P-8

雪氷環境における好冷性微細藻類の共⽣細菌
Hymenobacter nivisのゲノム解析及び代謝機構の解明
寺島美亜 （北海道大学低温科学研究所、微生物生態学分野）

氷河や高山雪原では雪解けが進む春、雪の表面をピンクに染める赤雪現象が起こる。この現象は藻類増殖によるものであり、低温、強光、高紫外線、低栄養、凍結による乾燥などのストレス下においても藻類が生育している。赤雪には微細藻類のほか、細菌も検出されており、藻類が生産した有機物を消費していると思われる。南極の赤雪からは真核緑藻と一緒に従属栄養性細菌の Hymenobacter nivisが特異的に検出されている。本研究では、赤雪に優占している H.
nivis が南極赤雪の厳しい環境へ適応するメカニズムを解明するため、ゲノム解析、プロテオーム解析及び培養実験を行った。その結果、光に反応するタンパク質（プロテオロドプシン、クリプトクロム等）の遺伝子がゲノム上に含まれていることが明らかになった。
さらに、光と暗環境下とで本菌の増殖を比較をすると、光環境下の方が速かった。また、 H. nivis のプロテオームを解析したところ、光反応のタンパク質が発現されていることがわかった。このうした
光反応タンパク質により、 H. nivis は光を認識し、利用することが可能であり、南極赤雪の低温・強光環境に適応し、優占していると考えられる。
Hymenobacter nivis was isolated from algae-rich red snow in Antarctica (Kojimaet al., 2016). H. nivis was found to be by far the most dominating bacterium in
red snow, suggesting that it is well-adapted to flourish in the snow surface
environment (Fujii et al., 2010). This study aims to understand the adaptive
strategy of H. nivis in low temperature and light-exposed environments throughgenome and proteome analyses as well as growth tests.

I. Analyzing the genome of Hymenobacter nivis P3T

III. H. nivis cells grown in the dark produce more EPS• Total carbohydrates and protein levels in the extracellular polymeric
substances (EPS) were determined with cells at stationary growth phase.
• EPS was higher under the dark conditions on a per cell basis.

Fig. 2. Total carbohydrate (panel A) and protein (panel B) concentrations
from extracellular polymeric substances (EPS) extracted from H. nivis
P3 T cells at stationary phase after growth at 5 ˚C. P=1.4x10 -4 (panel A)
and P=1.8x10 -6 (panel B) by non-paired two sample T-test.

IV. Light-reactive proteins are expressed in the proteome• Total protein was extracted from H. nivis P3 T cultured in the light and dark
conditions at 5 ˚C.
• The proteome was analyzed through mass spectrometry using a Q-Exactive
plus Orbitrap mass spectrometer (Thermo Fisher Scientific).
• The proteome confirmed the expression of light reactive proteins such asproteorhodopsin, phytochrome, photolyase and cryptochromes.

• The genome of H. nivis P3 T was sequenced using PacBio (Takara Bio).
• Genome sequencing reveals the presence of pathways adapted for cold andhigh light environments.
• H. nivis P3 T genome contains the carotenoid biosynthesis pathway and lightreactive proteins such as proteorhodopsin, cryptochromes, a phytochrome,deoxyribodipyrimidine, and photolyase.
Table 1. Genomic features of Hymenobacter nivis P3 TFeatures
Genome size (bp)5,027,597
Contig1
DNA GC content (%)
62.5
Total protein-coding genes
4604
Protein-coding genes with function prediction 2563
Protein-coding genes of hypothetical function 2041
rRNA9tRNA
43
Number of subsystems
364
Protein-coding genes in subsystems
1324

II. Growth of Hymenobacter nivis P3T is enhanced under light••••Growth of under light and dark conditions were compared at 5 ˚C and 15 ˚CCell densities were determined using flow cytometry
Both the speed and capacity of growth were enhanced under the light
These results indicate that H. nivis can utilize light positively for growthFig. 3. Proteome of H. nivis P3 T under dark and light growth conditions. 1724
proteins are plotted on the graph and the values plotted represent the % molar
emPAI for each protein identified with two or more unique peptides.

V. Conclusions

Fig. 1. Growth of H. nivis P3 T monitored under dark and light (200 µmol/m 2s)
conditions at 5 ˚C (panel A) and 15 ˚C (panel B).

The genome of a Antarctic heterotrophic bacterium Hymenobacter nivis P3 T wassequenced, revealing several genes encoding for putative light-reactive proteins.
Growth rates were enhanced under the light, while EPS accumulation was higher
in the dark. Further analysis of the proteome of dark- and light-grown cells at 5
˚C identified that the proteome remains similar between the two conditions.
Furthermore, the expression of putative light-reactive proteins were confirmed.
These identified proteins were: proteorhodopsin, cryptochrome, phytochrome,and deoxyribodipyrimidine photolyase. The light-enhanced growth of H. nivis
P3 T and the expression of proteorhodopsin and other putative light-reactive
proteins suggest that this bacterium is likely to sense and utilize light, resultingin a growth advantage under the light-exposed conditions of Antarctic red snow.
The results from this study has been published (Terashima et al. 2019).

Acknowledgements:
Many thanks to IFO for funding this study. I thank Manabu Fukui, Hisaya Kojima, Taichi Takasuka and Keisuke Ohashi for experimental and intellectual assistance.
References:
Fujii et al., 2010, Microb Ecol, 59(3):466-75; Kojima et al., 2016, Int J Syst Evol Microbiol, 66(11):4821-4825; Terashima et al., 2019, Environ Microbiol Rep, 11(2):227-235.