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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 08 September 2015, Vol.112(36), pp.11359-64
    Description: The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.
    Keywords: Eal Domain ; Pseudomonas Aeruginosa ; Biofilm ; Cyclic Diguanylate ; Oligoribonuclease ; Signal Transduction ; Bacterial Proteins -- Metabolism ; Cyclic Gmp -- Analogs & Derivatives ; Exoribonucleases -- Metabolism ; Pseudomonas Aeruginosa -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Journal of Bacteriology, August, 2012, Vol.194(15-16), p.4285(10)
    Description: Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103?fimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the [delta]fimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103?fimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle, type IV pili, over a broad range of c-di-GMP concentrations. Assembly of pili at low c-di-GMP concentrations requires a polarly localized c-di-GMP binding protein and phosphodiesterase, FimX; this requirement for FimX is bypassed at high c-di-GMP concentrations. Thus, P. aeruginosa can assemble the same surface organelle in distinct ways for motility or adhesion under very different environmental conditions.
    Keywords: Bacterial Adhesion -- Research ; Bacterial Motility -- Research ; Bacterial Proteins -- Structure ; Bacterial Proteins -- Chemical Properties ; Cell Regulation -- Research ; Pseudomonas Aeruginosa -- Physiological Aspects
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 3
    Language: English
    In: FEBS Letters, 20 October 2011, Vol.585(20), pp.3259-3262
    Description: ► Known human phosphodiesterases do not hydrolyze cCMP. ► Several human phosphodiesterases effectively hydrolyze cUMP. ► The study points to a role of cCMP and cUMP as second messengers. Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC–MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.
    Keywords: Phosphodiesterase ; Hplc–MS ; Cyclic Nucleotide ; Biology ; Chemistry ; Anatomy & Physiology
    ISSN: 0014-5793
    E-ISSN: 1873-3468
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  • 4
    Language: English
    In: The Journal of biological chemistry, 30 December 2016, Vol.291(53), pp.26970-26986
    Description: Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.
    Keywords: Staphylococcus Aureus (S. Aureus) ; Ybbr ; Bacterial Signal Transduction ; Ph Regulation ; Phosphodiesterases ; Stress ; Acids -- Metabolism ; Bacterial Proteins -- Metabolism ; Dinucleoside Phosphates -- Metabolism ; Staphylococcus Aureus -- Metabolism
    E-ISSN: 1083-351X
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  • 5
    Language: English
    In: The Journal of biological chemistry, 27 February 2015, Vol.290(9), pp.5826-39
    Description: Nucleotide-signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to external stimuli. The stringent response alarmones guanosine tetra- (ppGpp) and pentaphosphate (pppGpp) control a global response allowing cells to adapt to starvation conditions such as amino acid depletion. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that this signaling nucleotide is essential for the growth of Staphylococcus aureus, and its increased production during late growth phases indicates that c-di-AMP controls processes that are important for the survival of cells in stationary phase. By examining the transcriptional profile of cells with high levels of c-di-AMP, we reveal a significant overlap with a stringent response transcription signature. Examination of the intracellular nucleotide levels under stress conditions provides further evidence that high levels of c-di-AMP lead to an activation of the stringent response through a RelA/SpoT homologue (RSH) enzyme-dependent increase in the (p)ppGpp levels. This activation is shown to be indirect as c-di-AMP does not interact directly with the RSH protein. Our data extend this interconnection further by showing that the S. aureus c-di-AMP phosphodiesterase enzyme GdpP is inhibited in a dose-dependent manner by ppGpp, which itself is not a substrate for this enzyme. Altogether, these findings add a new layer of complexity to our understanding of nucleotide signaling in bacteria as they highlight intricate interconnections between different nucleotide-signaling networks.
    Keywords: Bacterial Signal Transduction ; Cyclic Nucleotide ; Gene Regulation ; Microarray ; Phosphodiesterases ; Stress Response ; Staphylococcus Aureus (S. Aureus) ; Signal Transduction ; Dinucleoside Phosphates -- Metabolism ; Guanosine Pentaphosphate -- Metabolism ; Guanosine Tetraphosphate -- Metabolism ; Staphylococcus Aureus -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 6
    Language: English
    In: Neuroscience Letters, Sept 5, 2014, Vol.579, p.183(5)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.neulet.2014.07.019 Byline: Christina Hartwig, Heike Bahre, Sabine Wolter, Ulrike Beckert, Volkhard Kaever, Roland Seifert Abstract: Author Affiliation: (a) Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany (b) Research Core Unit Metabolomics, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany Article History: Received 16 June 2014; Revised 12 July 2014; Accepted 14 July 2014
    ISSN: 0304-3940
    Source: Cengage Learning, Inc.
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  • 7
    Language: English
    In: Neuroscience Letters, 16 November 2015, Vol.609, pp.74-80
    Description: Lesch–Nyhan syndrome (LNS) is an X-chromosomal disorder with congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) as underlying defect. We determined the concentrations of dopamine, histamine and their metabolites in brains of HPRT knockout mice, which serve as an animal model for LNS, and compared the results to those obtained from wild-type controls. Analyses were performed by high performance liquid chromatography (HPLC)-coupled tandem mass spectrometry (MS/MS). Besides a decrease of dopamine and 3-methoxytyramine (3-MT) concentrations in the cerebral hemisphere, HPRT-deficient mice also exhibited significantly reduced 1-methylhistamine (1-MH) and 1-methylimidazole-4-acetic acid (1-MI4AA) concentrations in the brain hemisphere and medulla. Moreover, the amount of 1-MI4AA was significantly decreased in the cerebellum. Our findings show that neuronal perturbations caused by HPRT deficiency are not restricted to the dopamine system but also affect histaminergic neurotransmission. These new insights into the brain metabolism of an LNS mouse model may help to find new therapeutic strategies to improve the quality of life of LNS patients.
    Keywords: Lesch–Nyhan Syndrome ; Hprt Knockout Mice ; Dopamine ; Histamine ; Hplc ; Mass Spectrometry ; Medicine ; Anatomy & Physiology
    ISSN: 0304-3940
    E-ISSN: 1872-7972
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  • 8
    Language: English
    In: Biochemical and Biophysical Research Communications, 24 January 2014, Vol.443(4), pp.1195-1199
    Description: Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α β has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn but not Mg , the physiological relevance of these findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α β and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn is a physiological sGC cofactor.
    Keywords: Soluble Guanylyl Cyclase ; Nitric Oxide ; Manganese ; Cyclic Gmp ; Cyclic Cmp ; Cyclic Ump ; Biology ; Chemistry ; Anatomy & Physiology
    ISSN: 0006-291X
    E-ISSN: 1090-2104
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  • 9
    Language: English
    In: Biochemical and Biophysical Research Communications, 30 May 2014, Vol.448(2), pp.236-240
    Description: Intact HEK293 cells and B103 neuroblastoma cells possess high basal concentrations of the established second messengers cAMP and cGMP and of the emerging second messengers cCMP and cUMP. We asked the question which nucleotidyl cyclase accounts for the high basal cNMP concentrations. Activators and inhibitors of soluble guanylyl cyclase had no major effects on cNMPs, and the activator of membranous adenylyl cyclase forskolin increased only cAMP. Addition of bicarbonate to medium increased, whereas removal of bicarbonate decreased levels of all four cNMPs. The inhibitor of soluble adenylyl cyclase, 2-(1 -benzo[ ]imidazol-2-ylthio)- ′-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), reduced bicarbonate-stimulated cNMPs. In conclusion, bicarbonate-stimulated soluble adenylyl cyclase plays an important role in the regulation of basal cellular cNMP levels, most notably cCMP and cUMP.
    Keywords: Soluble Adenylyl Cyclase ; Membranous Adenylyl Cyclase ; Cyclic Amp ; Cyclic Cmp ; Cyclic Ump ; Biology ; Chemistry ; Anatomy & Physiology
    ISSN: 0006-291X
    E-ISSN: 1090-2104
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  • 10
    Language: English
    In: Journal of Bacteriology, 2016, Vol.198(17-18), p.2524(12)
    Description: DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-beta-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface.
    Keywords: Escherichia Coli – Research ; Escherichia Coli – Physiological Aspects ; Biosynthesis – Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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