Biology subjects

Harms, K.

Publications and source records attributed to Harms, K..

9 recordsLinked to original sources

bioRxiv2024

Plasmids modulate microindel mutations in Acinetobacter baylyi ADP1

Plasmids can impact the evolution of their hosts, e.g. due to carriage of mutagenic genes, through cross-talk with host genes or as result of SOS induction during transfer. Here we demonstrate that plasmids can cause microindel mutations in the host genome. These mutations are driven by the production of single-stranded DNA molecules that invade replication forks at microhomologies and subsequently get integrated into the genome. Using the gammaproteobacterial model organism Acinetobacter baylyi, we show that carriage of broad host range plasmids from different incompatibility groups can cause microindel mutations directly or indirectly. The plasmid pQLICE belonging to the incompatibility group Q (IncQ) and replicating by a characteristic strand displacement mechanism can generate chromosomal microindel mutations directly with short stretches of DNA originating from pQLICE. In addition, the presence of plasmids can increase microindel mutation frequencies indirectly (i.e., with chromosomal ectopic DNA) as shown with the IncP plasmid vector pRK415 (theta replication mechanism), presumably through plasmid-chromosome interactions that lead to DNA damages. These results provide new mechanistic insights into the microindel mutation mechanism, suggesting that single-stranded DNA repair intermediates are the causing agents. By contrast, the IncN plasmid RN3 appears to suppress host microindel mutations. The suppression mechanism remains unknown. Other plasmids in this study confer ambiguous or no quantifiable mutagenic effects.

microbiology↗

bioRxiv2023

The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated in the course of DNA replication, presumably acting as Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR mutations are tightly controlled by genome maintenance functions including RecA. In this study, we investigate the roles of DprA, RecFOR and RecBCD, which are cytoplasmic functions that load DNA single-strands with RecA. All three functions suppress SPDIR mutations in wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA alone, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest an antimutational function for DprA and offer an explanation for the ubiquity of dprA in the genomes of non-transformable bacteria.

molecular biology↗

bioRxiv2023

Escherichia coli plasmidome maps the game of clones

Escherichia coli is the most widely studied microbe in history, but its extrachromosomal elements known as plasmids remain poorly delineated. Here we used long-read technology to high-resolution sequence the entire plasmidome and the corresponding host chromosomes from an unbiased longitudinal survey covering two decades and over 2,000 E. coli isolates. We find that some plasmids have persisted in lineages even for centuries, demonstrating strong plasmid-lineage associations. Our analysis provides a detailed map of recent vertical and horizontal evolutionary events involving plasmids with key antibiotic resistance, competition and virulence determinants. We present genomic evidence of both chromosomal and plasmid-driven success strategies that represent convergent phenotypic evolution in distant lineages, and use in vitro experiments to verify the importance of bacteriocin-producing plasmids for clone success. Our study has general implications for understanding plasmid biology and bacterial evolutionary strategies.

microbiology↗

bioRxiv2023

Testing for the fitness benefits of natural transformation during community-embedded evolution

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the "sex hypothesis." Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wildtype Acinetobacter baylyi and a transformation-deficient {Delta}comA mutant were evolved for five weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wildtype populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

microbiology↗

bioRxiv2021

Evolutionary instability of collateral susceptibility networks in ciprofloxacin resistant clinical Escherichia coli strains

Collateral sensitivity and resistance occur when resistance development towards one antimicrobial either potentiates or deteriorates the effect of others, respectively. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression is the main driver destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks.

microbiology↗

bioRxiv2021

Sugar beet cold-induced PMT5a and STP13 carriers are poised for taproot proton-driven plasma membrane sucrose and glucose import

Sugar beet (Beta vulgaris) is the major sugar-producing crop in Europe and Northern America, as the taproot stores sucrose at a concentration of around 20%. Genome sequence analysis together with biochemical and electrophysiological approaches led to the identification and characterization of the TST sucrose transporter driving vacuolar sugar accumulation in the taproot. However, the sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown. As with glucose, sucrose stimulation of taproot parenchyma cells caused inward proton fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. To decipher the nature of the corresponding proton-driven sugar transporters, we performed transcriptomic taproot profiling and identified the cold-induced PMT5a and STP13 transporters. When expressed in Xenopus laevis oocytes, BvPMT5a was characterized as a voltage- and H+-driven low-affinity glucose transporter, which does not transport sucrose. In contrast, BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose better than sucrose, and being more cold-tolerant than BvPMT5a. Modeling of the BvSTP13 structure with bound mono- and disaccharides suggests plasticity of the binding cleft to accommodate the different saccharides. The identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of sugar beet to provide a sustainable energy crop. Significance StatementIn vivo electrophysiological studies with sugar beet taproots provide clear evidence for proton-coupled glucose and sucrose uptake into taproot parenchyma cells. In search for the molecular entities, the taproot-expressed BvPMT5a and BvSTP13 carriers were studied in detail, because they mediate proton-driven import of glucose and sucrose and thus provide proper candidates for sugar beet plasma membrane sugar-proton cotransporters.

plant biology↗

bioRxiv2021

Cold-triggered induction of ROS- and raffinose metabolism in freezing-sensitive taproot tissue of sugar beet

Sugar beet (Beta vulgaris subsp. vulgaris) is the exclusive source of sugar in the form of sucrose in temperate climate zones. There, sugar beet is grown as an annual crop from spring to autumn because of the damaging effect of freezing temperatures to taproot tissue. Natural and breeded varieties display variance in the degree of tolerance to freezing temperatures and genotypes with elevated tolerance to freezing have been isolated. Here we compare initial responses to frost between genotypes with either low and high winter survival rates. The selected genotypes differed in the severity of frost injury. We combined transcriptomic and metabolite analyses of leaf- and taproot tissues from such genotypes to elucidate mechanisms of the early freezing response and to dissect genotype- and tissue-dependent responses. Freezing temperatures induced drastic downregulation of photosynthesis-related genes in leaves but upregulation of genes related to minor carbohydrate metabolism, particularly of genes involved in raffinose metabolism in both, leaf and taproot tissue. In agreement with this, it has been revealed that raffinose and the corresponding intermediates, inositol and galactinol, increased markedly in these tissues. We found that genotypes with improved tolerance to freezing, showed higher accumulation of raffinose in a defined interior region within the upper part of the taproot, the pith, representing the tissue most susceptible to freeze damages. This accumulation was accompanied by specific upregulation of raffinose synthesizing enzymes in taproots, suggesting a protective role for raffinose and its precursors for freezing damage in sugar beet.

plant biology↗

bioRxiv2020

Cryptic β-lactamase evolution is driven by low β-lactam concentrations

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary {beta}-lactamases is limited. Using the wide-spread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to develop clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-minimum inhibitory concentrations (sub-MIC) of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MIC of ceftazidime, they showed strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies towards ceftazidime. These changes are likely caused by enhanced flexibility of the {Omega}- and {beta}5-{beta}6 loops. In conclusion, low-level concentrations of {beta}-lactams can drive the evolution of {beta}-lactamases through cryptic phenotypes which may act as stepping-stones towards clinical resistance.

evolutionary biology↗

bioRxiv2020

Vernalization alters sugar beet (Beta vulgaris) sink and source identities and reverses phloem translocation from taproots to shoots

During vegetative growth, biennial sugar beets maintain a steep gradient between the shoot (source) and the sucrose-storing taproot (sink). To shift from vegetative to generative growth, they require a chilling phase, called vernalization. Here, we studied sugar beet sink-source dynamics upon cold temperature-induced vernalization and revealed a pre-flowering taproot sink to source reversal. This transition is induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in long distance transport system, the phloem. As a key process for this transition, vacuolar sucrose importers and exporters, BvTST2;1 and BvSUT4, are oppositely regulated, leading to re-mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that the now deciphered processes are a prerequisite for bolting. Our data may thus serve dissecting metabolic and developmental triggers for bolting, which are potential targets for genome editing or breeding approaches.

plant biology↗