BACKGROUND & AIMS:
:The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta- and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC.IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.
背景与目标:
: 第二信使环二聚体GMP (c-di-GMP) 和c-di-GMP转换蛋白几乎在细菌中无处不在,它们介导了运动性和稳健性之间的过渡。EAL结构域蛋白已被表征为c-二GMP特异性磷酸二酯酶。虽然大多数EAL结构域蛋白都包含额外的 (通常是N末端) 结构域,但有一个独特的具有独立EAL结构域的蛋白质家族,例如沙门氏菌肠道血清型鼠伤寒杆菌蛋白STM3611 (YhjH/PdeH),一种c-di-GMP特异性磷酸二酯酶,以及酶促失活的STM1344 (YdiV/CdgR) 和STM1697,它们通过与鞭毛主调节剂FlhDC相互作用来调节细菌的运动。我们已经分析了仅EAL蛋白的系统发育分布及其潜在功能。在各种细菌门中都发现了仅编码EAL蛋白的基因,尽管其中大多数在变形杆菌中,尤其是肠杆菌中。基于活性位点残基的保守性,几乎所有由变形杆菌以外的门基因组编码的独立EAL结构域似乎都代表功能性磷酸二酯酶。在肠杆菌中,发现仅EAL蛋白与YhjH或与YdiV相关蛋白的亚科之一聚集。测试了福氏志贺氏菌,肺炎克雷伯菌和小肠结肠炎耶尔森氏菌的仅EAL蛋白调节游泳和蜂群运动以及形成红色,干燥和粗糙 (rdar) 生物膜形态的能力。在这些测试中,YhjH相关蛋白S4210,KPN_01159,KPN_03274和YE4063显示出酶活性磷酸二酯酶的典型特性,而S1641和YE1324的表现类似于YdiV/STM1697亚家族的成员,小肠结肠炎耶尔森氏菌蛋白YE1324显示出下调其天然宿主的运动。在两种密切相关的仅EAL蛋白中,YE2225是一种活性磷酸二酯酶,而YE1324似乎与FlhD相互作用。这些结果表明,在携带 β-和gammaproteobacteria的FlhDC中,一些仅EAL蛋白通过与FlhDC相互作用而进化为催化失活并调节运动和生物膜形成。重要性EAL结构域超家族主要由具有环二聚体GMP特异性磷酸二酯酶活性的蛋白质组成,但是,根据其功能和保守的氨基酸特征,将各个结构域分为三类。仅由独立的EAL结构域组成的蛋白质不能依靠其他结构域来形成催化活性二聚体,并且它们大多数属于两个不同的类别之一: 具有活性位点和二聚化环的保守残基的催化活性磷酸二酯酶,和催化失活的YdiV/CdgR样蛋白,通过与鞭毛主调节剂FlhDC结合来调节细菌的运动,主要在肠杆菌中发现。在不表达FlhD的细菌中存在明显无活性的仅EAL蛋白,这表明存在其他EAL相互作用伙伴。