The Lrs14 family of DNA-binding proteins as nucleoid-associated proteins in the Crenarchaeal order Sulfolobales.

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions.

YbiB: a novel interactor of the GTPase ObgE.

Obg is a widely conserved and essential GTPase in bacteria, which plays a central role in a large range of important cellular processes, such as ribosome biogenesis, DNA replication, cell division and bacterial persistence. Nevertheless, the exact function of Obg in these processes and the interactions it makes within the associated pathways remain largely unknown. Here, we identify the DNA-binding TrpD2 protein YbiB as an interactor of the Escherichia coli Obg (ObgE). We show that both proteins interact with high affinity in a peculiar biphasic fashion, and pinpoint the intrinsically disordered and highly negatively charged C-terminal domain of ObgE as a main driver for this interaction. Molecular docking and X-ray crystallography, together with site-directed mutagenesis, are used to map the binding site of this ObgE C-terminal domain within a highly positively charged groove on the surface of the YbiB homodimer. Correspondingly, ObgE efficiently inhibits the binding of DNA to YbiB, indicating that ObgE competes with DNA for binding in the positive clefts of YbiB. This study thus forms an important step for the further elucidation of the interactome and cellular role of the essential bacterial protein Obg.

In Vivo Screening Method for the Identification and Characterization of Prokaryotic, Metabolite-Responsive Transcription Factors.

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion. Here, an approach is described for the selection and cloning of TF-promoter pairs, the development of a reporter system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is illustrated together with prospective adaptations to modify a reporter system's output signal. The given approach can be used for the investigation of native, heterologous, or even artificially created TFs in Escherichia coli, and can be extended toward use in other microorganisms.