REC domain stabilizes the active heptamer of σ54-dependent transcription factor, FleR from Pseudomonas aeruginosa.

Motility in Pseudomonas aeruginosa is mediated through a single, polar flagellum, which is essential for virulence, colonization, and biofilm formation. FleSR, a two-component system (TCS), serves as a critical checkpoint in flagellar assembly. FleR is a σ54-dependent response regulator that undergoes phosphorylation via cognate sensor kinase FleS for the assembly of the functionally active form. The active form remodels the σ54-RNAP complex to initiate transcription. Small-angle X-ray scattering, crystallography, and negative staining electron microscopy reconstructions of FleR revealed that it exists predominantly as a dimer in the inactive form with low ATPase activity and assembles into heptamers upon phosphorylation with amplified ATPase activity. We establish that receiver (REC) domain stabilizes the heptamers and is indispensable for assembly of the functional phosphorylated form of FleR. The structural, biochemical, and in vivo complementation assays provide details of the phosphorylation-mediated assembly of FleR to regulate the expression of flagellar genes.

N-Terminus Plays a Critical Role for Stabilizing the Filamentous Assembly and the Antifungal Activity of Bg_9562.

Bg_9562, a prophage tail-like protein was earlier shown to be required for bacterial mycophagy by Burkholderia gladioli strain NGJ1. The purified protein exhibited broad-spectrum antifungal activity; however, the structural and mechanistic details vis-à-vis its activity remained elusive. In this study, we have structurally characterized the protein Bg_9562 using negatively stained transmission electron microscopy, molecular modeling and mutagenesis. We find that Bg_9562 shows structural similarity to Gp13, a tail assembly chaperone. The transmission electron microscopy revealed that, Bg_9562 forms long flexible tubular structures. Molecular modeling of the filament like structure divulges that the inter subunit contacts are meditated largely through hydrophobic interactions. Using mutagenesis, we demonstrate that the N-terminal residues of the protein when deleted results in reduced activity and destabilization of filament formation. Overall, structure-function analysis opens up avenues for further utilization of the protein as a potent antifungal molecule. IMPORTANCE Burkholderia gladioli strain NGJ1, isolated from healthy rice seedling, was earlier demonstrated to have mycophagous properties on a broad range of fungi, including Rhizoctonia solani, a causal agent of deadly sheath blight disease of rice. The purified Bg_9562 protein exerts broad-spectrum antifungal activity. The protein also inhibits the growth of laboratory strain of Candida, an opportunistic human pathogen. In this study, we structurally characterize Bg_9562 using a combination of negative staining transmission electron microscopy, molecular modeling, mutagenesis, and functional antifungal assay. We show that the protein assembles into long filament like structures stabilized by N-terminus residues and this region is important for its activity. Our study has implications in utilizing Bg_9562 or its derivatives as antifungal molecule(s) which will provide environmentally friendly control of fungal diseases of plants and animals.

Molecular and structural facets of c-di-GMP signalling associated with biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen and is the primary cause of nosocomial infections. Biofilm formation by this organism results in chronic and hard to eradicate infections. The intracellular signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a secondary messenger in bacterial cells crucial for motile to sessile transition. The signalling pathway components encompass two classes of enzymes with antagonistic activities, the diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that regulate the cellular levels of c-di-GMP at distinct stages of biofilm initiation, maturation and dispersion. This review summarizes the structural analysis and functional studies of the DGCs and PDEs involved in biofilm regulation in P. aeruginosa. In addition, we also describe the effector proteins that sense the perturbations in c-di-GMP levels to elicit a functional output. Finally, we discuss possible mechanisms that allow the dynamic levels of c-di-GMP to regulate cognate cellular response. Uncovering the details of the regulation of the c-di-GMP signalling pathway is vital for understanding the behaviour of the pathogen and characterization of novel targets for anti-biofilm interventions.

Transcriptional Fidelity of Mitochondrial RNA Polymerase RpoTm from Arabidopsis thaliana.

Fidelity of RNA synthesis is essential for the faithful transfer of information from DNA to RNA. A comprehensive analysis of the nucleotide selectivity by the mitochondrial RNA polymerase (RNAP) RpoTm, from Arabidopsis thaliana, has been carried out. The kinetic parameters for the incorporation of cognate, noncognate, and oxidized bases have been determined. The results establish high fidelity of mitochondrial transcription resembling those of replicative polymerases in the absence of repair. In addition, RpoTm incorporates oxidized nucleotides with similar efficiency compared with mismatches and is capable of extending the RNA beyond the insertion of the oxidized base. Furthermore, lesion bypass study on RpoTm demonstrates that the enzyme bypasses 8-oxo-guanine by insertion of adenine leading to C to A mutations in RNA. Homology modeling of RpoTm elongation complex allows delineation of the residues necessary for stabilizing the incoming NTP substrate and for posing the template nucleotide residue. Substitution of these residues leads to compromise in the activity of the enzyme corroborating their importance in RNA synthesis. Comparison of the data with T7 RNAPs indicates that low efficiency of misincorporation is a universal strategy used by single-subunit RNAPs for maintaining high fidelity in the absence of proofreading and repair activity in mitochondria.