A corepressor participates in LexA-independent regulation of error-prone polymerases in Acinetobacter

The DNA damage response of the multidrug-resistant pathogen Acinetobacter baumannii, which induces mutagenic UmuD′2C error-prone polymerases, differs from that of many bacteria. Acinetobacter species lack a LexA repressor, but induce gene transcription after DNA damage. One regulator, UmuDAb, binds to and represses the promoters of the multiple A. baumannii ATCC 17978 umuDC alleles and the divergently transcribed umuDAb and ddrR genes. ddrR is unique to the genus Acinetobacter and of unknown function. 5\u27 RACE (rapid amplification of cDNA ends) PCR mapping of the umuDAb and ddrR transcriptional start sites revealed that their −35 promoter elements overlapped the UmuDAb binding site, suggesting that UmuDAb simultaneously repressed expression of both genes by blocking polymerase access. This coordinated control of ddrR and umuDAb suggested that ddrR might also regulate DNA damage-inducible gene transcription. RNA-sequencing experiments in 17 978 ddrR− cells showed that ddrR regulated approximately 25 % (n=39) of the mitomycin C-induced regulon, with umuDAb coregulating 17 of these ddrR-regulated genes. Eight genes (the umuDC polymerases, umuDAb and ddrR) were de-repressed in the absence of DNA damage, and nine genes were uninduced in the presence of DNA damage, in both ddrR and umuDAb mutant strains. These data suggest ddrR has multiple roles, both as a co-repressor and as a positive regulator of DNA damage-inducible gene transcription. Additionally, 57 genes were induced by mitomycin C in the ddrR mutant but not in wild-type cells. This regulon contained multiple genes for DNA replication, recombination and repair, transcriptional regulators, RND efflux, and transport. This study uncovered another regulator of the atypical DNA damage response of this genus, to help describe how this pathogen acquires drug resistance through its expression of the error-prone polymerases under DdrR and UmuDAb control

Tracking down carbon inputs underground from an arid zone Australian calcrete.

Freshwater ecosystems play a key role in shaping the global carbon cycle and maintaining the ecological balance that sustains biodiversity worldwide. Surficial water bodies are often interconnected with groundwater, forming a physical continuum, and their interaction has been reported as a crucial driver for organic matter (OM) inputs in groundwater systems. However, despite the growing concerns related to increasing anthropogenic pressure and effects of global change to groundwater environments, our understanding of the dynamics regulating subterranean carbon flows is still sparse. We traced carbon composition and transformations in an arid zone calcrete aquifer using a novel multidisciplinary approach that combined isotopic analyses of dissolved organic carbon (DOC) and inorganic carbon (DIC) (δ13CDOC, δ13CDIC, 14CDOC and 14CDIC) with fluorescence spectroscopy (Chromophoric Dissolved OM (CDOM) characterisation) and metabarcoding analyses (taxonomic and functional genomics on bacterial 16S rRNA). To compare dynamics linked to potential aquifer recharge processes, water samples were collected from two boreholes under contrasting rainfall: low rainfall ((LR), dry season) and high rainfall ((HR), wet season). Our isotopic results indicate limited changes and dominance of modern terrestrial carbon in the upper part (northeast) of the bore field, but correlation between HR and increased old and 13C-enriched DOC in the lower area (southwest). CDOM results show a shift from terrestrially to microbially derived compounds after rainfall in the same lower field bore, which was also sampled for microbial genetics. Functional genomic results showed increased genes coding for degradative pathways-dominated by those related to aromatic compound metabolisms-during HR. Our results indicate that rainfall leads to different responses in different parts of the bore field, with an increase in old carbon sources and microbial processing in the lower part of the field. We hypothesise that this may be due to increasing salinity, either due to mobilisation of Cl- from the soil, or infiltration from the downstream salt lake during HR. This study is the first to use a multi-technique assessment using stable and radioactive isotopes together with functional genomics to probe the principal organic biogeochemical pathways regulating an arid zone calcrete system. Further investigations involving extensive sampling from diverse groundwater ecosystems will allow better understanding of the microbiological pathways sustaining the ecological functioning of subterranean biota

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

A Toxicological Framework for the Prioritization of Children’s Safe Product Act Data

In response to concerns over hazardous chemicals in children’s products, Washington State passed the Children’s Safe Product Act (CSPA). CSPA requires manufacturers to report the concentration of 66 chemicals in children’s products. We describe a framework for the toxicological prioritization of the ten chemical groups most frequently reported under CSPA. The framework scores lifestage, exposure duration, primary, secondary and tertiary exposure routes, toxicokinetics and chemical properties to calculate an exposure score. Four toxicological endpoints were assessed based on curated national and international databases: reproductive and developmental toxicity, endocrine disruption, neurotoxicity and carcinogenicity. A total priority index was calculated from the product of the toxicity and exposure scores. The three highest priority chemicals were formaldehyde, dibutyl phthalate and styrene. Elements of the framework were compared to existing prioritization tools, such as the United States Environmental Protection Agency’s (EPA) ExpoCast and Toxicological Prioritization Index (ToxPi). The CSPA framework allowed us to examine toxicity and exposure pathways in a lifestage-specific manner, providing a relatively high throughput approach to prioritizing hazardous chemicals found in children’s products

Prophage Induction and Differential RecA and UmuDAb Transcriptome Regulation in the DNA Damage Responses of <i>Acinetobacter baumannii</i> and <i>Acinetobacter baylyi</i>

<div><p>The SOS response to DNA damage that induces up to 10% of the prokaryotic genome requires RecA action to relieve LexA transcriptional repression. In <i>Acinetobacter</i> species, which lack LexA, the error-prone polymerase accessory UmuDAb is instead required for <i>ddrR</i> induction after DNA damage, suggesting it might be a LexA analog. RNA-Seq experiments defined the DNA damage transcriptome (mitomycin C-induced) of wild type, <i>recA</i> and <i>umuDAb</i> mutant strains of both <i>A. baylyi</i> ADP1 and <i>A. baumannii</i> ATCC 17978. Of the typical SOS response genes, few were differentially regulated in these species; many were repressed or absent. A striking 38.4% of all ADP1 genes, and 11.4% of all 17978 genes, were repressed under these conditions. In <i>A. baylyi</i> ADP1, 66 genes (2.0% of the genome), including a CRISPR/Cas system, were DNA damage-induced, and belonged to four regulons defined by differential use of <i>recA</i> and <i>umuDAb</i>. In <i>A. baumannii</i> ATCC 17978, however, induction of 99% of the 152 mitomycin C-induced genes depended on <i>recA</i>, and only 28 of these genes required <i>umuDAb</i> for their induction. 90% of the induced <i>A. baumannii</i> genes were clustered in three prophage regions, and bacteriophage particles were observed after mitomycin C treatment. These prophages encoded <i>esvI</i>, <i>esvK1</i>, and <i>esvK2</i>, ethanol-stimulated virulence genes previously identified in a <i>Caenorhabditis elegans</i> model, as well as error-prone polymerase alleles. The induction of all 17978 error-prone polymerase alleles, whether prophage-encoded or not, was <i>recA</i> dependent, but only these DNA polymerase V-related genes were de-repressed in the <i>umuDAb</i> mutant in the absence of DNA damage. These results suggest that both species possess a robust and complex DNA damage response involving both <i>recA-</i>dependent and <i>recA</i>-independent regulons, and further demonstrates that although <i>umuDAb</i> has a specialized role in repressing error-prone polymerases, additional regulators likely participate in these species' transcriptional response to DNA damage.</p></div

RT-qPCR experiments indicate that <i>umuDAb</i> is required for repression of error-prone polymerase components, not all DNA damage-induced genes.

<p>Delta Cq values from RT-qPCR experiments measuring expression of selected <i>A. baumannii</i> ATCC 17978 genes demonstrates the repressing activity of UmuDAb only for error prone polymerase components. The expression of each gene in both wild type and <i>umuDAb</i> null mutant is shown, with gene identity and A1S number listed on the x axis. Each gene was assayed in one RT-qPCR experiment (plate), with error bars indicating standard error of the mean from technical triplicates of biological triplicates.</p

Mitomycin-C induced and repressed SOS genes in ADP1 and 17978 differentially require RecA and UmuDAb.

<p>Gene induction ratios obtained from RNASeq transcriptome experiments are shown, with the induction (panel A) and repression (panel B) of canonical SOS genes. Gene names prefaced by “AD” indicate ADP1 genes; “AB” indicates 17978 genes, with A1S numbers of 17978 genes listed for <i>umuDC</i> alleles. The placement of the horizontal axis in each panel represents the cutoff level for a gene to be considered induced (A) or repressed (B). Bars above the horizontal axis indicate induced genes (panel A), and bars below the horizontal axis indicate repressed genes (panel B), with bars rising either below (panel A) or above (panel B) this axis to have lost their induction or repression, respectively, in the <i>umuDAb</i> and/or <i>recA</i> mutant strains. (A) Induced genes did not require <i>umuDAb</i> in either ADP1 or 17978, except for the category of <i>umuDC</i> alleles, and <i>recA</i> was required for all induced 17978 genes but only some induced ADP1 genes (<i>ruvA</i> and <i>uvrA</i>). (B) Repressed genes required only <i>umuDAb</i> in ADP1 but required both <i>umuDAb</i> and <i>recA</i> in 17978.</p

Distribution of regulation mechanisms for mitomycin C-induced and repressed transcriptome in ADP1 and 17978.

<p>The absolute number of genes induced (A) or repressed (panel B) by MMC in the transcriptome of ADP1 and 17978 is shown. The designation of regulon is represented by the following terms: Neither (genes requiring neither <i>umuDAb</i> nor <i>recA</i> for regulation), Both (genes requiring both <i>umuDAb</i> and <i>recA</i> for regulation), RecA (genes requiring only <i>recA</i> for regulation), or UmuDAb (genes requiring only <i>umuDAb</i> for regulation). (A) Many more repressed genes were observed in ADP1 than 17978, with UmuDAb sufficing for this repression in most genes; 17978 repressed genes required either UmuDAb or both UmuDAb and RecA. (B) A greater number of induced genes was observed in 17978 than ADP1, and these genes required either RecA or both RecA and UmuDAb. In comparison, ADP1 induced genes belong to four regulons (Neither, Both, RecA, or UmuDAb).</p

Mitomycin C treatment induces production of bacteriophage particles in 17978.

<p>(A) Overnight LB cultures of 17978 cells were diluted into fresh LB medium and grown for 0.75 hours before addition of 2 μg/mL MMC. After approximately two hours of MMC treatment, the optical density leveled off and decreased slightly but continued to increase in the absence of MMC treatment. Error bars represent standard error of the mean from three independent experiments. (B) Electron micrograph of bacteriophage particles at 100,000× magnification, showing polyhedral capsid, long, flexible tail and tail fibers. Results shown are representative of three independent experiments producing and imaging bacteriophage particles.</p

Description of gene functions in order of appearance in each prophage in 17978.

<p>Description of gene functions in order of appearance in each prophage in 17978.</p