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Isolation and characterization of extreme halophilic archaea M. Franze, A. Cherkouk Extreme halophilic archaea from the family Halobac- A31_2 A32_2 salt rock isolates tereacea represent a dominant part of the microbial A34_2 Haloamina salifodinae JCM 18548 (AB935417) community present in saline soils as well as rock salts. Halobacterium hubeiense JI20-1(LN831302) Halobacterium sp. BAB-4204 (KJ794143) By using a culture-dependent approach different Halobacterium sp. Q37 (KJ644181) Uncultured haloarchaeon clone 10EY-Ar51 (FM946148) Haloarchaea could be isolated and were phylogenetic Uncultured Halobacteria clone B-72-11-LP6 (EF535044) analysed. Interestingly, isolates closely related to dif- A30_2 Halobacterium sp. A1 (AJ548827) ferent Halobacterium spp. were found in both envi- Halobacterium noricense JCM 15102 (NR 113426) Halobacterium noricense A1 (NR 028187) ronments. A22_2 Uncultured archaeon clone CK572 (HQ400559) Halosimplex sp. XZYJT29 (KX585242) Salt rock formations are considered as potential host rock Halosimplex carlsbadense 2-9-1 (NR 114594) A5_2 systems for the long-term storage of highly radioactive Halobacteriaceae archaeon LT61 (KM520759) Natrinema sp. YIM 93581 (JF449432) waste in a deep geological repository. To date, little is Natrinema altunense JCM 12890 (NR 113524) Halorubrum sp. FIC145 (EU308273) known about the habitat salt rock and the way of life of the A16_1 Uncultured archaeon clone H162 (HQ400434) microorganisms occurring there. Next to bacteria and fungi, Halobacteriaceae archaeon RO5-14 (FJ944013) extreme halophilic archaea are dominating this habitat. It is A11_1 of interest to know what kind of extreme halophilic archaea Halopelagius fulvigenes YIM 94188 (NR 109659) A15_2 are living there, how active they are under repository rele- Uncultured archaeon clone CK522 (HQ400542) Halopelagius inordinatus RO5-2 (NR 116491) vant conditions, and how these microorganisms can influ- Halopelagius inordinatus JCM 15773 (NR 113459) ence the safe storage of the waste. So far, microbial investi- Halopenitus persicus DC30 (JF979130) Uncultured archaeon clone ARC182 (JN185055) gations regarding the disposal of radioactive waste in salt Halobacteriaceae archaeon TH33 (KM496553) 0.020 Haloparvum sedimenti JCM 30891 (LC163946) rock were only carried out in the WIPP (Waste Isolation Pi- Halorubrum sp. DYS4 (KP202830) [1] Halophilic archaeon CLR-175-A1 (KF561231) lot Plant) in Carlsbad, New Mexico, U.S.A. Furthermore, A7_1 Uncultured archaeon clone B179 (KT216158) recently an extremely halophilic archaeon termed Halobac- Uncultured haloarchaeon clone ZB-A7 (AF505666) terium noricense was also isolated from the WIPP site, Halorubrum gandharaense MK13-1 (NR 136762) which is phylogenetic closely related to another Hbt . strain Halorubrum sp. YPL12(KT368145) [2] A13_2 DSM-15987 isolated from a salt mine in Austria. A15_1 A20_2 EXPERIMENTAL. The microbial diversity in salt rock Fig. 1: Phylogenetic dendrogram showing the position of isolated hal o- from Gorleben as well as saline soil sample from Arava De- archaeal species from two different time points of isolation (1,2) sert, Israel, was examined with culture-dependent methods. based on 16S rRNA gene sequences. The sequence data used were obtained from the NCBI database. Bar: 2 % sequence di- The latter was investigated in order to get experiences in vergence. different approaches to isolate halophilic archaea and to de- velop new methods to get isolates from our salt rock sam- sample only five colonies appeared after two months. After ples from Gorleben. A specific portion of the samples were transferring individual colonies to new plates and subse- incubated at room temperature in three different sodium quently cultivation in liquid MR2A medium, the isolates chloride con-centrations (2 M, 3 M and 4 M) of modified were further characterized by 16S rRNA gene analysis. The [3] R2A resuscitation buffer. After 24 h incubation time, gene sequences obtained from respective isolates were com- 300 µL of the respectively mixtures were spread on corre- pared with the sequences of the NCBI database by means of sponding agar plates containing modified R2A medium and BLAST algorithm [7] to get the phylogenetic closest relatives adapted for halophilic microorganisms and were incubated (Fig. 1). [4] at 37 °C in the dark. Selected colonies were transferred to The isolates of the saline soil sample can be assigned to dif- new plates and afterwards in MR2A liquid medium to get ferent genera Natrinema, Halorubrum, Halopelagius, individual isolates. For phylogenetic characterization of the Haloamina and Halobacterium . The closest relative of the obtained isolates, the 16S rRNA gene was amplified by insi- isolates from the salt rock is with a sequence similarity of tu PCR of the genomic DNA from cells lysed in purified more than 99 % Halobacterium hubeiense JI20-1, which water. The gene encoding 16S rRNA was amplified by PCR was isolated from a 123 million years old core from a salt [5] with the primer 21f and the reverse primer 958r were used mine in Qianjing, China.[8] One isolate of the soil sample for the PCR and the amplifications were performed with 30 namely A30_2 was also closely related to the same species cycles, each of which consisted of denaturation for 1 min at with 99 % sequence similarity. Furthermore, H. hubeiense 94 °C, annealing for 1 min at 55 °C, and polymerization for has a high sequence similarity to H. noricense , which was 2 min at 72 °C. The purified PCR products were Sanger se- isolated from the WIPP (Fig. 1). Therefore, the obtained iso- quenced by GATC Biotech. The 16S rRNA gene sequences lates could be used for further investigations, e. g. experi- were aligned using ClustalW and the phylogenetic tree was ments under repository relevant conditions as high tempera- constructed with MEGA 7 using the maximum-likelihood ture and radiation as well as interactions with radionuclides. [6] algorithm. [1] Swanson, J. S. et al. (2012) Statusreport Los Alamos National La- RESULTS. With the chosen approach extreme halophilic boratory LA-UR-12-22824 , p. 1. archaea could be isolated from both kinds of samples. Dif- [2] Gruber, C. et al. (2004) Extremophiles 8, 431 – 439. ferences were obvious in time when first visible colonies [3] Jankowski, U. (2009) Report FZD-511 , p. 35. [4] Robinson, J. L. et al. (2005) J. Bacteriol . 187 , 923 – 929. appeared and in amount of colonies. From the saline soil [5] DeLong, E. F. (1992) Proc. Natl. Acad. Sci . USA 89 , 5685 – 5689. sample much more colonies, which differ in shape and col- [6] Kumar, S. et al. (2016) Mol. Biol. Evol . 33 , 1870 – 1874. or, were visible after two weeks, whereas from salt rock [7] Altschul, S. F. et al. (1990) J. Mol. Biol . 215 , 403 – 410. [8] Jaakkola, S. T. et al. (2016) Environ. Microbiol . 18 , 565 – 579. 52 HZDR | Institute of Resource Ecology | Annual Report 2016 .
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