Detailed analysis of c-di-GMP mediated regulation of csgD expression in Salmonella typhimurium

Abstract

Background: 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. 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.

Results: Among 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.

Conclusion: The 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.

Keywords: CsgD; GGDEF/EAL domain proteins; Salmonella typhimurium; biofilm formation; c-di-GMP; rdar morphotype.

Fig. 1

Identification of novel GGDEF domain proteins regulating csgD expression. a Rdar morphotype formation of S. typhimurium UMR1 and STM1987 and STM4551 mutants after 48 h of growth on Congo red agar plates. b CsgD levels after 24 h of growth at 28°C on LB without salt agar plates. WT is wild type S. typhimurium UMR1. MAE50 is a csgD deletion mutant of UMR1 (negative control) whereas MAE52 strain is used as a CsgD positive control

Fig. 2

Cumulative effect of GGDEF proteins on rdar morphotype and csgD expression in S. typhimurium UMR1. a Rdar morphotype and CsgD 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 STM4551_E267A. Cells were grown for 24 h at 28°C on LB without salt agar plates supplemented with ampicillin (100 μg ml⁻¹) and 0.1% L-arabinose. VC = Vector control pBAD30, p4551 = STM4551 cloned in pBAD30, p4551_E267A = catalytic mutant STM4551_E267A cloned in pBAD30

Fig. 3

Complementation of the rdar morphotype and csgD expression phenotypes of GGDEF/EAL mutants of S. typhimurium UMR1. Complementation by the GGDEF protein STM4551 (a) and the EAL protein STM3611 (b) in respective mutants. Catalytically inactive proteins did not restore csgD expression and the rdar morphotype. c Rdar morphotype formation and CsgD levels of S. typhimurium UMR1 upon the chromosomal replacement of the EAL to AAL motif of EAL protein STM4264. Cells were grown for 24 h at 28°C on LB without salt agar plates supplemented with ampicillin (100 μg ml⁻¹) and 0.1% L-arabinose

Fig. 4

The GGDEF-EAL domain protein STM3388 promotes csgD expression through its diguanylate cyclase activity, whereas the GGDEF-EAL domain protein STM1703 suppresses csgD expression through its phosphodiesterase activity. a Rdar morphotype and CsgD levels upon expression of wild type STM3388 and catalytic GGDEF and EAL mutants of STM3388 in STM3388 deletion mutant of S. typhimurium UMR1. b Rdar morphotype and CsgD levels upon overexpression of STM1703 and catalytic mutants of STM1703 in the STM1703 deletion background of S. typhimurium UMR1. Cells were grown for 24 h at 28°C on LB without salt agar plates supplemented with ampicillin (100 μg ml⁻¹) and 0.1% L-arabinose. VC = Vector control pBAD30

Fig. 5

Corresponding GGDEF/EAL domain proteins. Rdar morphotype and CsgD levels of S. typhimurium UMR1 upon deletion of the EAL domain protein STM3611 (a), the GGDEF-EAL protein STM1703 (b), and the EAL protein STM4264 (c) in different diguanylate cyclase mutant backgrounds. Alteration in rdar morphotype upon adrA deletion is due to lack of cellulose synthesis. Stereomicroscopic image of the rdar morphotype formation of the STM4264 mutant upon the deletion of four diguanylate cyclases after 48 h of growth at 28°C on LB without salts plates supplemented with Congo red (d) and without Congo red (e). Captions (a) 1 = UMR1Δ2123, 2 = UMR1Δ3611Δ2123, 3 = UMR1Δ3388, 4 = UMR1Δ3611Δ3388, 5 = UMR1Δ4551Δ1987, 6 = UMR1Δ3611Δ4551Δ1987. b 1 = UMR1Δ1703, 2 = UMR1Δ4551Δ1987Δ2123Δ3388, 3 = UMR1Δ1703Δ4551 Δ1987Δ2123Δ3388, 4 = Δ1703ΔAdrA 5 = Δ1703ΔAdrAΔ1283Δ2672. c 1 = UMRI, 2 = UMR1Δ4264, 3 = UMR1 Δ2123Δ3388, 4 = UMR1Δ4264Δ2123Δ3388, 5 = UMR1Δ4551Δ1987. 6 = UMR1Δ4264Δ4551Δ1987, 7 = UMR1Δ4551Δ1987Δ2123Δ3388, 8 = UMR1Δ4264Δ4551Δ1987Δ2123Δ3388

Fig. 6

Effect of c-di-GMP on csgD transcription in the S. typhimurium csgD mutant background. a Schematic drawing of fusion constructs containing the csgD promoter region of different length. Transcriptional activity of the csgD promoter region was analyzed in pUGE13 [25, 36] (b) upon deletion of STM1703 and STM4264 and (c) upon deletion of four diguanylate cyclases (Δ4DGCs) compared to the respective ΔcsgD::Km background The transcriptional activity was not affected in the Δ4DGC mutant whereas enhanced β-galactosidase activity was observed in Δ1703 compared to ΔcsgD:101. d-f Identification of the STM1703 regulatory region. As enhanced activity was observed only for pUGE5, but not for other fusions, STM1703 acts via the upstream region between nt −340 and −208. Controls were ΔryeB with partial reduction of csgD transcription [28] and ΔompR mutant as negative control. β-galactosidase measurements were done in duplicates using at least three technical replicates. Statistical significance is indicated by *P < 0.05 as compared to the respective ΔcsgD wild type control using unpaired t-test (Prism 5, GraphPad Software)

Fig. 7

Overexpression of the PDE STM4264 from chromosome under lacUV5 promoter suppresses CsgD production and rdar morphotype formation. CsgD expression (a) and rdar morphotype formation (b) upon overexpressing csgD from pBAD30 in S. typhimurium strain MAE1900 where STM4264 is expressed from the constitutive lacUV5 promoter as compared to the ΔcsgD:101 deletion strain MAE50

Fig. 8

Schematic diagram showing the regulatory network of c-di-GMP signaling regulating csgD expression. At least three distinct groups of c-di-GMP turnover proteins regulate csgD expression. Regulation of csgD by STM1703 occurs on the transcriptional level, while the regulatory level by the other groups is unknown and drawn arbitrarily. The STM1987/STM4551/STM3611 group inversely regulates motility [40]. EAL-like proteins STM1344 and STM1697 affect the c-di-GMP signaling network through post-translational inhibition of FlhD₄C₂, the regulator of the flagellar cascade and regulation of STM1703 [41, 55]. Green represents a diguanylate cyclase, blue a phosphodiesterase and magenta represents a diguanylate cyclase/phosphodiesterase; light grey not directly investigated in this work