ABSTRACT
Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections.
Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/chemistry , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Catalytic Domain , Cyclic GMP/analogs & derivatives , Protein Multimerization , Pseudomonas aeruginosa/enzymology , Amino Acid Sequence , Biocatalysis , Cyclic GMP/metabolism , Cyclic GMP/pharmacology , Enzyme Activation/drug effects , Escherichia coli Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Phosphorus-Oxygen Lyases/metabolism , Protein Multimerization/drug effects , Protein Structure, QuaternaryABSTRACT
Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.
