LSPR-Based Biosensing Enables the Detection of Antimicrobial Resistance Genes.

The development of rapid, simple, and accurate bioassays for the detection of nucleic acids has received increasing demand in recent years. Here, localized surface plasmon resonance (LSPR) spectroscopy for the detection of an antimicrobial resistance gene, sulfhydryl variable ß-lactamase (blaSHV), which confers resistance against a broad spectrum of ß-lactam antibiotics is used. By performing limit of detection experiments, a 23 nucleotide (nt) long deoxyribonucleic acid (DNA) sequence down to 25 nm was detected, whereby the signal intensity is inversely correlated with sequence length (23, 43, 63, and 100 nt). In addition to endpoint measurements of hybridization events, the setup also allowed to monitor the hybridization events in real-time, and consequently enabled to extract kinetic parameters of the studied binding reaction. Performing LSPR measurements using single nucleotide polymorphism (SNP) variants of blaSHV revealed that these sequences can be distinguished from the fully complementary sequence. The possibility to distinguish such sequences is of utmost importance in clinical environments, as it allows to identify mutations essential for enzyme function and thus, is crucial for the correct treatment with antibiotics. Taken together, this system provides a robust, label-free, and cost-efficient analytical tool for the detection of nucleic acids and will enable the surveillance of antimicrobial resistance determinants.

Selective advantages favour high genomic AT-contents in intracellular elements.

Extrachromosomal genetic elements such as bacterial endosymbionts and plasmids generally exhibit AT-contents that are increased relative to their hosts' DNA. The AT-bias of endosymbiotic genomes is commonly explained by neutral evolutionary processes such as a mutational bias towards increased A+T. Here we show experimentally that an increased AT-content of host-dependent elements can be selectively favoured. Manipulating the nucleotide composition of bacterial cells by introducing A+T-rich or G+C-rich plasmids, we demonstrate that cells containing GC-rich plasmids are less fit than cells containing AT-rich plasmids. Moreover, the cost of GC-rich elements could be compensated by providing precursors of G+C, but not of A+T, thus linking the observed fitness effects to the cytoplasmic availability of nucleotides. Accordingly, introducing AT-rich and GC-rich plasmids into other bacterial species with different genomic GC-contents revealed that the costs of G+C-rich plasmids decreased with an increasing GC-content of their host's genomic DNA. Taken together, our work identifies selection as a strong evolutionary force that drives the genomes of intracellular genetic elements toward higher A+T contents.

Convergent Evolution in Intracellular Elements: Plasmids as Model Endosymbionts.

Endosymbionts are organisms that live inside the cells of other species. This lifestyle is ubiquitous across the tree of life and is featured by unicellular eukaryotes, prokaryotes, and by extrachromosomal genetic elements such as plasmids. Given that all of these elements dwell in the cytoplasm of their host cell, they should be subject to similar selection pressures. Here we show that strikingly similar features have evolved in both bacterial endosymbionts and plasmids. Since host and endosymbiont are often metabolically tightly intertwined, they are difficult to disentangle experimentally. We propose that using plasmids as tractable model systems can help to solve this problem, thus allowing fundamental questions to be experimentally addressed about the ecology and evolution of endosymbiotic interactions.

Impaired competence in flagellar mutants of Bacillus subtilis is connected to the regulatory network governed by DegU.

The competent state is a developmentally distinct phase, in which bacteria are able to take up and integrate exogenous DNA into their genome. Bacillus subtilis is one of the naturally competent bacterial species and the domesticated laboratory strain 168 is easily transformable. In this study, we report a reduced transformation frequency of B. subtilis mutants lacking functional and structural flagellar components. This includes hag, the gene encoding the flagellin protein forming the filament of the flagellum. We confirm that the observed decrease of the transformation frequency is due to reduced expression of competence genes, particularly of the main competence regulator gene comK. The impaired competence is due to an increase in the phosphorylated form of the response regulator DegU, which is involved in regulation of both flagellar motility and competence. Altogether, our study identified a close link between motility and natural competence in B. subtilis suggesting that hindrance in motility has great impact on differentiation of this bacterium not restricted only to the transition towards sessile growth stage.