Quantification of Cyclic Dinucleotides by Reversed-Phase LC-MS/MS

Burhenne, Heike; Kaever, Volkhard

doi:10.1007/978-1-62703-441-8_3

Cited by 68 publications

(66 citation statements)

References 11 publications

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“…The cells were immediately harvested at 4°C and washed twice by distilled water, respectively. Nucleotide was extracted from the cell pellets using the method previously reported (30). Detection of c-di-AMP and c-di-GMP were performed on a Thermo UltiMate3000 liquid chromatography system followed by a Bruker HD Impact tandem mass spectrum system.…”

Section: Determination Of Intracellular C-di-amp and C-di-gmp Concentmentioning

confidence: 99%

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

Wang

Liu

et al. 2016

Journal of Biological Chemistry

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The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å ), and its complex with hydrolysis intermediate 5-pApA (2.35 Å ). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

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Section: Determination Of Intracellular C-di-amp and C-di-gmp Concentmentioning

confidence: 99%

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

Wang

Liu

et al. 2016

Journal of Biological Chemistry

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“…Quantification of intracellular cdG of bacterial cells was performed as previously described (42). Briefly, nucleotides were extracted from cell pellets with 40% acetonitrile, 40% methanol, and 20% water; samples were dried and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS).…”

Section: Methodsmentioning

confidence: 99%

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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“…Currently, very few methods for quantifying c-di-AMP have been reported and they rely primarily on h igh- p erformance l iquid c hromatography (HPLC) (Bai et al , 2012, Oppenheimer-Shaanan et al , 2011), l iquid c hromatography–mass s pectrometry (LC-MS) (Barker et al , 2013, Burhenne & Kaever, 2013, Corrigan et al , 2011, Mehne et al , 2013), or complex formation between c-di-AMP and coralyne (Zhou et al , 2014). HPLC and LC-MS both have a relatively short analysis time for single samples, strong resolving power, reproducibility, and acceptable sensitivity of results.…”

Section: Resultsmentioning

confidence: 99%

Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein

Underwood

Zhang

Metzger

et al. 2014

Journal of Microbiological Methods

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Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling.

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Quantification of Cyclic Dinucleotides by Reversed-Phase LC-MS/MS

Cited by 68 publications

References 11 publications

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

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

Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein

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