Non-thermal atmospheric pressure plasmas have an antiseptic activity beneficial in different medical applications. In a genome-wide screening, hydrogen peroxide and superoxide were identified as key species contributing to the antibacterial effects of plasma while [FeS] cluster proteins emerged as potential cellular targets. We investigated the impact of plasma treatment on [FeS] cluster homeostasis inEscherichia colitreated for 1 min with the effluent of a microscale atmospheric pressure plasma jet (μAPPJ). Mutants defective in [FeS] cluster synthesis and maintenance lacking the SufBC2D scaffold protein complex or desulfurase IscS were hypersensitive to plasma treatment. Monitoring the activity of [FeS] cluster proteins of the tricarboxylic acid cycle (aconitase, fumarase, succinate dehydrogenase) and malate dehydrogenase (no [FeS] clusters), we identified cysteine, iron, superoxide dismutase, and catalase as determinants of plasma sensitivity. Survival rates, enzyme activity, and restoration of enzyme activity after plasma treatment were superior in mutants with elevated cysteine levels and in the wildtype under iron replete conditions. Mutants with elevated hydrogen peroxide and superoxide detoxification capacity over-expressingsodAandkatEshowed full protection from plasma-induced enzyme inactivation and survival rates increased from 34% (controls) to 87%. Our study indicates that metabolic and genetic adaptation of bacteria may result in plasma tolerance and resistance, respectively.

Effects of atmospheric pressure plasmas on proteins are studied to assess the quality of plasma decontamination and to gain insights into plasma-triggered molecular events underlying observations made in plasma medicine on the cellular, organ, and systemic level. Atmospheric pressure plasma treatment has been reported to cause protein degradation. Degradation products, however, have not been characterized. Treating different model proteins in aqueous solution with a DBD plasma, we confirmed with different methods (Bradford assay, application of Lambert–Beer’s law on absorption measurements at 280 nm, ninhydrin assay, size exclusion chromatography, SDS-PAGE) that protein degradation takes place. Peptides of different sizes were detected by size exclusion chromatography. The ninhydrin assay indicated that peptide bonds are cleaved. In the presence of hydroxyl radical scavenger d -mannitol, the concentration of amino termini formed during the initial 10 min of plasma treatment was reduced by 96%, while at longer treatment times mannitol did no longer prevent the formation of amino termini, indicating that hydroxyl radicals play an important role in the initial cleaving of peptide bonds in the protein, but other mechanisms are at play in cleaving the peptide bonds in the resulting peptides. The generation of peptides has implications for plasma decontamination and plasma medicine. It is critical to verify that plasma decontamination processes do not result in protein fragments with undesired properties. In plasma medicine, plasma-generated protein fragments may act as molecular triggers in treated cells, tissues, or patients, e.g., regulating signaling cascades in a protease-like fashion.

A molecular-level understanding of the effects of atmospheric-pressure plasma on biological samples requires knowledge of the effects on proteins. Superoxide dismutases, which detoxify superoxide under oxidative stress conditions, play a key role in bacterial plasma resistance. Investigation of the impact of dielectric barrier discharge (DBD) treatment on purified superoxide dismutases SodA and SodB of Escherichia coli showed that DBD treatment caused a rapid protein degradation, with only 8% of protein remaining after 10 min. The affinity of SodA for the metal cofactor Mn2+ was reduced. Mass spectrometry, in conjunction with coupled-cluster calculations, revealed that modifications of amino acid residues in the active site can explain the decreased metal affinity and a distortion of the coordination geometry responsible for the activity loss.

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

Non-equilibrium atmospheric-pressure plasmas are an alternative means to sterilize and disinfect. Plasma-mediated protein aggregation has been identified as one of the mechanisms responsible for the antibacterial features of plasma. Heat shock protein 33 (Hsp33) is a chaperone with holdase function that is activated when oxidative stress and unfolding conditions coincide. In its active form, it binds unfolded proteins and prevents their aggregation. Here we analyse the influence of plasma on the structure and function of Hsp33 of Escherichia coli using a dielectric barrier discharge plasma. While most other proteins studied so far were rapidly inactivated by atmospheric-pressure plasma, exposure to plasma activated Hsp33. Both, oxidation of cysteine residues and partial unfolding of Hsp33 were observed after plasma treatment. Plasma-mediated activation of Hsp33 was reversible by reducing agents, indicating that cysteine residues critical for regulation of Hsp33 activity were not irreversibly oxidized. However, the reduction yielded a protein that did not regain its original fold. Nevertheless, a second round of plasma treatment resulted again in a fully active protein that was unfolded to an even higher degree. These conformational states were not previously observed after chemical activation with HOCl. Thus, although we could detect the formation of HOCl in the liquid phase during plasma treatment, we conclude that other species must be involved in plasma activation of Hsp33. E. coli cells over-expressing the Hsp33-encoding gene hslO from a plasmid showed increased survival rates when treated with plasma while an hslO deletion mutant was hypersensitive emphasizing the importance of protein aggregation as an inactivation mechanism of plasma.

Non-thermal atmospheric pressure plasmas are investigated as augmenting therapy to combat bacterial infections. The strong antibacterial effects of plasmas are attributed to the complex mixture of reactive species, (V)UV radiation and electric fields. The experience with antibiotics is that upon their introduction as medicines, resistance occurs in pathogens and spreads. To assess the possibility of bacterial resistance developing against plasma, we investigated intrinsic protective mechanisms that allow Escherichia coli to survive plasma stress. We performed a genome-wide screening of single-gene knockout mutants of E. coli and identified 87 mutants that are hypersensitive to the effluent of a microscale atmospheric pressure plasma jet. For selected genes ( cysB, mntH, rep and iscS) we showed in complementation studies that plasma resistance can be restored and increased above wild-type levels upon over-expression. To identify plasma-derived components that the 87 genes confer resistance against, mutants were tested for hypersensitivity against individual stressors (hydrogen peroxide, superoxide, hydroxyl radicals, ozone, HOCl, peroxynitrite, NO•, nitrite, nitrate, HNO3, acid stress, diamide, heat stress and detergents). k-means++ clustering revealed that most genes protect from hydrogen peroxide, superoxide and/or nitric oxide. In conclusion, individual bacterial genes confer resistance against plasma providing insights into the antibacterial mechanisms of plasma.

The bacterial phytochrome of Pseudomonas aeruginosa (PaBphP) is an in vitro-active red/far-red light sensor histidine kinase of a two-component regulatory system. Despite solid biochemical data, its function in this heterotrophic, opportunistic pathogen is still unknown. Previous studies established that the genes encoding the two necessary phytochrome components BphO, a chromophore-producing haem oxygenase, and BphP, the apo-phytochrome, are co-transcribed in a bicistronic operon. Transcription has been shown to be induced in the stationary phase and to be dependent on the alternative sigma factor RpoS. Here we show an additional regulation of bphP expression through the quorum-sensing (QS) regulator LasR. This regulation is also reflected in a combination of expression profile experiments and proteome analyses of wild-type and phytochrome-deficient strains. While PaBphP has a pleiotropic effect on global gene expression, 66 % of the downregulated genes in the phytochrome mutant display a link to the Las QS system. Most of these genes seem to be indirectly regulated by LasR through BphP and the unknown response regulator BphR. A model of phytochrome function within the Las QS network is presented.

Rhodobacter capsulatus can efficiently grow with taurine as the sole sulfur source. The products of the tpa-tauR-xsc gene region are essential for this activity. TauR, a MocR-like member of the GntR superfamily of transcriptional regulators, activates tpa transcription, as shown by analysis of wild-type and tauR mutant strains carrying a tpa-lacZ reporter fusion. Activation of the tpa promoter requires taurine but is not inhibited by sulfate, which is the preferred sulfur source. TauR directly binds to the tpa promoter, as demonstrated by DNA mobility shift assays. As expected for a transcriptional activator, the TauR binding site is located upstream of the transcription start site, which has been determined by primer extension. Site-directed promoter mutations reveal that TauR binds to direct repeats, an unusual property that has to date been shown for only one other member of the MocR subfamily, namely, GabR from Bacillus subtilis. In contrast, all other members of the GntR family analyzed so far bind to inverted repeats.

Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.

ABSTRACT Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.