Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

Background: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence

Mycobacterium tuberculosis DosR Regulon Gene Rv0079 Encodes a Putative, ‘Dormancy Associated Translation Inhibitor (DATIN)’

Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN

Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination

<p>Abstract</p> <p>Background</p> <p>Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle".</p> <p>Results</p> <p>Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, <it>Aceros waldeni </it>and <it>Penelopides panini</it>. The mt genomes are characterized by a tandemly duplicated region encompassing part of <it>cytochrome b</it>, 3 tRNAs, <it>NADH6</it>, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (<it>A. waldeni</it>) and 22,737 bp (<it>P. panini</it>), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation.</p> <p>Conclusions</p> <p>The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB is supposed to halt replication, it offers a potential mechanistic explanation for frequent recombination in mitochondrial genomes.</p

The SuperCam Instrument Suite on the Mars 2020 Rover: Science Objectives and Mast-Unit Description

On the NASA 2020 rover mission to Jezero crater, the remote determination of the texture, mineralogy and chemistry of rocks is essential to quickly and thoroughly characterize an area and to optimize the selection of samples for return to Earth. As part of the Perseverance payload, SuperCam is a suite of five techniques that provide critical and complementary observations via Laser-Induced Breakdown Spectroscopy (LIBS), Time-Resolved Raman and Luminescence (TRR/L), visible and near-infrared spectroscopy (VISIR), high-resolution color imaging (RMI), and acoustic recording (MIC). SuperCam operates at remote distances, primarily 2-7 m, while providing data at sub-mm to mm scales. We report on SuperCam's science objectives in the context of the Mars 2020 mission goals and ways the different techniques can address these questions. The instrument is made up of three separate subsystems: the Mast Unit is designed and built in France; the Body Unit is provided by the United States; the calibration target holder is contributed by Spain, and the targets themselves by the entire science team. This publication focuses on the design, development, and tests of the Mast Unit; companion papers describe the other units. The goal of this work is to provide an understanding of the technical choices made, the constraints that were imposed, and ultimately the validated performance of the flight model as it leaves Earth, and it will serve as the foundation for Mars operations and future processing of the data.In France was provided by the Centre National d'Etudes Spatiales (CNES). Human resources were provided in part by the Centre National de la Recherche Scientifique (CNRS) and universities. Funding was provided in the US by NASA's Mars Exploration Program. Some funding of data analyses at Los Alamos National Laboratory (LANL) was provided by laboratory-directed research and development funds

Atrial fibrillation genetic risk differentiates cardioembolic stroke from other stroke subtypes

AbstractObjectiveWe sought to assess whether genetic risk factors for atrial fibrillation can explain cardioembolic stroke risk.MethodsWe evaluated genetic correlations between a prior genetic study of AF and AF in the presence of cardioembolic stroke using genome-wide genotypes from the Stroke Genetics Network (N = 3,190 AF cases, 3,000 cardioembolic stroke cases, and 28,026 referents). We tested whether a previously-validated AF polygenic risk score (PRS) associated with cardioembolic and other stroke subtypes after accounting for AF clinical risk factors.ResultsWe observed strong correlation between previously reported genetic risk for AF, AF in the presence of stroke, and cardioembolic stroke (Pearson’s r=0.77 and 0.76, respectively, across SNPs with p &lt; 4.4 × 10−4 in the prior AF meta-analysis). An AF PRS, adjusted for clinical AF risk factors, was associated with cardioembolic stroke (odds ratio (OR) per standard deviation (sd) = 1.40, p = 1.45×10−48), explaining ∼20% of the heritable component of cardioembolic stroke risk. The AF PRS was also associated with stroke of undetermined cause (OR per sd = 1.07, p = 0.004), but no other primary stroke subtypes (all p &gt; 0.1).ConclusionsGenetic risk for AF is associated with cardioembolic stroke, independent of clinical risk factors. Studies are warranted to determine whether AF genetic risk can serve as a biomarker for strokes caused by AF.</jats:sec

Toxin Secretion and Tail Autotomy by Irritated Oxynoe panamensis (Opisthobranchiata; Sacoglossa)

The green sacoglossan gastropod Oxynoe panamensis occurs in mangrove swamps on the coasts of Baja Californi a, Mexico, apparently feeding exclusively on the green siphonaceous alga Caulerpa sertularioides . When irritated, it secretes an astringent, milky mucus, which contains a toxin lethal to fish. Continued molestation may induce autotomy of the tail