Substrat-spezifische Induktion der anaeroben Abbauwege von aromatischen Verbindungen in Aromatoleum aromaticum EbN1

Das denitrifizierende Bakterium Aromatoleum aromaticum EbN1 kann aromatische Kohlenwasserstoffe sowie phenolische Verbindungen anaerob über verschiedene Abbauwege metabolisieren. Diese distinkte Degradierung war im Vergleich zu aeroben Abbauern wie Pseudomonas putida überraschend und weckte das Interesse, den Abbau dieser umweltschädigenden Verbindungen in den tiefliegenden, anaeroben Bodenschichten zu verstehen. Die genetische Zugänglichkeit von Aromatoleum aromaticum EbN1 macht es zu einem guten Modellorganismus, um diese Stoffwechselwege besser nachvollziehen zu können. Außerdem sind die Abbaumechanismen der chemisch sehr ähnlichen Verbindungen Toluol, Ethylbenzol, Phenol und 4-Ethylphenol alle separat reguliert. Basierend auf zuvor gemachten Beobachtungen, Sequenzvergleichen und Vergleichen zu anderen Kohlenwasserstoff-abbauenden Organismen wurden putative Regulatoren für die jeweiligen Abbau-Operons ermittelt. Es wurde postuliert, dass der Abbau von Toluol, Ethylbenzol und Acetophenon unter der Kontrolle von verschiedenen Zweikomponenten-Systemen steht, die jeweils durch die Gene tdiRS, ediRS und adiRS kodiert werden. Andererseits sind wahrscheinlich σ54-abhängige Regulatoren verantwortlich für die Aktivierung des Abbaus von 4-Ethylphenol (EtpR) und Phenol (PdeR). Alle postulierten Regulatoren sind dabei direkt neben den bekannten Abbau-Operons kodiert. In dieser Arbeit wurden die Funktionen von EtpR und PdeR näher untersucht. Dafür wurden zwei Disruptions-Mutanten erstellt und auf ihren Wachstumsphänotyp getestet. Die Ergebnisse unterstützen die These, dass die Gene etpR bzw. pdeR für Aktivatoren kodieren, die die anliegenden Gencluster induzieren. Außerdem wurden erste Studien zur Bestimmung möglicher Induktoren der 4-Ethylphenol- und Phenol-Operons, sowie Analysen von transkriptionellen Startpunkten und Operator-Bindestellen, gestartet. Neuste Ergebnisse haben eine komplexe Interaktion zwischen den Abbauwegen von 4-Ethylphenol und Ethylbenzol und ihren vorhergesagten Regulationssystemen gezeigt. Um ein besseres Verständnis dafür zu bekommen, wurde Aromatoleum aromaticum EbN1 mit verschiedenen Kohlenstoffquellen kultiviert und eine Methode zur Transkriptom-Sequenzierung über reverse Transkription und Hochdurchsatz-Sequenzierung angereicherter mRNA etabliert. Dadurch wurden viele neue Einblicke in das komplexe Regulationsnetzwerk des anaeroben Abbaus aromatischer Kohlenwasserstoffe und phenolischer Verbindungen gewonnen und zusätzlich ergaben sich Hinweise auf ungeklärte Schritte in den jeweiligen Abbauwegen

The Industrial Organism Corynebacterium glutamicum Requires Mycothiol as Antioxidant to Resist Against Oxidative Stress in Bioreactor Cultivations

In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO(2)) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient Delta mshC mutant was impaired in growth in bioreactor experiments performed at pO(2) values of 30%. At a pO(2) level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (E-MSH) at pO(2) values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes

The alpha-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

alpha-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial alpha-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the alpha-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial alpha-glucan phosphorylases

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

Chi BK, Busche T, Van Laer K, et al. Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress. Antioxidants & Redox Signaling. 2014;20(4):589-605.Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation. Results: Transcriptomics revealed the strong upregulation of the disulfide stress sigma(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH. Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives

Are Host Genetics the Predominant Determinant of Persistent Nasal Staphylococcus aureus Carriage in Humans?

Background. Staphylococcus aureus nasal carriage is influenced by multifactorial interactions which are difficult to study in open populations. Therefore, we concomitantly assessed the epidemiological, microbiological, and human-genetic carriage-related factors in a nearly closed population. Methods. In 2006 and 2008, we collected nasal S. aureus strains, human DNA, and epidemiological data from 154 adult Wayampi Amerindians living in an isolated village in the Amazonian forest. The genetics of the strains (multilocus sequence type, spa type, and toxin-content type), epidemiological risk factors, antibiotic exposure, and allelic polymorphism of human genes putatively involved in carriage of the persistent carriers were compared with those of other volunteers. Results. Overall carriage prevalence was 41.7% in 2006 and 57.8% in 2008, but the overall prevalence of persistent carriage was only 26%. The rare and phylogenetically distant multilocus sequence type ST1223 was present in 18.5% of the carriers in 2006 and 34.8% in 2008. No epidemiological factors or antibiotic exposure were significantly associated with persistent carriage, but single nucleotide polymorphism distribution in C-reactive proteins C2042T and C1184T and interleukin-4 C524T genes was significantly associated (P=.02, by global test). Conclusion. Host genetic factors appeared to be the predominant determinant for S. aureus persistent nasal carriage in humans

Are Host Genetics the Predominant Determinant of Persistent Nasal Staphylococcus aureus Carriage in Humans?

International audienceStaphylococcus aureus nasal carriage is influenced by multifactorial interactions which are difficult to study in open populations. Therefore, we concomitantly assessed the epidemiological, microbiological, and human-genetic carriage-related factors in a nearly closed population