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Auto Tag: DNA damage

September 22, 2019  |  

The plasmid-encoded transcription factor ArdK contributes to the repression of the IMP-6 metallo-ß-lactamase gene blaIMP-6, leading to a carbapenem-susceptible phenotype in the blaIMP-6-positive Escherichia coli strain A56-1S.

Carbapenemase-producing Enterobacteriaceae (CPE) are a global concern because these bacteria are resistant to almost all ß-lactams. Horizontal interspecies gene transfer via plasmid conjugation has increased the global dissemination of CPE. Recently, an Enterobacteriaceae strain positive for carbapenemase gene but showing a carbapenem-susceptible phenotype was identified, suggesting that these susceptible strains may be challenging to detect solely via antimicrobial susceptibility tests without molecular analysis. Here, we isolated a blaIMP-6 carbapenemase-gene positive but imipenem- and meropenem-susceptible Escherichia coli (ISMS-E) strain A56-1S (imipenem and meropenem minimum inhibitory concentration, = 0.125 mg/L), from a human urine specimen in Japan. A56-1S was carbapenemase negative by the Carba NP test, suggesting that IMP-6 production was low or undetectable. Thus, to characterize the mechanism of this phenotype, a meropenem-resistant E. coli A56-1R strain was obtained using meropenem-selection. A56-1R was positive for carbapenemase production by the Carba NP test, and blaIMP-6 transcription in A56-1R was 53-fold higher than in A56-1S, indicating that blaIMP-6 in A56-1S is negatively regulated at the transcriptional level. Comparative genomic analysis between the two strains revealed that the alleviation of restriction of DNA (ardK) gene encoding a putative transcription factor is disrupted by the IS26 insertion in A56-1R. A cotransformation assay of ardK and the regulatory element upstream of blaIMP-6 showed repression of blaIMP-6 transcription, indicating that ArdK negatively modulates blaIMP-6 transcription. ArdK binding and affinity assays demonstrated that ArdK directly binds to the regulatory element upstream of blaIMP-6 with dissociation constant values comparable to those of general transcription factors. The IMP-6 carbapenemase showed low hydrolytic activity against imipenem, resulting in an imipenem-susceptible and meropenem-resistant (ISMR) phenotype (previously reported as a stealth phenotype). However, the low expression of IMP-6 in the A56-1S strain could be a typical characteristic of ISMS-E due to gene repression, indicating that conventional antimicrobial susceptibility tests might be unable to detect such strains even when using both imipenem and meropenem. Bacteria that exhibit the ISMS phenotype could play a potential role as undetectable reservoirs and might facilitate gene transfer to other organisms while avoiding detection.

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September 22, 2019  |  

Approaches for surveying cosmic radiation damage in large populations of Arabidopsis thaliana seeds-Antarctic balloons and particle beams.

The Cosmic Ray Exposure Sequencing Science (CRESS) payload system is a proof of concept experiment to assess the genomic impact of space radiation on seeds. CRESS was designed as a secondary payload for the December 2016 high-altitude, high-latitude, and long-duration balloon flight carrying the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) experimental hardware. Investigation of the biological effects of Galactic Cosmic Radiation (GCR), particularly those of ions with High-Z and Energy (HZE), is of interest due to the genomic damage this type of radiation inflicts. The biological effects of upper-stratospheric mixed radiation above Antarctica (ANT) were sampled using Arabidopsis thaliana seeds and were compared to those resulting from a controlled simulation of GCR at Brookhaven National Laboratory (BNL) and to laboratory control seed. The payload developed for Antarctica exposure was broadly designed to 1U CubeSat specifications (10cmx10cmx10cm, =1.33kg), maintained 1 atm internal pressure, and carried an internal cargo of four seed trays (about 580,000 seeds) and twelve CR-39 Solid-State Nuclear Track Detectors (SSNTDs). The irradiated seeds were recovered, sterilized and grown on Petri plates for phenotypic screening. BNL and ANT M0 seeds showed significantly reduced germination rates and elevated somatic mutation rates when compared to non-irradiated controls, with the BNL mutation rate also being significantly higher than that of ANT. Genomic DNA from mutants of interest was evaluated with whole-genome sequencing using PacBio SMRT technology. Sequence data revealed the presence of an array of genome structural variants in the genomes of M0 and M1 mutant plants.

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September 22, 2019  |  

Genomic and genetic insights into a cosmopolitan fungus, Paecilomyces variotii (Eurotiales).

Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, Paecilomyces variotii, was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of P. variotii, including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on P. variotii on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.

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September 22, 2019  |  

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

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September 22, 2019  |  

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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September 21, 2019  |  

Direct detection of DNA methylation during single-molecule, real-time sequencing.

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule, real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenine, 5-methylcytosine and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We used these kinetic signatures to identify adenine methylation in genomic samples and found that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns in even highly repetitive genomic regions.

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