Halolysin SptA有助于嗜盐古菌Natrinema sp.J7-2长期生存

Halolysin SptA boosts the long-term survival of haloarchaea Natrinema sp. J7-2

摘要:

【背景】嗜盐古菌可以在盐沉积物中存活长达几百万年，是著名的长寿菌。许多嗜盐古菌分泌胞外蛋白酶，大多数分泌的胞外蛋白酶被称为Halolysin，具有以下特征：属于枯草杆菌蛋白酶类蛋白酶；在胞内折叠后经Tat途径高效分泌至胞外；可自加工形成成熟酶；尤其在天然宿主中大多数Halolysin在对数生长后期表达并在稳定期达到最高水平。目前Halolysin的酶学性质、加工成熟及分泌机制已被广泛研究，然而其生理功能的研究较少。Halolysin SptA是嗜盐古菌Natrinema sp.J7-2的主要胞外蛋白酶，前期研究发现多个顺式调控元件协同调节SptA的生长期依赖性表达，使SptA参与J7-2菌株不同生长期之间的转变，而且在衰亡期之后SptA有助于J7-2菌株继续生存。【目的】研究Halolysin SptA对Natrinema sp.J7-2长期生存的作用。【方法】将J7-2菌株和突变体ΔsptA1分别在寡营养、无外源营养物质（液体）及营养丰富（固体）条件下长期培养，通过比较二者的生长、生存和SptA的表达分泌情况进一步探讨SptA的作用。【结果】J7-2菌株在寡营养条件下产生更多SptA，培养后期（33 d） J7-2菌株活细胞数显著高于ΔsptA1。在无外源营养物质情况下长期温育，J7-2菌株和ΔsptA1经历多次细胞分裂和细胞死亡，在延长温育期间（73—200 d）存活的J7-2菌株细胞数量均显著多于存活的ΔsptA1细胞数量。在营养丰富的固体平板上培养的后期（160 d），由于营养物质消耗，J7-2菌株通过SptA吸收和利用来源于死细胞蛋白的降解产物，帮助其群体长期生存。【结论】SptA介导的细胞死亡和死细胞蛋白降解，促进J7-2菌株利用来源于死细胞的营养物质，从而有助于菌株群体在营养缺乏条件下长期存活。本研究提供了关于Halolysin生理作用的新见解。

关键词:嗜盐古菌;Halolysin;SptA;长期生存

Abstract:

[Background] Haloarchaea are able to survive in salt deposits for millions of years. Many haloarchaea produce extracellular subtilisin-like proteases (Halolysins). The Halolysins are folded in cells and transported across cytoplasmic or thylakoid membranes by Tat pathway. In addition, they mature automatically and most of them are produced at the late log phase and peak when the culture enters stationary phase. At the moment, the enzymatic properties, autocatalytic activation, and secretion mechanisms of Halolysins have been widely characterized. However, the physiological functions are rarely studied. Halolysin SptA is the major extracellular protease of Natrinema sp. J7-2. Previous studies showed that the growth phase-dependent production of SptA relies on the cooperative action of multiple cis-acting elements, allowing SptA to participate in the growth-phase transition of strain J7-2. SptA also contributes to the continued survival of strain J7-2 after the death phase. [Objective] To study the effect of SptA on long-term survival of strain J7-2. [Methods] strain J7-2 and ΔsptA1 mutant were cultured under nutrient-deficient, non-exogenous nutrient (liquid), and nutrient-rich (solid) conditions for a long time, respectively. The growth, survival, and SptA expression of the two strains were compared to further investigate the role of SptA. [Results] J7-2 strain produced more SptA under nutrient-deficient conditions, and number of viable J7-2 strain cells was significantly larger than that of viable ΔsptA1 cells after 33 days of growth under such conditions. strain J7-2 and ΔsptA1 underwent multiple events of cell division and cell death during long-term incubation in the absence of exogenous nutrients. The number of viable J7-2 strain cells was significantly larger than that of viable ΔsptA1 cells during the prolonged incubation (73-200 days). At the late stage of culture (160 days) on nutrient-rich solid plates, due to nutrient depletion, J7-2 strain benefited from SptA in terms of long-term survival by assimilating and utilizing the degradation products of proteins derived from dead cells.[Conclusion] The SptA-mediated cell death and degradation of dead cell-derived proteins enhance the long-term survival of J7-2 strain in response to nutrient starvation by helping J7-2 strain to scavenge dead cell-derived nutrients. This study provides new insight into the physiological role of Halolysins.

Key words:haloarchaea;Halolysin;SptA;long-term survival