Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference.
Functional diversification of gram-negative intermembrane phospholipid transporters by intrinsic substrate preference.
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通过内在底物偏好实现革兰氏阴性膜间磷脂转运蛋白的功能多样化。
DOI：
10.1101/2023.06.21.545913
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Mitchell,AngelaM
中科院分区：
文献类型：
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作者：
Rai,AshutoshK;Sawasato,Katsuhiro;Bennett,HaleyC;Kozlova,Anastasiia;Sparagna,GenevieveC;Bogdanov,Mikhail;Mitchell,AngelaM
革兰氏阴性菌的外膜是化学和物理应力的屏障。磷脂在内膜和外膜之间的转运一直是研究的热点。coliK-12，它最近已被证明是介导的YhdP，TamB，和YdbH，这被认为是提供疏水通道的磷脂扩散，YhdP和TamB发挥主要作用。然而，YhdP和TamB具有不同的表型，表明不同的功能。我们使用合成的yhdP缺失菌株的冷敏感性（在30 °C下）来研究这些功能，但不是BorydbH和fadR，fadR是一种控制脂肪酸降解和不饱和脂肪酸产生的转录调节因子。删除oftamB，将磷脂转运重定向至YdbH，抑制ΔyhdPΔ fadR冷敏感性，表明该表型与磷脂转运直接相关。ΔyhdPΔ fadR菌株在转移到非允许温度时显示心磷脂的更大增加，并且遗传降低心磷脂水平可以抑制冷敏感性。这些数据还揭示了E中心磷脂酶之间的质的差异。大肠杆菌，因为clsA和clsC的缺失抑制冷敏感性，但clsB的缺失并不抑制，尽管心磷脂水平较低。除了增加心磷脂，增加脂肪酸饱和度是冷敏感性所必需的，通过遗传或补充油酸降低该水平可抑制ΔyhdPΔ fadR菌株的冷敏感性。一个吝啬的解释，我们的数据是，YhdP和TamB有不同的底物运输偏好，最有可能与YhdP优先运输更多的饱和磷脂和TamB优先运输更多的不饱和磷脂。我们设想心磷脂有助于这种运输偏好的空间堵塞TamB介导的饱和磷脂的运输。因此，我们的数据提供了一个潜在的机制，独立控制的磷脂组成的内外膜在不断变化的conditions.Author SummaryGram-negative细菌拥有一个不可渗透的外膜，保护免受环境压力和抗生素。外膜磷脂转运一直是个谜，直到最近YhdP、TamB和YdbH参与了E.杆菌YhdP和/或TamB的类似作用已经在密切相关和远亲的革兰氏阴性细菌中提出。在这里，考虑到转运蛋白的明显部分冗余，我们研究了YhdP和TamB之间的功能差异。我们的数据表明，YhdP和TamB有差异参与脂肪酸和磷脂代谢。事实上，TamB在非允许温度下转运高于正常水平的心磷脂和饱和磷脂是致命的。从这些数据中，我们提出了一个模型，其中YhdP和TamB的功能是由他们的偏好与YhdP优先运输更饱和的磷脂和TamB运输更不饱和的磷脂磷脂运输区分。心磷脂头基特异性可能有助于运输抑制，因为其庞大的性质抑制其他磷脂的通过。YhdP和TamB之间功能的多样化提供了在变化的环境条件下调节磷脂组成的机制，并且可能调节外膜的机械强度和渗透性，从而调节细胞的内在抗生素抗性。
The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, inE. coliK-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 °C) of a strain with deletion ofyhdP, but nottamBorydbH, andfadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production. Deletion oftamB, redirecting phospholipid transport to YdbH, suppresses the ΔyhdPΔfadRcold sensitivity suggesting this phenotype is directly related to phospholipid transport. The ΔyhdPΔfadRstrain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases inE. coli, as deletion ofclsA and clsCsuppresses cold sensitivity but deletion ofclsBdoes not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdPΔfadRstrain. A parsimonious explanation for our data is that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions.Author SummaryGram-negative bacteria possess an impermeable outer membrane, which protects against environmental stress and antibiotics. Outer membrane phospholipid transport remained mysterious until recently when YhdP, TamB, and YdbH were implicated in phospholipid transport between the inner and outer membranes ofE. coli. Similar roles for YhdP and/or TamB have been suggested in both closely and distantly related gram-negative bacteria. Here, given the transporters’ apparent partial redundancy, we investigated functional differentiation between YhdP and TamB. Our data demonstrate that YhdP and TamB have differential involvement with fatty acid and phospholipid metabolism. In fact, transport of higher than normal levels of cardiolipin and saturated phospholipids by TamB at a non-permissive temperature is lethal. From these data, we suggest a model where the functions of YhdP and TamB are distinguished by their preference for phospholipid transport with YhdP preferentially transporting more saturated phospholipids and TamB transporting more unsaturated phospholipids. Cardiolipin headgroup specificity may contribute transport inhibition due to its bulky nature inhibiting the passage of other phospholipids. Diversification of function between YhdP and TamB provides a mechanism for regulation of phospholipid composition, and possibly the mechanical strength and permeability of the outer membrane, and so the cell’s intrinsic antibiotic resistance, in changing environmental conditions.