarctica, and on their cell membrane proper- ties. The aims of the study were: (i) to determine the FA composition E-mail address: [email protected] http://dx.doi.org/10.1016/j.syapm.2017.06.001 0723-2020/© 2017 Elsevier GmbH. All rights reserved. 330 S. Králová / Systematic and Applied Microbiology 40 (2017) 329–333 of Antarctic Flavobacterium spp., (ii) to determine their cold- re-cultivated three times and their FAs were analysed. The cultiva- adaptation mechanisms to low temperatures due to alteration of tion temperatures were initially set up according to optimal growth ◦ ◦ ◦ ◦ the FA composition, (iii) to compare the FAs of Antarctic flavobac- temperatures (20 C, 25 C, 30 C and 50 C) and, using standard teria to mesophilic and thermophilic reference strains of the genus deviation values, more stable FA profiles were obtained after cul- Flavobacterium, and (iv) to compare the cold-shock response of all tivation for 48 h. The same process was repeated with the strains ◦ ◦ ◦ three of these thermotypes. cultivated under lower cultivation temperatures (10 C, 15 C, 20 C ◦ and 40 C). In this case, the FA values varied less when the cultiva- tion of Antarctic strains was prolonged for 72 h, since the growth Materials and methods rate decreased significantly at lower temperature. The indicator of the late exponential growth phase required for harvesting the Bacterial strains bacterial biomass was confluent growth in the third sector of the agar plates, which was reached after 72 h cultivation. FA profiles Two groups of Flavobacterium spp. were used in this study. The T of mesophilic flavobacteria and F. thermophilum CCM 3496 were first group included 28 unique psychrophilic gliding Flavobacterium again most reproducible after cultivation for 48 h. In general, the FA spp. isolated from terrestrial abiotic sources in Antarctica (vari- amounts varied by less than 2%, which indicated high reproducibil- ous water sources, rocks and regolith from James Ross Island). The ity and stability of FA composition patterns. strains used are listed and detailed in Table S1. These psychrophilic species exhibited the main characteristics of the genus Flavobac- Fatty acid composition of Antarctic Flavobacterium strains terium: Gram-negative rods with a yellow pigment exhibiting gliding motility. The assignment of these bacteria to the genus A total of 42 FAs were detected in the FAME profiles for all the 28 Flavobacterium was based on sequencing of their 16S rRNA gene Antarctic flavobacteria cultivated under optimal growth conditions and MALDI-TOF MS analysis (data not shown). All strains grew well ◦ ± ◦ (20 2 C, 48 h). Nineteen FAs were uniformly present among all under aerobic conditions at a temperature of 20 C. strains, with 10 of them present at values greater than 4% for most The second group was comprised of mesophilic flavobacte- strains, and these were assigned as the main FAs of the Antarctic ria reference cultures and one thermophilic reference culture of T gliding Flavobacterium spp. (Fig. S1; bold emphasized in Table S3). Flavobacterium thermophilum CCM 3694 with optimal growth ◦ ◦ According to Bernardet and Bowman [3], it is clear that members temperatures of 25–30 C and 50 C, respectively. The reference of the genus Flavobacterium have typically high amounts of C strains were obtained from the Czech Collection of Microorganisms 15:0 iso, and significant amounts of C , C iso 3OH, C anteiso, (http://www.sci.muni.cz/ccm/) and are detailed in Table S2. 15:0 15:0 15:0 C15:1 iso G, C15:1 ω6c, C16:0 iso 3OH, C17:0 iso 3OH, C15:0 iso 2OH and/or C16:1 ω7c and/or C16:1 ω7t. In this study, the highest quanti- Cultivation for fatty acid methyl ester (FAME) analysis ties were found among branched FAs, namely C15:0 iso (18.6%), C15:0 anteiso (6.0%), C15:0 iso 3OH (8.4%), C17:0 iso 3OH (8.2%), C15:1 iso All strains used in the study were cultured under optimal growth G (9.6%) and summed feature 3 containing C16:1 ω7c and/or C16:1 conditions for 48 h, and at temperatures that decreased by approx- ◦ ω6c (12.6%). Slightly lower quantities were detected for C17:1 ω6c imately 10 C (for 48 or 72 h) in order to investigate the cold-shock (3.7%) and C16:0 iso 3OH (5.3%). These major components matched response of their cell membranes. The psychrophilic strains used ◦ ◦ the taxa-specific FAs mentioned above, except for summed fea- for FA analysis were grown on R2A agar at 20 ± 2 C and 10 ± 2 C ture 9 that included C16:0 10-methyl and/or C17:1 iso ω9c (4.3%), for 48 and 72 h, respectively. The mesophilic reference strains used ◦ ◦ ◦ which were found among all strains in quantities varying from were grown on R2A agar at 30 ± 2 C and 20 ± 2 C, 25 ± 2 C and ◦ 0.8 to 7.7%. These FAs made up approximately 83% of the over- 15 ± 2 C for 48 h, and one thermophilic strain of F. thermophilum ◦ ◦ all FA membrane content. Thus, the results clearly showed that was grown at 50 ± 2 C and 40 ± 2 C for 48 h.