7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

PD Dr.

Chair

Microbial Sulfur and Persulfide Oxygenases

Microbial metabolism & biochemistry

Introduction: Iron-containing persulfide dioxygenases (PDO) catalyze the oxidation of glutathione persulfide (GSSH) to sulfite and GSH. They are abundant in Bacteria and mitochondria.Sulfur oxygenase reductases (SOR) catalyze an O2-dependent elemental sulfur (S0) dismutation to sulfite and H2S. They occur in chemolithotrophic sulfur oxidizers and are structurally unrelated to PDOs. Both enzymes contain mononuclear non-heme Fe active sites, each with two His and one carboxylate ligands. Here, we compare structural and spectroscopic properties of the AcPDO from Acidithiobacillus caldus and three different SORs from Bacteria and Archaea in order to better understand the reaction mechanisms.Results: GSSH binds to the PDO active site in a cleft at the enzyme's surface with the iron at its bottom. Incubation with GSSH resulted in the formation of S-Fe charge transfer absorption spectra; which was not observed in the SORs. Modeling of GSSH binding to the AcPDO crystal structure suggested that a transient persulfide-peroxo intermediate is formed, which is resolved by proton shuffling with a second coordination sphere His. Cysteines do not participate in catalysis, a disulfide present at the AcPDO surface stabilizes its C-terminus.In contrast, SORs have a spacious and deeply embedded active site cavity with a narrow entrance pore comprising 3 conserved cysteines. X-ray crystallography of SORs showed that each of the cysteines could be persulfurated and that SOR mutants can become stuck in that state. Free and/or cysteine-bound polysulfide chains seem to be binding to Fe with the terminal S atom. WE suggest that sulfane S dismutation occurs, when the Fe-bound peroxo oxygen attacks the penultimate sulfane S atom of the chain, thus splitting off the terminal sulfide, while the oxidation steps proceeds in a similar way as in PDO.Conclusion: The results suggest that the glutathione moiety positions the persulfide for terminal sulfane sulfur oxidation at the open PDO active site, whereas the closed cavity of the SORs is required to keep the more mobile free persulfides trapped in the vicinity of the Fe atom in order to get S0 dismutation to work.

Oral Presentation

Prof. Dr.

Investigating the regulation of the carbon switch in central metabolism of Synechocystis sp. PCC 6803

Dependent on the light/nutrient conditions Synechocystis is able to grow auto-, hetero-, and mixotrophically for which the reversibility of sugar metabolism particularly the Embden-Meyerhof-Parnas pathway (EMP) is essential. To switch between anabolic and catabolic direction sophisticated regulation is required and also the interplay of e.g. EMP and Calvin cycle for CO2 fixation must be tightly balanced. The antagonistic PFK and FBPase operate exclusively in the catabolic or anabolic direction of the EMP, respectively, and PFK is the classical control point of glycolysis. Two paralogous copies of both enzymes are present in Synechocystis. Furthermore, the reversible phosphoglucose isomerase (PGI) has been discussed to direct fluxes between EMP and CBB under different growth conditions. However, the control points and the function and regulatory capacities of the enzymes are not established in Synechocystis.Herein, the regulatory functions of the PFK and FBPase isoenzymes as well as the PGI were analysed. Pfk-A1, Pfk-A2, Fbpase1 and Pgi from Synechocystis were overproduced in E. coli, purified and characterized with respect to substrate specificity, kinetic and regulatory properties.Both PFKs are ADP dependent, thereby defining a new class of ADP-dependent PFK-A superfamily. PFK-A1 is inhibited by 3-phosphoglycerate (3PG), and PFK2 by ATP. FBPase1 is not regulated by various effectors. The PGI displays a preference for the gluconeogenic direction and is inhibited by erythrose 4-phosphate (E4P).Our investigations reveal that both PFKs are active under conditions of low energy-charge, facilitating the breakdown of glycogen/D-glucose. The inhibition of PFK-A1 and PFK-A2 by 3PG and ATP, respectively, enables rapid generation of CO2 acceptor molecule. The sustained FBPase1 activity in presence of various effectors suggests its housekeeping role to enable reversibility of the EMP pathway in response to substrate availability/growth conditions. E4P enables PGI function as a valve to balance the non-oxidative pentose phosphate pathway intermediates and glycogen synthesis.Feng et al., FEBS J (2014) 281.3: 916-926Schulze et al., Microb Cell Fact (2022); 21:699

A hexameric NADP+-reducing hydrogenase from Moorella thermoacetica: First insights into a unique hydrogenase

Introduction: Moorella thermoacetica reduces CO2 with H2 as reductant via the Wood-Ljungdahl pathway (WLP). The enzymes of the WLP in M. thermoacetica require NADH, NADPH and reduced ferredoxin as reductant. Whereas an electron-bifurcating, ferredoxin- and NAD+-reducing hydrogenase HydABC had been described, the enzyme that reduces NADP+ remained to be identified. A likely candidate is the HydABCDEF hydrogenase encoded by M. thermoacetica.Goals: In order to clarify the electric connectivity between the oxidative and reductive branches in acetogenesis in M. thermoacetica, we attempted to isolate the NADP+-reducing hydrogenase. Since cells grow poorly on H2 + CO2 and the activity was not present in glucose-grown cells we first identified conditions under which the enzyme was produced and cell mass could be obtained easily.Materials &amp; Methods: Growth experiments, enzyme purification, enzyme assays, genome-wide expression analyses and bioinformatic analysis were performed.Results: Cell-free extract of H2 + CO2-grown cells catalyzed H2-dependent NADP+ reduction whereas this activity was not observed in glucose-grown cells. Cells grown on glucose + DMSO also produced the NADP+-reducing hydrogenase. We have purified the hydrogenase to apparent homogeneity from cells grown on glucose + DMSO. The enzyme had six subunits encoded by hydABCDEF, contained 58 mol iron and one mol FMN. The enzyme reduced methyl viologen with H2 as reductant and of the physiological acceptors tested only NADP+ was reduced. Electron bifurcation to pyridine nucleotides and ferredoxin was not observed. Hydrogen-dependent NADP+ reduction was optimal at pH 8 and 60°C; the specific activity was 8.5 U/mg and the Km for NADP+ was 0.086 mM.Summary: The NADP+-dependent hydrogenase HydABCDEF from M. thermoacetica has been purified and characterized. Despite its similarity to the electron-bifurcating hydrogenase HydABC, the HydABCDEF hydrogenase did not bifurcate electrons and NADP+ is the only physiological electron acceptor. The enzyme provides the WLP with NADPH.

Professor

Exploring the genomic landscape: identification of aminoglycoside fitness-genes in resistant Escherichia coli

Aminoglycosides resistance is common worldwide in pathogenic bacteria, and helper-drugs are urgently needed for this class of antimicrobials. The objective of this study was to identify genes that are advantageous for growth of E. coli in the presence of aminoglycosides, as these genes may represent targets for helper-drugs. We generated three highly saturated transposon mutant libraries in E. coli MG1655 containing streptomycin (aph(3"")-Ib/aph(6)-Id), gentamicin (aac(3)-IV), or neomycin (aph(3"")-Ia) resistance gene(s). Transposon Directed Insertion-site Sequencing (TraDIS) revealed 106 genes, where transposon insertions caused reduced fitness in the presence of one or more of the antibiotics. Fifty-six, 39 and 32 genes were identified as fitness-genes under streptomycin, gentamicin and neomycin stress (Fig. 1 A). ATP-synthesis, cell division, and stress response genes dominated among streptomycin and gentamicin fitness-genes, while the neomycin fitness-genes were associated with enterobacterial common antigen biosynthesis or magnesium sensing/transport (Fig 1 B). To confirm the importance of the fitness-genes, the genes/gene clusters minCDE, hflCK, clsA and cpxR were deleted among the streptomycin and gentamicin fitness-genes, and phoPQ, wecA, lpp and pal among the neomycin resistance ones. Phenotypic characterization including growth curves, MIC testing and a novel sequencing-based competition approach revealed that all deletion mutants exhibited an attenuated growth in the presence of the corresponding aminoglycoside(s). In summary, we report a set of aminoglycoside fitness-genes which may be putative targets for helper-drugs. Moreover, our study has expanded our understanding of changes that occur in aminoglycoside-resistant E. coli in response to antibiotic exposure.Figure 1: Aminoglycoside fitness-genes (a) and their functional classification (b) in E. coli MG1655 in the presence of STREP, GEN or NEO.This INNOTARGETS project has received funding from the European Union"s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement number 956154.

In situ electrophysiological profiling of Shewanella oneidensis MR-1 in a novel single-chamber microfluidic BES featuring a transparent gas diffusion anode

Oxygen plays a crucial role in the operation of bioelectrochemical systems (BESs). Not all microorganisms and bioprocesses thrive in oxygen-rich environments; however, it is crucial for many in supporting their growth and metabolism. Oxygen also competes with an anode as an electron acceptor in certain systems1. In conventional BES setups, spatial segregation of planktonic and biofilm forming communities occur when oxygen is supplied via headspace or sparging, complicating the physiological investigations. Moreover, visualizing electroactive biofilms in BES in vivo is challenging, as microbial characterization typically occurs post-experiment.This study aimed at development of a microfluidic BES offering the unique advantage of real time biofilm growth visualization, even while dynamically switching between oxygen concentration (composition ranging from 0.1% to 21% v/v) and an anode as electron acceptors. This was achieved by a channel-type microfluidic gas diffusion layer, separated from the BES anode by a transparent Polydimethylsiloxane (PDMS) membrane, enabling controlled aeration (or anaerobic) conditions at the BES anode. To verify the accurate gas dosing function of the PDMS membrane and validate the overall infrastructure in the Micro-BES, we investigated the aerobic and anodic metabolism of the model electroactive microorganism Shewanella oneidensis MR-1 in the Micro-BES. An electrochemical, secondary metabolite and mediator analysis was performed. Additionally, a fluorescently labelled ATP reporter2 strain of S. oneidensis was used to record different growth phases of the electroactive biofilm.The successful construction of a 0.3 ml BES was presented, allowing for online imaging under diverse operational conditions, including variations in oxygen concentration, working electrode potentials, electrode configurations and mediator concentrations. This comprehensive approach facilitated a thorough functional validation of the system and gave new options for physiological insight into the model electroactive bacterium S. oneidensis.Lu, M., et al. (2017). Biotechnology and bioengineering 114(1): 96-105Deng, Y., et al. (2021). BMC biology 19(1): 101

Exploring the physiological and metabolic effects of rpsL mutations in Streptomyces albidoflavus J1074

Introduction Well-known antibiotic-producing genus Streptomyces remains a source of hidden chemical diversity. The introduction of point mutations in the rpsL gene for r-protein S12 is one of the tools that has shown great potential for manipulating the metabolism of these bacteria. One of the currently most popular heterologous expression hosts, Streptomyces albidoflavus J1074, was chosen to study the effect of the engineered rpsL mutations (1). Goals This work aims to study the properties of rpsL mutants K88E, K88R, and R94G. Methods Scanning electron microscopy; CFU, dry biomass, protein content measurement in TSB medium; fermentation in R5 and SDB media and subsequent antibiotic extraction; c-di-GMP extraction from TSB; HPLC-MS; bioactivity assays; qPCR; RNA-seq. Results Strains S. albidoflavus K88E and K88R exhibited altered morphology and a reduced number of sporulating hyphae compared to SAM2 and R94G. Similar levels of total protein accumulation and CFU counts were observed in all strains. However, the dry biomass weight of the original strain SAM2 was significantly increased. The c-di-GMP level, a key regulator of specialized metabolite production, was notably reduced in the K88R mutant, and all strains showed a decrease in the expression of genes for c-di-GMP synthesis. K88E and R94G produced, on average, more specialized metabolites than SAM2 and K88R, as confirmed by bioassays and HPLC-MS analysis of extracts. Summary The rpsL mutants exhibit distinct morphological and growth differences, highlighting the pleiotropic effects of missense mutations within genes for ribosomal proteins. To the best of our knowledge, our work for the first time links rpsL mutations to c-di-GMP metabolism in streptomycetes. This offers fresh insight into the mechanisms of pleiotropicity of rpsL mutations. References 1. Lopatniuk M, Myronovskyi M, Nottebrock A, Busche T, Kalinowski J, Ostash B, et al. Effect of "ribosome engineering" on the transcription level and production of S. albus indigenous secondary metabolites. Applied Microbiology and Biotechnology. 2019 Sep 19;103(17):7097–110.

Oral Session
Microbial Metabolism & Biochemistry 1

Termin

Chair

Thema

Programm

Oral SessionMicrobial Metabolism & Biochemistry 1

Termin

Chair

Thema

Programm

Oral Session
Microbial Metabolism & Biochemistry 1