Biology subjects

Paczia, N.

Publications and source records attributed to Paczia, N..

28 records · Page 2Linked to original sources

bioRxiv2024

Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described {beta}-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. While the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, as well as the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.

microbiology↗

bioRxiv2024

Polyketide synthase-derived sphingolipids determine microbiota-mediated protection against pathogens in C. elegans

Protection against pathogens is a major function of the gut microbiota. Although bacterial natural products have emerged as crucial components of host-microbiota interactions, their exact role in microbiota-mediated protection is largely unexplored. We addressed this knowledge gap with the nematode Caenorhabditis elegans and its microbiota isolate Pseudomonas fluorescens MYb115 that is known to protect against Bacillus thuringiensis (Bt) infection. We find that MYb115-mediated protection depends on sphingolipids that are derived from an iterative type I polyketide synthase (PKS), thereby describing a noncanonical pathway of bacterial sphingolipid production. We provide evidence that MYb115-derived sphingolipids affect C. elegans tolerance to Bt infection by altering host sphingolipid metabolism. This work establishes sphingolipids as structural outputs of bacterial PKS and highlights the role of microbiota-derived sphingolipids in host protection against pathogens.

molecular biology↗

bioRxiv2024

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

microbiology↗

bioRxiv2024

MprF-mediated immune evasion is necessary for Lactiplantibacillus plantarum resilience in Drosophila gut during inflammation

BackgroundMultiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. While MprF has been proven to be crucial for the virulence of various pathogens, its role in commensal gut bacteria remains uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. ResultsThe L. plantarum {Delta}mprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on {Delta}mprF mutants ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the {Delta}mprF mutant after infection-induced inflammation in the guts of wild-type flies but not flies lacking AMPs. These results demonstrate that host AMPs reduce the abundance of the {Delta}mprF mutant during infection. We found in addition that the {Delta}mprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. ConclusionOverall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important resilience factor in maintaining stable microbiota-host interactions during intestinal inflammation.

microbiology↗

bioRxiv2023

An interdependent Metabolic and Genetic Network shows emergent properties in vitro

A hallmark of all living organisms is their ability for self-regeneration which requires a tight integration of metabolic and genetic networks. Here we constructed a metabolic and genetic linked in vitro network (MGLN) that shows life-like behavior outside of a cellular context and generates its own building blocks from non-living matter. To this end, we integrated the metabolism of the crotonyl-CoA/ethyl-malonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle with cell-free protein synthesis using recombinant elements (PURE). We demonstrate that the MGLN produces the essential amino acid glycine from inorganic carbon (CO2), and incorporates it into target proteins following DNA-encoded instructions. By programming genetically encoded response into metabolic networks our work opens new avenues for the development of advanced biomimetic systems with emergent properties, including decision-making, self-regeneration and evolution.

synthetic biology↗

bioRxiv2023

NAD+ metabolism is a key modulator of bacterial respiratory epithelial infections

1.Lower respiratory tract infections caused by StreptococcusOpneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD+ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD+. Knockdown of NAD+ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD+ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD+ production increased NAD+ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD+ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD+. Thus, we identified the NAD+ salvage pathway as an antibacterial cascade in Spn infections, predicting a novel antibacterial mechanism of NAD+.

immunology↗

bioRxiv2023

Ketosynthase engineering enhances activity and shifts specificity towards non-native extender units in type I linear polyketide synthase

Engineering modular type I polyketide synthases (PKS) for the targeted incorporation of non-natural substrates to create variations in the polyketide backbone is a long-standing goal of PKS research. Thus far, most approaches focused on engineering the acyltransferase domain (AT) of PKS, whereas the effects of other ubiquitous domains such as the ketosynthase domain (KS) have received much less attention. In this work, we investigated the effects of thirteen active site substitutions in the module 3 KS (KS3) of the 6-deoxyerythronolide B synthase (DEBS) on incorporation of non-natural extender units in vitro. Using a truncated and a complete DEBS assembly line, we show that substitutions of F263 in KS3 invert specificity up to 1,250-fold towards incorporation of non-natural extender units in the terminal position. In contrast, substitutions of I444 in KS3 show up to 8-fold increased production of 6-deoxyerythonolide B (6-dEB) analogues with non-natural extender units at internal positions. The latter notably without compromising overall productivity of the assembly line. Our study further elucidates the underlying mechanisms for these different behaviors, highlighting the potential of KS engineering for the production of designer polyketides in the future.

biochemistry↗

bioRxiv2022

Identification of NAD-RNAs and ADPR-RNA decapping in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii

NAD is a coenzyme central to metabolism that was also found to serve as a 5-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5[->]3 exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells. ImportanceThis study reports the first characterization of 5-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.

molecular biology↗

bioRxiv2019

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinsons disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.

biochemistry↗

bioRxiv2018

NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells

NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both the S- and R-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivatives in vivo and their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD+ and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair deficient children.

biochemistry↗