The AGXX (R) Antimicrobial Coating Causes a Thiol-Specific Oxidative Stress Response and Protein S-bacillithiolation in Staphylococcus aureus

Van Loi V, Busche T, Preuss T, Kalinowski J, Bernhardt J, Antelmann H. The AGXX (R) Antimicrobial Coating Causes a Thiol-Specific Oxidative Stress Response and Protein S-bacillithiolation in Staphylococcus aureus. FRONTIERS IN MICROBIOLOGY. 2018;9: 3037.Multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) pose an increasing health burden and demand alternative antimicrobials to treat bacterial infections. The surface coating AGXX (R) is a novel broad-spectrum antimicrobial composed of two transition metals, silver and ruthenium that can be electroplated on various surfaces, such as medical devices and implants. AGXX (R) has been shown to kill nosocomial and waterborne pathogens by production of reactive oxygen species (ROS), but the effect of AGXX (R) on the bacterial redox balance has not been demonstrated. Since treatment options for MRSA infections are limited, ROS-producing agents are attractive alternatives to combat multi-resistant strains. In this work, we used RNA-seq transcriptomics, redox biosensor measurements and phenotype analyses to study the mode of action of AGXX (R) microparticles in S. aureus USA300. Using growth and survival assays, the growth-inhibitory amount of AGXX (R) microparticles was determined as 5 mu g/ml. In the RNA-seq transcriptome, AGXX (R) caused a strong thiol-specific oxidative stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, and HrcA regulons. The derepression of the Fur, Zur, and CsoR regulons indicates that AGXX (R) also interferes with the metal ion homeostasis inducing Fe-2(+)- and Zn-2(+)-starvation responses as well as export systems for toxic Ag+ ions. The induction of the SigB and GraRS regulons reveals also cell wall and general stress responses. AGXX (R). stress was further shown to cause protein S-bacillithiolation, protein aggregation and an oxidative shift in the bacillithiol (BSH) redox potential. In phenotype assays, BSH and the HypR-controlled disulfide reductase MerA were required for protection against ROS produced under AGXX (R) stress in S. aureus. Altogether, our study revealed a strong thiol-reactive mode of action of AGXX (R) in S. aureus USA300 resulting in an increased BSH redox potential and protein S-bacillithiolation

Analyse von Regulationsnetzwerken der Extracytoplasmic Function (ECF)-Sigmafaktoren in Corynebacterium glutamicum

Busche T. Analyse von Regulationsnetzwerken der Extracytoplasmic Function (ECF)-Sigmafaktoren in Corynebacterium glutamicum. Bielefeld: Universitätsbibliothek Bielefeld; 2013.Die Anpassung an sich ändernde Umwelteinflüsse ist eine wesentliche Voraussetzung für das Leben allgemein, und im speziellen für das Überleben von Bakterien. Dies geschieht unter anderem über die Regulation der Genexpression. Der erste Schritt der Genexpression, die Transkiptionsinitiation, ist der am häufigsten und am stärksten regulierte Schritt in Bakterien. Hierfür verwenden Bakterien unter anderem verschieden Sigmafaktoren. Sigmafaktoren sind Untereinheiten der bakteriellen RNA-Polymerase und sorgen zusammen mit dem RNA-Polymerase Core-Enzym als Holo-Enzyme für die Erkennung spezifischer Promotorsequenzen. Sie kontrollieren oft große Netzwerke von Genen. Gerade die ECFSigmafaktoren sind in Bakterien weit verbreitet und erfüllen wichtige Funktionen in der Stressantwort. Der Gram-positive Modellorganismus Corynebacterium glutamicum besitzt sieben Sigmafaktoren, darunter den Housekeeping Sigmafaktor SigA, den alternativen SigAähnlichen Sigmafaktor SigB und fünf ECF (Extracytoplasmic Function)-Sigmafaktoren (SigC, SigD, SigE, SigH und SigM). Im Rahmen dieser Arbeit wurden die ECF-Sigmafaktoren SigH und SigE und deren Anti-Sigmafaktoren RshA bzw. CseE näher untersucht. Für SigH war bekannt, dass es in C. glutamicum und in anderen Actinomyzeten die Antwort auf Hitze- und oxidativen Stress reguliert. Für SigE war eine Beteiligung an der Antwort auf Oberflächen-Stress vermutet worden. Durch die in vivo Transkriptomanalyse von Sigmafaktor- und Antsigmafaktorgen-Mutanten mittels Microrarray-Analyse und RNA-Sequenzierung in Kombination mit der Analyse spezifischer Sigmafaktor-RNAP-Komplexe in vitro durch ROSE (Run-Off Transcription/RNASequencing)konnten die ECF-Sigmafaktor-Netzwerke von SigH und SigE und ihre Interaktionen, die Funktion der Anti-Sigmafaktoren und die transkriptionelle Organisation der beiden Sigmafaktor/Anti-Sigmafaktor Operons erstmalig und detailliert beschrieben werden. Die ROSE-Methodik stellt dabei eine im Rahmen dieser Arbeit erfolgte Neuentwicklung zur genomweiten in-vitro Transkription mit Einzelnukleotidauflösung dar. Das SigH-Regulon war bereits durch frühere Arbeiten untersucht worden, konnte aber um Gene der SOS-Antwort (uvrA, uvrC und uvrD3), der Proteinqualitätskontrolle (pup und diverse Proteasen) und einer Reihe von Genen, die Membranproteine kodieren, erweitert werden. Daneben wurde das Spektrum der Proteine des SigH-Regulons erweitert, die der Aufrechterhaltung der Redox-Homöostase dienen. Es konnte weiter gezeigt werden, dass RshA in C. glutamicum die Aufgabe als Anti-Sigmafaktor besitzt und das das rshA-Gen SigHabhängig transkribiert wird. Durch diese Arbeit konnte das SigE-Regulon erstmalig umfassend beschrieben werden. Bei den Genfunktionen des SigE-Netzwerks handelt es sich hauptsächlich um Membranproteine,was im Einklang mit dem Einfluß des SigE-Netzwerkes auf die Antwort von Membran-Stress steht. Daneben konnte interessanterweise das Gen für den globalen Regulator des Stickstoffmetabolismus AmtR als Mitglied des SigE-Netzwerkes identifiziert werden. Es konnte weiter gezeigt werden, dass der Großteil des SigE-Regulons bis auf AmtR ebenfalls Bestandteil des SigH-Regulon ist. Das sigE-Gen liegt, wie sigH-rshA, in einem Operon mit dem Gen für den Anti-Sigmafaktor, rseA. Auch hier wird das Anti-Sigmafaktorgen in Abhängigkeit des entsprechenden Sigmafaktors transkribiert. Der rseA-Promotor wird allerdings zusätzlich von SigH erkannt. Es konnte, wie für Mycobacterium tuberculosis beschrieben, gezeigt werden, dass die ECFSigmafaktoren SigH und SigE in C. glutamicum ähnliche Promotoren erkennen. Dabei ist der 3`-Bereich des -35 Promotormotivs für die Unterscheidung von SigH und zusätzlich SigEabhängigen Promotoren verantwortlich. Somit konnte klar gestellt werden, dass die Regulationsnetzwerke von SigH und SigE auf verschiedenen Ebenen hierarchisch verflochten sind. Während das SigH-Netzwerk vermutlich der Bekämpfung der unter vielen Stressbedingungen auftretenden Auslenkung der Redox- Homöostase dient, integriert das partiell untergeordnete SigE-Netzwerk vermutlich die spezielle Reaktion auf Oberflächenstress und ein Herunterfahren des Stickstoffmetabolismus unter den spezifischen Induktionsbedingungen für SigE. Da Oberflächenstress sicher auch ohne masive Störung der Redox-Homöostase vorkommen kann, erlaubt die Verknüpfung der Netzwerke so eine flexiblere Antwort. Zusammenfassend konnten die erzielten Ergebnisse dazu genutzt werden das Sigmafaktor-Netzwerk in C. glutamicum näher zu charakterisieren. Es zeigen sich bei der Verknüpfung viele Parallelen zu orthologen Sigmafaktoren in den verwandten Actinobakterien M. tuberculosis und Streptomyces coelicolor. Die entwickelten komplementären in vivo und in vitro-Methoden haben ein großes Potential für zukünftige Studien von bakteriellen Regulationsnetzwerken

Optimal Detection for Diffusion-Based Molecular Timing Channels

This work studies optimal detection for communication over diffusion-based molecular timing (DBMT) channels. The transmitter simultaneously releases multiple information particles, where the information is encoded in the time of release. The receiver decodes the transmitted information based on the random time of arrival of the information particles, which is modeled as an additive noise channel. For a DBMT channel without flow, this noise follows the L\'evy distribution. Under this channel model, the maximum-likelihood (ML) detector is derived and shown to have high computational complexity. It is also shown that under ML detection, releasing multiple particles improves performance, while for any additive channel with $\alpha$-stable noise where $\alpha<1$ (such as the DBMT channel), under linear processing at the receiver, releasing multiple particles degrades performance relative to releasing a single particle. Hence, a new low-complexity detector, which is based on the first arrival (FA) among all the transmitted particles, is proposed. It is shown that for a small number of released particles, the performance of the FA detector is very close to that of the ML detector. On the other hand, error exponent analysis shows that the performance of the two detectors differ when the number of released particles is large.Comment: 16 pages, 9 figures. Submitted for publicatio

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

Ruwe M, Persicke M, Busche T, Müller B, Kalinowski J. Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum. Frontiers in Microbiology. 2019;10: 2769.The alarmone species ppGpp and pppGpp are elementary components of bacterial physiology as they both coordinate the bacterial stress response and serve as fine-tuners of general metabolism during conditions of balanced growth. Since the regulation of (p)ppGpp metabolism and the effects of (p)ppGpp on cellular processes are highly complex and show massive differences between bacterial species, the underlying molecular mechanisms have so far only been insufficiently investigated for numerous microorganisms. In this study, (p)ppGpp physiology in the actinobacterial model organism Corynebacterium glutamicum was analyzed by phenotypic characterization and RNAseq-based transcriptome analysis. Total nutrient starvation was identified as the most effective method to induce alarmone production, whereas traditional induction methods such as the addition of serine hydroxamate (SHX) or mupirocin did not show a strong accumulation of (p)ppGpp. The predominant alarmone in C. glutamicum represents guanosine tetraphosphate, whose stress-associated production depends on the presence of the bifunctional RSH enzyme Rel. Interestingly, in addition to ppGpp, another substance yet not identified accumulated strongly under inducing conditions. A C. glutamicum triple mutant (Δrel,ΔrelS,ΔrelH) unable to produce alarmones [(p)ppGpp0 strain] exhibited unstable growth characteristics and interesting features such as an influence of illumination on its physiology, production of amino acids as well as differences in vitamin and carotenoid production. Differential transcriptome analysis using RNAseq provided numerous indications for the molecular basis of the observed phenotype. An evaluation of the (p)ppGpp-dependent transcriptional regulation under total nutrient starvation revealed a complex interplay with the involvement of ribosome-mediated transcriptional attenuation, the stress-responsive sigma factors σB and σH and transcription factors such as McbR, the master regulator of sulfur metabolism. In addition to the differential regulation of genes connected with various cell functions, the transcriptome analysis revealed conserved motifs within the promoter regions of (p)ppGpp-dependently and independently regulated genes. In particular, the representatives of translation-associated genes are both (p)ppGpp-dependent transcriptionally downregulated and show a highly conserved and so far unknown TTTTG motif in the −35 region, which is also present in other actinobacterial genera

The neutrophil oxidant hypothiocyanous acid causes a thiol-specific stress response and an oxidative shift of the bacillithiol redox potential in Staphylococcus aureus

During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (EBSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced EBSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of EBSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in EBSH and increased S-bacillithiolation in S. aureus

The MarR-Type Repressor MhqR Confers Quinone and Antimicrobial Resistance in Staphylococcus aureus

Aims: Quinone compounds are electron carriers and have antimicrobial and toxic properties due to their mode of actions as electrophiles and oxidants. However, the regulatory mechanism of quinone resistance is less well understood in the pathogen Staphylococcus aureus. Results: Methylhydroquinone (MHQ) caused a thiol-specific oxidative and electrophile stress response in the S. aureus transcriptome as revealed by the induction of the PerR, QsrR, CstR, CtsR, and HrcA regulons. The SACOL2531-29 operon was most strongly upregulated by MHQ and was renamed as mhqRED operon based on its homology to the Bacillus subtilis locus. Here, we characterized the MarR-type regulator MhqR (SACOL2531) as quinone-sensing repressor of the mhqRED operon, which confers quinone and antimicrobial resistance in S. aureus. The mhqRED operon responds specifically to MHQ and less pronounced to pyocyanin and ciprofloxacin, but not to reactive oxygen species (ROS), hypochlorous acid, or aldehydes. The MhqR repressor binds specifically to a 9–9 bp inverted repeat (MhqR operator) upstream of the mhqRED operon and is inactivated by MHQ in vitro, which does not involve a thiol-based mechanism. In phenotypic assays, the mhqR deletion mutant was resistant to MHQ and quinone-like antimicrobial compounds, including pyocyanin, ciprofloxacin, norfloxacin, and rifampicin. In addition, the mhqR mutant was sensitive to sublethal ROS and 24 h post-macrophage infections but acquired an improved survival under lethal ROS stress and after long-term infections. Innovation: Our results provide a link between quinone and antimicrobial resistance via the MhqR regulon of S. aureus. Conclusion: The MhqR regulon was identified as a novel resistance mechanism towards quinone-like antimicrobials and contributes to virulence of S. aureus under long-term infections

Identifying the Growth Modulon of Corynebacterium glutamicum

The growth rate (μ) of industrially relevant microbes, such as Corynebacterium glutamicum, is a fundamental property that indicates its production capacity. Therefore, understanding the mechanism underlying the growth rate is imperative for improving productivity and performance through metabolic engineering. Despite recent progress in the understanding of global regulatory interactions, knowledge of mechanisms directing cell growth remains fragmented and incomplete. The current study investigated RNA-Seq data of three growth rate transitions, induced by different pre-culture conditions, in order to identify transcriptomic changes corresponding to increasing growth rates. These transitions took place in minimal medium and ranged from 0.02 to 0.4 h-1 μ. This study enabled the identification of 447 genes as components of the growth modulon. Enrichment of genes within the growth modulon revealed 10 regulons exhibiting a significant effect over growth rate transition. In summary, central metabolism was observed to be regulated by a combination of metabolic and transcriptional activities orchestrating control over glycolysis, pentose phosphate pathway, and the tricarboxylic acid cycle. Additionally, major responses to changes in the growth rate were linked to iron uptake and carbon metabolism. In particular, genes encoding glycolytic enzymes and the glucose uptake system showed a positive correlation with the growth rate

Enhancing stability of recombinant CHO cells by CRISPR/Cas9-mediated site-specific integration into regions with distinct histone modifications

Chinese hamster ovary (CHO) cells are the most important platform for producing biotherapeutics. Random integration of a transgene into epigenetically instable regions of the genome results in silencing of the gene of interest and loss of productivity during upstream processing. Therefore, cost- and time-intensive long-term stability studies must be performed. Site-specific integration into safe harbors is a strategy to overcome these limitations of conventional cell line design. Recent publications predict safe harbors in CHO cells based on omics data sets or by learning from random integrations, but those predictions remain theory. In this study, we established a CRISPR/Cas9-mediated site-specific integration strategy based on ChIP-seq data to improve stability of recombinant CHO cells. Therefore, a ChIP experiment from the exponential and stationary growth phase of a fed-batch cultivation of CHO-K1 cells yielded 709 potentially stable integration sites. The reporter gene eGFP was integrated into three regions harboring specific modifications by CRISPR/Cas9. Targeted Cas9 nanopore sequencing showed site-specific integration in all 3 cell pools with a specificity between 23 and 73%. Subsequently, the cells with the three different integration sites were compared with the randomly integrated donor vector in terms of transcript level, productivity, gene copy numbers and stability. All site-specific integrations showed an increase in productivity and transcript levels of up to 7.4-fold. In a long-term cultivation over 70 generations, two of the site-specific integrations showed a stable productivity (>70%) independent of selection pressure

Deciphering the Transcriptional Response Mediated by the Redox-Sensing System HbpS-SenS-SenR from Streptomycetes

Busche T, Winkler A, Wedderhoff I, Rückert C, Kalinowski J, Lucana DO de O. Deciphering the Transcriptional Response Mediated by the Redox-Sensing System HbpS-SenS-SenR from Streptomycetes. PLOS ONE. 2016;11(8): e0159873.The secreted protein HbpS, the membrane-embedded sensor kinase SenS and the cytoplasmic response regulator SenR from streptomycetes have been shown to form a novel type of signaling pathway. Based on structural biology as well as different biochemical and biophysical approaches, redox stress-based post-translational modifications in the three proteins were shown to modulate the activity of this signaling pathway. In this study, we show that the homologous system, named here HbpSc-SenSc-SenRc, from the model species Streptomyces coelicolor A3(2) provides this bacterium with an efficient defense mechanism under conditions of oxidative stress. Comparative analyses of the transcriptomes of the Streptomyces coelicolor A3(2) wild-type and the generated hbpSc-senSc-senRc mutant under native and oxidative-stressing conditions allowed to identify differentially expressed genes, whose products may enhance the anti-oxidative defense of the bacterium. Amongst others, the results show an up-regulated transcription of genes for biosynthesis of cysteine and vitamin B-12, transport of methionine and vitamin B-12, and DNA synthesis and repair. Simultaneously, transcription of genes for degradation of an anti-oxidant compound is down-regulated in a HbpSc-SenSc-SenRc-dependent manner. It appears that HbpSc-SenSc-SenRc controls the non-enzymatic response of Streptomyces coelicolor A3(2) to counteract the hazardous effects of oxidative stress. Binding of the response regulator SenRc to regulatory regions of some of the studied genes indicates that the regulation is direct. The results additionally suggest that HbpSc-SenSc-SenRc may act in concert with other regulatory modules such as a transcriptional regulator, a two-component system and the Streptomyces B-12 riboswitch. The transcriptomics data, together with our previous in vitro results, enable a profound characterization of the HbpS-SenS-SenR system from streptomycetes. Since homologues to HbpS-SenS-SenR are widespread in different actinobacteria with ecological and medical relevance, the data presented here will serve as a basis to elucidate the biological role of these homologues