Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli

Yizhi Song; Micha?l L. Cartron; Philip J. Jackson; Paul A. Davison; Mark J. Dickman; Di Zhu; Wei E. Huang; C. Neil Hunter

摘要

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli. Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ～148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis. We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.KEYWORDS: proteorhodopsin, Escherichia coli, membrane, Raman spectroscopy, single cells, cell viability, marine bacteria, cell membranesINTRODUCTIONMicrobial rhodopsins are ubiquitous in nature and comprise a diverse, abundant, and widespread protein superfamily of photoreactive proteins that either drive metabolic reactions or act as photosensors that carry out signaling and regulatory roles (1). Despite their varied roles, rhodopsins all comprise seven transmembrane α-helices that form a binding pocket for retinal (1). In 2000, Béjà et al. reported the existence of proteorhodopsin (PR), a rhodopsin found in several uncultured species of gammaproteobacteria (2). Subsequently, genes encoding PR were found in many different marine bacterial species (3, 4) and even viruses (5). PR is now believed to represent globally the most widespread phototrophic system at the genetic level (4). Thus, PR and chlorophyll-based photosystems represent two routes for harvested solar energy to power the biosphere (4).The first report of PR established its function by expressing the PR-encoding gene, obtained from SAR86, in Escherichia coli. Exogenously supplied retinal was sequestered by the recombinant PR and formed a photoactive protein capable of initiating a photocycle and generating a transmembrane proton gradient (2). The means to produce functional PR, both natural and engineered variants, led to a proliferation of structural and biophysical studies, which established its oligomeric state, structural organization, and photocycle and established new fields of research that now encompass electrophysiology and optogenetics (reviewed in references 1 and 6).Once it became possible to cultivate a marine PR-containing bacterium, the highly abundant SAR11 strain HTCC1062 (“Pelagibacter ubique”), it was shown that PR is a light-dependent proton pump, although it appeared to confer no increase in growth rate in the light (7). The effect of harboring PR on promoting cell growth has not been fully confirmed. PR-based phototrophy was demonstrated in the marine bacterium Dokdonia sp. strain MED134 (8), whereas the closely related Dokdonia sp. strain PRO95 had no growth advantage in the light, even though the PR gene was expressed at levels 10-fold higher in the light than in the dark (9). Deletion of the PR gene sho

机译：编码光反应性蛋白质蛋白质（PR）的基因已在广泛的海洋细菌种类中发现，反映了PR在海洋生态系统中的能量通量和碳循环的显着贡献。 PR还可以赋予优势以增强海洋细菌在饥饿期内生存的能力。在这里，我们研究了异源产生的PR对大肠杆菌的可行性的影响。定量质谱表明，随后用视网膜辅因子供应的大肠杆菌，每个细胞组装多达187,000个Holo-Pr分子，占膜面积的约47％;甚至没有视网膜的细胞均每种细胞没有合成约148,000 APO-PR分子。我们展示了含有PR的大肠杆菌细胞的群体在多周内显示出显着延伸的活力，并且我们使用单细胞拉曼光谱（SCR）来检测9个月大的细胞中的Holo-Pr。 SCRS表明，这种细胞甚至在暗中孵育，因此具有无活性Pr，维持DNA和RNA的细胞水平，并避免细胞质膜的劣化，这是延伸的可存活率的可能基础。所需的大部分大肠杆菌膜的大部分大肠杆菌膜可能促进了广泛的分子间触点，建议物理稳定细胞膜，并在黑暗中赋予显现为延伸的活力的长期益处。我们提出，海洋细菌可以与高PR含量类似地受益，当那些细菌经历的营养素或海洋中的浓度或光限制的细菌经历时，稳定的细胞膜延伸生存。分为纹理光（PR）是多元化，丰富的一部分普遍的超级小家族的光反应性蛋白质，微生物紫红蛋白。 Pr，一种光驱动的质子泵，增强了海洋细菌振动菌株和4从饥饿期生存和恢复的能力，异构生产的PR延长了营养有限的雪松onidensis的可行性。我们表明，异源产生的PR在几周内长期增强了大肠杆菌培养物的活力，并使用单细胞拉曼光谱（SCR）检测9个月大的细胞中的PR。我们鉴定了一种密集的填充和因此稳定的细胞膜，其可能是延伸的活力的可能性。建议申请类似的考虑因素，该细菌似乎高PR水平代表稀缺代谢资源的重大投资。提出了在船舶细菌中的PR稳定的细胞膜，以使人口在延长的光或营养限制期间可行，直至条件改善。蛋白字：蛋白晶体，大肠杆菌，膜，拉曼光谱，单细胞，细胞活力，海洋细菌，细胞膜介质罗经血蛋白本质上是无处不在的，包括不同的，丰富，蛋白质超级蛋白质的光致反应性蛋白质，其促进代谢反应或充当进行信号传导和调节作用（1）的光电传感器。尽管有变化的作用，但罗摩蛋白全部包括七个跨膜α-螺旋，其形成用于视网膜（1）的粘合剂。 2000年，Béjà等人。据报道，存在蛋白质红豆瓣（PR），在几种未露药的γ曲线杆菌（2）中发现的洛越蛋白酶（2）。随后，在许多不同的海洋细菌物种（3,4）甚至病毒（5）中发现编码Pr的基因。 PR现在被认为在遗传水平（4）中全球最广泛的光养系统。因此，Pr和基于叶绿素的光系统代表了用于为生物圈的收获的太阳能的两条航线（4）。PR的第一报告通过表达从SAR86获得的PR编码基因在大肠杆菌中建立了其功能。通过重组PR一起隔离外源供应视网膜，形成能够引发光循环并产生跨膜质子梯度（2）的光活性蛋白。生产功能公关的方法，自然和工程变体，导致结构和生物物理学研究的增殖，该研究是建立了其低聚体状态，结构组织和光循环，并建立了现在涵盖电生理学和视科学的新研究领域（参考文献审查1和6）。培养含有海洋PR的细菌，高度丰厚的SAR11菌株HTCC1062（“Pelagibacter Ubique”）变得可以培养，结果表明，PR是光依赖性的质子泵，但它似乎达成了增加光的生长速度（7）。疏松PR对促进细胞生长的影响尚未得到完全证实。在海洋细菌的Dokdonia sp中证明了Pr基光萎缩。菌株Med134（8），而密切相关的Dokdonia sp。菌株PRO95在光中没有生长优势，即使PR基因在光的含量高于暗中在暗中（9）的水平高于10倍。删除PR基因笑

Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli

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