Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

• Published in: Microbiology (Society for General Microbiology) Vol. 160; no. Pt 7; pp. 1427 - 1439
• Main Authors: Tan, H, West, J A, Ramsay, J P, Monson, R E, Griffin, J L, Toth, I K, Salmond, G P C
• Format: Journal Article
• Language: English
• Published: England 01.07.2014
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Computational Biology; Cyclic GMP - analogs & derivatives; Cyclic GMP - analysis; Cyclic GMP - metabolism; Gene Expression; Gene Expression Regulation, Bacterial; Pectobacterium - genetics; Pectobacterium - pathogenicity; Pectobacterium - physiology; Phenotype; Plant Diseases - microbiology; Plant Tubers - microbiology; Recombinant Fusion Proteins; Signal Transduction; Solanum tuberosum - microbiology; Virulence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.076828-0

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.