Biology subjects

Gescher, J.

Publications and source records attributed to Gescher, J..

12 recordsLinked to original sources

bioRxiv2026

Restriction-site-based enrichment coupled to adaptive sampling enables long-read transposon-insertion sequencing

Long-read Transposon insertion sequencing is essential for linking genotypes to phenotypes, as short-read approaches struggle with complex genomic regions. Long-read sequencing demands high sequencing depth or targeted sequencing, which either requires extensive sample preparation or suffer from low net efficiency. We developed a simple, low-cost workflow combining enzymatic cleavage with adaptive sampling to drastically improve target recovery and simplify analysis. I-SceI restriction site was introduced into a mini-Tn5 transposon to generate a mutant library in Cupriavidus necator. Prior to Nanopore sequencing, gDNA was digested to introduce double-strand breaks precisely at insertion sites, followed by sequencing with adaptive sampling. The workflow was validated via a biofilm selection screen. Combining I-SceI digestion with adaptive sampling yielded an effective 11.3-fold enrichment (after normalizing for pore occupancy). High-depth sequencing covered 97.1% of all genes. Because reads consistently began at the exact insertion site, bioinformatic trimming was bypassed. The biofilm screen successfully identified enriched integration loci, uniquely mapped insertions within repetitive rRNA operons, and revealed co-selected, spontaneous secondary mutations.This method adds less than one hour to library preparation, reduces consumable costs by over 90%, and establishes a budget-friendly, simple and high-resolution long-read Tn-seq pipeline ideal for complex and multipartite genomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/733934v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@e1533aorg.highwire.dtl.DTLVardef@11f3ebborg.highwire.dtl.DTLVardef@4f94b3org.highwire.dtl.DTLVardef@ab7d29_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Identification of chaperone-independent outer membrane proteins and MtrA-assisted MtrB folding in Shewanella oneidensis

Extracellular electron transfer is a respiratory process conducted by a number of microorganisms in order to access insoluble or membrane impermeable electron acceptors or donors. The process has wide implication for the biogeochemistry of our planet and offers many opportunities for biotechnological applications. Outer membrane spanning electron transfer is conducted by the model organism Shewanella oneidensis MR-1 by a stable trimeric protein complex. The electron conduit consists of two c-type cytochromes on either site of the outer membrane and a {beta}-barrel protein in the middle that seems to facilitate interaction of the two heme containing proteins. This study reveals that the periplasmic c-type cytochrome MtrA is not only part of the electron conduit, but also assists in the periplasmic transport of the unfolded outer membrane protein MtrB, a function that was so far believed to be conducted for all outer membrane {beta}-barrel proteins by one of the canonical chaperones SurA, Skp or DegP. However, three more {beta}-barrel proteins were identified that are independent of the canonical chaperones as well but still rely on the BAM complex in the outer membrane, suggesting that many more solutions for periplasmic transfer of {beta}-barrel protein towards the outer membrane exist in Gram-negative microorganisms.

microbiology↗

bioRxiv2023

Oxic microbial electrosynthesis can be more energy efficient for biomass production than knallgas or photosynthesis based processes

Data on the efficiency and development of continuous processes are scarce in the emerging field of oxic microbial electrosynthesis (OMES). Therefore, the recently isolated knallgas bacterium Kyrpidia spormannii was observed in a bioelectrochemical flow cell setup to study biomass formation and energy efficiency of cathode dependent growth. The study revealed that a potential of -500 mV vs. the standard hydrogen electrode (SHE) caused differences in the structure of the biofilm developing on the cathode, but had almost no impact on biomass growth behavior compared to -375 mV vs. SHE. No growth was observed at 0 mV vs SHE. Coulombic efficiency (CE) was calculated for the cultivation at -500 mV vs. SHE. The process can be conducted with the same electron efficiency as traditional gas fermentation. The solar energy demand with 67.89 kWh kg-1 dry biomass is highly competitive to alternative and already established processes for converting (solar) energy to biomass. Additionally, with suggestions for a biomass harvesting method and subsequent recultivation, proof of principle for a continuously operable process was provided. The results pave the way for a new concept in carbon dioxide-based biotechnology. Significance statementTo mitigate global climate change, it is imperative to transition the human economy to a different resource foundation, moving away from fossil fuels and reducing atmospheric carbon dioxide levels. Biotechnological production based on CO2 necessitates a supply of energy and electrons. This study reveals that an oxic process, wherein bacteria are directly cultivated as a biofilm on the cathode surface of a bioelectrochemical system, can exhibit higher energy efficiency than plant-based systems or systems reliant on hydrogen generated through water electrolysis. This technology could be instrumental in establishing carbon dioxide and renewable energy as the foundation for feed, food, and platform chemical production.

bioengineering↗

bioRxiv2022

A scalable, Rotating Disc Bioelectrochemical Reactor (RDBER) suitable for the cultivation of both cathodic and anodic biofilms

This study discusses the construction and operation of a membrane-less bioelectrochemical reactor that employs rotating working electrodes with a surface area of up to 1 m2. As a proof-of-principle for an aerobic microbial electrosynthesis process, Kyrpidia spormannii was cultivated in the reactor. Optical coherence tomography was used to examine the spatial distribution of the cathodic biofilm. After 24 days 87% of the cathode surface was covered with biofilm that was characterized by a radial increase in its biovolume towards the circumcenter of the electrodes reaching up to 92.13 m3 m-2. To demonstrate the versatility of the system, we further operated the reactor as a microbial electrolysis cell employing a co-culture of Shewanella oneidensis and Geobacter sulfurreducens. Anodic current densities of up to 130 A cm-2 were measured during these batch experiments. This resulted in a maximum production rate of 0.43 liters of pure hydrogen per liter reactor volume and day. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/507646v3_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@102fbecorg.highwire.dtl.DTLVardef@e96345org.highwire.dtl.DTLVardef@7476aaorg.highwire.dtl.DTLVardef@1ac358_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIConstruction of a 10 L membrane-less, pressurizable bioelectrochemical reactor C_LIO_LIRotating working electrodes with up to 1 m2 electrode surface C_LIO_LIElectroautotrophic cultivation and quantification of K. spormannii biofilms C_LIO_LIInitial cell density crucial for successful K. spormannii biofilm formation C_LIO_LIAnodic operation as MEC with Shewanella / Geobacter coculture C_LI

microbiology↗

bioRxiv2021

Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. "Candidatus Micrarchaeum harzensis A_DKE", for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKEs isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for bio-chemical characterisation. MhIDH appeared to be specific for NADP+, yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03{+/-}5.63 {micro}M and 1.94{+/-}0.12 mM and kcat-values of 38.48{+/-}1.62 s-1 and 43.99{+/-}1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDHs exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely attributed to the presence of a proline residue in the NADP+ binding-pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.

biochemistry↗

bioRxiv2021

Unraveling the critical growth factors for stable cultivation of (nano-sized) Micrarchaeota

Micrarchaeota are members of the archaeal DPANN superphylum. These so far poorly characterized archaea have been found to have reduced genomes and likely depend on interactions with host organisms for growth and survival. Here we report on the enrichment of the first stable co-culture of a member of the Micrarchaeota together with its host, as well as the isolation of the latter. Electron microscopic analysis suggest that growth is dependent on the physical interaction of the two organisms within a biofilm. The interaction seems to be ensured by the necessity to grow in form of a biofilm. Furthermore, transcriptomic analyses indicate a shift towards biofilm formation of the host as a result of co-cultivation. Finally, genomic, metabolomic, extracellular polymeric substance (EPSs) and lipid content analyses reveal that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host and thereby provide first insights into the basis of symbiont-host interactions.

microbiology↗

bioRxiv2021

Micrarchaeota are covered by a proteinaceous S-Layer

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proofs that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in A_DKE and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologs of this protein were found in other Micrarchaeota genomes, which raises the question, whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeum expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of A_DKE. Both organisms are clearly distinguishable by cell size, shape and surface structure.

microbiology↗

bioRxiv2021

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis

Some microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalysed by the terminal reductases and as endogenous electron shuttles. In the model organism Shewanella oneidensis, riboflavin production and excretion triggers a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescin decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations which by far exceed the necessary threshold value. ImportanceFerric iron is the fourth most abundant element of the earth crust. It occurs at neutral pH in the form insoluble iron minerals. The dissimilatory reduction of these minerals is an import part of global geological cycles and is catalyzed by microorganisms through extended respiratory chains to the cell surface. Shewanella oneidensis is one of the best understood model organisms for this kind of extracellular respiration. Flavins are important for the reduction of extracellular electron acceptors by S. oneidensis. since they have a function as (I) cofactors of the terminal reductases and (II) electron shuttles. In this study we reveal that flavin molecules are further employed as quorum sensing molecules. They are excreted by the organisms in a growth dependent manner and lead to anaerobic biofilm formation as a specific response at a certain threshold concentration. Although we know multiple examples of quorum sensing mechanisms, the use of riboflavin was so far not described and at least in S. oneidensis proceeds via a new regulatory routine that proceeds on the trancriptomic and posttranscriptomic level.

microbiology↗

bioRxiv2021

Seasonal Dynamics in the Number and Composition of Coliform Bacteria in Drinking Water Reservoirs

Worldwide, surface waters like lakes and reservoirs are one of the major sources for drinking water production, especially in regions with water scarcity. In the last decades, they have undergone significant changes due to climate change. This includes not only an increase of the water temperature but also microbiological changes. In recent years, increased numbers of coliform bacteria have been observed in these surface waters. In our monitoring study we analyzed two drinking water reservoirs (Klingenberg and Kleine Kinzig Reservoir) over a two-year period in 2018 and 2019. We detected high numbers of coliform bacteria up to 2.4 x 104 bacteria per 100 ml during summer months, representing an increase of four orders of magnitude compared to winter. Diversity decreased to one or two species that dominated the entire water body, namely Enterobacter asburiae and Lelliottia spp., depending on the reservoir. Interestingly, the same, very closely related strains have been found in several reservoirs from different regions. Fecal indicator bacteria Escherichia coli and enterococci could only be detected in low concentrations. Furthermore, fecal marker genes were not detected in the reservoir, indicating that high concentrations of coliform bacteria were not due to fecal contamination. Microbial community revealed Frankiales and Burkholderiales as dominant orders. Enterobacterales, however, only had a frequency of 0.04% within the microbial community, which is not significantly affected by the extreme change in coliform bacteria number. Redundancy analysis revealed water temperature, oxygen as well as nutrients and metals (phosphate, manganese) as factors affecting the dominant species. We conclude that this sudden increase of coliform bacteria is an autochthonic process that can be considered as a mass proliferation or "coliform bloom" within the reservoir. It is correlated to higher water temperatures in summer and is therefore expected to occur more frequently in the near future, challenging drinking water production. HighlightsO_LIColiform bacteria proliferate in drinking water reservoirs to values above 104 per 100 ml C_LIO_LIThe genera Lelliottia and Enterobacter can form these "coliform blooms" C_LIO_LIMass proliferation is an autochthonic process, not related to fecal contaminations C_LIO_LIIt is related to water temperature and appears mainly in summer C_LIO_LIIt is expected to occur more often in future due to climate change C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/428560v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@7f63a0org.highwire.dtl.DTLVardef@1659ea7org.highwire.dtl.DTLVardef@932434org.highwire.dtl.DTLVardef@2e12d7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Cultivation of Exoelectrogenic Bacteria in Conductive DNA Nanocomposite Hydrogels Yields a Programmable Biohybrid Materials System

The use of living microorganisms integrated within electrochemical devices is an expanding field of research, with applications in microbial fuel cells, microbial biosensors or bioreactors. We describe the use of porous nanocomposite materials prepared by DNA polymerization of carbon nanotubes (CNT) and silica nanoparticles (SiNP) for the construction of a programmable biohybrid system containing the exoelectrogenic bacterium Shewanella oneidensis. We initially demonstrate the electrical conductivity of the CNT-containing DNA composite by employment of chronopotentiometry, electrochemical impedance spectroscopy, and cyclic voltammetry. Cultivation of Shewanella oneidensis in these materials shows that the exoelectrogenic bacteria populate the matrix of the composite, while non-exoelectrogenic Escherichia coli remain on its surface. Moreover, the ability to use extracellular electron transfer pathways is positively correlated with number of cells within the conductive synthetic biofilm matrix. The Shewanella containing composite remains stable for several days. Programmability of this biohybrid material system is demonstrated by on-demand release and degradation induced by a short-term enzymatic stimulus. The perspectives of this approach for technical applications are being discussed.

biochemistry↗

bioRxiv2017

Machine-assisted cultivation and analysis of biofilms

Biofilms are the natural form of life of the majority of microorganisms. These multispecies consortia are intensively studied not only for their effects on health and environment but also because they have an enormous potential as tools for biotechnological processes. Further exploration and exploitation of these complex systems will benefit from technical solutions that enable integrated, machine-assisted cultivation and analysis. We here introduce a microfluidic platform, where readily available microfluidic chips are connected by automated liquid handling with analysis instrumentation, such as fluorescence detection, microscopy, chromatography and optical coherence tomography. The system is operable under oxic and anoxic conditions, allowing for different gases as feeding sources and offers high spatiotemporal resolution in the analysis of metabolites and biofilm composition. We demonstrate the platforms performance by monitoring the self-organized separation of mixed cultures along autonomously created gradients, the productivity of biofilms along the microfluidic channel and the enrichment of microbial nanoorganisms.

microbiology↗

bioRxiv2017

Laboratory cultivation of acidophilic nanoorganisms. Physiological and bioinformatic dissection of a stable laboratory co-culture.

This study describes the laboratory cultivation of ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms). After 2.5 years of successive transfers in an anoxic medium containing ferric sulfate as an electron acceptor, a consortium was attained that is comprised of two members of the order Thermoplasmatales, a member of a proposed ARMAN group, as well as a fungus. The 16S rRNA of one archaeon is only 91.6% identical to Thermogymnomonas acidicola as most closely related isolate. Hence, this organism is the first member of a new genus. The enrichment culture is dominated by this microorganism and the ARMAN. The third archaeon in the community seems to be present in minor quantities and has a 100% 16S rRNA identity to the recently isolated Cuniculiplasma divulgatum. The enriched ARMAN species is most probably incapable of sugar metabolism because the key genes for sugar catabolism and anabolism could not be identified in the metagenome. Metatranscriptomic analysis suggests that the TCA cycle funneled with amino acids is the main metabolic pathway used by the archaea of the community. Microscopic analysis revealed that growth of the ARMAN is supported by the formation of cell aggregates. These might enable cross feeding by other community members to the ARMAN.

microbiology↗