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-step Escherichia coli mutant screen. The initial screen of E. coli for 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 and cpxA, 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. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was 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, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.

中文翻译：

---

细胞壁完整性基因cpxA和mrcB的缺失导致大肠杆菌中的絮凝

在酿造和废水处理中，絮凝已有数百年的历史了，是一种重要的现象。但是，潜在的分子机制仍然难以捉摸。缺乏区分慢生长突变体和絮凝物形成突变体的独特表型，阻止了通过常规突变体筛选分离絮凝物相关基因。为了克服这个问题，我们进行了两步法大肠杆菌突变体筛选。对高盐处理过程中产生絮凝物的大肠杆菌进行初步筛选，得到了一个包含61个基因中点突变的突变体。下面的相应单基因突变体筛选结果确定了两个基因mrcB，它们编码肽聚糖合成酶和cpxA，编码两组分信号转导系统的组氨酸激酶，有助于盐耐受性和絮凝预防。两个单一突变体在高盐冲击下均形成絮凝物，这些絮凝物含有胞质蛋白。ΔcpxA随絮凝物产量的增加而显示出减少的生长，并且向ΔcpxA添加镁可有效抑制絮凝物的产生。相反，在高盐条件下，ΔmrcB的生长不一致。在两种菌株中，絮凝伴随着含有内膜和外膜蛋白的膜囊泡的释放。在测试的25个组氨酸激酶突变体中，ΔcpxA在絮凝物中产生的蛋白质量最高。高盐度在ΔcpxA中上调了cpxP的表达，表明高盐度和CpxR的激活可能促进絮凝物的形成。