Biology subjects

Gebhard, S.

Publications and source records attributed to Gebhard, S..

11 recordsLinked to original sources

bioRxiv2025

Decoding the functional role of the calcium ATPase YloB in microbially induced calcite precipitation and sporulation in Solibacillus silvestris

Calcium homeostasis is essential for bacterial physiology, yet its regulation and role in specialized processes such as microbially induced calcite precipitation (MICP) and sporulation remain poorly understood. Here, we characterized the sole annotated P-type calcium ATPase in Solibacillus silvestris, which was previously shown to be upregulated under MICP conditions. We named the protein YloB based on homology to the Bacillus subtilis protein. Structural modeling confirmed that YloB possesses the conserved domains and motifs typical of calcium binding P-type ATPases. Deletion of yloB did not affect growth under elevated calcium levels nor did it reduce MICP yields, indicating that YloB is dispensable for calcium detoxification and biomineralization. In contrast, {Delta}yloB cells produced only partially dehydrated spores with severely reduced heat resistance, a defect rescued by complementation. A YloB-mNeonGreen fluorescent fusion localized specifically to the cell membrane surrounding the forespore during early engulfment, and was retained at this site throughout the remainder of sporulation. A transcriptional PyloB-msfGFP reporter demonstrated that yloB transcription was induced in the early stages of sporulation, but did not respond to a calcium stimulus. These results show that YloB does not act to detoxify calcium from the cytoplasm of S. silvestris, but rather functions to pump calcium into the forespore, enabling Ca-dipicolinic acid accumulation and proper spore maturation. Our study reveals a specialized role for a bacterial calcium ATPase in sporulation, distinguishing calcium transport for endospore development from biomineralization and global calcium homeostasis.

microbiology↗

bioRxiv2024

Metabolic insights into microbially induced calcite formation by Bacillaceae for application in bio-based construction materials

Microbially induced calcite precipitation (MICP) offers promising solutions for sustainable, low-cement infrastructure materials. While it is known how urea catabolism leads to biomineralization, the non-ureolytic pathways of MICP are less clear. This limits the use of the latter in biotechnology, despite its clear benefit of avoiding toxic ammonia release. To address this knowledge gap, the present study explored the interdependence between carbon source utilization and non-ureolytic MICP. We show that acetate can serve as the carbon source driving calcite formation in several environmental Bacillaceae isolates. This effect was particularly clear in a Solibacillus silvestris strain, which could precipitate almost all provided calcium when provided with a 2:1 acetate-to-calcium molar ratio, and we show that this process was independent of active cell growth. Genome sequencing and gene expression analyses revealed an apparent link between acetate catabolism and calcite precipitation in this species, suggesting MICP may be a calcium stress response. Development of a simple genetic system for S. silvestris led to deletion of a proposed calcium binding protein, although this showed minimal effects on MICP. Taken together, this study provides insights into the physiological processes leading to non-ureolytic MICP, paving the way for targeted optimization of biomineralization for sustainable materials development.

microbiology↗

bioRxiv2023

Adrenal extramedullary hematopoiesis as an inducible model of the adult hematopoietic niche

Hematopoietic Stem and Progenitor Cells (HSPCs) reside in the hematopoietic niche, a structure that regulates the balance of cellular quiescence, self-renewal and commitment in a demand-adapted manner. The bone marrow (BM) hematopoietic niche is formed by several cellular players, mainly endothelial cells, osteoblasts, adipocytes, and stromal cells. While the BM niche forms a complex structure, evidence exists for simpler, albeit functional, extramedullary hematopoietic niches. However, the composition of what constitutes the simplest unit of an HSPC supportive microenvironment remains largely unknown. Here, we show that the adult adrenal gland can be transformed into a hematopoietic supportive environment. Upon splenectomy and hormonal stimulation, the adult adrenal gland can be induced to recruit and host HSPC function, including serial transplantation. Furthermore, the adrenal stroma contains a CXCL12+ population, reminiscent of BM CXCL12-Abundant Reticular (CAR) cells. Mirroring this, we found CXCL12+ cells in patient samples obtained from a local cohort of myelolipoma, a benign adrenal tumor composed of adipose and hematopoietic tissue that constitutes the most common site of extramedullary hematopoiesis specific to the adult. We present our model as a novel tool to increase our understanding of the physiology of hematopoietic support and to facilitate the development of a boneless niche model.

cell biology↗

bioRxiv2022

The two-component system CroRS regulates isoprenoid flux to mediate antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis

Antimicrobial tolerance is the ability of a microbial population to survive, but not proliferate, during antimicrobial exposure. Significantly, it has been shown to precede the development of bona fide antimicrobial resistance. We have previously identified the two-component system CroRS as a critical regulator of tolerance to antimicrobials like teixobactin in the bacterial pathogen Enterococcus faecalis. To understand the molecular mechanism of this tolerance, we carried out RNA-seq analyses in the E. faecalis wild-type and isogenic {Delta}croRS mutant to determine the teixobactin-induced CroRS regulon. We identified a 132 gene CroRS regulon and show CroRS upregulates expression of all major components of the enterococcal cell envelope in response to teixobactin challenge. To gain further insight into the function of this regulon we isolated and characterized {Delta}croRS mutants recovered for wild-type growth and tolerance. We show introduction of a single stop codon in a heptaprenyl diphosphate synthase (hppS), a key enzyme in the synthesis of the quinone electron carrier demethylmenaquinone (DMK), is sufficient to rescue loss of cell envelope integrity in the croRS deletion strain. Based on these findings, we propose a model where CroRS acts as a gate-keeper of isoprenoid biosynthesis, mediating flux of isoprenoids needed for cell wall synthesis (undecaprenyl pyrophosphate; UPP) and respiration (DMK) to maintain cell wall homeostasis upon antimicrobial challenge. Dysregulation of this flux in the absence of croRS leads to a loss of tolerance, which is rescued by loss of function mutations in HppS, allowing an increase in isoprenoid flow to UPP and subsequently cell wall synthesis. ImportanceAntimicrobial tolerance is the ability of a microorganism to survive, but not grow upon antimicrobial challenge, and is an important precursor to the development of antimicrobial resistance (the ability to profilerate). Understanding the molecular mechanisms that underpin tolerance will therefore aid in hampering the development of resistance to novel antimicrobials such as teixobactin. CroRS is an essential two-component regulator of antimicrobial tolerance in the bacterial pathogen Enterococcus faecalis. We have determined the antimicrobial-induced CroRS regulon and identified key mutations in a heptaprenyl diphosphate synthase to uncover a novel mechanism of antimicrobial tolerance.

microbiology↗

bioRxiv2022

Novel regulatory logic in the antibiotic resistance response of Enterococcus faecalis against cell envelope targeting antibiotics

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how E. faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

microbiology↗

bioRxiv2022

Chimeric MerR-Family Regulators and Logic Elements for the Design of Metal Sensitive Genetic Circuits in Bacillus subtilis

Whole-cell biosensors are emerging as promising tools for monitoring environmental pollutants such as heavy metals. These sensors constitute a genetic circuit comprising a sensing module and an output module, such that a detectable signal is produced in the presence of the desired analyte. The MerR family of metal-responsive regulators offers great potential for the construction of metal sensing circuits, due to their high sensitivity, tight transcription control and large diversity in metal-specificity. However, the sensing diversity is broadest in Gram-negative systems, while chassis organisms are often selected from Gram-positive species, particularly sporulating bacilli. This can be problematic, because Gram-negative biological parts, such as promoters, are frequently observed to be non-functional in Gram-positive hosts. Herein, we combined construction of synthetic genetic circuits and chimeric MerR regulators, supported by structure-guided design, to generate metal-sensitive biosensor modules that are functional in the biotechnological work-horse species Bacillus subtilis. These chimeras consist of a constant Gram-positive derived DNA-binding domain fused to variable metal binding domains of Gram-negative origins. To improve the specificity of the whole-cell biosensor, we developed a modular AND gate logic system based on the B. subtilis natively split {sigma}-factor, SigO-RsoA, designed to maximise future use for synthetic biology applications in B. subtilis. This work provides insights into the use of modular regulators, such as the MerR family, in the design of synthetic circuits for the detection of heavy metals, with potential wider applicability of the approach to other systems and genetic backgrounds.

synthetic biology↗

bioRxiv2021

Genetic Optimisation of Bacteria-Induced Calcite Precipitation in Bacillus subtilis

BackgroundMicrobially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation. ResultsIn this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity. ConclusionsOur findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

microbiology↗

bioRxiv2020

How to make a dial not a switch: Control of histidine kinase conformation by an ABC-transporter facilitates need-based activation of antibiotic resistance

Bacteria closely control gene expression to ensure optimal physiological responses to their environment. Such careful gene expression can minimize the fitness cost associated with antibiotic resistance. We previously described a novel regulatory logic in Bacillus subtilis enabling the cell to directly monitor its need for detoxification. This cost-effective strategy is achieved via a two-component regulatory system (BceRS) working in a sensory complex with an ABC-transporter (BceAB), together acting as a flux-sensor where signaling is proportional to transport activity. How this is realized at the molecular level has remained unknown. Using experimentation and computation we here show that the histidine kinase is activated by piston-like displacements in the membrane, which are converted to helical rotations in the catalytic core via an intervening HAMP-like domain. Intriguingly, the transporter was not only required for kinase activation, but also to actively maintain the kinase in its inactive state in the absence of antibiotics. Such coupling of kinase activity to that of the transporter ensures the complete control required for transport flux-dependent signaling. Moreover, we show that the transporter likely conserves energy by signaling with sub-maximal sensitivity. These results provide the first mechanistic insights into transport flux-dependent signaling, a unique strategy for energy-efficient decision making.

microbiology↗

bioRxiv2019

In-depth profiling of calcite precipitation by environmental bacteria reveals fundamental mechanistic differences with relevance to application

Microbial-induced calcite precipitation (MICP) has not only helped to shape our planets geological features, but is also a promising technology to address environmental concerns in civil engineering applications. However, limited understanding of the biomineralization capacity of environmental bacteria impedes application. We therefore surveyed the environment for different mechanisms of precipitation across bacteria. The most fundamental difference was ureolytic ability, where urease-positive bacteria caused rapid, widespread increases in pH, while non-ureolytic strains produced such changes slowly and locally. These pH shifts correlated well with patterns of precipitation on solid media. Strikingly, while both mechanisms led to high levels of precipitation, we observed clear differences in the precipitate. Ureolytic bacteria produced homogenous, inorganic fine crystals, whereas the crystals of non-ureolytic strains were larger with a mixed organic/inorganic composition. When representative strains were tested in application for crack healing in cement mortars, non-ureolytic bacteria gave robust results, while ureolytic strains showed more variation. This may be explained by our observation that urease activity varied between growth conditions, or by the different nature and therefore material performance of the precipitate. Our results shed light on the breadth of biomineralization activity among environmental bacteria, an important step towards the rational design of bacteria-based engineering solutions.

microbiology↗

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↗