Biology subjects

Fritz, G.

Publications and source records attributed to Fritz, G..

23 records · Page 2Linked to original sources

bioRxiv2021

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens

Vibrio natriegens is known as the worlds fastest growing organism with a doubling time of less than 10 minutes. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection - a highly flexible Golden Gate Assembly-based cloning toolbox optimized for the emerging chassis organism V. natriegens. The Marburg Collection overcomes the paradigm of plasmid construction - integrating inserts into a backbone - by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organismspecific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

synthetic biology↗

bioRxiv2020

Co-evolutionary analysis reveals a conserved dual binding interface between extracytoplasmic function (ECF) σ factors and class I anti-σ factors

Extracytoplasmic function {sigma} factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Amongst the diverse regulators of ECF activity, class I anti-{sigma} factors are the most important signal transducers in response to internal and external stress conditions. Despite the conserved secondary structure of the class I anti-{sigma} factor domain (ASDI) that binds and inhibits the ECF under non-inducing conditions, the binding interface between ECFs and ASDIs is surprisingly variable between the published co-crystal structures. In this work, we provide a comprehensive computational analysis of the ASDI protein family and study the different contact themes between ECFs and ASDIs. To this end, we harness the co-evolution of these diverse protein families and predict covarying amino acid residues as likely candidates of an interaction interface. As a result, we find two common binding interfaces linking the first -helix of the ASDI to the DNA binding region in the {sigma}4 domain of the ECF, and the fourth -helix of the ASDI to the RNA polymerase (RNAP) binding region of the {sigma}2 domain. The conservation of these two binding interfaces contrasts with the apparent quaternary structure diversity of the ECF/ASDI complexes, partially explaining the high specificity between cognate ECF and ASDI pairs. Furthermore, we suggest that the dual inhibition of RNAP- and DNA-binding interfaces are likely a universal feature of other ECF anti-{sigma} factors, preventing the formation of non-functional trimeric complexes between {sigma}/anti-{sigma} factors and RNAP or DNA. SignificanceIn the bacterial world, extracytoplasmic function {sigma} factors (ECFs) are the most widespread family of alternative {sigma} factors, mediating many cellular responses to environmental cues, such as stress. This work uses a computational approach to investigate how these {sigma} factors interact with class I anti-{sigma} factors - the most abundant regulators of ECF activity. By comprehensively classifying the anti-{sigma}s into phylogenetic groups and by comparing this phylogeny to the one of the cognate ECFs, the study shows how these protein families have co-evolved to maintain their interaction over evolutionary time. These results shed light on the common contact residues that link ECFs and anti-{sigma}s in different phylogenetic families and set the basis for the rational design of anti-{sigma}s to specifically target certain ECFs. This will help to prevent the cross-talk between heterologous ECF/anti-{sigma} pairs, allowing their use as orthogonal regulators for the construction of genetic circuits in synthetic biology.

bioinformatics↗

bioRxiv2019

Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family

Extracytoplasmic function {sigma} factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As result, we identified [~]10 ECFs per bacterial genome on average and classified them into 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands the number of unique ECF sequences [~]50-fold relative to previous classification efforts, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences, enables the application of in silico tools for the prediction of important functional residues, and serves as a powerful hypothesis-generator to guide future research in the field.

bioinformatics↗

bioRxiv2019

BceAB-type antibiotic resistance transporters appear to act by target protection of cell wall synthesis

Resistance against cell wall-active antimicrobial peptides in bacteria is often mediated by transporters. In low GC-content Gram-positive bacteria, a wide-spread type of such transporters are the BceAB-like systems, which frequently provide a high level of resistance against peptide antibiotics that target intermediates of the lipid II cycle of cell wall synthesis. How a transporter can offer protection from drugs that are active on the cell surface, however, has presented researchers with a conundrum. Multiple theories have been discussed, ranging from removal of the peptides from the membrane, internalisation of the drug for degradation, to removal of the cellular target rather than the drug itself. To resolve this much-debated question, we here investigated the mode of action of the transporter BceAB of Bacillus subtilis. We show that it does not inactivate or import its substrate antibiotic bacitracin. Moreover, we present evidence that the critical factor driving transport activity is not the drug itself, but instead the concentration of drug-target complexes in the cell. Our results, together with previously reported findings, lead us to propose that BceAB-type transporters act by transiently freeing lipid II cycle intermediates from the inhibitory grip of antimicrobial peptides, and thus provide resistance through target protection of cell wall synthesis. Target protection has so far only been reported for resistance against antibiotics with intracellular targets, such as the ribosome. However, this mechanism offers a plausible explanation for the use of transporters as resistance determinants against cell wall-active antibiotics in Gram-positive bacteria where cell wall synthesis lacks the additional protection of an outer membrane.

microbiology↗

bioRxiv2019

From modules to networks: A systems-level analysis of the bacitracin stress response in Bacillus subtilis

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic, and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of B. subtilis towards the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild-type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response towards this stressor.\n\nImportanceAntibiotic resistance poses a major threat to global health and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combine experiments and theoretical modelling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. In response to bacitracin stress we find a high level of cross-regulation between the two major resistance modules and quantify their effects on bacterial resistance. To rationalize our experimental data, we expand a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This leads us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

systems biology↗