Biology subjects

Seibold, G. M.

Publications and source records attributed to Seibold, G. M..

8 recordsLinked to original sources

bioRxiv2022

Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies

Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Towards versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.

synthetic biology↗

bioRxiv2022

Combined sensor-based monitoring of mycothiol redox potential and DNA-damage response in Corynebacterium glutamicum

Excessive amounts of reactive oxygen species (ROS) can cause irreversible damages to essential cellular components such as DNA. Genetically encoded biosensors targeting oxidative stress and DNA-stress have emerged to a powerful analytical tool to assess physiological states in a non-invasive manner. In this study, we aimed to combine the redox biosensor protein Mrx1-roGFP2 with a transcriptional biosensor for DNA-damage based on the PrecA promoter fused to a reporter gene (e2-crimson) in Corynebacterium glutamicum. Therefore, the redox biosensor strains C. glutamicum WT_Mrx1-roGFP2 and the mycothiol (MSH)-deficient mutant strain C. glutamicum {Delta}mshC_Mrx1-roGFP2 were equipped with the DNA-stress reporter plasmid pJC1_PrecA_e2-crimson. Exposure of the double-sensor equipped C. glutamicum WT strain to hypochlorite resulted in an oxidative redox shift, accompanied by an induction of the DNA-stress reporter system. In absence of the major non-enzymatic antioxidant MSH, the induction of the DNA-stress response was even more pronounced. This confirms the linkage of oxidative stress and DNA-damage response, and therefore making antioxidants a crucial player to protect DNA. Furthermore, exposure of the double biosensor strains to a DNA-damage inducing agent resulted in an oxidative redox shift. These results suggest a direct link of DNA-damage and oxidative stress response in C. glutamicum. Finally, we observed that inhibition of cell wall biosynthesis by penicillin caused both an oxidative redox shift and a DNA-damage response in C. glutamicum. The excellent compatibility of Mrx1-roGFP2 with E2-Crimson shown here provides a powerful combinatorial biosensor concept for in-depth studies of redox-related physiology in future studies.

microbiology↗

bioRxiv2021

Impedance flow cytometry for viability analysis of Corynebacterium glutamicum

Corynebacterium glutamicum efficiently produces glutamate when growth is inhibited. Analyses of viability in this non-growing state requires time consuming plating and determination of colony forming units. We here establish impedance flow cytometry measurements to assess the viability of non-growing, glutamate producing C. glutamicum cultures within minutes.

microbiology↗

bioRxiv2021

Angicin, a novel bacteriocin of Streptococcus anginosus

As a conserved defense mechanism, many bacteria produce antimicrobial peptides, called bacteriocins, which give a colonization advantage in a multispecies environment. Here the first bacteriocin of Streptococcus anginosus, designated Angicin, is described. S. anginosus is commonly described as a commensal, however it also possesses a high pathogenic potential. Therefore, understanding factors contributing to its host colonization and persistence are important. A radial diffusion assay was used to identify S. anginosus BSU 1211 as a potent bacteriocin producer. By genetic mutagenesis the background of bacteriocin production and the bacteriocin gene itself were identified. Synthetic Angicin shows high activity against closely related streptococci, listeria and vancomycin resistant enterococci. It has a fast mechanism of action and causes a membrane disruption in target cells. Angicin, present in cell free supernatant, is insensitive to changes in temperature from -70 to 90 {degrees}C and pH values from 2-10, suggesting that it represents an interesting compound for potential applications in food preservation or clinical settings.

microbiology↗

bioRxiv2021

Towards improved resistance of Corynebacterium glutamicum against nisin

The bacteriocin nisin is one of the best studied antimicrobial peptides. It is widely used as a food preservative due to its antimicrobial activity against various Gram-positive bacteria including human pathogens such as Listeria monocytogenes and others. The receptor of nisin is the universal cell wall precursor lipid II, which is present in all bacteria. Thus, nisin has a broad spectrum of target organisms. Consequently, heterologous production of nisin with biotechnological relevant organisms including Corynebacterium glutamicum is difficult. Nevertheless, bacteria have evolved several mechanisms of resistance against nisin and other cationic antimicrobial peptides (CAMPs). Here, we transferred resistance mechanisms described in other organisms to C. glutamicum with the aim to improve nisin resistance. The presented approaches included: expression of (i) nisin immunity genes nisI and/or nisFEG or (ii) nisin ABC-transporter genes of Staphylococcus aureus and its homologues of C. glutamicum, (iii) genes coding for enzymes for alanylation or lysinylation of the cell envelope to introduce positive charges, and/or (iv) deletion of genes for porins of the outer membrane. None of the attempts alone increased resistance of C. glutamicum more than two-fold. To increase resistance of C. glutamicum to levels that will allow heterologous production of active nisin at relevant titers, further studies are needed.

microbiology↗

bioRxiv2021

Visualizing the pH in Escherichia coli colonies via the sensor protein mCherryEA allows high-throughput screening of mutant libraries

Cytoplasmic pH is tightly regulated by diverse active mechanisms and interconnected regulatory processes in bacteria. Many processes and regulators underlying pH-homeostasis have been identified via phenotypic screening of strain libraries towards non-growth at low or high pH values. Direct screens with respect to changes of the internal pH in mutant strain collections are limited by laborious methods including fluorescent dyes or radioactive probes. Genetically encoded biosensors equip single organisms or strain libraries with an internal sensor molecule already during the generation of the strain. In this study, we used the pH-sensitive mCherry variant mCherryEA as ratiometric pH biosensor. We visualized the internal pH of E. coli colonies on agar plates by the use of a Gel-Doc imaging system. Combining this imaging technology with robot-assisted colony picking and spotting allowed us to screen and select mutants with altered internal pH values from a small transposon mutagenesis derived E. coli library. Identification of the TN- insertion sites in strains with altered internal pH levels revealed that the transposon was inserted into trkH (encoding a transmembrane protein of the potassium uptake system) or the rssB gene (encoding the anti-adaptor protein RssB which mediates the proteolytic degradation of the general stress response regulator RpoS), two genes known to be associated with pH-homeostasis and pH stress adaptation. This successful screening approach demonstrates that the pH- sensor based analysis of arrayed colonies on agar plates is a sensitive approach for the fast identification of genes involved in pH-homeostasis or pH stress adaptation in E. coli. ImportancePhenotypic screening of strain libraries on agar plates has become a versatile tool to understand gene functions and to optimize biotechnological platform organisms. Screening is supported by genetically encoded biosensors that allow to easily measure intracellular processes. For this purpose, transcription-factor-based biosensors have emerged as the sensor-type of choice. Here, the target stimulus initiates the activation of a response gene (e.g. a fluorescent protein) followed by transcription, translation and maturation. Due to this mechanistic principle, biosensor readouts are delayed and cannot report the actual intracellular state of the cell in real-time. To capture fast intracellular processes adequately, fluorescent reporter proteins are extensively applied. But these sensor-types are not utilized for phenotypic screenings so far. To take advantage of their properties, we here established an imaging method, which allows to apply a fast ratiometric sensor protein for assessing the internal pH of colonies in a high-thoughput manner.

microbiology↗

bioRxiv2021

Evolving a new efficient mode of fructose utilization for improved bioproduction in Corynebacterium glutamicum

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (~10 %) compared to glucose (~60 %). Consequently, the biosynthesis of NADPH demanding products, e.g. L-lysine, by C. glutamicum is largely decreased, when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and consequently NADPH availability. Here, we generated deletion strains either lacking in the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose specific PTS, as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased biomass formation, reduced side-product accumulation, and increased L-lysine production by 50 %.

synthetic biology↗

bioRxiv2019

Immediate inhibition of sucrose uptake in Corynebacterium glutamicum in response to intracellular glucose-6-phosphate accumulation requires the ptsG encoded EII-permease

Corynebacterium glutamicum co-metabolizes most carbon sources, such as glucose and sucrose. Uptake of those sugars by the PTS involves a glucose- and a sucrose-specific permease EIIGlc (ptsG) and EIISuc (ptsS), respectively. Block of glycolysis by deletion of pgi (encodes phosphoglucoisomerase) redirects glucose-driven carbon flux towards pentose phosphate pathway. C. glutamicum {Delta}pgi grows poorly with glucose but has unaffected, good growth with sucrose. However, addition of glucose to sucrose-cultivated C. glutamicum {Delta}pgi immediately arrested growth via inhibition of the EIISuc-mediated sucrose uptake and reduction of ptsS-mRNA amounts. Kinetic analyses revealed that sucrose uptake inhibition in C. glutamicum {Delta}pgi took place within 15 s after glucose addition. We show that inhibition of PTS-mediated sucrose uptake occurs as direct response to glucose-6-P accumulation. Moreover, addition of non-PTS substrates, which are metabolized to glucose-6-P such as maltose or glucose-6-P itself (uptake was enabled by heterologously produced UhpT), led to similar growth and sucrose uptake inhibition as glucose addition. Despite EIIGlc not being involved in uptake of these substrates, negative effects on sucrose uptake after addition of maltose and glucose-6-P were absent in the EIIGlc-deficient strain C. glutamicum {Delta}pgi{Delta}ptsG. These results show that the ptsG-encoded EIIGlc is part of a novel mechanism for perception of intracellular glucose-6-P accumulation and instantaneous inhibition of EIISuc-mediated sucrose uptake in C. glutamicum. This novel mode of control of PTS activity by an early glycolytic metabolite probably allows efficient adaptation of sugar uptake to the capacity of the central metabolism during co-metabolization, which is characteristic for C. glutamicum.\n\nIMPORTANCECoordination of substrate uptake and metabolism are a prerequisite for efficient co-utilization of substrates, a trait typical for the Gram-positive C. glutamicum. Sucrose uptake via the PTS permease EIISuc in this organism immediately was inhibited in response to intracellular accumulation of the glycolysis intermediate glucose-6-phosphate. This inhibition depends exclusively on the presence but not activity of the PTS permease EIIGluc. Thus, C. glutamicum possesses a novel, immediate, and PTS-dependent way to control and coordinate both uptake and metabolization of multiple substrates by monitoring of their metabolic levels in the cell. This offers new insights and interesting concepts for a further rational engineering of this industrially important production organism and exemplifies a putative general strategy of bacteria for the coordination of sugar uptake and central metabolism.

microbiology↗