The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag 1؉ , AsO 2 1؊ , Cd 2؉ , SbO 2 1؊ , and Ni 2؉ , but most of the strains were less sensitive to Pb 2؉ (6 to 8 mM), CrO 4 2؊ (1.0 to 1.75 mM), AsO 4 3؊ (>50 mM), and SeO 2 2؊ (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu 2؉ , four strains were resistant to elevated levels of Cu 2؉ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO 2 2؊ resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb 2؉ resistance and Cu 2؉ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.Frankia, a member of the order Actinomycetales, forms a symbiotic nitrogen-fixing association with a variety of woody dicotyledonous plants (for reviews see references 3 and 28). The members of this bacterial genus are known to be associated with over 200 species of plants representing eight plant families. These bacteria fix N 2 from the atmosphere and produce a significant amount of the fixed nitrogen on the planet. Actinorhizal plants are ecologically important as pioneer community plants and have economic value in land reclamation, reforestation, and soil stabilization.The lack of a well-established genetic system is a major obstacle in the elucidation of the mechanism of actinorhizal nitrogen fixation and plant-microbe interactions (for reviews see references 22, 23, and 25). There is a paucity of genetic markers for Frankia. Some of the most useful genetic markers include resistance to antibiotics, resistance to antimetabolites, and resistance to heavy metals. These directly selectable traits provide a mechanism for positive selection in genetic studies and are also useful in the development of cloning vectors. For example, metal resistance has been useful in the development of cloning vectors for Rhodococcus, another member of the Actinomycetales (8). Previously, we developed a growth inhibition assay that was used to screen several Frankia strains for resistance to antibiotics (27). Although several important antibiotic resistance markers were described in that study, we were interested in identifying other selectable genetic markers. Since actinorhizal plants have been used for land reclamation in strip-mined areas, we reasoned that it is possible that the bacteria are resistant to heavy metals. The purpose of this study was to extend the use of our growth inhibition assay to identify new selectable markers, resistance to heavy metals. Frankia strains ACN1AG (14), CcI3 (29), Cc1.17 (18), CN3 (20), CpI1 succinate variant (CpI1-S) (5, 26), CpI1 propionate variant (CpI1-P) (5, 26), DC12 (1), EI5c (17), EAN1pec (16), EuI1c (2), EUN1f (14), and QA3 (12) were grown and maintained in basal growth medium with NH 4 Cl as the nitrogen source, as described previously (26, 27). For the heavy metal s...

The XENON1T experiment uses a time projection chamber (TPC) with liquid Xenon to search for Weakly Interacting Massive Particles (WIMPs), a proposed Dark Matter particle, via direct detection. As this experiment relies on capturing rare events, the focus is on achieving a high recall of WIMP events. Hence the ability to distinguish between WIMP and the background is extremely important. To accomplish this, we suggest using Convolutional Neural Networks (CNNs); a Machine Learning procedure mainly used in image recognition tasks. To explore this technique we use XENON collaboration open-source software to simulate the TPC graphical output of Dark Matter signals and main backgrounds. A CNN turns out to be a suitable tool for this purpose, as it can identify features in the images that differentiate the two types of events without the need to manipulate or remove data in order to focus on a particular region of the detector. We find that the CNN can distinguish between the dominant background events (ER) and 500 GeV WIMP events with an accuracy of 87.0% and a recall of 88.2%.

A major hurdle in the development of a genetic system for Frankia is the lack of genetic markers. To identify potential genetic markers, 12 strains of Frankia were screened for resistance to antibiotics by the use of a growth inhibition assay. All of the strains demonstrated sensitivity to tested antibiotics. Several strains had distinctive patterns of antibiotic resistance that are potentially useful as genetic markers. Novobiocin was the antibiotic to which the most strains were resistant.Key words: genetics, genetic markers, Frankia, actinorhizal, nitrogen fixation, vesicles.

No abstract

Previous genetic analysis of Haemophilus influenzae revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene acrR and in the fmt gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 g/ml versus 4 g/ml against the parent strain NB65044) that lacks mutations in the acrR or fmt structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in def transcript titer. No mutations were found in the region upstream of def that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing def had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the def gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.In recent years, there has been a growing appreciation of the need for new antibiotics targeting novel bacterial functions, to combat the spread of resistance to currently used antibiotics. The result of one effort to develop such a compound is LBM415, a potent low-molecular-weight inhibitor of bacterial peptide deformylase that shows promising antibacterial activities against many drug-resistant bacteria (9,12,34).Two main mechanisms mediating resistance to peptide deformylase inhibitors in bacteria have been previously described. The first is amino acid substitutions within the target protein (Def) (17), and the second is "FMT bypass" (4, 18), which results from mutational loss of methionyl tRNA formyltransferase (Fmt). Loss of the Fmt function reduces or eliminates formylation of the initiating methionyl-tRNA fMet . In many bacteria, the initiation of protein synthesis still occurs in the absence of the Fmt function (1,18,20,33), but the deformylation step mediated by peptide deformylase is then unnecessary (i.e., the formylation-deformylation cycle is bypassed), leading to insusceptibility to peptide deformylase inhibitors (4, 18). Recently, amino acid substitutions in the FolD component of the folate biosynthesis pathway have been shown to confer resistance to the peptide deformylase inhibitor actinonin in Salmonella enterica, presumably as a result of reduced formylation of methionyl-tRNA fMet due to interference with synthesis of the formyl group (22).Given the potency of LBM415 against the respiratory pathogen Streptococcus pneumonia...