Structure-specific binding of MeCP2 to four-way junction DNA through its methyl CpG-binding domain

MeCP2, whose methylated DNA-binding domain (MBD) binds preferentially to DNA containing 5Me-CpG relative to linear unmethylated DNA, also binds preferentially, and with similar affinity, to unmethylated four-way DNA junctions through the MBD. The Arg133Cys (R133C) mutation in the MBD, a Rett syndrome mutation that abolishes binding to methylated DNA, leads to only a slight reduction in the affinity of the MBD for four-way junctions, suggesting distinct but partially overlapping modes of binding to junction and methylated DNA. Binding to unmethylated DNA junctions is likely to involve a subset of the interactions that occur with methylated DNA. High-affinity, methylation-independent binding to four-way junctions is consistent with additional roles for MeCP2 in chromatin, beyond recognition of 5Me-CpG

Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions

BACKGROUND: Mycobacteria inhabit diverse niches and display high metabolic versatility. They can colonise both humans and animals and are also able to survive in the environment. In order to succeed, response to environmental cues via transcriptional regulation is required. In this study we focused on the TetR family of transcriptional regulators (TFTRs) in mycobacteria. RESULTS: We used InterPro to classify the entire complement of transcriptional regulators in 10 mycobacterial species and these analyses showed that TFTRs are the most abundant family of regulators in all species. We identified those TFTRs that are conserved across all species analysed and those that are unique to the pathogens included in the analysis. We examined genomic contexts of 663 of the conserved TFTRs and observed that the majority of TFTRs are separated by 200 bp or less from divergently oriented genes. Analyses of divergent genes indicated that the TFTRs control diverse biochemical functions not limited to efflux pumps. TFTRs typically bind to palindromic motifs and we identified 11 highly significant novel motifs in the upstream regions of divergently oriented TFTRs. The C-terminal ligand binding domain from the TFTR complement in M. tuberculosis showed great diversity in amino acid sequence but with an overall architecture common to other TFTRs. CONCLUSION: This study suggests that mycobacteria depend on TFTRs for the transcriptional control of a number of metabolic functions yet the physiological role of the majority of these regulators remain unknown. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1696-9) contains supplementary material, which is available to authorized users

Characterisation of the Putative Effector Interaction Site of the Regulatory HbpR Protein from Pseudomonas azelaica by Site-Directed Mutagenesis

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition

A GFP-lacZ Bicistronic Reporter System for Promoter Analysis in Environmental Gram-Negative Bacteria

Here, we describe a bicistronic reporter system for the analysis of promoter activity in a variety of Gram-negative bacteria at both the population and single-cell levels. This synthetic genetic tool utilizes an artificial operon comprising the gfp and lacZ genes that are assembled in a suicide vector, which is integrated at specific sites within the chromosome of the target bacterium, thereby creating a monocopy reporter system. This tool was instrumental for the complete in vivo characterization of two promoters, Pb and Pc, that drive the expression of the benzoate and catechol degradation pathways, respectively, of the soil bacterium Pseudomonas putida KT2440. The parameterization of these promoters in a population (using β-galactosidase assays) and in single cells (using flow cytometry) was necessary to examine the basic numerical features of these systems, such as the basal and maximal levels and the induction kinetics in response to an inducer (benzoate). Remarkably, GFP afforded a view of the process at a much higher resolution compared with standard lacZ tests; changes in fluorescence faithfully reflected variations in the transcriptional regimes of individual bacteria. The broad host range of the vector/reporter platform is an asset for the characterization of promoters in different bacteria, thereby expanding the diversity of genomic chasses amenable to Synthetic Biology methods

Achievements and new knowledge unraveled by metagenomic approaches

Metagenomics has paved the way for cultivation-independent assessment and exploitation of microbial communities present in complex ecosystems. In recent years, significant progress has been made in this research area. A major breakthrough was the improvement and development of high-throughput next-generation sequencing technologies. The application of these technologies resulted in the generation of large datasets derived from various environments such as soil and ocean water. The analyses of these datasets opened a window into the enormous phylogenetic and metabolic diversity of microbial communities living in a variety of ecosystems. In this way, structure, functions, and interactions of microbial communities were elucidated. Metagenomics has proven to be a powerful tool for the recovery of novel biomolecules. In most cases, functional metagenomics comprising construction and screening of complex metagenomic DNA libraries has been applied to isolate new enzymes and drugs of industrial importance. For this purpose, several novel and improved screening strategies that allow efficient screening of large collections of clones harboring metagenomes have been introduced

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed

Advantages of the metagenomic approach for soil exploration: reply from Vogel et al.

Correspondence : original article http://www.nature.com/nrmicro/journal/v7/n4/full/nrmicro2119.htmlInternational audienc