Comparison of two extreme halophilic Halobacterium noricense strains on DNA and protein level M. Bader, K. Flemming, J. S. Swanson,1 A. Cherkouk 1Los Alamos National Laboratory, Carlsbad, U.S.A.

Two strains of the halophilic archaeon Halobacterium noricense isolated from rock salt of different locations A B were used for interaction studies with uranium. It was found that both strains showed similar, atypical bioas- sociation kinetics accompanied by cell agglomeration as a stress response. The 16S rRNA gene sequences of both strains had a high similarity (> 99 %). However, 50 µm 20 µm differences in the whole protein pattern were appar- ent. Fig. 1: Fluorescence micrographs of H. noricense cells incubated with uranium (100 µM) for 48 h, at pC H+ 6. Red fluorescence shows Halobacterium noricense is a halophilic archaeon common- dead cells, green fluorescence living cells. ly found in salt rock. Investigations into its bioassociation H. noricense DSM-15987 (A) and H. noricense WIPP (B). behavior with uranium have been conducted with two dif- Differences between H. noricense DSM-15987 and ferent strains. Halobacterium noricense DSM-15987 was [1] H. noricense isolated from the WIPP are particularly appar- isolated from an Austrian salt mine and Halobacterium ent in the whole protein pattern (Fig. 2). Due to the fact that putatively noricense WIPP strain was isolated from the salt [2] the same amount of biomass was loaded to the gel, a direct basin in New Mexico, U.S.A., which hosts the Waste Iso- comparison is possible. It can be seen that bands are not on- lation Pilot Plant (WIPP) – a final repository for transuranic ly different in their intensity (Fig. 2, white arrow) but also waste. Exposure of both strains to uranium over time result- different in size. For example, the WIPP-strain shows an ed in similar multistage bioassociation kinetics and an in- [3] additional band at 78 Da (Fig. 2, gray arrow). Other bands crease in cell agglomeration. Nevertheless, small differ- are only shifted, like the one at 50 Da, which is shifted to ences were apparent, especially regarding the size of the ag- 53 Da for the DSM-strain (Fig. 2, black arrow). No differ- glomerates (Fig. 1). Hence, a comparison of the strains at ence in protein pattern of samples exposed to uranium for the DNA and protein level is of interest. up to three weeks (data not shown) were visible. Further proteomics analyses are ongoing. EXPERIMENTAL. Both H. noricense strains were culti- vated in DSM372 medium in the dark with shaking at 30 °C. Cells were harvested in the mid exponential phase and washed three times with 3 M NaCl, pC H+ 6.0 (corrected pH due to high ionic strength). [4] Bioassociation. A defined amount of cells (5 mg dry bio- mass) was resuspended in 10 mL of filter-sterilized 3 M NaCl solution with 100 µM uranium adjusted to pC H+ 6. Af- ter 48 h, cells were washed (3 M NaCl, pC H+ 6) and a stain- ing with the LIVE/DEAD® BacLight TM Bacterial Viability Kit L7012, Molecular Probes TM , Inc., Eugene, OR, U.S.A. was performed. Protein level. To compare both strains on a protein level, sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), using a 10 % polyacrylamide separation gel, was performed.[5] The gels were loaded with an amount of cells corresponding to 0.1 mg dry biomass and stained with Fig. 2: SDS -PAGE image colloidal Coomassie brilliant blue. of the whole pro- DNA level. For comparison on a DNA level, washed cells tein pattern of were lysed in water to exclude the high content of NaCl be- both H. noricense fore an in situ polymerase chain reaction (PCR) was per- strains. formed. Therefore, the Arch 21f and Arch 958r primer were [6] TM In conclusion, despite the fact that both investigated strains used. The amplified product was purified (QuickStep 2 show the same atypical bioassociation kinetics,[3] differ- PCR Purification Kit, Edge Bio, BD) and sequenced from ences on DNA and protein level exist. GATC Biotech AG. ACKNOWLEDGEMENTS. The authors would like to thank RESULTS. The 16S rRNA gene sequences of both M. Dudek and S. Kluge for the preparation of the SDS-PAGE gels. H. noricense strains have a 99.9 % similarity. Out of 853 bases only one was different. At this position, DSM-strain [1] Gruber, C. et al. (2004) Extremophiles 8, 431 – 439. has an A and the WIPP strain a G. Interestingly, the G in [2] Swanson, J. S. et al. (2012) Report LA-UR-12-22824 , p. 1. this position is also present in other isolates belonging to the [3] Bader, M. et al. (2017) J. Hazard. Mater. 32 , 225 – 232. genus of Halobacterium . The archaeon isolated from the [4] Borkowski, M. et al. (2009) Report LA-14360 , p. 26 – 27. [5] Laemmli, U. K. (1970) Nature 227 , 680 – 700. WIPP shows a 100 % similarity in 835 bases of the [6] DeLong, E. F. (1992) Proc. Natl. Acad. Sci. USA 89 , 5685 – 5689. 16S rRNA gene sequence of Halobacterium sp. YI80-2 iso- [7] Jaakkola, S. T. et al. (2014) PLoS ONE 9, e110533. lated from Yunying salt mine in Hubei Province, China.[7]

HZDR | Institute of Resource Ecology | Annual Report 2016 51