Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.
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.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
H
H
Tan
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
J A
JA
West
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
J P
JP
Ramsay
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
R E
RE
Monson
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
J L
JL
Griffin
Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK.
I K
IK
Toth
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.
G P C
GPC
Salmond
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019295:Computational Biology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D044043:Pectobacterium / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology*; D010641:Phenotype; D010935:Plant Diseases / Q000382:microbiology*; D035281:Plant Tubers / Q000382:microbiology; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D011198:Solanum tuberosum / Q000382:microbiology*; D014774:Virulence
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D006152:Cyclic GMP