Institut für Mikrobiologie - Abteilung für Genetik & Biochemie
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Glutathione (GSH) was initially identified and characterized for its redox properties andlater for its contributions to detoxification reactions. Over the past decade, however, the essentialcontributions of glutathione to cellular iron metabolism have come more and more into focus. GSH isindispensable in mitochondrial iron-sulfur (FeS) cluster biosynthesis, primarily by co-ligating FeSclusters as a cofactor of the CGFS-type (class II) glutaredoxins (Grxs). GSH is required for the exportof the yet to be defined FeS precursor from the mitochondria to the cytosol. In the cytosol, it is anessential cofactor, again of the multi-domain CGFS-type Grxs, master players in cellular iron and FeStrafficking. In this review, we summarize the recent advances and progress in this field. The mosturgent open questions are discussed, such as the role of GSH in the export of FeS precursors frommitochondria, the physiological roles of the CGFS-type Grx interactions with BolA-like proteins andthe cluster transfer between Grxs and recipient proteins
Acidobacteria represents one of the most dominant bacterial groups across diverse ecosystems. However, insight into their ecology and physiology has been hampered by difficulties in cultivating members of this phylum. Previous cultivation efforts have suggested an important role of trace elements for the proliferation of Acidobacteria, however, the impact of these metals on their growth and metabolism is not known. In order to gain insight into this relationship, we evaluated the effect of trace element solution SL10 on the growth of two strains (5B5 and WH15) of Acidobacteria belonging to the genus Granulicella and studied the proteomic responses to manganese (Mn). Granulicella species had highest growth with the addition of Mn, as well as higher tolerance to this metal compared to seven other metal salts. Variations in tolerance to metal salt concentrations suggests that Granulicella sp. strains possess different mechanisms to deal with metal ion homeostasis and stress. Furthermore, Granulicella sp. 5B5 might be more adapted to survive in an environment with higher concentration of several metal ions when compared to Granulicella sp. WH15. The proteomic profiles of both strains indicated that Mn was more important in enhancing enzymatic activity than to protein expression regulation. In the genomic analyses, we did not find the most common transcriptional regulation of Mn homeostasis, but we found candidate transporters that could be potentially involved in Mn homeostasis for Granulicella species. The presence of such transporters might be involved in tolerance to higher Mn concentrations, improving the adaptability of bacteria to metal enriched environments, such as the decaying wood-rich Mn environment from which these two Granulicella strains were isolated.
Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.
Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.
Invasion of the bacterial pathogen Listeria monocytogenes into human host cells requires specialized surface molecules for attachment and induction of phagocytosis. However, efficient invasion is also dependent on factors with house-keeping functions, such as SecA2-dependent secretion of autolysins for post-divisional segregation of daughter cells. Mutations in this pathway prevent degradation of peptidoglycan cross-walls, so that long cell chains are formed that cannot be phagocytosed. The extreme chaining of such mutants manifests as rough colony phenotype. One rough clone was isolated from a transposon library with a transposon insertion in the uncharacterized lmo0720 gene (lftS) together with a spontaneous point mutation in the secA2 gene. We separated both mutations and demonstrated that this point mutation in the intramolecular regulator 2 domain of SecA2 was sufficient to inactivate the protein. In contrast, lftS deletion did not cause a ΔsecA2-like phenotype. lftS is located in an operon with lftR (lmo0719), encoding a PadR-like transcriptional regulator, and lftR deletion affected growth, invasion and day-light dependent coordination of swarming. Inactivation of lftS partially suppressed these phenotypes, suggesting a functional relationship between LftR and LftS. However, the invasion defect of the ΔlftR mutant was only marginally suppressed by lftS removal. LftR regulates expression of the lmo0979–0980 (lieAB) operon, encoding a putative multidrug resistance transporter and lieAB transcription was strongly upregulated in the absence of LftR. Deletion of lieAB in the ΔlftR background restores wild type-like invasion levels. Hence, we conclude that tight transcriptional repression of the lieAB operon is essential for efficient listerial host cell invasion.
Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods.
Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.
Background: The association of polyomaviruses BK and JC with other opportunistic infections and graft-versus-host disease (GvHD) in allogeneic stem cell transplantation is controversially discussed. Methods: We conducted a retrospective study of 64 adult patients who received their first allogeneic stem cell transplantation between March 2010 and December 2014; the follow-up time was 2 years. Results: Acute leukemia was the most frequent underlying disease (45.3%), and conditioning included myeloablative (67.2%) and nonmyeloablative protocols (32.8%). All patients received 10 mg of alemtuzumab on day -2 (20 mg in case of mismatch) as GvHD prophylaxis. Twenty-seven patients (41.5%) developed cytomegalovirus (CMV) reactivation. BKPyV-associated hemorrhagic cystitis was diagnosed in 10 patients (15.6%). Other opportunistic infections caused by viruses or protozoa occurred rarely (<10%). There was no association of BKPyV or JCPyV with CMV reactivation, Epstein-Barr virus reactivation, human herpes virus 6, or parvovirus B19 infection requiring treatment. There was a significant correlation of BKPyV-associated hemorrhagic cystitis with toxoplasmosis (p = 0.013). Additionally, there was a significant link of simultaneous BKPyV and JCPyV viruria with toxoplasmosis (p = 0.047). BKPyV and JCPyV were not associated with GvHD, relapse, or death. Conclusion: We found no association of BKPyV or JCPyV with viral infections or GvHD. Only the correlation of both polyomaviruses with toxoplasmosis was significant. This is a novel and interesting finding.