Biology subjects

Frankenberg-Dinkel, N.

Publications and source records attributed to Frankenberg-Dinkel, N..

4 recordsLinked to original sources

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↗

bioRxiv2021

A bacterial signal transduction phosphorelay in the methanogenic archaeon Methanosarcina acetivorans

Signal transduction via two-component systems is a powerful tool for microorganisms to respond to environmental changes. Histidine kinases originating from Bacteria are the most common signaling enzymes and are also present in Archaea, but not in all phyla. A total of 124 bacterial-type histidine kinases and/or regulators were identified in a screen of 149 Euryarchaeota genomes, but little is known about the signal transfer and molecular regulation of these systems (1). In this work, the hybrid kinase MA4377 from the methanogenic archaeon Methanosarcina acetivorans was investigated (2, 3). MA4377 is a multidomain protein resembling a bacterial-type histidine kinase with two additional receiver domains at the C-terminus. Recombinant protein was employed to investigate the intra- and intermolecular phosphorelay in vitro. The kinase displays autophosphorylation activity of histidine residue 497. While no intramolecular phosphorelay was observed, the CheY-like receiver protein MA4376 was identified as part of the multi-component system that also seems to include the Msr-type transcription factor MA4375. This study reveals the presence and in vitro function of a bacterial-type hybrid histidine kinase integrated into an archaeal phosphorelay system. ImportanceSignal transduction enables organisms to rapidly react to changes in their surroundings. Different systems containing one-, two- or multiple-components are employed to sense and react to environmental changes. Most commonly, external perceived stimuli are converted into internal signals through protein phosphorylation. These systems are found in all domains of life but understood at different levels of complexity, the least being those from the domain of Archaea. By better elucidating the function of these early signal transduction systems, we will gain insight and understanding of the selection pressures on signal transduction pathways, and their evolution within the Archaea.

microbiology↗

bioRxiv2020

Discovery of a novel small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded. This degradation is tightly coordinated, follows a genetic program and involves small proteins serving as proteolysis adaptors. Here, we describe the role of NblD, a novel factor in this process in cyanobacteria. NblD is a cysteine-rich, 66-amino acid small protein that becomes rapidly induced upon nitrogen starvation. Deletion of the nblD gene in the cyanobacterium Synechocystis prevents the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype. Competition experiments provided direct evidence for the physiological importance of NblD. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type. Overexpression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. Transcriptome analysis revealed that nitrogen starvation correctly induces nblA1/2 transcription in the {Delta}nblD strain implying that NblD does not act as a transcriptional (co-)regulator. However, fractionation and coimmunoprecipitation experiments indicated the presence of NblD in the phycobilisome fraction and identified the {beta}-phycocyanin subunit as its target. These data add NblD as a new factor to the genetically programmed response to nitrogen starvation and demonstrate that it plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. Significance StatementDuring genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the small protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells run low in nitrogen, a condition that frequently occurs in nature. Similar to the NblA proteins that label phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration.

microbiology↗

bioRxiv2020

Exchange of a single amino acid residue in the cryptophyte phycobiliprotein lyase GtCPES expands its substrate specificity

Cryptophyte algae are among the few eukaryotes that employ phycobiliproteins (PBP) for light harvesting during oxygenic photosynthesis. In contrast to the cyanobacterial PBP that are organized in large membrane-associated super complexes, the phycobilisomes, those from cryptophytes are soluble within the chloroplast thylakoid lumen. Their light-harvesting capacity is due to covalent linkage of several open-chain tetrapyrrole chromophores (phycobilins). Guillardia theta utilizes the PBP phycoerythrin PE545 with 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB) as chromophores. Thus far, the assembly of cryptophyte PBPs is not yet completely understood but involves the action of PBP-lyases as shown for cyanobacterial PBP. PBP-lyases facilitate the attachment of the chromophore in the right configuration and stereochemistry. Here we present the functional characterization of eukaryotic S-type PBP lyase GtCPES from G. theta. We show GtCPES mediated transfer and covalent attachment of PEB to the conserved Cys82 of the acceptor PBP {beta}-subunit (PmCpeB) of Prochlorococcus marinus MED4. Based on the previously solved crystal structure, the GtCPES binding pocket was investigated using site-directed mutagenesis. Thereby, amino acid residues involved in phycobilin binding and transfer were identified. Interestingly, exchange of a single amino acid residue Met67 to Ala extended the substrate specificity to phycocyanobilin (PCB) likely by enlarging the substrate-binding pocket. Variant GtCPES_M67A binds both PEB and PCB forming a stable, colorful complex in vitro and in vivo produced in Escherichia coli. GtCPES_M67A is able to mediate PCB transfer to Cys82 of PmCpeB. Based on our data we postulate that a single amino acid residue determines the bilin-specificity of phycoerythrin S-type lyases but that additional factors regulate hand over to the target protein.

biochemistry↗