Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Gao, Xiaohui; Dong, Xiao; Subramanian, Sundharraman; Matthews, Paige M.; Cooper, Caleb A.; Kearns, Daniel B.; Dann, Charles E.

doi:10.1128/aem.01638-14

Cited by 33 publications

(37 citation statements)

References 64 publications

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“…c-di-GMP consistently represses flagellum-mediated motility in diverse species, such as P. aeruginosa and B. subtilis, and appears to repress TFP-based twitching motility in P. aeruginosa (20,(77)(78)(79). However, we have found that c-di-GMP promotes TFP-dependent surface motility in C. difficile.…”

Section: Fig 6 Growth On Surfaces Increases Intracellular C-di-gmp Incontrasting

confidence: 45%

“…C. difficile 630 encodes a total of 37 predicted diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes, which synthesize and degrade, respectively, c-di-GMP (17,18). The catalytic abilities of these enzymes have been independently assessed through heterologous expression in Vibrio cholerae and Bacillus subtilis, which indicated that the majority (21 and 27 enzymes, respectively) are catalytically active (19,20). The remaining proteins may be active in C. difficile or under conditions not tested.…”

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confidence: 99%

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Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

et al. 2016

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The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called "hypervirulent" epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile. We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile. The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCEHow Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile. TFP are also required for surface motility, which has not previously been demonstrated for C. difficile. Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.

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Section: Fig 6 Growth On Surfaces Increases Intracellular C-di-gmp Incontrasting

confidence: 45%

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confidence: 99%

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

et al. 2016

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“…The genome of C. difficile 630 codes for 18 predicted DGCs and 19 predicted PDEs, many of which have confirmed enzymatic activity (29,30). Sudarsan et al (26) revealed the presence of a c-di-GMP-I riboswitch, Cd1, upstream of the flgB gene, which is part of one of two loci encoding the flagellum genes in C. difficile.…”

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confidence: 99%

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

et al. 2015

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Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation under high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile. The Gram-positive spore-forming bacterium Clostridium difficile is the leading cause of nosocomial diarrhea and antibioticassociated colitis in hospital settings (1). Illness caused by C. difficile infections (CDI) may range from mild diarrhea to lifethreatening, fulminant colitis. Throughout its life cycle, C. difficile has to cope with multiple changing environments. In the early steps of CDI, in order to colonize the colon and produce toxins, C. difficile spores first germinate in the intestine in response to stimuli such as the presence of bile salts (2, 3). Vegetative cells need to reach the colon, likely attaching to and colonizing the gut mucosa, where they proliferate and produce toxins. Hence, C. difficile arguably needs to sense and integrate multiple environmental stimuli in order to coordinate the expression of its colonization and virulence factors during its journey through the gastrointestinal tract. The mechanisms allowing this adaptability in C. difficile have been the focus of many recent studies, including those on the agr quorum-sensing system, alternative sigma factors, two-component systems, the global transcription regulator CodY, and the sigma factor TcdR, which promotes TcdA and TcdB toxin expression (4-9).One way by which bacteria sense and respond to changes in their environment is through signal transduction involving secondary messenger molecules. The bacterial second messenger 3=-5= cyclic diguanosine monophosphate (c-di-GMP) plays multiple key roles in bacterial physiology and virulence (10-12). c-di-GMP has been shown to antagonistically control the...

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“…Mutants lacking all enzymes involved in c-di-GMP metabolism have been described (e.g., see references [15][16][17]. A complementary approach involving the use of the constitutive or inducible overexpression of diguanylate cyclases or phosphodiesterase has also been used (e.g., see reference 2).…”

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confidence: 99%

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense

et al. 2017

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Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger c-di-GMP to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect the metabolic status of the cells to the sensory activity of chemotaxis receptors.

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Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

Cited by 33 publications

References 64 publications

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense

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