CorE from Myxococcus xanthus Is a Copper-Dependent RNA Polymerase Sigma Factor

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators

Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs

Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus

Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family

Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Built Shallow to Maintain Homeostasis and Persistent Infection: Insight into the Transcriptional Regulatory Network of the Gastric Human Pathogen Helicobacter pylori

Transcriptional regulatory networks (TRNs) transduce environmental signals into coordinated output expression of the genome. Accordingly, they are central for the adaptation of bacteria to their living environments and in host–pathogen interactions. Few attempts have been made to describe a TRN for a human pathogen, because even in model organisms, such as Escherichia coli, the analysis is hindered by the large number of transcription factors involved. In light of the paucity of regulators, the gastric human pathogen Helicobacter pylori represents a very appealing system for understanding how bacterial TRNs are wired up to support infection in the host. Herein, we review and analyze the available molecular and “-omic” data in a coherent ensemble, including protein–DNA and protein–protein interactions relevant for transcriptional control of pathogenic responses. The analysis covers ∼80% of the annotated H. pylori regulators, and provides to our knowledge the first in-depth description of a TRN for an important pathogen. The emerging picture indicates a shallow TRN, made of four main modules (origons) that process the physiological responses needed to colonize the gastric niche. Specific network motifs confer distinct transcriptional response dynamics to the TRN, while long regulatory cascades are absent. Rather than having a plethora of specialized regulators, the TRN of H. pylori appears to transduce separate environmental inputs by using different combinations of a small set of regulators

MicroMotility: State of the art, recent accomplishments and perspectives on the mathematical modeling of bio-motility at microscopic scales

Mathematical modeling and quantitative study of biological motility (in particular, of motility at microscopic scales) is producing new biophysical insight and is offering opportunities for new discoveries at the level of both fundamental science and technology. These range from the explanation of how complex behavior at the level of a single organism emerges from body architecture, to the understanding of collective phenomena in groups of organisms and tissues, and of how these forms of swarm intelligence can be controlled and harnessed in engineering applications, to the elucidation of processes of fundamental biological relevance at the cellular and sub-cellular level. In this paper, some of the most exciting new developments in the fields of locomotion of unicellular organisms, of soft adhesive locomotion across scales, of the study of pore translocation properties of knotted DNA, of the development of synthetic active solid sheets, of the mechanics of the unjamming transition in dense cell collectives, of the mechanics of cell sheet folding in volvocalean algae, and of the self-propulsion of topological defects in active matter are discussed. For each of these topics, we provide a brief state of the art, an example of recent achievements, and some directions for future research