Abstract
BackgroundThe secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty- two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development.ResultsAmong twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region.ConclusionThe enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.
Highlights
The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium
Identification of novel GGDEF domain proteins promoting csgD expression Multicellular behavior as expressed by the rdar biofilm morphotype in S. typhimurium UMR1 correlates with expression of the response regulator CsgD, a major target of c-di-GMP signalling
STM4551 is an established diguanylate cyclase [30, 39], reportedly its catalytic activity is not required to restore most of the phenotypes levels of double and quadruple mutants of GGDEF proteins after 24 h of growth at 28°C on LB without salt agar plates. b Overexpression of the diguanylate cyclase STM4551 in the Δ4DGC mutant restored rdar morphotype and csgD expression, in contrast to catalytically inactive STM4551E267A
Summary
The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. We dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development. The ubiquitous second messenger bis- (3′–5′)-cyclic dimeric GMP (c-di-GMP), plays a major role in the transition from the motile to the sessile life style on the single cell level [3,4,5,6]. C-di-GMP is synthesized by diguanylate cyclases (DGCs), GGDEF domain proteins [7,8,9,10], and degraded by c-di-GMP phosphodiesterases (PDEs), EAL or HD-GYP domain proteins [11,12,13]. CsgD is central in regulating the transition between biofilm formation and virulence. csgD expression is usually regulated by environmental stimuli such as temperature
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