Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli
Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli
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细胞壁完整性基因 cpxA 和 mrcB 的缺失导致大肠杆菌絮凝
DOI：
10.1042/bcj20200723
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发表时间：
2021
影响因子：
4.1
通讯作者：
Uozumi Nobuyuki
中科院分区：
文献类型：
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作者：
Sugawara Keita;Toyoda Hayato;Kimura Mami;Hayasaka Shunsuke;Saito Hiromi;Kobayashi Hiroshi;Ihara Kunio;Ida Tomoaki;Akaike Takaaki;Ando Eiji;Hyodo Mamoru;Hayakawa Yoshihiro;Hamamoto Shin;Uozumi Nobuyuki
数百年来，絮凝一直被认为是酿造和废水处理中的重要现象。然而，潜在的分子机制仍然难以捉摸。由于缺乏一个明显的表型来区分生长缓慢的突变体和絮凝形成的突变体，阻碍了絮凝相关基因的分离通过常规的突变体筛选。为了克服这一点，我们进行了两步大肠埃希氏菌大肠杆菌筛选。E.在高盐处理期间赋予絮凝物产生的大肠杆菌突变体产生了在61个基因中含有点突变的突变体。以下筛选相应的单基因突变体确定了两个基因，mrcB，编码肽聚糖合成酶和cpxA，编码组氨酸激酶的双组分信号转导系统，有助于耐盐性和絮凝预防。这两个单突变体在高盐休克过程中形成絮状物，这些絮状物含有胞浆蛋白。随着絮体生成量的增加，Δ cpxA的生长速度下降，添加镁能有效抑制絮体的生成。相反，在高盐条件下，Δ mrcB的生长不一致。在这两种菌株中，絮凝伴随着释放的膜囊泡含有内外膜蛋白。在25个组氨酸激酶突变体中，Δ cpxA在絮凝物中产生的蛋白量最高。高盐对ΔcpxA中cpxP的表达有上调作用，提示高盐和CpxR的激活可能促进了絮体的形成。发现Δ mrcB或Δ cpxA赋予絮凝物的产生表明，不利的环境条件触发的细胞被膜应激导致E中絮凝的开始。杆菌
Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-stepEscherichia colimutant screen. The initial screen ofE. colifor mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes,mrcB, encoding a peptidoglycan-synthesizing enzyme andcpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxAexhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxAsuppressed floc production effectively. In contrast, the growth of ΔmrcBwas inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxAproduced the highest amount of proteins in floc. Expression ofcpxPwas up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcBor ΔcpxAconferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation inE. coli.