Horizontal gene transfer via plasmid conjugation enables antimicrobial resistance (AMR) to spread among bacteria and is a major health concern. The range of potential transfer hosts of a particular conjugative plasmid is characterised by its mobility (MOB) group, which is currently determined based on the amino acid sequence of the plasmid-encoded relaxase. To facilitate prediction of plasmid MOB groups, we have developed a bioinformatic procedure based on analysis of the origin-of-transfer (oriT), a merely 230 bp long non-coding plasmid DNA region that is the enzymatic substrate for the relaxase. By computationally interpreting conformational and physicochemical properties of the oriT region, which facilitate relaxase-oriT recognition and initiation of nicking, MOB groups can be resolved with over 99% accuracy. We have shown that oriT structural properties are highly conserved and can be used to discriminate among MOB groups more efficiently than the oriT nucleotide sequence. The procedure for prediction of MOB groups and potential transfer range of plasmids was implemented using published data and is available at http://dnatools.eu/MOB/plasmid.html.
COBISS.SI-ID: 31226663
In mammalian organisms liquid tumors such as acute myeloid leukemia (AML) are related to spontaneous chromosomal translocations ensuing in gene fusions. We previously developed a system named bridge-induced translocation (BIT) that allows linking together two different chromosomes exploiting the strong endogenous homologous recombination system of the yeast Saccharomyces cerevisiae. The BIT system generates a heterogeneous population of cells with different aneuploidies and severe aberrant phenotypes reminiscent of a cancerogenic transformation. In this work, thanks to a complex pop-out methodology of the marker used for the selection of translocants, we succeeded by BIT technology to precisely reproduce in yeast the peculiar chromosome translocation that has been associated with AML, characterized by the fusion between the human genes NUP98 and TOP2B. To shed light on the origin of the DNA fragility within NUP98, an extensive analysis of the curvature, bending, thermostability, and B-Z transition aptitude of the breakpoint region of NUP98 and of its yeast ortholog NUP145 has been performed. On this basis, a DNA cassette carrying homologous tails to the two genes was amplified by PCR and allowed the targeted fusion between NUP145 and TOP2, leading to reproduce the chimeric transcript in a diploid strain of S. cerevisiae. The resulting translocated yeast obtained through BIT appears characterized by abnormal spherical bodies of nearly 500 nm of diameter, absence of external membrane and defined cytoplasmic localization. Since Nup98 is a well-known regulator of the post-transcriptional modification of P53 target genes, and P53 mutations are occasionally reported in AML, this translocant yeast strain can be used as a model to test the constitutive expression of human P53. Although the abnormal phenotype of the translocant yeast was never rescued by its expression, an exogenous P53 was recognized to confer increased vitality to the translocants, in spite of its usual and well-documented toxicity to wild-type yeast strains. These results obtained in yeast could provide new grounds for the interpretation of past observations made in leukemic patients indicating a possible involvement of P53 in cell transformation toward AML.
COBISS.SI-ID: 1540168644
Whole-cell biosensors are still the method of choice when measuring bioavailable mercury, though their implementation in environmental monitoring is limited by low sensitivity, lack of portability and use of environmentally irrelevant bacteria. To address these issues, we have engineered a new luminescence-based whole-cell mercury biosensor, as part of a standalone fully automated portable device. Our method allows the incorporation of any environmentally relevant bacterial cell, which has been modified to translate the concentration of biologically available mercury into a dose-dependent luminescent signal. The use of environmentally relevant bacteria, Pseudomonas putida for fresh waters and Aliivibrio fischeri for salt waters, demonstrated that environmental samples will not exhibit toxic effects, when appropriate microorganisms are implemented. Additionally, by assuring efficient aeration of the medium and thus sufficient oxygenation of sensor cells during generation of the luminescence signal, we obtained a clear dose-dependent response and observed an increased sensitivity of the method up to 100 times (the limit of detection [LOD] was determined to be as low as ∼10 ng L−1). Finally, using our automated device, we demonstrated that in the environment the biologically available fraction of mercury can (1) represent an important part of the total mercury content (40–70%) and (2) it can correspond to the changes of total mercury content, which results in higher bioavailability of mercury closer to the source of mercury contamination.
COBISS.SI-ID: 30060071
Scientific poster presented at the conference ASM Microbe. Increased incidence of antimicrobial resistance (AMR) in bacteria has raised global awareness and will continue to do so, due to the decreased rate of introduction of new antibiotics. One approach to treat AMR infections is to use the most appropriate antibiotic combinations that inhibit the transfer of AMR genes, since inappropriate treatments increase AMR incidence. In human pathogens, AMR genes spread frequently by transfer of mobile conjugative plasmids from the large environmental AMR pool. Each plasmid can be hosted in a particular repertoire of appropriate bacterial hosts, characterized by the plasmid’s MOB group. Currently to determine the MOB group, a mobile plasmid must be sequenced, the sequence of its relaxase must be determined and conjugation experiments must performed. We have presently considered the following. The most important components for plasmid transfer are oriT regions and conjugative relaxases. The oriT site is a substrate for the relaxase, which nicks the DNA and initiates transfer. Since each relaxase is specific to one type of oriT, DNA in each oriT region must have specific physicochemical properties that most efficiently attract and enable activity of the particular relaxase. We thus developed a bioinformatic framework, incorporating prediction of DNA physicochemical properties of oriT regions according to current models and machine learning algorithms. Based on our computational analysis of physicochemical properties of oriT regions, we were able to successfully sort 64 mobile plasmids into their corresponding MOB groups (classification accuracy of 99%). By using this method we only needed to obtain the identity of a DNA sequence up to 240 bp long, with which the most probable hosts can be determined. In the future, this approach will help clinicians determine the right antibiotic treatment based on the present mobile elements and their host ranges, as well as develop new approaches for inhibition of horizontal AMR gene transfer within polymicrobial infections. Objavljeno v
COBISS.SI-ID: 1539178948
Scientific poster presented at the conference ASM Microbe. Metagenomic analyses are crucial to study the diversity, activity and dynamics of uncultivable microbes. An integral part in the analyses is the polymerase chain reaction (PCR), which can also present a bottleneck, such as when amplifying parts of 16S rRNA genes. Often in agarose electrophoresis gels we see smeared DNA bands of wrong size, which are considered to be non-specific PCR errors and are eliminated. However, if these amplicons are not errors, then a part of the sample’s true diversity is lost. Therefore, we explored if band smearing after PCR is in fact caused by imperfectly paired strands of the amplified DNA. Using synthetic oligonucleotides that mimic 16S rRNA in PCR, we determined that the amount of smear in agarose gels was proportional to DNA sequence heterogeneity of the 16S rRNA variable regions. Since in denaturing alkaline gels, lack of smear showed that amplified DNA had a uniform size, we suspected that two separate groups of structures had formed - correctly and imperfectly paired DNA strands. This was confirmed, by isolating and sequencing the two groups of structures using a newly developed electroelution procedure and characterizing the pairing of the sequenced DNA strands using a bioinformatics approach. When amplifying highly heterogeneous target DNA, such as 16S rRNA, imperfect pairing of the amplified DNA can lead to band smearing in agarose gels, which is not an indicator of low specificity of the PCR. Since the smear in agarose gels is only a structural part of the correctly amplified DNA, it carries important information about the richness and diversity of the analysed microbial communities. Incorrect handling of 16S rRNA samples, e.g. eliminating smearing by increasing PCR amplification stringency or excision of amplified DNA, thus leads to an underestimation of the richness and diversity of microbial species.
COBISS.SI-ID: 1539178692