Tan H, West J A, Ramsay J P, Monson R E, Griffin J L, Toth I K, Salmond G P C
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK.
Microbiology (Reading). 2014 Jul;160(Pt 7):1427-1439. doi: 10.1099/mic.0.076828-0. Epub 2014 Apr 23.
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.
双（3'-5'）-环二鸟苷单磷酸（c-di-GMP）是一种普遍存在的细菌信号分子，由GGDEF结构域家族的双鸟苷酸环化酶产生。在许多细菌物种中，c-di-GMP水平升高或GGDEF蛋白表达增加通常与固着和生物膜形成的开始有关。相反，EAL和HD-GYP结构域蛋白通过磷酸二酯酶依赖性降低c-di-GMP水平，通常伴随着运动性和毒力的增加。在本研究中，我们分别过表达了植物病原体黑胫果胶杆菌SCRI1043菌株编码的23种预测的GGDEF、EAL或HD-GYP结构域蛋白。基于质谱检测这些菌株中的c-di-GMP和5'-磷酸鸟苷酰-（3'-5'）-鸟苷，结果显示，除了GGDEF结构域蛋白ECA0659和ECA3374分别诱导c-di-GMP细胞水平增加1290倍和7660倍外，大多数基因的过表达促进了c-di-GMP细胞水平适度的1至10倍变化。大多数EAL结构域蛋白的过表达增加了运动性，而大多数GGDEF结构域蛋白的过表达降低了运动性并增加了聚-β-1,6-N-乙酰葡糖胺依赖性絮凝。与基于结构域的预测相反，EAL蛋白ECA3549或HD-GYP蛋白ECA3548的过表达增加了c-di-GMP浓度并降低了运动性。大多数过表达构建体改变了分泌的纤维素酶、果胶酶和蛋白酶的水平，证实了c-di-GMP对黑胫果胶杆菌毒力的调节作用。然而，毒力因子诱导与表达的结构域类别或细胞c-di-GMP水平之间没有明显的相关性，这表明调节是对网络内特定效应物的反应，而不是总c-di-GMP浓度。最后，我们证明了GGDEF/EAL/HD-GYP蛋白的细胞定位模式差异很大，这表明它可能是限制c-di-GMP网络内特定相互作用的一个因素。
