Biology subjects

Kadowaki, T.

Publications and source records attributed to Kadowaki, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Host cell identity shapes the replication and evolution of Drosophila C virus

RNA viruses encounter diverse cellular environments within their hosts, yet how different host cell types influence viral evolution remains poorly understood. Here, we investigated the replication and experimental evolution of Drosophila C virus (DCV) in glial, mesodermal, and hemocyte-derived S2 cells. Mesodermal cells supported substantially higher viral RNA accumulation, viral protein production, and infectivity than glial or S2 cells, demonstrating pronounced cell type-dependent permissiveness. Comparative transcriptomic and proteomic analyses revealed distinct host responses to DCV infection despite the common mesodermal origin of mesodermal and S2 cells. Functional analyses showed that RNA interference, cGAS-STING signaling, viral binding and entry, and cellular ATP abundance could not account for the enhanced replication of DCV in mesodermal cells, whereas S2 cells displayed stronger cGAS-STING and JAK- STAT responses. Serial passage of DCV for 15 generations in mesodermal or S2 cells resulted in rapid phenotypic adaptation, with evolved viruses exhibiting improved replication in the corresponding cell type without altering infectivity in adult flies. Genome sequencing revealed distinct evolutionary patterns between nonstructural and structural protein-coding regions. Nonstructural proteins remained highly conserved with limited evidence of parallel evolution, whereas structural proteins accumulated more mutations and exhibited broader signatures of positive selection. A recurrent VP3 mutation (G8089A; R270H) independently emerged in three S2-passaged lineages, representing the only mutation consistently associated with adaptation to a specific cell type. Structural modeling suggested that this substitution is unlikely to substantially alter VP2-VP3 interactions, implying more subtle effects on capsid function. Together, our findings demonstrate that different Drosophila cell types impose distinct intracellular selective pressures that shape DCV replication and evolutionary trajectories, highlighting host cell identity as an important determinant of RNA virus evolution.

microbiology↗

bioRxiv2026

The endosomal trafficking protein Myopic differentially regulates Drosophila C virus infection in cultured cells and adult flies

Positive-sense RNA viruses extensively exploit host membrane trafficking pathways to establish intracellular replication organelles required for efficient viral replication. However, the contribution of endosomal trafficking proteins to infection by Drosophila C virus (DCV), a natural dicistrovirus of Drosophila melanogaster, remains poorly understood. Here, we investigated the role of Myopic (Mop), a conserved ESCRT-associated endosomal trafficking protein, during DCV infection. Knockdown of mop in cultured S2 cells significantly increased DCV RNA and viral protein accumulation without affecting viral binding or entry, indicating that Mop restricts DCV replication at a post-entry stage. In addition, depletion of mop altered the intracellular distribution of viral proteins, suggesting that Mop may influence the organization of DCV-associated intracellular membrane compartments. Unexpectedly, fat body-specific knockdown of mop in adult flies produced the opposite phenotype, resulting in reduced viral accumulation and prolonged survival following systemic DCV infection. This antiviral phenotype was not associated with enhanced activation of the STING or JAK-STAT pathways. Instead, transcriptomic and proteomic analyses revealed extensive remodeling of gene and protein expression, including altered abundance of proteins associated with autophagy and RNA interference. Together, our findings identify Mop as a previously unrecognized regulator of DCV infection and reveal distinct cell-autonomous and systemic functions of this conserved trafficking protein. These results highlight the importance of endosomal trafficking in antiviral defense and demonstrate that host membrane trafficking factors can exert fundamentally different effects on virus infection at the cellular and organismal levels.

microbiology↗

bioRxiv2026

Stochastic Growth Modeling of Vascular Plaque Dynamics and Derivation of Optimal Dosing Curves

Targeted drug delivery offers a promising approach for personalized medicine in treating vascular stenosis. However, biomechanical constraints, such as drug washout by high-velocity central blood flow and unintended absorption by healthy vascular walls, complicate the determination of optimal dosing locations. Conventional three-dimensional computational fluid dynamics (CFD) provides precise flow analysis but incurs prohibitive computational costs, making long-term tracking of plaque growth and reverse-engineering of optimal delivery highly inefficient. In this study, we propose a pseudo-3D stochastic growth model that dramatically reduces computational load while capturing the essential dynamics of plaque progression and regression. By modeling the advection-diffusion of lipid and drug particles as a discrete Markov process within a Stokes flow field, we simulate the morphological evolution of plaques under continuous and interrupted targeted therapies. Furthermore, by formulating the drug transport process as an absorbing Markov chain with boundaries at the healthy walls and vessel outlet, we calculate the exact reaching probability and mean first passage time (MFPT) to the plaque. Based on these probability distributions, we discover continuous "Optimal Dosing Curves", which indicate the most effective spatial coordinates for catheter-based drug release to maximize therapeutic efficacy. This mathematical framework not only elucidates the stochastic nature of vascular plaque dynamics but also provides a scalable, computationally efficient foundation for optimizing targeted drug delivery in personalized medicine.

bioengineering↗

bioRxiv2026

An arthropod-specific TMEM16 protein accelerates olfactory response termination in Drosophila

Rapid termination of odor responses is essential for accurate olfactory coding in insects, where odorant receptors function as ligand-gated ion channels rather than G protein-coupled receptors. However, the mechanisms that restore olfactory receptor neuron (ORN) excitability after stimulation remain poorly understood. Here, we identify DmTMEM16O (CG6938), an arthropod-specific member of the TMEM16 (Anoctamin) family, as a key regulator of ORN response termination in Drosophila melanogaster. DmTMEM16O is highly enriched in Orco-positive ORNs in both larval and adult olfactory organs. Loss of DmTMEM16O prolongs odor-evoked neuronal activity, increasing decay time constants and causing persistent depolarization. DmTMEM16O mutant ORNs fail to resolve repeated odor stimulation and show impaired temporal coding, accompanied by reduced behavioral responses to both attractive and aversive odorants. Cell-specific rescue demonstrates that DmTMEM16O acts within ORNs to accelerate response termination. Although DmTMEM16O does not exhibit detectable Ca2+-activated chloride channel activity in heterologous cells, our results support a model in which it increases membrane conductance during the decay phase of the response, thereby shortening the membrane time constant and promoting rapid repolarization. This function is consistent with a role for chloride influx in insect ORNs, in contrast to mammalian systems where TMEM16B-mediated chloride efflux amplifies depolarization. Together, our findings identify DmTMEM16O as a lineage-specific regulator of ORN dynamics that enables precise temporal coding in insect olfaction.

physiology↗

bioRxiv2025

Linking MSMEG_1353 to lipid metabolism and envelope integrity in Mycolicibacterium smegmatis

Mycobacterium tuberculosis (Mtb) poses a significant global health burden. Rv0647c, an essential Mtb cell wall protein, is a potential drug target. We studied its Mycolicibacterium smegmatis (MSMEG) homologue, MSMEG_1353, using a CRISPRi conditional knockdown. MSMEG_1353 depletion increased cell width and volume, delayed log-phase initiation, slowed aggregation, reduced biofilm formation, and heightened susceptibility to antibiotics and sodium dodecyl sulfate (SDS). AlphaFold, sequence alignment, and UNIPROT analyses suggest MSMEG_1353 functions as a protein kinase, with conserved residues in intermediate high-confidence regions potentially forming an ATP-binding site. Investigating its role in cell envelope biosynthesis, mass spectrometry revealed elevated levels of mycolic acid biosynthesis proteins upon MSMEG_1353 knockdown. RT-qPCR confirmed upregulation of the fabD-acpM-kasA-KasB-accD6 operon, encoding key mycolic acid synthesis enzymes. Notably, a strong negative correlation with MSMEG_0911, the predominant isocitrate lyase, important in the glyoxylate cycle, was observed via both MS and RT-qPCR. Collectively, MSMEG_1353 deficiency compromises cell wall integrity, likely due to altered lipid composition resulting from dysregulated mycolic acid biosynthesis and lipid metabolism. These findings support the development of models explaining MSMEG_1353s involvement in these pathways.

microbiology↗

bioRxiv2025

Essential roles of HGT-derived sterol biosynthesis in trypanosomatid growth and parasitism

Horizontal gene transfer (HGT) has played a major role in the evolution of kinetoplastids, but its scope and functional significance remain incompletely understood. We performed a comparative genomic analysis of 12 trypanosomatid parasites together with the free-living kinetoplastid Bodo saltans as an outgroup, using the DarkHorse algorithm to identify putative HGT candidates. B. saltans encoded the largest number of HGT-derived genes (980; 5.2% of coding sequences), whereas parasitic Leishmania species retained the fewest (67-71; 0.8-0.9%), consistent with extensive gene loss accompanying the transition to parasitism. Dollo parsimony reconstruction indicated that the kinetoplastid ancestor acquired numerous horizontally transferred genes, many of which were subsequently lost, but several functionally important families were retained in trypanosomatids. Among these, we identified a bacterial-derived aspartate ammonia ligase that, together with aspartate ammonia lyase, likely supports redox balance and succinate fermentation under anaerobic conditions in the insect gut. Phylogenetic analyses also revealed bacterial or archaeal origins of multiple enzymes in ergosterol biosynthesis, an essential pathway in trypanosomatids. Functional characterization in Lotmaria passim demonstrated that sterol C-8 isomerase is indispensable, whereas sterol C-5 desaturase is nonessential in vitro but critical for parasite growth at low temperature, amphotericin B sensitivity, and successful colonization of the honey bee hindgut. These findings illustrate the dual evolutionary trajectory of HGT in kinetoplastids: extensive gene loss during the transition to parasitism, coupled with the retention of lineage-specific innovations essential for survival in host environments. Significance StatementKinetoplastids include both free-living species such as Bodo saltans and parasitic trypanosomatids, the latter responsible for devastating human, animal, and insect diseases. While trypanosomatids have streamlined their genomes during host adaptation, free-living relatives retained a broader genetic repertoire shaped by environmental gene acquisition. By systematically tracing horizontally transferred genes (HGTs) across kinetoplastids, we reveal how free-living B. saltans accumulated diverse HGT candidates, whereas parasitic trypanosomatids lost many of these genes but selectively retained or acquired others critical for their specialized niches. We show that HGT contributed to the evolution of sterol biosynthesis and metabolic pathways supporting parasite survival in anaerobic insect guts. Functional analyses in Lotmaria passim, a honey bee parasite, demonstrate that HGT-dependent ergosterol biosynthesis is essential for gut colonization and influence sensitivity to amphotericin B. These findings highlight how horizontal gene transfer not only shaped the metabolic architecture of kinetoplastids but also underpins parasite adaptation to host environments.

microbiology↗

bioRxiv2024

Functional diversification of the BBSome: Insights from honey bee trypanosomatid parasite, Lotmaria passim

The BBSome is an octameric protein complex crucial for ciliary transport, though it also participates in multiple other cellular processes. These diverse functions may result from the co-option of its ancestral roles. Studying the BBSome in flagellated protists can provide insights into these ancestral functions and their subsequent adaptations. Here, we examine the function of the BBSome in Lotmaria passim, a monoxenous trypanosomatid parasite infecting honey bee. Parasites deficient in LpBBS2 are smaller and less motile compared to wild-type. Although intraflagellar transport of a marker membrane protein is only mildly impaired, its association with lipid rafts is significantly disrupted in the mutants. This suggests that the BBSome is essential for maintaining lipid raft integrity in L. passim. Transcriptomic comparisons between wild-type and LpBBS2-deficient parasites reveal that the BBSome may also influence processes related to metabolism, membrane localization of specific proteins, DNA repair, microtubules, and mitochondria. In contrast to Leishmania mexicana, the BBSome in L. passim is crucial for efficient infection of the honey bee gut, demonstrating that its cellular functions vary between related trypanosomatid species. The BBSome is likely an adaptor that links multiple proteins in a species-specific manner under various cellular contexts.

cell biology↗

bioRxiv2023

Protein subcellular relocalization and function of duplicated flagellar calcium binding protein genes in honey bee trypanosomatid parasite

The honey bee trypanosomatid parasite, Lotmaria passim, contains two genes that encode the flagellar calcium binding protein (FCaBP) through tandem duplication in its genome. FCaBPs localize in the flagellum and cell body of L. passim through specific N-terminal sorting sequences. This finding suggests that this is an example of protein subcellular relocalization resulting from gene duplication, altering the intracellular localization of FCaBP. However, this phenomenon may not have occurred in Leishmania, as one or both of the duplicated genes have become pseudogenes. Multiple copies of the FCaBP gene are present in several Trypanosoma species and Leptomonas pyrrhocoris, indicating rapid evolution of this gene in trypanosomatid parasites. The N-terminal flagellar sorting sequence of L. passim FCaBP1 interacts with the BBSome complex, while those of Trypanosoma brucei and Leishmania donovani FCaBPs do not direct GFP to the flagellum in L. passim. These results suggest that the N-terminal flagellar sorting sequence of FCaBP1 has co-evolved with the BBSome complex in each trypanosomatid species. Deletion of the two FCaBP genes in L. passim affected growth and impaired flagellar morphogenesis and motility, but it did not impact host infection. Therefore, FCaBP represents a duplicated gene with a rapid evolutionary history that is essential for flagellar structure and function in a trypanosomatid parasite.

microbiology↗

bioRxiv2023

Aspartyl protease in the secretome of honey bee trypanosomatid parasite is essential for the efficient infection of host

Exoproteome represents the proteome consisting of all secreted proteins and proteins derived from the cell surface and lysed cell. The exoproteome of the trypanosomatid parasite should interact with the host cells and the associated microbiota; however, the roles of infecting insect hosts are not yet understood. To uncover the functions of exoproteome, we identified the exoproteome of honey bee trypanosomatid parasite, Lotmaria passim, and found that approximately 28 % are shared with that common between Leishmania spp. It demonstrates a core exoproteome with conserved functions exists in the Leishmaniinae lineage. The bioinformatic characterization suggests that L. passim exoproteome may interact with the host and its microbiota as well as their metabolites. Deletion of genes encoding two secretome proteins revealed that an aspartyl protease but not chitinase affects the development of L. passim under the culture condition and is necessary for the efficient infection in the honey bee gut. Our results demonstrate that the exoproteome represents a resource to uncover the mechanisms of trypanosomatid parasites to infect the insect host by interacting with the gut environment.

microbiology↗

bioRxiv2022

Evolutionary history of metazoan TMEM16 family

Transmembrane protein 16 (TMEM16) functions as either a Ca2+-activated Cl- channel (CaCC) or phospholipid scramblase (CaPLSase) and plays diverse physiological roles. It is well conserved in eukaryotes; however, the origin and evolution of different subfamilies in Metazoa are not yet understood. To uncover the evolutionary history of the TMEM16 family, we analyzed 412 proteins from 74 invertebrate species using evolutionary genomics. We found that the TMEM16C-F and J subfamilies are vertebrate-specific, but the TMEM16A/B, G, H, and K subfamilies are ancient and present in many, but not all metazoan species. The most ancient subfamilies in Metazoa, TMEM16L and M, are only maintained in limited species. TMEM16N and O are Cnidaria- and Ecdysozoa-specific subfamilies, respectively, and Ctenophora, Xenacoelomorpha, and Rotifera contain species-specific proteins. We also identified TMEM16 genes that are closely linked together in the genome, suggesting that they have been generated via recent gene duplication. The anoctamin domain structures of invertebrate-specific TMEM16 proteins predicted by AlphaFold2 contain conserved Ca2+-binding motifs and permeation pathways with either narrow or wide inner gates. The inner gate distance of TMEM16 protein may have frequently switched during metazoan evolution, and thus determined the function of the protein as either CaCC or CaPLSase. These results demonstrate that TMEM16 family has evolved by gene gain and loss in metazoans, and the genes have been generally under purifying selection to maintain protein structures and physiological functions.

evolutionary biology↗

bioRxiv2022

DWV 3C protease uncovers the diverse catalytic triad in insect RNA viruses

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3Cpro), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. We found that the 3Cpros of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3Cpro by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cpros of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3Cpro with RNA-dependent RNA polymerase and mature 3Cpro are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3Cpro is the first 3Cpro characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cpros of Picornavirales. Furthermore, the specific inhibitors of DWV 3Cpro could be used to control DWV infection in honey bees.

microbiology↗

bioRxiv2020

DWV infection and replication at the early stage in vitro using honey bee pupal cells

Deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known about the mechanisms of viral infection and replication due to the lack of honey bee cell lines. To resolve this problem, we established in vitro system to reconstitute DWV binding and entry to the host cell followed by translation of the genome RNA and the polyprotein processing with honey bee pupal cells. Using this system, P-domain of VP1 was found to be essential for DWV infection/replication but not binding/entry to the cell. DWV efficiently infects/replicates in cells derived from early but not late pupa, suggesting that the undifferentiated cells are targeted for the viral infection/replication. Furthermore, we found that inhibitors for mammalian picornavirus 3C-Protease, Rupintrivir and Quercetin suppress DWV infection/replication, indicating that this in vitro system is also useful for screening a compound to modify the viral infection/replication. Our in vitro system should help to understand the mechanisms of DWV infection and replication at the early stage. ImportanceRecent decline of managed honey bee colonies has been driven by the pathogens and parasites. However, studying the mechanisms of pathogen infection and replication in honey bee at molecular and cellular levels has been challenging. DWV is the most prevalent virus in honey bee across the globe and we established in vitro system to reconstitute the viral infection and replication with the primary pupal cells. Using RNA-dependent RNA polymerase (RdRP) and the negative strand of DWV genome RNA as markers, we show that the pupal cells can support DWV infection and at least replication at the early stage. The results shown in this report indicate that our in vitro system helps to uncover the mechanisms of DWV infection and replication. Furthermore, it is also feasible to conduct a large scale screening for compounds to inhibit or stimulate DWV infection/replication.

microbiology↗

bioRxiv2020

Structural basis of ethnic-specific variants of PAX4 associated with type 2 diabetes

Recently, we conducted genome-wide association studies of type 2 diabetes (T2D) in a Japanese population, which identified 20 novel T2D loci that were not associated with T2D in Europeans. Moreover, nine novel missense risk variants, such as those of PAX4, were not rare in the Japanese population, but rare in Europeans. We report in silico structural analysis of ethnic-specific variants of PAX4, which suggests the pathogenic effect of these variants.

genetics↗

bioRxiv2020

Honey bee suppresses the parasitic mite Vitellogenin by antimicrobial peptide

The negative effects of honey bee parasitic mites and deformed wing virus (DWV) on honey bee and colony health have been well characterized. However, the relationship between DWV and mites, particularly viral replication inside the mites, remains unclear. Furthermore, the physiological outcomes of honey bee immune responses stimulated by DWV and the mite to the host (honey bee) and perhaps the pathogen/parasite (DWV/mite) are not yet understood. To answer these questions, we studied the tripartite interactions between the honey bee, Tropilaelaps mercedesae, and DWV as the model. T. mercedesae functioned as a vector for DWV without supporting active viral replication. Thus, DWV negligibly affected mite fitness. Mite infestation induced mRNA expression of antimicrobial peptides (AMPs), Defensin-1 and Hymenoptaecin, which correlated with DWV copy number in honey bee pupae and mite feeding, respectively. Feeding T. mercedesae with fruit fly S2 cells heterologously expressing honey bee Hymenoptaecin significantly downregulated mite Vitellogenin expression, indicating that the honey bee AMP manipulates mite reproduction upon feeding on bee. Our results provide insights into the mechanism of DWV transmission by the honey bee parasitic mite to the host, and the novel role of AMP in defending against mite infestation.

microbiology↗

bioRxiv2019

Honey bee trypanosomatid parasite dynamically changes the transcriptome during the infection of honey bee and modifies the host physiology

Although there are many honey bee pathogens/parasites, it is still not understood how they change their gene expression to adapt to the host environment or how the host simultaneously responds to pathogen/parasite infection by modifying its own gene expression. Such interactions must lead to changes in the physiological states of both host and parasite. To address this question, we studied a trypanosomatid, Lotmaria passim, which can be cultured in medium and inhabit the honey bee hindgut. We found that L. passim dynamically modifies the expression of mRNAs associated with protein translation and the electron transport chain to adapt to the anaerobic and nutritionally poor honey bee hindgut at early stages of infection, and to become dormant at late stages of infection. Meanwhile, several genes are continuously up- or down-regulated during infection, including GP63 as well as genes coding for host cell signaling pathway modulators (up-regulated), and those involved in detoxification of radical oxygen species as well as flagellar formation (down-regulated). L. passim infection only slightly increases honey bee mortality and does not affect the number of microorganisms in the gut microbiota; but it induces honey bee innate immune response. Upon infection, the host appears to be in poor nutritional status, indicated by the increase in the levels of mRNAs for take-out and facilitated trehalose transporter and the decrease of vitellogenin mRNA level. Simultaneous gene expression profiling of L. passim and honey bee during infection provided insight into how both parasite and host modify their gene expressions. This study presents one of the best models to understand host-parasite interactions at the molecular and cellular levels in honey bee.

microbiology↗

bioRxiv2018

Honey bee parasitic mite contains the sensory organ expressing ionotropic receptors with conserved functions

Honey bee parasitic mites (Tropilaelaps mercedesae and Varroa destructor) detect temperature, humidity, and odor but the underlying sensory mechanisms are poorly understood. To uncover how T. mercedesae responds to environmental stimuli inside a hive, we identified the sensilla-rich sensory organ on the foreleg tarsus. The organ contained four types of sensilla, which may respond to different stimuli based on their morphology. We found the forelegs were enriched with mRNAs encoding sensory proteins such as ionotropic receptors (IRs) and gustatory receptors (GRs), as well as proteins involved in ciliary transport. We also found that T. mercedesae and Drosophila melanogaster IR25a and IR93a are functionally equivalent. These results demonstrate that the structures and physiological functions of ancient IRs have been conserved during arthropod evolution. Our study provides insight into the sensory mechanisms of honey bee parasitic mites, as well as potential targets for methods to control the most serious honey bee pest.

physiology↗

bioRxiv2018

Gene knock out of honey bee trypanosomatid parasite, Lotmaria passim, by CRISPR/Cas9 system

Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by antibiotics, we first established an L. passim clone expressing tdTomato, GFP, or Cas9. We also successfully knocked out the endogenous miltefosine transporter and tyrosine amino transferase genes by replacement with antibiotics (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clones expressing fluorescent markers, as well as the simple method for knocking out specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.

microbiology↗

bioRxiv2018

Global analysis of the RpaB regulon based on the positional distribution of HLR1 sequences and comparative differential RNA-Seq data

The transcription factor RpaB regulates the expression of genes encoding photosynthesis-associated proteins during light acclimation. The binding site of RpaB is the HLR1 motif, a pair of imperfect octameric direct repeats, separated by two random nucleotides. Here, we used high-resolution mapping data of transcriptional start sites (TSSs) in the model Synechocystis sp. PCC 6803 in conjunction with the positional distribution of HLR1 sites for the global prediction of the RpaB regulon. The results demonstrate that RpaB regulates the expression of more than 150 promoters, driving the transcription of protein-coding and non-coding genes and antisense transcripts under low light and upon the shift to high light when DNA binding activity is lost. Transcriptional activation by RpaB is achieved when the HLR1 motif is located 66 to 45 nt upstream, repression occurs when it is close to or overlapping the TSS. Selected examples were validated by multiple experimental approaches, including chromatin affinity purification, reporter gene, northern hybridization and electrophoretic mobility shift assays. We found that RpaB controls ssr2016/pgr5, which is involved in cyclic electron flow and state transitions; six out of nine ferredoxins; three of four FtsH proteases; gcvP/slr0293, encoding a crucial photorespiratory protein; and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA, respectively. In addition to photosynthetic gene functions, RpaB contributes to the control of genes affiliated with nitrogen assimilation, cofactor biosyntheses, the CRISPR system and the circadian clock, making it one of the most versatile regulators in cyanobacteria.\n\nSignificance StatementRpaB is a transcription factor in cyanobacteria and in the chloroplasts of several lineages of eukaryotic algae. Like other important transcription factors, the gene encoding RpaB cannot be deleted, making the study of deletion mutants impossible. Based on a bioinformatic approach, we increased the number of known genes controlled by RpaB by a factor of 5. Depending on the distance to the TSS, RpaB mediates transcriptional activation or repression. The high number and functional diversity among its target genes and co-regulation with other transcriptional regulators characterize RpaB as a regulatory hub.

microbiology↗