Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip™ based mass spectrometry for the identification of proteins in nucleoprotein interactions

We compared coupling approaches of SPR to LC-MS and ProteinChip™-based mass spectrometry (SELDI™) as a means of identifying proteins captured on DNA surfaces. The approach we outline has the potential to allow multiple, quantitative analysis of macromolecular interactions followed by rapid mass spectrometry identification of retained material

High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing

The nucleoid-associated proteins H-NS and FIS modulate the DNA supercoiling response of the pel genes, the major virulence factors in the plant pathogen bacterium Dickeya dadantii

Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Previously we found that the pel gene expression is modulated by H-NS and FIS, two nucleoid-associated proteins (NAPs) modulating the DNA topology. Here, we show that relaxation of the DNA in growing D. dadantii cells decreases the expression of pel genes. Deletion of fis aggravates, whereas that of hns alleviates the negative impact of DNA relaxation on pel expression. We further show that H-NS and FIS directly bind the pelE promoter and that the response of D. dadantii pel genes to stresses that induce DNA relaxation is modulated, although to different extents, by H-NS and FIS. We infer that FIS acts as a repressor buffering the negative impact of DNA relaxation on pel gene transcription, whereas H-NS fine-tunes the response of virulence genes precluding their expression under suboptimal conditions of supercoiling. This novel dependence of H-NS effect on DNA topology expands our understanding of the role of NAPs in regulating the global bacterial gene expression and bacterial pathogenicity

Removing nucleic acids from nucleoid-Associated proteins purified by affinity column

In bacteria, DNA is tightly compacted in a supercoiled organization, which is mediated in part by nucleoid-associated proteins (NAPs). NAPs are well characterized for their ability to bind nucleic acids and for their involvement in gene regulation. A method commonly used to study protein-nucleic acid interactions involves immunoprecipitation of the protein of interest which is subsequently incubated with nucleic acids. A common cause of artifact is due to nucleic acids that remains bound to the protein of interest during the whole purification process. We developed an optimized method for the purification of tagged NAPs on affinity columns. The combination of three known methods allows removal of most of the nucleic acids bound to proteins during the purification process. This protocol is designed to improve the quality and specificity of results of in vitro experiments involving nucleic acid binding tests on purified NAPs. It can be used for in vitro studies of other RNA/DNA binding proteins

The degree of oligomerization of the H-NS nucleoid structuring protein is related to specific binding to DNA.

International audienceAt several E. coli promoters, initiation of transcription is repressed by a tight nucleoprotein complex formed by the assembly of the H-NS protein. In order to characterize the relationship between the structure of H-NS oligomers in solution and on relevant DNA fragments, we have compared wild-type H-NS and several transdominant H-NS mutants using gel shift assays, DNase I footprinting, analytical ultracentrifugation, and reactivity toward a cross-linking reagent. In solution, oligomerization occurs through two protein interfaces, one necessary to construct a dimeric core (and involving residues 1-64) and the other required for subsequent assembly of these dimers. We show that, as well as region 64-95, residues present in the NH(2)-terminal coiled coil domain also participate in this second interface. Our results support the view that the same interacting interfaces are also involved on the DNA. We propose that the dimeric core recognizes specific motifs, with the second interface being critical for their correct head to tail assembly. The COOH-terminal domain of the protein contains the DNA binding motif essential for the discrimination of this specific functional assembly over competitive nonspecific H-NS polymers

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

International audienceThe secretion of bacterial toxin proteins is achieved by dedicated machineries called secretion systems. The type VI secretion system (T6SS) is a widespread versatile machine used for the delivery of protein toxins to both prokaryotic and eukaryotic cells. In Salmonella enterica serovar Typhimurium, the expression of the T6SS genes is activated during macrophage or mouse infection. Here, we show that the T6SS gene cluster is silenced by the histone-like nucleoid structuring H-NS protein using a combination of reporter fusions, electrophoretic mobility shift assays, DNase footprinting, and fluorescence microscopy. We further demonstrate that derepression of the S. Typhimurium T6SS genes induces T6SS-dependent intoxication of competing bacteria. Our results suggest that relieving T6SS H-NS silencing may be used as a sense-and-kill mechanism that will help S. Typhimurium to homogenize and synchronize the microbial population to gain efficiency during infection

Bacterial-chromatin structural proteins regulate the bimodal expression of the locus of enterocyte effacement (LEE) pathogenicity island in enteropathogenic Escherichia coli

No commentInternational audienceIn enteropathogenic Escherichia coli (EPEC), the locus of enterocyte effacement (LEE) encodes a type 3 secretion system (T3SS) essential for pathogenesis. This pathogenicity island comprises five major operons (LEE1 to LEE5), with the LEE5 operon encoding T3SS effectors involved in the intimate adherence of bacteria to enterocytes. The first operon, LEE1, encodes Ler (LEE-encoded regulator), an H-NS (nucleoid structuring protein) paralog that alleviates the LEE H-NS silencing. We observed that the LEE5 and LEE1 promoters present a bimodal expression pattern, depending on environmental stimuli. One key regulator of bimodal LEE1 and LEE5 expression is ler expression, which fluctuates in response to different growth conditions. Under conditions in vitro considered to be equivalent to nonoptimal conditions for virulence, the opposing regulatory effects of H-NS and Ler can lead to the emergence of two bacterial subpopulations. H-NS and Ler share nucleation binding sites in the LEE5 promoter region, but H-NS binding results in local DNA structural modifications distinct from those generated through Ler binding, at least in vitro. Thus, we show how two nucleoidbinding proteins can contribute to the epigenetic regulation of bacterial virulence and lead to opposing bacterial fates. This finding implicates for the first time bacterialchromatin structural proteins in the bimodal regulation of gene expression. IMPORTANCE Gene expression stochasticity is an emerging phenomenon in microbiology. In certain contexts, gene expression stochasticity can shape bacterial epigenetic regulation. In enteropathogenic Escherichia coli (EPEC), the interplay between H-NS (a nucleoid structuring protein) and Ler (an H-NS paralog) is required for bimodal LEE5 and LEE1 expression, leading to the emergence of two bacterial subpopulations (with low and high states of expression). The two proteins share mutual nucleation binding sites in the LEE5 promoter region. In vitro, the binding of H-NS to the LEE5 promoter results in local structural modifications of DNA distinct from those generated through Ler binding. Furthermore, ler expression is a key parameter modulating the variability of the proportions of bacterial subpopulations. Accordingly, modulating the production of Ler into a nonpathogenic E. coli strain reproduces the bimodal expression of LEE 5. Finally, this study illustrates how two nucleoid-binding proteins can reshape the epigenetic regulation of bacterial virulence