Function and mechanism of action of the small regulatory RNA ArcZ in Enterobacterales.

ArcZ is a small regulatory RNA conserved in Enterobacterales It is a Hfq-dependent RNA that is cleaved by RNase E in a processed form of 55 to 60 nucleotides. This processed form is highly conserved for controlling the expression of target mRNAs. ArcZ expression is induced by abundant oxygen levels and reaches its peak during the stationary growth phase. This control is mediated by the oxygen-responsive two-component system ArcAB, leading to the repression of arcZ transcription under low-oxygen conditions in most bacteria in which it has been studied. ArcZ displays multiple targets, and it can control up to 10% of a genome and interact directly with more than 300 mRNAs in Escherichia coli and Salmonella enterica ArcZ displays a multi-faceted ability to regulate its targets through diverse mechanisms such as RNase recruitment, modulation of ribosome accessibility on the mRNA and interaction with translational enhancing regions. By influencing stress response, motility and virulence through the regulation of master regulators such as FlhDC or RpoS, ArcZ emerges as a major orchestrator of cell physiology within Enterobacterales.

Genetic dissection of the bacterial Fe-S protein biogenesis machineries.

Iron­sulfur (Fe-S) clusters are one of the most ancient and versatile inorganic cofactors present in the three domains of life. Fe-S clusters are essential cofactors for the activity of a large variety of metalloproteins that play crucial physiological roles. Fe-S protein biogenesis is a complex process that starts with the acquisition of the elements (iron and sulfur atoms) and their assembly into an Fe-S cluster that is subsequently inserted into the target proteins. The Fe-S protein biogenesis is ensured by multiproteic systems conserved across all domains of life. Here, we provide an overview on how bacterial genetics approaches have permitted to reveal and dissect the Fe-S protein biogenesis process in vivo.

Expanding the Staphylococcus aureus SarA Regulon to Small RNAs.

SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.

Use of nuclear receptor luciferase-based bioassays to detect endocrine active chemicals in a biosolids-biochar amended soil.

Biosolids are a potentially valuable source of carbon and nutrients for agricultural soils; however, potential unintended impacts on human health and the environment must be considered. Virtually all biosolids contain trace amounts endocrine-disrupting chemicals derived from human use of pharmaceuticals and personal care products (PPCPs). One potential way to reduce the bioavailability of PPCPs is to co-apply biosolids with biochar to soil, because biochar's chemical (e.g., aromaticity) and physical properties (e.g., surface area) give it a high affinity to bind many organic chemicals in the environment. We developed a soil-specific extraction method and utilized a luciferase-based bioassay (CALUX) to detect endocrine active chemicals in a biosolids-biochar co-amendment soil greenhouse study. Both biochar (walnut shell, 900 °C) and biosolids had positive impacts on carrot and lettuce biomass accumulation over our study period. However, the walnut shell biochar stimulated aryl hydrocarbon receptor activity, suggesting the presence of potential endocrine active chemicals in the biochar. Since the biochar rate tested (100 t ha-1) is above the average agronomic rate (10-20 t ha-1), endocrine effects would not be expected in most environmental applications. The effect of high temperature biochars on endocrine system pathways must be explored further, using both quantitative analytical tools to identify potential endocrine active chemicals and highly sensitive bioanalytical assays such as CALUX to measure the resulting biological activity of such compounds.