The use of disc diffusion susceptibility tests to determine the antibacterial activity of engineered nanoparticles (ENPs) is questionable because their low diffusivity practically prevents them from penetrating through the culture media. In this study, we investigate the ability of such a test, namely the Kirby-Bauer disc diffusion test, to determine the antimicrobial activity of Au and Ag ENPs having diameters from 10 to 40 nm on Escherichia coli cultures. As anticipated, the tests did not show any antibacterial effects of Au nanoparticles (NPs) as a result of their negligible diffusivity through the culture media. Ag NPs on the other hand exhibited a strong antimicrobial activity that was independent of their size. Considering that Ag, in contrast to Au, dissolves upon oxidation and dilution in aqueous solutions, the apparent antibacterial behavior of Ag NPs is attributed to the ions they release. The Kirby-Bauer method, and other similar tests, can therefore be employed to probe the antimicrobial activity of ENPs related to their ability to release ions rather than to their unique size-dependent properties. [Figure not available: see fulltext.].@en

Studies of the spatiotemporal protein dynamics within live bacterial cells impose a strong demand for multi-color imaging. Despite the increasingly large collection of fluorescent protein (FP) variants engineered to date, only a few of these were successfully applied in bacteria. Here, we explore the performance of recently engineered variants with the blue (TagBFP), orange (TagRFP-T, mKO2), and far-red (mKate2) spectral colors by tagging HU, LacI, MinD, and FtsZ for visualizing the nucleoid and the cell division process. We find that, these FPs outperformed previous versions in terms of brightness and photostability at their respective spectral range, both when expressed as cytosolic label and when fused to native proteins. As this indicates that their folding is sufficiently fast, these proteins thus successfully expand the applicable spectra for multi-color imaging in bacteria. A near-infrared protein (eqFP670) is found to be the most red-shifted protein applicable to bacteria so far, with brightness and photostability that are advantageous for cell-body imaging, such as in microfluidic devices. Despite the multiple advantages, we also report the alarming observation that TagBFP directly interacts with TagRFP-T, causing interference of localization patterns between their fusion proteins. Our application of diverse FPs for endogenous tagging provides guidelines for future engineering of fluorescent fusions in bacteria, specifically: (1) The performance of newly developed FPs should be quantified in vivo for their introduction into bacteria; (2) spectral crosstalk and inter-variant interactions between FPs should be carefully examined for multi-color imaging; and (3) successful genomic fusion to the 5'-end of a gene strongly depends on the translational read-through of the inserted coding sequence.@en

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.@en