Protein/DNA interactions in complex DNA topologies: expect the unexpected

DNA supercoiling results in compacted DNA structures that can bring distal sites into close proximity. It also changes the local structure of the DNA, which can in turn influence the way it is recognised by drugs, other nucleic acids and proteins. Here, we discuss how DNA supercoiling and the formation of complex DNA topologies can affect the thermodynamics of DNA recognition. We then speculate on the implications for transcriptional control and the three-dimensional organisation of the genetic material, using examples from our own simulations and from the literature. We introduce and discuss the concept of coupling between the multiple length-scales associated with hierarchical nuclear structural organisation through DNA supercoiling and topology

Chromosomal "stress-response" domains govern the spatiotemporal expression of the bacterial virulence program

International audienceRecent studies strongly suggest that the gene expression sustaining both normal and pathogenic bacterial growth is governed by the structural dynamics of the chromosome. However, the mechanistic device coordinating the chromosomal configuration with selective expression of the adaptive traits remains largely unknown. We used a holistic approach exploring the inherent relationships between the physicochemical properties of the DNA and the expression of adaptive traits, including virulence factors, in the pathogen Dickeya dadantii (formerly Erwinia chrysanthemi). In the transcriptomes obtained under adverse conditions encountered during bacterial infection, we explored the patterns of chromosomal DNA sequence organization, supercoil dynamics, and gene expression densities, together with the long-range regulatory impacts of the abundant DNA architectural proteins implicated in pathogenicity control. By integrating these data, we identified transient chromosomal domains of coherent gene expression featuring distinct couplings between DNA thermodynamic stability, supercoil dynamics, and virulence traits.IMPORTANCE We infer that the organization of transient chromosomal domains serving specific functions acts as a fundamental device for versatile adjustment of the pathogen to environmental stress. We believe that the identification of chromosomal "stress-response" domains harboring distinct virulence traits and mediating the cellular adaptive behavior provides a breakthrough in understanding the control mechanisms of bacterial pathogenicity

A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti

A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli

Two-step chromosome segregation in the stalked budding bacterium Hyphomonas neptunium

Chromosome segregation typically occurs after replication has finished in eukaryotes but during replication in bacteria. Here, we show that the alphaproteobacterium Hyphomonas neptunium, which proliferates by bud formation at the tip of a stalk-like cellular extension, segregates its chromosomes in a unique two-step process. First, the two sister origin regions are targeted to opposite poles of the mother cell, driven by the ParABS partitioning system. Subsequently, once the bulk of chromosomal DNA has been replicated and the bud exceeds a certain threshold size, the cell initiates a second segregation step during which it transfers the stalk-proximal origin region through the stalk into the nascent bud compartment. Thus, while chromosome replication and segregation usually proceed concurrently in bacteria, the two processes are largely uncoupled in H. neptunium, reminiscent of eukaryotic mitosis. These results indicate that stalked budding bacteria have evolved specific mechanisms to adjust chromosome segregation to their unusual life cycle

Gene order and chromosome dynamics coordinate spatiotemporal gene expression during the bacterial growth cycle

In Escherichia coli crosstalk between DNA supercoiling, nucleoid-associated proteins and major RNA polymerase σ initiation factors regulates growth phase-dependent gene transcription. We show that the highly conserved spatial ordering of relevant genes along the chromosomal replichores largely corresponds both to their temporal expression patterns during growth and to an inferred gradient of DNA superhelical density from the origin to the terminus. Genes implicated in similar functions are related mainly in trans across the chromosomal replichores, whereas DNA-binding transcriptional regulators interact predominantly with targets in cis along the replichores. We also demonstrate that macrodomains (the individual structural partitions of the chromosome) are regulated differently. We infer that spatial and temporal variation of DNA superhelicity during the growth cycle coordinates oxygen and nutrient availability with global chromosome structure, thus providing a mechanistic insight into how the organization of a complete bacterial chromosome encodes a spatiotemporal program integrating DNA replication and global gene expression