scholarly journals Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

Microbiology ◽  
2014 ◽  
Vol 160 (7) ◽  
pp. 1427-1439 ◽  
Author(s):  
H. Tan ◽  
J. A. West ◽  
J. P. Ramsay ◽  
R. E. Monson ◽  
J. L. Griffin ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Cellular Localization ◽  
Bacterial Species ◽  
Guanosine Monophosphate ◽  
Domain Family ◽  
Pectobacterium Atrosepticum ◽  
Diguanylate Cyclases ◽  
Signalling Molecule ◽  
Apparent Correlation ◽  
Ggdef Domain

Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5′-phosphoguanylyl-(3′-5′)-guanosine in these strains revealed that overexpression of most genes promoted modest 1–10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

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

2014 ◽  
Vol 80 (19) ◽  
pp. 6167-6174 ◽  
Author(s):  
Xiaohui Gao ◽  
Xiao Dong ◽  
Sundharraman Subramanian ◽  
Paige M. Matthews ◽  
Caleb A. Cooper ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Threshold Level ◽  
Metabolic Enzymes ◽  
Guanosine Monophosphate ◽  
Content Type ◽  
Diguanylate Cyclases ◽  
Rapid Characterization ◽  
Green Fluorescent

ABSTRACTMicrobial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite ofBacillus subtilisstrains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs inClostridium difficileusing parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putativeC. difficile630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility inBacillus subtilis.

Phosphodiesterase activity of NbdA from Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Anna Scherhag ◽  
Martina Rüger ◽  
Katrin Gerbracht ◽  
Jaqueline Rehner ◽  
Susanne Zehner ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Model Organism ◽  
Functional Complementation ◽  
Full Length ◽  
Protein Activity ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Ggdef Domain

<p>The molecule c-di-GMP is a bacterial second messenger that controls various processes such as motility or biofilm formation in bacteria [1]. To synthesize and degrade c-di-GMP, enzymes called diguanylate cyclases (DGC) containing a GGDEF-domain and phosphodiesterases (PDE) containing an EAL-domain or HD-GYP-domain are important [1, 2].<em> Pseudomonas aeruginosa</em>, a model organism for biofilm formation and dispersion, encodes for 18 GGDEF, 5 EAL, 16 GGDEF / EAL, and 3 HD-GYP-domain-containing proteins [3].<br />One of the GGDEF / EAL-containing proteins is NbdA. This protein also harbors an N-terminal membrane anchored MHYT-domain, that is predicted to be a sensor for NO, CO or O<sub>2</sub> [4]. In this work, recombinant and affinity purified NbdA was tested for its PDE activity. Three different methods were used to measure the PDE activity of NbdA: a bis-pNPP-assay in which the conversion of the pseudosubstrate bis-pNPP into p-nitrophenol was detected spectroscopically, an HPLC-analysis of an enzymatic assay with the native substrate c-di-GMP, and a MANT-c-di-GMP-assay in which a fluorescently labeled form of the presumed substrate c-di-GMP was utilized.<br />To establish these methods, the two known phosphodiesterases, PdeH from <em>Escherichia coli</em> [5] and RocR from <em>P. aeruginosa</em> [6], were also produced and tested. Subsequently, three variants of NbdA were investigated: the full-length version and two truncated versions of the protein. Activity was further assessed using functional complementation of an <em>E. coli</em> phosphodiesterase deficient strain with full-length and truncated NbdA variants confirming PDE activity <em>in vivo</em>.</p> <p> </p> <p> </p> <p>[1] Hengge, R. (2009) Nature Rev. Microbiol. 7: 263-273.</p> <p>[2] Römling, U., Gomelsky, M., Galperin, M.Y. (2005). Mol. Microbiol. 57: 629–639.</p> <p>[3] Valentini, M., Filloux, A. (2016). J. Biol. Chem. 291: 12547–12555.</p> <p>[4] Galperin, M.Y., Gaidenko, T.A., Mulkidjanian, A.Y., Nakano, M., und Price, C.W. (2001). FEMS Microbiol. Lett. 205, 17–23.</p> <p>[5] Pesavento, C., Becker, G., Sommerfeldt, N., Possling, A., Tschowri, N., Mehlis, A., Hengge, R. (2008). Genes Dev. 22: 2434–2446.</p> <p>[6] Chen et al. (2012) Chen, M.W., Kotaka, M., Vonrhein, C., Bricogne, G., Rao, F., Chuah, M.L.C., Svergun, D., Schneider, G., Liang, Z.-X., Lescar, J.  (2012). Signaling. J. Bacteriol. 194: 4837–4846</p> <p> </p>

Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

2003 ◽  
Vol 49 (7) ◽  
pp. 443-449 ◽  
Author(s):  
P Di Martino ◽  
R Fursy ◽  
L Bret ◽  
B Sundararaju ◽  
R S Phillips
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Klebsiella Oxytoca ◽  
Laboratory Strain ◽  
Luria Bertani ◽  
Dependent Manner ◽  
E Coli ◽  
Signalling Molecule

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria–Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.Key words: Escherichia coli, biofilm, indole, tryptophanase, signalling molecule.

scholarly journals π-Helix controls activity of oxygen-sensing diguanylate cyclases

2020 ◽  
Vol 40 (2) ◽  
Author(s):  
Johnnie A. Walker ◽  
Yuqi Wu ◽  
Jacob R. Potter ◽  
Emily E. Weinert
Keyword(s):  
Biofilm Formation ◽  
Guanosine Monophosphate ◽  
Diguanylate Cyclases ◽  
Cyclase Domain ◽  
Middle Domain ◽  
Binding Event ◽  
Domain Interactions ◽  
Liquid Chromatography Tandem Mass ◽  
Cellular Phenotypes ◽  
O2 Binding

Abstract The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

scholarly journals A Staphylococcal GGDEF Domain Protein Regulates Biofilm Formation Independently of Cyclic Dimeric GMP

2008 ◽  
Vol 190 (15) ◽  
pp. 5178-5189 ◽  
Author(s):  
Linda M. Holland ◽  
Sinéad T. O'Donnell ◽  
Dmitri A. Ryjenkov ◽  
Larissa Gomelsky ◽  
Shawn R. Slater ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Cyclase Activity ◽  
Mrna Levels ◽  
Diguanylate Cyclase ◽  
Diguanylate Cyclases ◽  
Exopolysaccharide Biosynthesis ◽  
Ggdef Domain ◽  
Domain Protein ◽  
Biofilm Development

ABSTRACT Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

scholarly journals Identification and Characterization of a Phosphodiesterase That Inversely Regulates Motility and Biofilm Formation in Vibrio cholerae

2010 ◽  
Vol 192 (18) ◽  
pp. 4541-4552 ◽  
Author(s):  
Xianxian Liu ◽  
Sinem Beyhan ◽  
Bentley Lim ◽  
Roger G. Linington ◽  
Fitnat H. Yildiz
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Genetic Interaction ◽  
Interaction Analysis ◽  
Lifestyle Changes ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Ggdef Domain

ABSTRACT Vibrio cholerae switches between free-living motile and surface-attached sessile lifestyles. Cyclic diguanylate (c-di-GMP) is a signaling molecule controlling such lifestyle changes. C-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a GGDEF domain and is degraded by phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain. We constructed in-frame deletions of all V. cholerae genes encoding proteins with GGDEF and/or EAL domains and screened mutants for altered motility phenotypes. Of 52 mutants tested, four mutants exhibited an increase in motility, while three mutants exhibited a decrease in motility. We further characterized one mutant lacking VC0137 (cdgJ), which encodes an EAL domain protein. Cellular c-di-GMP quantifications and in vitro enzymatic activity assays revealed that CdgJ functions as a PDE. The cdgJ mutant had reduced motility and exhibited a small decrease in flaA expression; however, it was able to produce a flagellum. This mutant had enhanced biofilm formation and vps gene expression compared to that of the wild type, indicating that CdgJ inversely regulates motility and biofilm formation. Genetic interaction analysis revealed that at least four DGCs, together with CdgJ, control motility in V. cholerae.

scholarly journals The Matrix Revisited: Opening Night for the Pel Polysaccharide Across Eubacterial Kingdoms

2021 ◽  
Vol 14 ◽  
pp. 117863612098858
Author(s):  
Gregory B Whitfield ◽  
P Lynne Howell
Keyword(s):  
Biofilm Formation ◽  
Genetic Locus ◽  
Bacterial Species ◽  
Guanosine Monophosphate ◽  
Formation Mechanisms ◽  
Variant Form ◽  
Gram Positive ◽  
Gram Negative ◽  
The Matrix ◽  
Biofilm Matrix

Bacteria synthesize and export adhesive macromolecules to enable biofilm formation. These macromolecules, collectively called the biofilm matrix, are structurally varied and often unique to specific bacterial species or subspecies. This heterogeneity in matrix utilization makes it difficult to facilitate direct comparison between biofilm formation mechanisms of different bacterial species. Despite this, some matrix components, in particular the polysaccharides poly-β-1,6- N-acetyl-glucosamine (PNAG) and bacterial cellulose, are utilized by many Gram-negative species for biofilm formation. However, there is a very narrow distribution of these components across Gram-positive organisms, whose biofilm matrix determinants remain largely undiscovered. We found that a genetic locus required for the production of a biofilm matrix component of P. aeruginosa, the Pel polysaccharide, is widespread in Gram-negative bacteria and that there is a variant form of this cluster present in many Gram-positive bacterial species. We demonstrated that this locus is required for biofilm formation by Bacillus cereus ATCC 10987, produces a polysaccharide that is similar to Pel, and is post-translationally regulated by cyclic-3′,5′-dimeric-guanosine monophosphate (c-di-GMP) in a manner identical to P. aeruginosa. However, while the proposed mechanism for Pel production appears remarkably similar between B. cereus and P. aeruginosa, we identified several key differences between Gram-negative and Gram-positive Pel biosynthetic components in other monoderms. In particular, 4 different architectural subtypes of the c-di-GMP-binding component PelD were identified, including 1 found only in Streptococci that has entirely lost the c-di-GMP recognition domain. These observations highlight how existing multi-component bacterial machines can be subtly tweaked to adapt to the unique physiology and regulatory mechanisms of Gram-positive organisms. Collectively, our analyses suggest that the Pel biosynthetic locus is one of the most phylogenetically widespread biofilm matrix determinants in bacteria, and that its mechanism of production and regulation is extraordinarily conserved across the majority of organisms that possess it.

scholarly journals Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate

2021 ◽  
Vol 12 ◽  
Author(s):  
Myles B. Poulin ◽  
Laura L. Kuperman
Keyword(s):  
Biofilm Formation ◽  
Bacterial Species ◽  
Bacterial Biofilm ◽  
Guanosine Monophosphate ◽  
Host Immune System ◽  
Bacterial Cells ◽  
Direct Role ◽  
Direct Targeting ◽  
Translational Activation ◽  
Time Required

Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly-N-acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c-di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.

Modulation of Gut Microbiota through Dietary Phytochemicals as a Novel Anti-infective Strategy

2020 ◽  
Vol 17 (4) ◽  
pp. 498-506 ◽  
Author(s):  
Pavan K. Mujawdiya ◽  
Suman Kapur
Keyword(s):  
Microbial Community ◽  
Quorum Sensing ◽  
Population Density ◽  
Gut Microbiota ◽  
Biofilm Formation ◽  
Chemical Interaction ◽  
Bacterial Species ◽  
Critical Role ◽  
Bacterial Cells ◽  
Gut Microbes

: Quorum Sensing (QS) is a phenomenon in which bacterial cells communicate with each other with the help of several low molecular weight compounds. QS is largely dependent on population density, and it triggers when the concentration of quorum sensing molecules accumulate in the environment and crosses a particular threshold. Once a certain population density is achieved and the concentration of molecules crosses a threshold, the bacterial cells show a collective behavior in response to various chemical stimuli referred to as “auto-inducers”. The QS signaling is crucial for several phenotypic characteristics responsible for bacterial survival such as motility, virulence, and biofilm formation. Biofilm formation is also responsible for making bacterial cells resistant to antibiotics. : The human gut is home to trillions of bacterial cells collectively called “gut microbiota” or “gut microbes”. Gut microbes are a consortium of more than 15,000 bacterial species and play a very crucial role in several body functions such as metabolism, development and maturation of the immune system, and the synthesis of several essential vitamins. Due to its critical role in shaping human survival and its modulating impact on body metabolisms, the gut microbial community has been referred to as “the forgotten organ” by O`Hara et al. (2006) [1]. Several studies have demonstrated that chemical interaction between the members of bacterial cells in the gut is responsible for shaping the overall microbial community. : Recent advances in phytochemical research have generated a lot of interest in finding new, effective, and safer alternatives to modern chemical-based medicines. In the context of antimicrobial research various plant extracts have been identified with Quorum Sensing Inhibitory (QSI) activities among bacterial cells. This review focuses on the mechanism of quorum sensing and quorum sensing inhibitors isolated from natural sources.

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