Biology subjects

Frankenberg-Dinkel, N.

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

17 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

Evolutionary insights into bilin biosynthesis: Functional characterization of pre-PcyA enzymes

Biosynthesis of the linear tetrapyrrole phycocyanobilin (PCB) by the ferredoxin-dependent bilin reductase (FDBRs) PcyA is essential for light-harvesting and regulatory processes in diverse photosynthetic organisms, yet its evolutionary origins are not fully understood. PcyA evolved from pre-PcyA proteins found in diverse bacteria. Three lineages of pre-PcyA proteins were identified: Pre-1, Pre-2 and Pre-3. Using an in vivo co-expression assay, Pre-2 and Pre-3 proteins were shown to be active FDBRs that did not synthesize PCB, whereas Pre-1 activity was apparently low. In refining these results, we noted a discrepancy between phycoerythrobilin populations generated by Pre-3 and by the distantly related FDBR PebS. We therefore examined the properties of pre-PcyA enzymes in vitro, using an updated pre-PcyA phylogeny to select an alternative pre-1 target. Biochemical analyses revealed that Pre-1 and Pre-2 catalyze the two-electron reduction of biliverdin (BV) to 3E-phytochromobilin (3E-P[FE]B), in contrast to the known synthesis of 3Z-phytobilins by other FDBRs. Pre-3 can also carry out an additional two-electron reduction to yield 3E-phycoerythrobilin (3E-PEB), again distinct from the 3Z-PEB produced by PebS. We then used comparative sequence and structure analysis to target candidate catalytic residues for site-directed mutagenesis. Variant Pre-1 exhibited altered product stereochemistry, but no effects on Pre-2 were observed and Pre-3 variants unexpectedly gained the ability to bind cyclic tetrapyrroles. These findings underscore the plasticity and promiscuity of this enzyme family. Together, this work illustrates how the flexible catalytic potential of ancestral enzymes shaped the evolution and diversification of bilin biosynthetic pathways.

biochemistry↗

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

Phylogenetic support of pebS as a phage-exclusive auxiliary metabolic gene

Marine picocyanobacteria, including the genera Prochlorococcus and Synechococcus, are major contributors to oceanic photosynthesis and global primary production. Their populations are influenced by T4-like cyanophages, which frequently encode auxiliary metabolic genes (AMGs) capable of altering host metabolism during infection. One such AMG, pebS, encodes a ferredoxin-dependent bilin reductase (FDBR) phycoerythrobilin (PEB) synthase, which converts biliverdin IX to PEB. In contrast, cyanobacteria perform a two-step reaction using the FDBR enzymes PebA (15,16-dihydrobiliverdin:ferredoxin oxidoreductase) and PebB (PEB:ferredoxin oxidoreductase), whereas pebS has not been reported in cyanobacterial genomes. Here, we re-evaluated whether pebS is truly restricted to cyanophages by searching the Ocean Gene Atlas and all available cyanobacterial genomes at NCBI using a cyanophage-derived PebS sequence as query. Using protein phylogenies, we find that most search hits group with PebA or PebB, while few sequences from cyanobacterial genome assemblies were confirmed to belong to PebS based on phylogenetic placement. However, genomic context analysis of these pebS sequences revealed that they are phage-derived, consistent with cyanophage infection at the time of sampling. In conclusion, our results support that pebS is absent in cyanobacterial genomes, raising questions about the evolutionary and biochemical rationale for the two-step reduction of biliverdin IX to PEB in these organisms.

microbiology↗

bioRxiv2025

Understanding PebS-Ferredoxin Recognition: A Structural Perspective on Viral and Host Redox Partners

Cyanophages play a critical role in shaping host metabolism during infection by encoding auxiliary metabolic genes (AMGs), including enzymes involved in phycobilin biosynthesis. PebS (phycoerythrobilin synthase) is a phage-encoded ferredoxin-dependent biliverdin reductase (FDBR) that catalyses the two-step reduction of biliverdin IX (BV) to phycoerythrobilin (PEB). Although high-resolution structures are available for both PebS and its electron donor ferredoxin (Fd), the structural details governing their interaction remain unclear. This study leverages the well-characterised structural models of PebS and a host-like ferredoxin from Thermosynechococcus elongatus (Te-Fd), whose NMR structure provides a reliable basis for probing protein- protein interactions. Using [{superscript 1}N]-labelled, gallium-substituted Te-Fd, we employed NMR spectroscopy to monitor chemical shift perturbations upon binding, enabling us to probe the interaction interface in solution. Based on these data, we conducted protein-protein docking with HADDOCK to predict the interaction interface between Te-Fd and PebS. PebS variants designed to disrupt this interface did indeed show corresponding alterations in enzymatic efficiency and product formation as determined by time resolved UV/Vis spectroscopy and HPLC analyses. Utilizing the Fd encoded in the cyanophage PSSM2-Fd in our assays, we could observe a significantly improved catalytic activity, suggesting an coevolution of phage enzyme and electron donor. A comparison of the two available X-ray structures of Te-Fd and PSSM2-Fd with an alphafold-model of the Fd of the natural host Prochlorococcus NATL1A (NATL1A-Fd) also supports this evolutionary adaptation and the role of both PSSM2-Fd and PebS as AMGs involved in viral infection by PSSM2.

molecular biology↗

bioRxiv2025

Phycocyanobilin biosynthesis in Galdieria sulphuraria requires isomerization of phycoerythrobilin synthesized by bilin reductases

Cyanobacteria and red algae (Rhodophyta) utilize phycobilisomes as light-harvesting antennae to carry out oxygenic photosynthesis. Phycobilisomes are large protein complexes comprised of phycobiliproteins and associated linker proteins, with phycobiliproteins carrying one or more linear tetrapyrrole (bilin) chromophores. Bilin biosynthesis is typically catalyzed by ferredoxin-dependent bilin reductases (FDBRs). Cyanobacterial genomes encode one FDBR, PcyA, for biosynthesis of phycocyanobilin (PCB), which is required both for harvesting red light and for coupling phycobilisomes to photosynthetic reaction centers via energy transfer. Some cyanobacteria also contain two additional FDBRs (PebA and PebB) for synthesis of the green-absorbing phycoerythrobilin (PEB). Most rhodophytes contain only PebA and PebB homologs (PEBA and PEBB), so the biosynthesis of PCB in such organisms is not understood. This process is especially relevant in the early-branching rhodophyte Galdieria sulphuraria, which contains PCB as the main light-harvesting chromophore yet contains the PEBA and PEBB FDBRs for PEB biosynthesis. In this organism, PCB biosynthesis is thought to be dependent both on FDBRs and on a yet-to-be identified bilin isomerase. Our current work reaffirms these findings. Phylogenetic analysis clearly places G. sulphuraria FDBRs in the PEBA and PEBB clades, in contrast to the PCYA found in Cyanidioschyzon merolae, another early-branching rhodophyte. All three rhodophyte FDBRs were found to have the expected substrate preferences and enzymatic activities after recombinant expression, affinity purification, and in vitro characterization. Therefore, bilin biosynthesis in G. sulphuraria results in biosynthesis of PEB rather than PCB. However, a previous procedure allowed partial purification of a PEB:PCB isomerase, clearly demonstrating the existence of such an activity and adding to our understanding of bilin biosynthesis and algal light harvesting.

plant biology↗

bioRxiv2024

Archaeal signalling networks - new insights into the structure and function of histidine kinases and response regulators of the methanogenic archaeon Methanosarcina acetivorans

The methanogenic archaeon Methanosarcina acetivorans has one of the largest known archaeal genomes. With 53 histidine kinases (HK), it also has the largest set of signal transduction systems. To gain insight into the hitherto not very well understood signal transduction in archaea and M. acetivorans in particular, we have categorized the predicted HK into four types based on their H-box using an in silico analysis. Representatives of three types were recombinantly produced in Escherichia coli and purified by affinity chromatography. All investigated kinases showed ATP binding and hydrolysis. The MA_type 2 kinase, which lacks the classical H-box, showed no autokinase activity. Furthermore, we could show that M. acetivorans possesses an above-average number of response regulators (RR), many of them consisting of only a REC domain (REC-only). Using the hybrid kinase MA4377 as an example we show that both intra-and intermolecular transphosphorylation to REC domains occur. These experiments are furthermore indicative of complex phosphorelay systems in M. acetivorans and suggest that REC-only proteins act as a central hub in signal transduction in M. acetivorans.

microbiology↗

bioRxiv2024

Early-branching cyanobacteria up-regulate superoxide dismutase activity under a simulated early Earth anoxic atmosphere.

The evolution of oxygenic photosynthesis during the Archean (4-2.5 Ga), required the presence of complementary reducing pathways to maintain the cellular redox balance. While the timing of the evolution of superoxide dismutases (SODs), enzymes that convert superoxide to hydrogen peroxide, within the Bacteria and Archaea is not resolved, SODs containing copper and zinc in the reaction centre (CuZnSOD) were the first SODs estimated to appear in photosynthetic cyanobacteria, [≥] 2.93 Ga. Here we analysed the SOD gene expression and activity in the deep branching strain, Pseudanabaena sp. PCC7367. It releases more O2 and exhibits significantly higher growth rates (p<0.001) and protein and glycogen contents (p<0.05) under anoxic conditions compared to control cultures grown under present oxygen rich atmospheres in low CO2 (LC) or high CO2 (HC), prompting the question as to whether this correlates to higher cellular SOD activity under anoxic Archean simulated conditions. Expression of sodB encoding an iron containing SOD (FeSOD) and sodC, encoding a CuZnSOD, strongly correlated with increased extracellular O2 levels (p<0.001), while transcription of sodA, encoding a manganese containing (MnSOD), correlated to SOD activity during the day (p=0.019), when medium O2 concentrations were the highest. Cytosolic SOD activity was significantly higher (p<0.001) in anoxic cultures, two hrs before nightfall compared to oxic growth conditions. Night-time combined sodABC transcription in stirred cultures was significantly reduced (p<0.05) under anoxic conditions at elevated CO2 levels, as were medium O2 levels (p[≤]0.001), when compared to cultures grown under present-day oxic conditions with low CO2. Total cytosolic SOD activity remained comparable, suggesting that the replacement rate of SOD is higher under modern-day conditions than on early Earth. Our data suggest that the early branching cyanobacterium Pseudanabaena sp. PCC7367 may have retained ancestral features permitting it to thrive in its ecological niche as a benthic mat in shallow water marine environments.

microbiology↗

bioRxiv2023

Darkness inhibits autokinase activity of bacterial bathy phytochromes

Bathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IX chromophore. In contrast to prototypical phytochromes that adopt a Pr ground state, the Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochrome PaBphP from Pseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly-enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 seconds to 50 minutes half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.

microbiology↗

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↗

bioRxiv2022

On the evolution of the plant phytochrome chromophore

Phytochromes are biliprotein photoreceptors present in plants, algae, certain bacteria and fungi. Land plant phytochromes use phytochromobilin (P{Phi}B) as the bilin chromophore. Phytochromes of streptophyte algae, the clade within which land plants evolved, employ phycocyanobilin (PCB), leading to a more blue-shifted absorption spectrum. Both chromophores are synthesized by ferredoxin-dependent bilin reductases (FDBRs) starting from biliverdin IX (BV). In cyanobacteria and chlorophyta, BV is reduced to PCB by the FDBR phycocyanobilin:ferredoxin oxidoreductase (PcyA), whereas, in land plants, BV is reduced to P[FE]B by phytochromobilin synthase (HY2). However, phylogenetic studies suggested the absence of any ortholog of PcyA in streptophyte algae and the presence of only P[FE]B biosynthesis related genes (HY2). The HY2 of the early diverging streptophyte alga Klebsormidium nitens (formerly Klebsormidium flaccidum) was already indirectly indicated to be involved in PCB biosynthesis. Here, we overexpressed and purified a His6-tagged variant of K. nitens HY2 (KflaHY2) in E. coli. Employing anaerobic bilin reductase activity assays and coupled phytochrome assembly assays, we were able to confirm the product and to identify intermediates of the reaction. Site-directed mutagenesis revealed two aspartate residues critical for catalysis. While it was not possible to convert KflaHY2 into a P{Phi}B-producing enzyme by simply exchanging the catalytic pair, the biochemical investigation of two additional members of the HY2 lineage enabled us to define two distinct clades, the PCB-HY2 and the P{Phi}B-HY2 clade. Overall, our study gives insight into the evolution of the HY2 lineage of FDBRs.

plant biology↗

bioRxiv2021

Lack of Fe(II) transporters in basal Cyanobacteria complicates iron uptake in ferruginous Archean oceans.

Introductory paragraphCyanobacteria oxygenated Earths atmosphere during the Great Oxygenation Event (GOE) through oxygenic photosynthesis. Their high iron requirement was presumed met by high levels of Fe(II) in the anoxic Archean ocean. Here we show that most basal Cyanobacteria cannot synthesize the primary Fe(II) transporter, FeoB. Relaxed molecular clock analyses estimate the arrival of FeoB, as well as the Fe(III) transporters, cFTR1 and FutB, in the Cyanobacteria after the GOE. Furthermore Pseudanabaena sp. PCC7367, a basal marine, benthic strain grown under simulated Archean conditions, constitutively expressed cftr1, even after the addition of Fe(II). By utilizing gene expression studies under a simulated Archean atmosphere, as well as comparative genomics, phylogenetics and molecular clock analyses, this study identified a need to reappraise iron uptake in ancestral Cyanobacteria, as genetic profiling suggests that scavenging of siderophore bound Fe(III), rather than Fe(II), appears to have been the means of iron acquisition prior to the GOE.

evolutionary 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↗

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↗