The cyclic di-GMP receptor YcgR links the second messenger with the putrescine quorum sensing system in modulation of Dickeya oryzae motility

Abstract

Dickeya oryzae is a prevalent pathogen capable of infecting a variety of crops and plants, and its cell motility plays a key role in invasion of host and subsequent systemic infection. We recently demonstrated that the bacterial second messenger c-di-GMP and the putrescine (PUT)-mediated quorum sensing (QS) system are, respectively, involved in negative and positive regulation of bacterial motility, and vice versa, biofilm formation. In this study, we aimed to investigate the potential interaction of these two signaling mechanisms in the modulation of bacterial motility. The results indicated that null mutation of the PUT system did not seem to have much effect on the cellular level of c-di-GMP; however, deletion of the genes encoding c-di-GMP degradation led to a significant reduction in PUT production. A subsequent study unveiled that the second messenger signaling system interacted with the putrescine QS system through the c-di-GMP receptor YcgR. This interaction enhanced the activity of SpeA, which is the rate-limiting enzyme in the putrescine biosynthesis pathway, resulting in increased intracellular putrescine level. Critically, this facilitative effect was inhibited by c-di-GMP molecules; thus, SpeA, YcgR, and c-di-GMP constitute a regulatory loop modulating D. oryzae motility by controlling the rate of putrescine biosynthesis. The findings from this study provide the first insight into the interaction mechanism between c-di-GMP and putrescine signaling systems in bacteria.IMPORTANCEDickeya oryzae is an important bacterial pathogen that can infect numerous plants and crops, leading to substantial economic losses, especially in rice and banana cultivation. Bacterial motility is a crucial pathogenic factor for D. oryzae as it enables the pathogen to compete for food resources and invade host plants. This motility is negatively regulated by the second messenger c-di-GMP and positively regulated by the quorum sensing signal putrescine (PUT). However, the potential connection between c-di-GMP and PUT signaling systems in regulating the motility of D. oryzae has not been understood. Here, we reveal the link and mechanism of the interaction between them, demonstrating that c-di-GMP interacts with the PUT system via its receptor YcgR. The significance of our research lies in providing the first insight into the molecular interaction between c-di-GMP and PUT signaling networks, both of which are widely conserved signaling mechanisms, and sheds light on the complex and sophisticated regulatory mechanisms that govern bacterial motility and virulence.

Keywords: Dickeya oryzae; YcgR; bacterial motility; c-di-GMP; putrescine.

Fig 1

Second messenger c-di-GMP and PUT QS system play a negative and positive role, respectively, in the regulation of D. oryzae swimming motility. (A) Swimming motility of D. oryzae EC1 and its derivatives defective in PUT biosynthesis (speA) and transportation (potF, plaP) in the absence or presence of 0.1 mM PUT. (B) Swimming motility of D. oryzae EC1 and its derivatives defective in c-di-GMP metabolism, including mutant 15∆DGC with all the c-di-GMP synthase genes being deleted and mutant 7∆PDE with all the c-di-GMP degradation genes being knocked out. The well-characterized DGC gene wspR and PDE gene rocR from P. aeruginosa were used in heterologous complementation analysis, as indicated. Dotted lines indicate the swimming motility of the wild-type level. The data shown are the mean ± standard deviations (n = 3). Statistics significance: ****, P < 0.0001; **, P < 0.01; ns, P > 0.05 (by one-way ANOVA with multiple comparisons).

Fig 2

Cellular c-di-GMP and putrescine levels in D. oryzae EC1 and its derivatives. (A) Quantitative measurement of cellular cyclic di-GMP levels of wild-type strain EC1 and mutant ∆speA∆potF∆plaP defective in PUT biosynthesis and transportation in MM. The dotted line indicates the wild-type c-di-GMP level. The data shown are the mean ± standard deviations (n = 3). Statistical significance: *, P < 0.05; ns, P > 0.05 (by Student’s unpaired t test). (B) Quantitative measurement of the cellular putrescine concentration of strain EC1 and derivatives defective in c-di-GMP synthesis (15∆DGC), degradation (7∆PDE), and signal transduction (∆ycgR) in MM. The data shown are the mean ± standard deviations (n = 3). Statistical significance: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (by one-way ANOVA with multiple comparisons). The dotted line indicates the wild-type PUT level. (C) Quantitative measurement of the cellular putrescine concentration of strain EC1, ∆ycgR, ∆ycgR(pB-ycgR), 7∆PDE, and 7∆PDE(pB-rocR) in MM. The data shown are the mean ± standard deviations (n = 3). Statistical significance: **, P < 0.01; *, P < 0.05; ns, P > 0.05 (by one-way ANOVA with multiple comparisons).

Fig 3

Transcriptional fusion assay of the genes encoding the c-di-GMP signaling system and PUT QS system in wild-type strain EC1 and corresponding mutants. (A) Transcriptional fusion assay of c-di-GMP system-related genes in wild-type strain EC1 and mutant ∆speA∆potF∆plaP. The following genes were quantified in this experiment: ycgR, encoding a PilZ domain c-di-GMP receptor; ddgcA (W909_14945), encoding the major diguanylate cyclase that synthesizes c-di-GMP; dpdeA (W909_14950), encoding the major phosphodiesterase that degrades c-di-GMP; dpdeB (W909_10355), encoding major bifunctional enzymes that can both synthesize and degrade c-di-GMP (8). (B and C) Transcriptional fusion assay of speA and speC encoding two key rate-limiting enzymes for putrescine synthesis in mutants ∆ycgR and 7∆PDE, respectively, compared to wild-type EC1. The bacterial cells were cultured in MM. The data shown are the mean ± standard deviations (n = 3). Statistical significance: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (by Student’s unpaired t test). The dotted line indicates the expression levels of these genes in the wild-type strain EC1.

Fig 4

Identification of proteins potentially interacting with YcgR. (A) Identification of YcgR-interacting proteins by bacterial two-hybridization. Four proteins that directly interact with YcgR have been identified as follows: SpeA, biosynthetic arginine decarboxylase; ArtP, arginine ABC transporter ATP-binding protein; ArgG, argininosuccinate synthase; and MetK, S-adenosylmethionine synthase. (B) GST pull-down analysis of the effect of c-di-GMP on the YcgR-SpeA interaction. c-di-GMP was added in 0, 1, and 4 molar ratios respectively to YcgR, as indicated in the pull-down assay.

Fig 5

Addition of YcgR promotes SpeA enzymatic activity. SpeA enzymatic activity in the absence or presence of YcgR in different ratios, as indicated. The product agmatine was extracted at 1 h or 2 h after the initiation of the reaction, and its absorbance value was measured. The data shown are the mean ± standard deviations (n = 3). Statistics significance: ****, P < 0.0001; *, P < 0.05; ns, P > 0.05 (by Student’s unpaired t test).

Fig 6

Relative strength of c-di-GMP and PUT signaling systems in regulation of bacterial motility. (A) Expression of rocR encoding c-di-GMP degradation restored and boosted the swimming motility of PUT signaling-defective mutant ∆speA∆potF∆plaP. The data shown are expressed as the mean ± standard deviations (n = 3). Statistical significance: ****, P < 0.0001; ***, P < 0.001 (by one-way ANOVA with multiple comparisons). (B) Expression of rocR encoding c-di-GMP degradation restored and boosted the swimming motility of fully PUT biosynthesis pathway-deficient mutant ∆speA∆speC. The data shown are expressed as the mean ± standard deviations (n = 3). Statistical significance: ****, P < 0.0001; ns, P > 0.05 (by one-way ANOVA with multiple comparisons). (C) Exogenous addition of PUT (0.1 mM) restored the swimming motility of mutant ∆speA but did not seem to affect the motility of strain EC1 and c-di-GMP mutants 15∆DGC and 7∆PDE. The data shown are expressed as the mean ± standard deviations (n = 3). Statistical significance: *, P < 0.05; ns, P > 0.05 (by Student’s unpaired t test). (D) Deletion of PUT synthase gene speA in c-di-GMP synthesis mutant 15∆DGC and degradation mutant 7∆PDE failed to generate significance on the bacterial swimming motility. The data shown are expressed as the mean ± standard deviations (n = 3). Statistical significance: ns, P > 0.05 (by one-way ANOVA with multiple comparisons).

Fig 7

c-di-GMP and PUT QS system co-regulation model on D. oryzae motility. SpeA, arginine decarboxylase; AguA, agmatine deiminase; AguB, N-carbamoylputrescine aminotransferase; YcgR, a PilZ domain receptor for c-di-GMP. In this model, at low c-di-GMP levels, YcgR interacts with SpeA to facilitate PUT signal production and thus promote bacterial motility and planktonic lifestyle; on the other hand, upon reaching a threshold level, c-di-GMP competes for YcgR and forms the YcgR-c-di-GMP complex to suppress bacterial motility and switch to biofilm lifestyle.