The alternative coproporphyrinogen III oxidase (CgoN) catalyzes the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III.

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in Bacillaceae, employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called ytpQ, encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from Priestia (Bacillus) megaterium. The recombinantly produced, purified, and monomeric YtpQ (CgoN) protein is shown to catalyze the oxygen-independent conversion of coproporphyrinogen III into coproporphyrin III. Minimal non-enzymatic conversion of coproporphyrinogen III was observed under the anaerobic test conditions employed in this study. FAD was identified as a cofactor, and menadione served as an artificial acceptor for the six abstracted electrons, with a KM value of 3.95 µmol/L and a kcat of 0.63 per min for the substrate. The resulting coproporphyrin III, in turn, acts as an effective substrate for the subsequent enzyme of the pathway, the coproporphyrin III ferrochelatase (CpfC). Under aerobic conditions, oxygen directly serves as an electron acceptor, but is replaced by the more efficient action of menadione. An AlphaFold2 model of the enzyme suggests that YtpQ adopts a compact triangular shape consisting of three domains. The N-terminal domain appears to be flexible with respect to the rest of the structure, potentially creating a ligand binding site that opens and closes during the catalytic cycle. A catalytic mechanism similar to the oxygen-independent protoporphyrinogen IX oxidase PgoH1 (HemG), based on the flavin-dependent abstraction of six electrons from coproporphyrinogen III and their potential quinone-dependent transfer to a membrane-localized electron transport chain, is proposed.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

Isolation and Quantification of Bacterial Membrane Vesicles for Quantitative Metabolic Studies Using Mammalian Cell Cultures.

Bacterial membrane vesicles (BMVs) are produced by most bacteria and participate in various cellular processes, such as intercellular communication, nutrient exchange, and pathogenesis. Notably, these vesicles can contain virulence factors, including toxic proteins, DNA, and RNA. Such factors can contribute to the harmful effects of bacterial pathogens on host cells and tissues. Although the general effects of BMVs on host cellular physiology are well known, the underlying molecular mechanisms are less understood. In this study, we introduce a vesicle quantification method, leveraging the membrane dye FM4-64. We utilize a linear regression model to analyze the fluorescence emitted by stained vesicle membranes to ensure consistent and reproducible vesicle-host interaction studies using cultured cells. This method is particularly valuable for identifying host cellular processes impacted by vesicles and their specific cargo. Moreover, it outcompetes unreliable protein concentration-based methods. We (1) show a linear correlation between the number of vesicles and the fluorescence signal emitted from the FM4-64 dye; (2) introduce the "vesicle load" as a new semi-quantitative unit, facilitating more reproducible vesicle-cell culture interaction experiments; (3) show that a stable vesicle load yields consistent host responses when studying vesicles from Pseudomonas aeruginosa mutants; (4) demonstrate that typical vesicle isolation contaminants, such as flagella, do not significantly skew the metabolic response of lung epithelial cells to P. aeruginosa vesicles; and (5) identify inositol monophosphatase 1 (SuhB) as a pivotal regulator in the vesicle-mediated pathogenesis of P. aeruginosa.

Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface.

Gut microbial production of trimethylamine (TMA) from l-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase, which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from Escherichia coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

Thiol Metabolism and Volatile Metabolome of Clostridioides difficile.

Clostridioides difficile (previously Clostridium difficile) causes life-threatening gut infections. The central metabolism of the bacterium is strongly influencing toxin production and consequently the infection progress. In this context, the composition and potential origin of the volatile metabolome was investigated, showing a large number of sulfur-containing volatile metabolites. Gas chromatography/mass spectrometry (GC/MS)-based headspace analyses of growing C. difficile 630Δerm cultures identified 105 mainly sulfur-containing compounds responsible of the typical C. difficile odor. Major components were identified to be 2-methyl-1-propanol, 2-methyl-1-propanethiol, 2-methyl-1-butanethiol, 4-methyl-1-pentanethiol, and as well as their disulfides. Structurally identified were 64 sulfur containing volatiles. In order to determine their biosynthetic origin, the concentrations of the sulfur-containing amino acids methionine and cysteine were varied in the growth medium. The changes observed in the volatile metabolome profile indicated that cysteine plays an essential role in the formation of the sulfur-containing volatiles. We propose that disulfides are derived from cysteine via formation of cystathionine analogs, which lead to corresponding thiols. These thiols may then be oxidized to disulfides. Moreover, methionine may contribute to the formation of short-chain disulfides through integration of methanethiol into the disulfide biosynthesis. In summary, the causative agents of the typical C. difficile odor were identified and first hypotheses for their biosynthesis were proposed.

ORFeome Phage Display Reveals a Major Immunogenic Epitope on the S2 Subdomain of SARS-CoV-2 Spike Protein.

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.

Functionalization of an extended-gate field-effect transistor (EGFET) for bacteria detection.

Traditional sensing technologies have drawbacks as they are time-consuming, cost-intensive, and do not attain the required accuracy and reproducibility. Therefore, new methods of measurements are necessary to improve the detection of bacteria. Well-established electrical measurement methods can connect high sensitive sensing systems with biological requirements. One approach is to functionalize an extended-gate field-effect transistor's (EGFET) sensing area with modified porphyrins containing two different linkers. One linker connects the electrode surface with the porphyrin. The other linker bonds bacteria on the functional layer through a specific peptide chain. The negative charge on the surface of the cells regulates the surface potential which has an impact on the electrical behavior of the EGFET. The attendance of attached bacteria on the functionalized sensing area could successfully be detected.

Cellular adaptation of Clostridioides difficile to high salinity encompasses a compatible solute-responsive change in cell morphology.

Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrhoeas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt-stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of l-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress.

PRODORIC: state-of-the-art database of prokaryotic gene regulation.

PRODORIC is worldwide one of the largest collections of prokaryotic transcription factor binding sites from multiple bacterial sources with corresponding interpretation and visualization tools. With the introduction of PRODORIC2 in 2017, the transition to a modern web interface and maintainable backend was started. With this latest PRODORIC release the database backend is now fully API-based and provides programmatical access to the complete PRODORIC data. The visualization tools Genome Browser and ProdoNet from the original PRODORIC have been reintroduced and were integrated into the PRODORIC website. Missing input and output options from the original Virtual Footprint were added again for position weight matrix pattern-based searches. The whole PRODORIC dataset was reannotated. Every transcription factor binding site was re-evaluated to increase the overall database quality. During this process, additional parameters, like bound effectors, regulation type and different types of experimental evidence have been added for every transcription factor. Additionally, 109 new transcription factors and 6 new organisms have been added. PRODORIC is publicly available at https://www.prodoric.de.

Radical SAM Enzymes Involved in Tetrapyrrole Biosynthesis and Insertion.

The anaerobic biosyntheses of heme, heme d 1, and bacteriochlorophyll all require the action of radical SAM enzymes. During heme biosynthesis in some bacteria, coproporphyrinogen III dehydrogenase (CgdH) catalyzes the decarboxylation of two propionate side chains of coproporphyrinogen III to the corresponding vinyl groups of protoporphyrinogen IX. Its solved crystal structure was the first published structure for a radical SAM enzyme. In bacteria, heme is inserted into enzymes by the cytoplasmic heme chaperone HemW, a radical SAM enzyme structurally highly related to CgdH. In an alternative heme biosynthesis route found in archaea and sulfate-reducing bacteria, the two radical SAM enzymes AhbC and AhbD catalyze the removal of two acetate groups (AhbC) or the decarboxylation of two propionate side chains (AhbD). NirJ, a close homologue of AhbC, is required for propionate side chain removal during the formation of heme d 1 in some denitrifying bacteria. Biosynthesis of the fifth ring (ring E) of all chlorophylls is based on an unusual six-electron oxidative cyclization step. The sophisticated conversion of Mg-protoporphyrin IX monomethylester to protochlorophyllide is facilitated by an oxygen-independent cyclase termed BchE, which is a cobalamin-dependent radical SAM enzyme. Most of the radical SAM enzymes involved in tetrapyrrole biosynthesis were recognized as such by Sofia et al. in 2001 (Nucleic Acids Res.2001, 29, 1097-1106) and were biochemically characterized thereafter. Although much has been achieved, the challenging tetrapyrrole substrates represent a limiting factor for enzyme/substrate cocrystallization and the ultimate elucidation of the corresponding enzyme mechanisms.

The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology.

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

What's a Biofilm?-How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile.

The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile's V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.

A Point Mutation in the Transcriptional Repressor PerR Results in a Constitutive Oxidative Stress Response in Clostridioides difficile 630Δerm.

The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.

Adaptation of Dinoroseobacter shibae to oxidative stress and the specific role of RirA.

Dinoroseobacter shibae living in the photic zone of marine ecosystems is frequently exposed to oxygen that forms highly reactive species. Here, we analysed the adaptation of D. shibae to different kinds of oxidative stress using a GeLC-MS/MS approach. D. shibae was grown in artificial seawater medium in the dark with succinate as sole carbon source and exposed to hydrogen peroxide, paraquat or diamide. We quantified 2580 D. shibae proteins. 75 proteins changed significantly in response to peroxide stress, while 220 and 207 proteins were differently regulated by superoxide stress and thiol stress. As expected, proteins like thioredoxin and peroxiredoxin were among these proteins. In addition, proteins involved in bacteriochlophyll biosynthesis were repressed under disulfide and superoxide stress but not under peroxide stress. In contrast, proteins associated with iron transport accumulated in response to peroxide and superoxide stress. Interestingly, the iron-responsive regulator RirA in D. shibae was downregulated by all stressors. A rirA deletion mutant showed an improved adaptation to peroxide stress suggesting that RirA dependent proteins are associated with oxidative stress resistance. Altogether, 139 proteins were upregulated in the mutant strain. Among them are proteins associated with protection and repair of DNA and proteins (e. g. ClpB, Hsp20, RecA, and a thioredoxin like protein). Strikingly, most of the proteins involved in iron metabolism such as iron binding proteins and transporters were not part of the upregulated proteins. In fact, rirA deficient cells were lacking a peroxide dependent induction of these proteins that may also contribute to a higher cell viability under these conditions.