Rïngo Schwabe

VpStyA1/VpStyA2B of Variovorax paradoxus EPS: An Aryl Alkyl Sulfoxidase Rather than a Styrene Epoxidizing Monooxygenase

Herein we describe the first representative of an E2-type two-component styrene monooxygenase of proteobacteria. It comprises a single epoxidase protein (VpStyA1) and a two domain protein (VpStyA2B) harboring an epoxidase (A2) and a FAD-reductase (B) domain. It was annotated as VpStyA1/VpStyA2B of Variovorax paradoxus EPS. VpStyA2B serves mainly as NADH:FAD-oxidoreductase. A Km of 33.6 ± 4.0 µM for FAD and a kcat of 22.3 ± 1.1 s−1 were determined and resulted in a catalytic efficiency (kcat Km−1) of 0.64 s−1 μM−1. To investigate its NADH:FAD-oxidoreductase function the linker between A2- and B-domain (AREAV) was mutated. One mutant (AAAAA) showed 18.7-fold higher affinity for FAD (kcat Km−1 of 5.21 s−1 μM−1) while keeping wildtype NADH-affinity and -oxidation activity. Both components, VpStyA2B and VpStyA1, showed monooxygenase activity on styrene of 0.14 U mg−1 and 0.46 U mg−1, as well as on benzyl methyl sulfide of 1.62 U mg−1 and 3.11 U mg−1, respectively. The high sulfoxidase activity was the reason to test several thioanisole-like substrates in biotransformations. VpStyA1 showed high substrate conversions (up to 95% in 2 h) and produced dominantly (S)-enantiomeric sulfoxides of all tested substrates. The AAAAA-mutant showed a 1.6-fold increased monooxygenase activity. In comparison, the GQWCSQY-mutant did neither show monooxygenase nor efficient FAD-reductase activity. Hence, the linker between the two domains of VpStyA2B has effects on the reductase as well as on the monooxygenase performance. Overall, this monooxygenase represents a promising candidate for biocatalyst development and studying natural fusion proteins.

Gallium Mobilization in Soil by Bacterial Metallophores

In the present study we explore the idea of biotechnologically produced metallophore mixtures as selective chelating compounds for economically valuable metals from various sources. A complex soil matrix with natural levels of metal mineralization was employed as a potential source of metals. We focused on gallium-chelating metallophore preparations of two soil bacteria (Gordonia rubripertincta CWB2 and Paracoccus denitrificans PD1222) which were compared to the commercially available desferrioxamine B (DFOB). As a reference, the binding of iron was analyzed. The herein described successful mobilization of metals such as gallium from soil provides first hints towards alternative strategies, such as phytomining, sensor development, or solvent extraction based on metallophores. The metallophore mixture produced by the strains showed best results at pH 8 and allowed to mobilize gallium about three times better as the pure commercially available DFOB.

On the Immobilization of Desferrioxamine-Like Siderophores for Selective Metal Binding

Gordonia rubripertincta CWB2 produces hydroxamate-type siderophores. Therefore it was cultivated under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth. The elution profile and its absorbance spectrum were similar to those of desferrioxamine B. The latter is a commercial available metal chelating agent which is of interest for industries. We successfully developed an HPLC protocol to separate metal-free and metal-loaded desferrioxamines. Further, we aimed to increase the re-usability of desferrioxamines as metal chelators by immobilization on silica based carriers. The siderophores of strain CWB2 have been covalently linked to the carrier with a high yield (up to 95%). Metal binding studies demonstrated that metals can be bound to non-immobilized as well as to the covalently linked desferrioxamines.

Revisiting the Chrome Azurol S Assay for Various Metal Ions

Siderophores play an important role in the solubilisation and mobilization of iron (III) and various metal ions. To have a useful method to test siderophores in culture supernatants for their metal binding affinity, we redesigned and optimized the liquid CAS-assay for selected metal ions. CAS-assay solutions were calibrated with desferrioxamine B in different concentrations to calculate DFOB-equivalents to get a semi-quantitative evaluation. With these assay solutions, we were able to test siderophores in culture supernatants for their ability to chelate with Fe, Al, Ga, Cu, V and As.

Siderophore Purification via Immobilized Metal Affinity Chromatography

Siderophores are low-molecular weight compounds that are produced by organisms to assimilate vital Fe3+ out of iron-deficient environments. They are of interest for several (bio-) technological applications because of their high selectivity for several metal ions. Unfortunately, the concentration in supernatants is often low and thus it is challenging to purify or even enrich these compounds. We applied different types of siderophores onto an immobilized metal-resin that was loaded with either Ni2+, Co2+ or Fe3+. Elution was done with ethanol to reduce salt load and facilitate downstream processing. Thus, it is possible to enrich as well as desalt a sample within one-step from culture supernatant, which allows faster characterization and application of siderophores.

Genomic Characterization of the Arsenic-Tolerant Actinobacterium, Rhodococcus erythropolis S43

Rhodococcus erythropolis S43 is an actinobacterium isolated from an arsenic-contaminated soil sample, collected from an old smelter site, including an arsenic smelter, in Germany. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of arsenic. Here, we present the microbiological features and genomic properties of this biotechnologically relevant strain. The 6,812,940 bp draft genome is arranged into 264 scaffolds of 848 contigs. It possesses 62.5% of CG content and comprises 6,040 coding sequences and 49 tRNA genes. Bioinformatic genome analysis showed the presence of arsenic-resistance genes. A complete ars operon was found containing the arsACDR cluster coding for ArsA (efflux pump ATPase), ArsC (arsenate reductase), ArsD (chaperone) and ArsR (ars operon regulator). Our results show that the arsC mRNA level significantly increased in response to arsenite and arsenate exposure, suggesting its involvement in the arsenic resistance phenotype of strain S43. In addition, this strain showed to have a plethora of genes coding for proteins involved in oxidative-stress response, including catalase, super-oxide dismutase, glutathione peroxidase-related genes, thioredoxin and thioredoxin reductase, suggesting it is highly tolerant to oxidative conditions. Finally, genes for radiation resistance, biodesulfurization, and oil and phenol degrading pathways were also detected. Altogether this data make R. erythropolis S43 a good candidate microorganism for bioremediation of highly contaminated environments and other industrial applications.

VpStyA1 and VpStyA2B of Variovorax paradoxus EPS: Rather an Aryl Alkyl Sulfoxidase than a Styrene Epoxidizing Monooxygenase

VpStyA1 and VpStyA2B of Variovorax paradoxus EPS is annotated and characterized as the 14 first representative of an E2-type styrene monooxygenase of proteobacteria. It comprises a single 15 epoxidase (VpStyA1) and a fusion protein (VpStyA2B) which serves mainly as NADH:FAD16 oxidoreductase. VpStyA2B had a Km of 33.6 ± 4.0 μM for FAD and a kcat of 22.3 ± 1.1 s-1. VpStyA2B 17 and VpStyA1 showed monooxygenase activity on styrene of 0.14 U mg-1 and 0.46 U mg-1 as well as 18 on benzyl methyl sulfide of 1.62 U mg-1 and of 3.11 U mg-1. A putative fusion region at position 408 19 (AREAV) was mutated to provide insights on VpStyA2B-function. The best mutant (408-AAAAA) 20 obtained showed a 6.6-times higher affinity for FAD while keeping the NADH-affinity and 21 oxidation activity. Corresponding epoxidase activity increased (1.6-times). But, other mutants 22 showed still NADH:FAD-oxidoreductase activity, but lost mostly their epoxidase activity indicating 23 effects on the monooxygenase-part as well. Thus, this monooxygenase system represents an 24 interesting candidate for biocatalyst development. 25

Detection of arsenic-binding siderophores in arsenic-tolerating Actinobacteria by a modified CAS assay

The metalloid arsenic is highly toxic to all forms of life, and in many countries decontamination of water and soil is still required. Some bacteria have mechanisms to detoxify arsenic and can live in its presence. Actinobacteria are well known for their ability to produce a myriad of biologically-active compounds. In the present study, we isolated arsenic-tolerant Actinobacteria from contaminated water in Saxony, Germany, and determined their ability to produce siderophores able to bind arsenic. The binding capacity of different siderophore-like compounds was determined by a modified chrome azurol S (As-mCAS) assay with As(III) at high pH and using CAS decolorization as a readout. Arsenic-tolerant isolates from three actinobacterial genera were identified by 16 S rRNA gene sequence analysis: Rhodococcus, Arthrobacter and Kocuria. The isolated Actinobacteria showed a high As(III)-binding activity by siderophore-like compounds, resulting in 82-100% CAS decolorization, as compared to the results with EDTA. The interaction between As(III) and siderophore-like compounds was also detected at neutral pH. In summary, our results suggest that the isolated arsenic-tolerant Actinobacteria produce siderophores that bind arsenic, and open new perspectives on potential candidates for decontaminating environments with arsenic and for other biotechnological applications.

Thermochelin, a Hydroxamate Siderophore from Thermocrispum agreste DSM 44070

Siderophores are produced by microorganisms in iron-deficient environments. They are classified by structure as hydroxamate, catecholate, carboxylate or mixed type siderophores. These differences are also reflected in the selectivity for other valuable elements than iron, which allows designating them as “metallophores”, and makes them of interest for several industrial and medical applications. Thus, it is essential to understand the biosynthesis of these molecules to increase the set of available metallophores that are stable and suited for the respective applications. The probable structure of the metallophore from T. agreste DSM 44070 was predicted by similarity search and gene annotation. An N-hydroxylating monooxygenase (NMO: TheA) of T. agreste DSM 44070 that catalyzes an initial step was synthesized and characterized in detail. The respective metallophore was synthesized, purified and studied. The structure prediction suggested a hydroxamate-type (Erythrochelin-like) metallophore that contains L-N5-hydroxyornithine. This precursor is synthesized by TheA. The siderophore designated as “Thermochelin” is produced, extracted and purified successfully. Complexation was confirmed by CAS-assay. In this study, we expanded the scope of siderophores and the knowledge towards their biosynthetic pathways. Thermochelin is the second siderophore, which was purified from a thermophilic organism, and TheA is the first NMO, which was characterized from an extremophile.