1.Synthesis of c-di-GMP analogs with thiourea, urea, carbodiimide, and guanidinium linkages.
Gaffney BL1, Jones RA. Org Lett. 2014 Jan 3;16(1):158-61. doi: 10.1021/ol403154w. Epub 2013 Dec 6.
The first syntheses of neutral thiourea, urea, and carbodiimide analogs, along with two guanidinium analogs, of the bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) are reported. The key intermediate, obtained in nine steps, is a 3'-amino-5'-azido-3',5'-dideoxy derivative. The 5'-azide serves as a masked amine from which the amine is obtained by Staudinger reduction, while the 3'-amine is converted to an isothiocyanate that, while stable to chromatography, and Staudinger conditions, nevertheless reacts well with the 5'-amine.
2.Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic Di-GMP level in the bacteria.
Christensen LD1, van Gennip M, Rybtke MT, Wu H, Chiang WC, Alhede M, Høiby N, Nielsen TE, Givskov M, Tolker-Nielsen T. Infect Immun. 2013 Aug;81(8):2705-13. doi: 10.1128/IAI.00332-13. Epub 2013 May 20.
Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms.
3.LC/MS/MS-based quantitative assay for the secondary messenger molecule, c-di-GMP.
Irie Y1, Parsek MR. Methods Mol Biol. 2014;1149:271-9. doi: 10.1007/978-1-4939-0473-0_22.
The secondary messenger molecule, 3',5'-cyclic diguanosine monophosphate (c-di-GMP), controls various cellular processes in bacteria. Direct measurement of intracellular concentration of c-di-GMP is fast becoming an important tool for studying prokaryotic biology. Here, we describe a comprehensive extraction protocol from live bacteria and quantitative analysis using LC/MS/MS.
4.Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis.
Sharma IM1, Prakash S1, Dhanaraman T2, Chatterji D3. Microbiology. 2014 Oct;160(Pt 10):2304-18. doi: 10.1099/mic.0.080200-0. Epub 2014 Jul 18.
We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M.
