Biology subjects

Zehner, S.

Publications and source records attributed to Zehner, S..

6 recordsLinked to original sources

bioRxiv2026

Comparative study on chromatic acclimation in the two Synechococcus species WH8109 and RS9916

Marine Synechococcus cyanobacteria can modify their light-harvesting phycobilisome antennae in response to spectral changes through chromatic acclimation (CA). CA type IV (CA4) involves adjustment of the ratio of the blue-absorbing phycourobilin (PUB) to the green-absorbing phycoerythrobilin (PEB). Although CA4 is associated with a genomic island occurring in two configurations (CA4-A and CA4-B), some strains carrying this island, including Synechococcus sp. WH8109, have been reported to lack CA4 activity. Here, we investigated whether WH8109 undergoes CA4 under blue light (BL) and green light (GL) conditions, using the CA4-positive strain RS9916 as a reference. Whole-cell fluorescence, flow cytometry, isolated phycobilisome analysis, and sequencing of the CA4-B genomic island were used to assess pigment acclimation and identify potential genetic differences. WH8109 showed a stable PUB:PEB ratio of approximately 0.71 under BL and GL, with no detectable changes in PUB content or phycobilisome chromophore composition. In contrast, RS9916 exhibited clear CA4, with PUB:PEB ratios of 1.57 under BL and 0.72 under GL. Sequencing revealed no insertions, deletions, or other sequence anomalies in the WH8109 CA4-B genomic island compared to the public sequence. These results demonstrate that WH8109 does not undergo detectable CA4 under the conditions tested, despite possessing an intact CA4-B genomic island, highlighting a genotype-phenotype discrepancy that warrants further investigation.

microbiology↗

bioRxiv2026

The phosphodiesterase NbdA links c-di-GMP signaling to type IV pili function in Pseudomonas aeruginosa PAO1

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

microbiology↗

bioRxiv2026

Inosine Monophosphate Dehydrogenases are key players for functional development in Arabidopsis

In plants, nucleotide de novo synthesis is required throughout development to provide building blocks for the synthesis of DNA, RNA and nucleotide-derived cofactors. Inosine Monophosphate Dehydrogenases (IMPDH) are key enzymes in de novo guanylate synthesis. The genome of Arabidopsis thaliana encodes two IMPDHs and we identify IMPDH2 as the major isoform, being enzymatically active in the plant cytosol. Bimolecular fluorescence complementation (BIFC) analysis suggests that IMPDH1 and IMPDH2 interact with themselves and with Cytidine Triphosphate Synthetase (CTPS) isoforms, connecting purine and pyrimidine metabolism. Nucleotide quantification identifies IMPDH2 as bottleneck in guanylate biosynthesis, and plants lacking this isoform suffer from nucleotide imbalance and limitation, causing reductions in ribosomal RNAs by reducing Target of Rapamycin (TOR) activity. Impaired photosynthesis and growth were further consequences thereof. In contrast, strong overexpression of IMPDH1 supported growth and led to altered organ development in 10% of corresponding plants presumably due to altered auxin metabolism during embryo development. Significance statementThis study reveals that Inosine Monophosphate Dehydrogenase 2 (IMPDH2) is required for early Arabidopsis development and growth. Knock-out mutants exhibit reduced amounts of metabolites (e.g. XMP, GMP, GTP) accompanied by reduced expression of genes in the GO-Terms Ribosome and Photosynthesis at seedling stage. This leads to reduced chlorophyll levels, impaired photosynthesis, and reduced growth. Marked overexpression of IMPDH1 provoked abnormal organ development in 10% of cases presumably due to altered auxin metabolism.

plant biology↗

bioRxiv2023

Virus-encoded Shemin pathway highlights the importance of tetrapyrrole metabolism during host infection

Tetrapyrroles such as heme, chlorophyll, or vitamin B12 are among the most complex molecules synthesised by nature and play a crucial role in many vital processes such as respiration, photosynthesis, methanogenesis, and catalysis. All modified tetrapyrroles are derived from the common precursor molecule 5-aminolevulinic acid (5-ALA). However, 5-ALA can be synthesised in two different ways. While alphaproteobacteria, mammals, and birds use the so-called Shemin pathway, all other bacteria, archaea, and plants use the C5 pathway. Here, we present evidence for the presence of 5-ALA synthase genes of the Shemin pathway in the genomes of viruses (valaS) that infect bacteria (bacteriophages) in marine and freshwater environments. These genes either occur in a three-gene cassette with two heme catabolising enzymes involved in linear tetrapyrrole biosynthesis, as broken cassettes or as individual genes. valaS encodes a functional enzyme, as shown by the enzymatic turnover of the co-substrates succinyl-Coenzyme A and glycine in an Escherichia coli strain overexpressing valaS. Phylogenetic analysis in combination with three-dimensional structure prediction supported our data that the viral sequences encode active enzymes. Interestingly, the viral valaS sequence is capable of functionally complementing a 5-ALA-auxotrophic Escherichia coli strain, which lacks one of the two enzymes of the C5 pathway. Host prediction suggests that different valaS carrying phages might infect bacteria that either utilise the Shemin or the C5 pathway for 5-ALA biosynthesis. Our results thus support the hypothesis that metabolic reprogramming and maintenance of the host bacteriums energy metabolism are crucial during bacteriophage infection and that tetrapyrroles play a key role in this process.

microbiology↗

bioRxiv2023

Expanding the toolbox for phycobiliprotein assembly: phycoerythrobilin biosynthesis in Synechocystis

Phycobiliproteins (PBPs) play a vital role in light harvesting by cyanobacteria, which enables efficient utilization of photon energy for oxygenic photosynthesis. The PBPs carry phycobilins, open-chain tetrapyrrole chromophores derived from heme. The structure and chromophore composition of PBPs is dependent on the organisms ecological niche. In cyanobacteria, these holo-proteins typically form large, macromolecular antenna complexes called phycobilisomes (PBSs). The PBS of Synechocystis sp. PCC 6803 (hereafter Synechocystis) consists of allophycocyanin (APC) and phycocyanin (PC), which exclusively harbor phycocyanobilin (PCB) as a chromophore. Investigations into heterologous PBP biosynthesis in E. coli have proven limiting with respect to PBP assembly and their functional characterization. Consequently, we wanted to engineer a platform for the investigation of heterologously produced PBPs, focusing on unusual, phycoerythrobilin (PEB)-containing light-harvesting proteins called phycoerythrins (PEs) in Synechocystis. As a first step, a gene encoding for the synthesis of the natural cyanobacterial chromophore, PEB, was introduced into Synechocystis. We provide spectroscopic evidence for heterologous PEB formation and show covalent attachment of PEB to the -subunit of PC, CpcA, by HPLC and LC-MS/MS analyses. Fluorescence microscopy and PBS isolation demonstrate a cellular dispersal of PBPs with modified phycobilin content. However, these modifications have minor effects on physiological responses, as demonstrated by growth rates, oxygen evolution, nutrient accumulation, and PBP content analyses. As a result, Synechocystis demonstrates the capacity to efficiently manage PEB biosynthesis and therefore reflects a promising platform for both biochemical and physiological investigations of foreign and unusual PEs.

synthetic biology↗

bioRxiv2021

The Pseudomonas aeruginosa phosphodiesterase gene nbdA is transcriptionally regulated by RpoS and AmrZ

Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function. IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa possesses a network of genes encoding proteins for the turnover of the second messenger c-di-GMP involved in regulating-among others-the lifestyle switch between planktonic, motile cells and sessile biofilms. Insight into the transcriptional regulation of these genes is important for the understanding of the protein function within the cell. Determination of the transcriptional start site of the phosphodiesterase gene nbdA revealed a new promoter region and consequently a shortened open reading frame for the corresponding protein. Binding sites for RpoS and AmrZ were identified in silico and confirmed experimentally. Previously reported regulation by nitric oxide was reevaluated and a strong influence of the moonlighting protein NirS identified.

microbiology↗