Interaction networks of Escherichia coli replication proteins under different bacterial growth conditions.

In this work we analyzed protein-protein interactions (PPIs) formed by E. coli replication proteins under three disparate bacterial growth conditions. The chosen conditions corresponded to fast exponential growth, slow exponential growth and growth cessation at the stationary phase. We performed affinity purification coupled with mass spectrometry (AP-MS) of chromosomally expressed proteins (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD, NrdB), tagged with sequential peptide affinity (SPA) tag. Composition of protein complexes was characterized using MaxQuant software. To filter out unspecific interactions, we employed double negative control system and we proposed qualitative and quantitative data analysis strategies that can facilitate hits identification in other AP-MS datasets. Our motivation to undertake this task was still insufficient understanding of molecular mechanisms coupling DNA replication to cellular growth. Previous works suggested that such control mechanisms could involve physical interactions of replication factors with metabolic or cell envelope proteins. However, the dynamic replication protein interaction network (PIN) obtained in this study can be used to characterize links between DNA replication and various cellular processes in other contexts.

What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii.

DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters.

DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the 'spacer' between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model's predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.

Mapping the Complex Transcriptional Landscape of the Phytopathogenic Bacterium Dickeya dadantii.

Dickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide and a model organism for studying virulence gene regulation. The present study provides a comprehensive and annotated transcriptomic map of D. dadantii obtained by a computational method combining five independent transcriptomic data sets: (i) paired-end RNA sequencing (RNA-seq) data for a precise reconstruction of the RNA landscape; (ii) DNA microarray data providing transcriptional responses to a broad variety of environmental conditions; (iii) long-read Nanopore native RNA-seq data for isoform-level transcriptome validation and determination of transcription termination sites; (iv) differential RNA sequencing (dRNA-seq) data for the precise mapping of transcription start sites; (v) in planta DNA microarray data for a comparison of gene expression profiles between in vitro experiments and the early stages of plant infection. Our results show that transcription units sometimes coincide with predicted operons but are generally longer, most of them comprising internal promoters and terminators that generate alternative transcripts of variable gene composition. We characterize the occurrence of transcriptional read-through at terminators, which might play a basal regulation role and explain the extent of transcription beyond the scale of operons. We finally highlight the presence of noncontiguous operons and excludons in the D. dadantii genome, novel genomic arrangements that might contribute to the basal coordination of transcription. The highlighted transcriptional organization may allow D. dadantii to finely adjust its gene expression program for a rapid adaptation to fast-changing environments. IMPORTANCE This is the first transcriptomic map of a Dickeya species. It may therefore significantly contribute to further progress in the field of phytopathogenicity. It is also one of the first reported applications of long-read Nanopore native RNA-seq in prokaryotes. Our findings yield insights into basal rules of coordination of transcription that might be valid for other bacteria and may raise interest in the field of microbiology in general. In particular, we demonstrate that gene expression is coordinated at the scale of transcription units rather than operons, which are larger functional genomic units capable of generating transcripts with variable gene composition for a fine-tuning of gene expression in response to environmental changes. In line with recent studies, our findings indicate that the canonical operon model is insufficient to explain the complexity of bacterial transcriptomes.

Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain.

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.

Role of the Discriminator Sequence in the Supercoiling Sensitivity of Bacterial Promoters.

DNA supercoiling acts as a global transcriptional regulator that contributes to the rapid transcriptional response of bacteria to many environmental changes. Although a large fraction of promoters from phylogenetically distant species respond to superhelical variations, the sequence or structural determinants of this behavior remain elusive. Here, we focus on the sequence of the "discriminator" element that was shown to modulate this response in several promoters. We develop a quantitative thermodynamic model of this regulatory effect, focusing on open complex formation during transcription initiation independently from promoter-specific regulatory proteins. We analyze previous and new expression data and show that the model predictions quantitatively match the in vitro and in vivo supercoiling response of selected promoters with mutated discriminator sequences. We then test the universality of this mechanism by a statistical analysis of promoter sequences from transcriptomes of phylogenetically distant bacteria under conditions of supercoiling variations (i) by gyrase inhibitors, (ii) by environmental stresses, or (iii) inherited in the longest-running evolution experiment. In all cases, we identify a robust and significant sequence signature in the discriminator region, suggesting that supercoiling-modulated promoter opening underpins a ubiquitous regulatory mechanism in the prokaryotic kingdom based on the fundamental mechanical properties of DNA and its basal interaction with RNA polymerase. IMPORTANCE In this study, we highlight the role of the discriminator as a global sensor of supercoiling variations and propose the first quantitative regulatory model of this principle, based on the specific step of promoter opening during transcription initiation. It defines the predictive rule by which DNA supercoiling quantitatively modulates the expression rate of bacterial promoters, depending on the G/C content of their discriminator and independently from promoter-specific regulatory proteins. This basal mechanism affects a wide range of species, which is tested by an extensive analysis of global high-throughput expression data. Altogether, ours results confirm and provide a quantitative framework for the long-proposed notion that the discriminator sequence is a significant determinant of promoter supercoiling sensitivity, underpinning the ubiquitous regulatory action of DNA supercoiling on the core transcriptional machinery, in particular in response to quick environmental changes.

The nucleoid-associated protein IHF acts as a 'transcriptional domainin' protein coordinating the bacterial virulence traits with global transcription.

Bacterial pathogenic growth requires a swift coordination of pathogenicity function with various kinds of environmental stress encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study, we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated heterodimeric protein IHF. We inactivated the IHFα subunit of IHF thus precluding the IHFαß heterodimer formation and determined both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. We show that ihfA mutation reorganises the genomic expression by modulating the distribution of chromosomal DNA supercoils at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF heterodimer is a 'transcriptional domainin' protein, the lack of which impairs the spatiotemporal organisation of transcriptional stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

Coherent Domains of Transcription Coordinate Gene Expression During Bacterial Growth and Adaptation.

Recent studies strongly suggest that in bacteria, both the genomic pattern of DNA thermodynamic stability and the order of genes along the chromosomal origin-to-terminus axis are highly conserved and that this spatial organization plays a crucial role in coordinating genomic transcription. In this article, we explore the relationship between genomic sequence organization and transcription in the commensal bacterium Escherichia coli and the plant pathogen Dickeya. We argue that, while in E. coli the gradient of DNA thermodynamic stability and gene order along the origin-to-terminus axis represent major organizational features orchestrating temporal gene expression, the genomic sequence organization of Dickeya is more complex, demonstrating extended chromosomal domains of thermodynamically distinct DNA sequences eliciting specific transcriptional responses to various kinds of stress encountered during pathogenic growth. This feature of the Dickeya genome is likely an adaptation to the pathogenic lifestyle utilizing differences in genomic sequence organization for the selective expression of virulence traits. We propose that the coupling of DNA thermodynamic stability and genetic function provides a common organizational principle for the coordinated expression of genes during both normal and pathogenic bacterial growth.

DNA Supercoiling: an Ancestral Regulator of Gene Expression in Pathogenic Bacteria?

DNA supercoiling acts as a global and ancestral regulator of bacterial gene expression. In this review, we advocate that it plays a pivotal role in host-pathogen interactions by transducing environmental signals to the bacterial chromosome and coordinating its transcriptional response. We present available evidence that DNA supercoiling is modulated by environmental stress conditions relevant to the infection process according to ancestral mechanisms, in zoopathogens as well as phytopathogens. We review the results of transcriptomics studies obtained in widely distant bacterial species, showing that such structural transitions of the chromosome are associated to a complex transcriptional response affecting a large fraction of the genome. Mechanisms and computational models of the transcriptional regulation by DNA supercoiling are then discussed, involving both basal interactions of RNA Polymerase with promoter DNA, and more specific interactions with regulatory proteins. A final part is specifically focused on the regulation of virulence genes within pathogenicity islands of several pathogenic bacterial species.

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling.

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.