The response threshold of <i><scp>S</scp>almonella</i><scp><scp>PilZ</scp></scp> domain proteins is determined by their binding affinities for c‐di‐<scp>GMP</scp>

Pultz, Ingrid Swanson; Christen, Matthias; Kulasekara, Hemantha D.; Kennard, Andrew S.; Kulasekara, Bridget R.; Miller, Samuel I.

doi:10.1111/mmi.12066

Cited by 90 publications

(101 citation statements)

References 69 publications

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“…This observation is in line with a bipartite role of cdG in flagellar-based motility regulation proposed for S. Typhimurium and C. crescentus in that cdG may coordinate flagellar assembly and rotation (16,73,75). In a strikingly similar manner, overproduction of a heterologous DGC rendered an S. Typhimurium mutant in the PilZ domain-encoding ycgR gene hypermotile, and combined overproduction of this DGC and YcgR provoked a nonmotile phenotype in the same way as combined overexpression of pleD and mcrA in S. meliloti (73). An additional similarity is probably a conformational change triggered upon binding of cdG to YcgR (73) and McrA.…”

Section: Discussionsupporting

confidence: 69%

“…In a strikingly similar manner, overproduction of a heterologous DGC rendered an S. Typhimurium mutant in the PilZ domain-encoding ycgR gene hypermotile, and combined overproduction of this DGC and YcgR provoked a nonmotile phenotype in the same way as combined overexpression of pleD and mcrA in S. meliloti (73). An additional similarity is probably a conformational change triggered upon binding of cdG to YcgR (73) and McrA.…”

Section: Discussionmentioning

confidence: 75%

“…PilZ proteins were previously reported to undergo conformational changes upon cdG binding (4,73). We tested for in vivo binding of cdG to McrA and a conformational change upon binding by employing a modified version of the fluorescence resonance energy transfer (FRET)-based cdG biosensor designed by Christen et al (20).…”

Section: Strong Reduction Of Eps I and Eps Ii Biosynthesis In Rm2011 mentioning

confidence: 99%

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Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

et al. 2016

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Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3=,5=-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG 0 ) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCEWe present the first systematic genome-wide investigation of the role of 3=,5=-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG 0 ) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.

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Section: Discussionsupporting

confidence: 69%

Section: Discussionmentioning

confidence: 75%

Section: Strong Reduction Of Eps I and Eps Ii Biosynthesis In Rm2011 mentioning

confidence: 99%

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Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

et al. 2016

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“…Studies focused on disrupting the I-site have previously found that deregulation of a DGC leads to increased production of c-di-GMP and a concomitant increase in c-di-GMPrelated phenotypes, including increases in biofilm formation and decreases in motility (7,8). Additionally, effectors have been thought to provide one mechanism of signaling specificity by differentially activating at various levels of c-di-GMP, as demonstrated in Salmonella enterica serovar Typhimurium (9), with a complementary study identifying various response curves for cdi-GMP-dependent processes in Caulobacter crescentus (10). The apparent function of the I-site is therefore 2-fold.…”

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confidence: 99%

The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein

et al. 2016

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Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity. IMPORTANCESome bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.A major method of intracellular signaling in bacteria revolves around the second messenger cyclic diguanylate (c-di-GMP). c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) in networks that may range from one or a few such proteins to several dozen (1). Effector proteins then sense c-di-GMP by binding it and promote some specific cellular action as a result. Importantly, many DGCs studied to date promote only one or a small number of such processes (2-6), indicating that there must be mechanisms in place that allow for specificity in signaling among the many members of a c-di-GMP network.A key regulatory element in signalin...

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“…In B. subtilis, evidence suggests that the YcgR homolog YpfA (now called DgrA) (20) interacts with MotA (16). The P. aeruginosa genome encodes seven PilZ domain-containing proteins that have been shown to bind to c-di-GMP and an eighth PilZ domain protein that lacks c-di-GMP binding, but no link between these proteins and flagellar motility has been established in this organism (12,(21)(22)(23).…”

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confidence: 99%

PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

et al. 2016

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The second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. IMPORTANCEThe regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa. We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities. Cyclic diguanylate (c-di-GMP) is a ubiquitous bacterial second messenger responsible for regulating a range of cellular processes, including motility and biofilm formation (1). In general, low intracellular c-di-GMP levels are associated with motile lifestyles, while elevated levels of c-di-GMP promote surface attachment and sessile lifestyles (2, 3). c-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and degraded by c-di-GMP-specific phosphodiesterases (PDEs) (1, 4). Many DGCs and PDEs involved in motility regulation have been characterized, but the mechanisms by which c-di-GMP regulates motility are poorly understood in Pseudomonas aeruginosa and in other bacterial species.To regulate numerous biological functions, c-di-GMP binds to specific effector proteins or RNA (reviewed in reference 5). Recent studies have focused on identifying these c-di-GMP effectors and their mechanisms for regulating c-di-GMP-dependent processes. One class of effectors is the PilZ domain-containing protein family, which is characterized by conserved ...

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The response threshold of SalmonellaPilZ domain proteins is determined by their binding affinities for c‐di‐GMP

Cited by 90 publications

References 69 publications

Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein

PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

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