Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ

Baraquet, Claudine; Harwood, Caroline S.

doi:10.1073/pnas.1318972110

Cited by 168 publications

(206 citation statements)

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“…We also note, however, that P. (36)(37)(38)(39)(40)(41). As with other EBPs, FleQ has an ATPase activity (12), and this activity is required for flagellar gene expression. Here, we determined that ATP hydrolysis was not required for either FleQ-mediated repression or activation of pel operon expression (Fig.…”

Section: Discussionmentioning

confidence: 64%

“…This mutation has previously been shown to abolish FleQ ATPase activity (12). pel expression was low in this mutant strain in the absence of c-di-GMP and high in the presence of c-di-GMP (Fig.…”

Section: Figmentioning

confidence: 59%

“…In conjunction with 54 , FleQ activates the expression of the twocomponent regulatory genes fleSR, as well as genes required for flagellar export apparatus assembly and flagellar basal body assembly (6,11). c-di-GMP inhibits FleQ ATPase activity (12), which explains the modest (1.5-to 2-fold) downregulation of flagellar gene expression that is seen in the presence of c-di-GMP (4,8). By inversely regulating biofilm and flagellar gene expression in response to c-di-GMP, FleQ helps to control the planktonic-tobiofilm lifestyle transition of P. aeruginosa.…”

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confidence: 99%

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FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

2016

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The transcription factor FleQ from Pseudomonas aeruginosa derepresses expression of genes involved in biofilm formation when intracellular levels of the second messenger cyclic diguanosine monophosphate (c-di-GMP) are high. FleQ also activates transcription of flagellar genes, and the expression of these genes is highest at low intracellular c-di-GMP. FleQ thus plays a central role in mediating the transition between planktonic and biofilm lifestyles of P. aeruginosa. Previous work showed that FleQ controls expression of the pel operon for Pel exopolysaccharide biosynthesis by converting from a repressor to an activator upon binding c-di-GMP. To explore the activity of FleQ further, we carried out DNase I footprinting at three additional biofilm gene promoters, those of psl, cdrAB, and PA2440. The expression of cdrAB, encoding a cell surface adhesin, was sufficiently responsive to FleQ to allow us to carry out in vivo promoter assays. The results showed that, similarly to our observations with the pel operon, FleQ switches from a repressor to an activator of cdrAB gene expression in response to c-di-GMP. From the footprinting data, we identified a FleQ DNA binding consensus sequence. A search for this conserved sequence in bacterial genome sequences led to the identification of FleQ binding sites in the promoters of the siaABCD operon, important for cell aggregation, and the bdlA gene, important for biofilm dispersal, in P. aeruginosa. We also identified FleQ binding sites upstream of lapA-like adhesin genes in other Pseudomonas species. IMPORTANCEThe transcription factor FleQ is widely distributed in Pseudomonas species. In all species examined, it is a master regulator of flagellar gene expression. It also regulates diverse genes involved in biofilm formation in P. aeruginosa when intracellular levels of the second messenger c-di-GMP are high. Unlike flagellar genes, biofilm-associated genes are not always easy to recognize in genome sequences. Here, we identified a consensus DNA binding sequence for FleQ. This allowed us to survey Pseudomonas strains and find new genes that are likely regulated by FleQ and possibly involved in biofilm formation. Cyclic diguanosine monophosphate (c-di-GMP) is an intracellular second messenger that is produced by bacteria and has diverse effects on bacterial physiology. c-di-GMP binds to riboswitches and effector proteins to modulate transcription, translation, and protein activities (1, 2). In the opportunistic pathogen Pseudomonas aeruginosa, the transcription factor FleQ binds c-di-GMP to derepress expression of genes for biofilm components (3, 4). Biofilms are surface-attached communities of bacteria embedded in a matrix made of exopolysaccharides, proteins, and DNA. Biofilm infections are particularly difficult to treat because bacteria are protected by the biofilm matrix, making them resistant to antimicrobial treatment compared to planktonic cells (5). Genes controlled by FleQ include pel genes, coding for Pel exopolysaccharide; psl genes, coding for Psl exopolys...

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Section: Discussionmentioning

confidence: 64%

Section: Figmentioning

confidence: 59%

mentioning

confidence: 99%

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FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

2016

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“…In P. aeruginosa, c-di-GMP regulates these processes through allosteric binding to many protein receptors to alter their function. c-di-GMP binds the FleQ transcriptional regulator (12), leading to the repression of the flagellum genes (13) and the activation of the psl and pel exopolysaccharide operons (14) as well as the surface adhesin CdrA (15). In addition to transcriptional regulation, c-di-GMP binds to PelD to activate the production of the Pel exopolysaccharide (16,17) and to FimX to repress type IV pilus-mediated twitching motility (18,19).…”

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confidence: 99%

Cyclic Di-GMP Signaling Contributes to Pseudomonas aeruginosa-Mediated Catheter-Associated Urinary Tract Infection

Cole

Lee

2016

J Bacteriol

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Bis-(3=-5=) cyclic dimeric GMP (c-di-GMP) controls the lifestyle transition between the sessile and motile states in many Gramnegative bacteria, including the opportunistic human pathogen Pseudomonas aeruginosa. Under laboratory conditions, high concentrations of c-di-GMP decrease motility and promote biofilm formation, while low concentrations of c-di-GMP promote motility and decease biofilm formation. Here we sought to determine the contribution of c-di-GMP signaling to biofilm formation during P. aeruginosa-mediated catheter-associated urinary tract infection (CAUTI). Using a murine CAUTI model, a decrease in CFU was detected in the bladders and kidneys of mice infected with strains overexpressing the phosphodiesterases (PDEs) encoded by PA3947 and PA2133 compared to those infected with wild-type P. aeruginosa. Conversely, overexpression of the diguanylate cyclases (DGCs) encoded by PA3702 and PA1107 increased the number of bacteria in bladder and significantly increased dissemination of bacteria to the kidneys compared to wild-type infection. To determine which of the DGCs and PDEs contribute to c-di-GMP signaling during infection, a panel of PA14 in-frame deletion mutants lacking DGCs and PDEs were tested in the CAUTI model. Results from these infections revealed five mutants, three containing GGDEF domains (⌬PA14_26970, ⌬PA14_72420, and ⌬siaD) and two containing dual GGDEF-EAL domains (⌬morA and ⌬PA14_07500), with decreased colonization of the bladder and dissemination to the kidneys. These results indicate that c-di-GMP signaling influences P. aeruginosa-mediated biofilms during CAUTI. IMPORTANCE Biofilm-based infections are an important cause of nosocomial infections, since they resist the immune response and traditional antibiotic treatment. Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in many Gram-negative pathogens, including Pseudomonas aeruginosa.Here we determined the contribution of c-di-GMP signaling to catheter-associated urinary tract infection (CAUTI), an animal model of biofilm-based infection. P. aeruginosa with elevated levels of c-di-GMP during the initial infection produces an increased bacterial burden in the bladder and kidneys. Conversely, low concentrations of c-di-GMP decreased the bacterial burden in the bladder and kidneys. We screened a library of mutants with mutations in genes regulating c-di-GMP signaling and found several mutants that altered colonization of the urinary tract. This study implicates c-di-GMP signaling during CAUTI. P seudomonas aeruginosa is an opportunistic human pathogen that is a leading cause of nosocomial infections, including catheter-associated urinary tract infection (CAUTI), central lineassociated bloodstream infection (CLABSI), ventilator-associated pneumonia (VAP), and surgical site infection (SSI) (1). In CAUTI, dissemination of bacteria from the catheter can lead to pyelonephritis and bacteremia. The common theme for these infections is the ability of P. aeruginosa to form biofilms on the surfaces of medica...

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“…Although the addition of nucleotides improved the specificity, it may at the same time prevent the capture of proteins that bind different nucleotides at the same site. For example the effector FleQ, which was recently shown to bind ATP and c-di-GMP 18 was fished specifically in the absence of ATP in our previous experiment 1 , but not anymore in the data presented here in the presence of an excess of ATP.…”

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Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Laventie

Nesper

Ahrné

et al. 2015

JoVE

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Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms 1 .Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling. Video LinkThe video component of this article can be found at

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Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ

Cited by 168 publications

References 35 publications

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

Cyclic Di-GMP Signaling Contributes to Pseudomonas aeruginosa-Mediated Catheter-Associated Urinary Tract Infection

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

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