阿塔卡馬沙漠是世界上最乾燥、最貧瘠的地區之一，其中的Salar Grande盆地位於智力北部海岸山脈的東側，數百萬年的乾旱和高強度太陽輻射造就了該地區獨特的鹽灘風貌。這裡的鹽灘是一個富含氯化鈉的礦床，巖鹽結核的內部和表面蘊藏了豐富的微生物資源，但這些微生物大部分不可培養。作者從巖鹽中成功分離出第一株富含菌紅素（bacterioruberin，BR）的極端嗜鹽古菌Haloterrigena sp. Strain SGH1。菌株SGH1是一種平均直徑為1-2 µm的革蘭氏陰性、球狀微生物，透射電鏡觀察古菌SGH1的超微結構，發現其細胞質中有幾個球狀顆粒，類似於古菌的氣泡結構（圖1）。菌株SGH1的最適生長條件為：25%(w/v) NaCl、45 ℃、pH 7-8。在25-35℃，25–30% NaCl條件下，菌株SGH1類胡蘿蔔素積累量約為400 µg/g乾重。在菌株SGH1中，類胡蘿蔔素積累是一個依賴於鹽度的可逆過程，將含色素的細胞從25% NaCl轉移到15%時色素含量降低約20倍，而將褪色的細胞再轉回高鹽度培養時色素會逐漸恢復至最高水平。除此之外，菌株SGH1對高氯酸鹽和氯化鎂具有較好的耐受性，最高耐受濃度分別為0.15 M和0.5 M。系統發育樹顯示菌株SGH1與Haloterrigena屬有較近的親緣關係，因此菌株SGH1初步定名為Haloterrigena sp.strain SGH1。
FIGURE 1 | (a, b) TEM micrographs of isolate Haloterrigena sp. strain SGH1. Cells are unicellular cocci with cytoplasmic electron-lucent globular structures resembling gas vacuoles. Bar: 0.2 µm.
作者採用色譜遷移、紫外可見光譜、光譜精細結構和質譜分析等方法對Haloterrigena sp. SGH1中的C50類胡蘿蔔素進行了鑑定。吸收光譜顯示菌株SGH1類胡蘿蔔素的甲醇粗提物具有典型的「三手指峰」（分別為468、494和526 nm）。HPLC/UHPLC分析結果顯示粗提物包含17個組分，並且通過HPLC分離獲得了9個組分(圖4)，其中組分FI含量最高。之後選取了含量較高的5個組分（FI~FV），採用UHPLC-MS系統進行二次分離，結果顯示從FI、FII、FIII、FIV和FV中分別獲得了3、4、4、3和3個亞組分（圖5），並且所有亞組分的吸收光譜均與BR類似，但最大吸收峰的波長略有不同。
FIGURE 4 | HPLC chromatogram of a methanolic extract from isolate Haloterrigena sp. strain SGH1, grown in Z8-MOD medium at 25% NaCl and 35℃. Cells were harvested, lyophilized and extracted with 100% methanol. An aliquot (50 µl) was injected in a HPLC Shimatzu Hitachi LC-20A Prominence, equipped with a reverse phase C18 column) and eluted with methanol: acetonitrile: water (85:10:5, v/v) at a flow rate of 1.0 ml/min, during 12 min. Five major fractions are observed and denominated FI to FV. Asterisks show the retention times used to compute the area of each fraction to obtain their relative abundance. Fraction collection was done at specific retention time avoiding the contamination between peaks.
FIGURE 5 | UHPLC chromatogram of Fraction FI from a methanolic extract of isolate Haloterrigena sp. strain SGH1. Lyophilized cells grown in Z8-MOD medium at 25% NaCl and 35℃ were extracted with 100% methanol and Fraction FI was recovered after separation by HPLC. An aliquot of 10 µL of Fraction FI was injected in a UHPLC Thermo Scientific Dionex Ultimate-3000 system, equipped with a C18 column and eluted with a gradient of methanol: acetonitrile. The inset shows the UV-Vis spectrum of Subfraction FI-1 (retention time: 10.39 min), the most abundant UHPLC signal.
通過最大吸收峰波長487-494 nm(II峰)、520-527 nm(III峰)和388 nm(順式或DII峰)來區分菌株SGH1BR的幾何異構體和脫水衍生物。根據分析結果和文獻報導推斷菌株SGH1細胞含有6個與BR家族相關的C50分子：4個幾何異構體(5-cis-BR、9-cis-BR、13-cis-BR和全反式BR)、兩個脫水衍生物(全反式脫水BR和順式脫水BR)，以及一個代謝中間體(全反式單脫水BR)。其中，全反式-BR含量最高，約佔BR總量的65%。
作者採用芬頓反應（Fenton Reaction）、ABTS 試驗(2,20-Azinobis-(3-Ethyl-Benzothiazole-6-Sulfonic Acid Assays)和FRAP 試驗(Ferric Ion Reducing Antioxidant Power Assay) 3種方法評估Haloterrigena sp.SGH1菌紅素的抗氧化能力。與抗氧化劑奎諾二甲基丙烯酸酯（Trolox）和β胡蘿蔔素（0.1 µg/µL）相比，即使是低濃度的菌株SGH1提取物（0.001 µg/µL）也能夠有效降低DNA氧化損傷（圖7）。ABTS 和FRAP 試驗表明菌株SGH1的甲醇提取物及純化的BR異構體均具有較強的清除自由基能力，其抗氧化能力是Trolox、β-胡蘿蔔素和蝦青素的幾到十幾倍。
FIGURE 7 | Dose-dependent DNA relaxation assay used to evaluate the antioxidant activity of the methanolic extract from Haloterrigena sp. SGH1. (A,B) Changes on the electrophoretic mobility of plasmid pUC19 were observed on 0.8% agarose gels after incubating the plasmid with water (as negative control: A1 and B1), Fenton reagent (as positive control: A2 and B2), Fenton reagent plus 0.001 µg/µL SGH1 extract (A3), Fenton reagent plus 0.002 µg/µL SGH1 extract (A4 and B5), Fenton reagent plus 0.1 µg/µL β-carotene (B3) and, Fenton reagent plus 0.1 µg/µL Trolox (B4)。
作者以源自1歲急性單核細胞白血病患兒血液的THP-1單核細胞系（ATCC® 30-2001）)為實驗材料，研究了菌株SGH1甲醇提取物及各純化組分I、II、III、IV、V是否對人細胞具有毒性。結果顯示，與對照組抗黴素A（55.83±1.49%）相比，實驗組的細胞存活率較高（圖8），表明它們對THP-1細胞沒有毒性。
FIGURE 8 | Toxicity of carotenoids from isolate Haloterrigena sp. strain SGH1 against THP-1 cells. The methanolic extract (SGH1 Extract) and purified fractions F-I, F-II, F-III, F-IV and F-V from Haloterrigena sp. strain SGH1 (500 µg/mL of each as final concentrations in the assays), were incubated with THP-1 cells for 18 hours. After that, cells were incubated with propidium iodide (PI) by 15 min and viable cells (PI negative) were quantified by flow cytometry. Methanol (MeOH) and Ethanol (EtOH) were used as negative control and 30 µM Antimycin A was used as positive control. The results were expressed as percentage of viable cells, 18 h post-treatment. T-test: +p < 0.5 compared with the control group (MeOH), n = 4.
綜上所述，Haloterrigena sp. SGH1是從阿塔卡馬沙漠巖鹽微生物群落中分離出的第一株極端嗜鹽古菌。菌株SGH1細胞中BR含量較高，且細胞內BR合成是一個受鹽度調控的可逆過程。菌株SGH1對高氯酸鹽具有較好的耐受性，可以作為新的實驗模型用於研究高氯酸鹽代謝相關的基因、酶及其生物去除方法。純化的BR異構體抗氧化活性均高於Trolox、β-胡蘿蔔素和蝦青素，且對培養的THP-1細胞無毒，因此它們在生物醫藥、食品和醫療用品等行業具有潛在的應用前景。