Kentaro Furukawa

Distributed biological computation with multicellular engineered networks

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.

Aspergillus nidulans HOG pathway is activated only by two-component signalling pathway in response to osmotic stress

Genome sequencing analyses revealed that Aspergillus nidulans has orthologous genes to all those of the high‐osmolarity glycerol (HOG) response mitogen‐activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae. A. nidulans mutant strains lacking sskA, sskB, pbsB, or hogA, encoding proteins orthologous to the yeast Ssk1p response regulator, Ssk2p/Ssk22p MAPKKKs, Pbs2p MAPKK and Hog1p MAPK, respectively, showed growth inhibition under high osmolarity, and HogA MAPK in these mutants was not phosphorylated under osmotic or oxidative stress. Thus, activation of the A. nidulans HOG (AnHOG) pathway depends solely on the two‐component signalling system, and MAPKK activation mechanisms in the AnHOG pathway differ from those in the yeast HOG pathway, where Pbs2p is activated by two branches, Sln1p and Sho1p. Expression of pbsB complemented the high‐osmolarity sensitivity of yeast pbs2Δ, and the complementation depended on Ssk2p/Ssk22p, but not on Sho1p. Pbs2p requires its Pro‐rich motif for binding to the Src‐homology3 (SH3) domain of Sho1p, but PbsB lacks a typical Pro‐rich motif. However, a PbsB mutant (PbsB(Pro)) with the yeast Pro‐rich motif was activated by the Sho1p branch in yeast. In contrast, HogA in sskAΔ expressing PbsB(Pro) was not phosphorylated under osmotic stress, suggesting that A. nidulans ShoA, orthologous to yeast Sho1p, is not involved in osmoresponsive activation of the AnHOG pathway. We also found that besides HogA, PbsB can activate another Hog1p MAPK orthologue, MpkC, in A. nidulans, although mpkC is dispensable in osmoadaptation. In this study, we discuss the differences between the AnHOG and the yeast HOG pathways.

MpkA-Dependent and -Independent Cell Wall Integrity Signaling in Aspergillus nidulans

ABSTRACT Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Δ and mpk1Δ mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Δ and swi6Δ mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmAΔ, Answi4Δ Answi6Δ, and mpkAΔ strains) and analyzed mpkA and CWG transcripts after treatment with a β-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The β-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkAΔ strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except α-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

Isolation and Functional Analysis of a Gene, tcsB, Encoding a Transmembrane Hybrid-Type Histidine Kinase from Aspergillus nidulans

ABSTRACT We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor synthetic lethal N-end rule protein 1 (SLN1), which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade in yeast cells in response to environmental osmotic stimuli. From an A. nidulans cDNA library, we isolated a positive clone containing a 3,210-bp open reading frame that encoded a putative protein consisting of 1,070 amino acids. The predicted tcsB protein (TcsB) has two probable transmembrane regions in its N-terminal half and has a high degree of structural similarity to yeast Sln1p, a transmembrane hybrid-type histidine kinase. Overexpression of the tcsB cDNA suppressed the lethality of a temperature-sensitive osmosensing-defective sln1-ts yeast mutant. However, tcsB cDNAs in which the conserved phosphorylation site His552 residue or the phosphorelay site Asp989 residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1Δ sho1Δ yeast double mutant, which lacked two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsBΔ) and examined its phenotype. However, unexpectedly, the tcsBΔ strain did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media. Our results suggest that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade.

The SskA and SrrA Response Regulators Are Implicated in Oxidative Stress Responses of Hyphae and Asexual Spores in the Phosphorelay Signaling Network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated ΔsskA and ΔsrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from ΔsskA were hypersensitive not only to treatment with H2O2, but also to treatment at aberrantly low (4 °C) and high (50 °C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from ΔsskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.

Characterization of the NikA Histidine Kinase Implicated in the Phosphorelay Signal Transduction of Aspergillus nidulans, with Special Reference to Fungicide Responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.

The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction

Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8 Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.

Disordered Cell Integrity Signaling Caused by Disruption of the kexB Gene in Aspergillus oryzae

ABSTRACT We isolated the kexB gene, which encodes a subtilisin-like processing enzyme, from a filamentous fungus, Aspergillus oryzae. To examine the physiological role of kexB in A. oryzae, we constructed a kexB disruptant (ΔkexB), which formed shrunken colonies with poor generation of conidia on Czapek-Dox (CD) agar plates and hyperbranched mycelia in CD liquid medium. The phenotypes of the ΔkexB strain were restored under high osmolarity in both solid and liquid culture conditions. We found that transcription of the mpkA gene, which encodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higher in ΔkexB cells than in wild-type cells. The ΔkexB cells also contained higher levels of transcripts for cell wall-related genes encoding β-1,3-glucanosyltransferase and chitin synthases, which is presumably attributable to cell integrity signaling through the increased gene expression of mpkA. As expected, constitutively increased levels of phosphorylated MpkA were observed in ΔkexB cells on the CD plate culture. High osmotic stress greatly downregulated the increased levels of both transcripts of mpkA and the phosphorylated form of MpkA in ΔkexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the ΔkexB strain is autoregulated by phosphorylated MpkA as the active form through cell integrity signaling. We think that KexB is required for precise proteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under transcriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.

Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation

Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data.

Expression of the yeast aquaporin Aqy2 affects cell surface properties under the control of osmoregulatory and morphogenic signalling pathways

Aquaporins mediate rapid and selective water transport across biological membranes. The yeast Saccharomyces cerevisiae possesses two aquaporins, Aqy1 and Aqy2. Here, we show that Aqy2 is involved in controlling cell surface properties and that its expression is controlled by osmoregulatory and morphogenic signalling pathways. Deletion of AQY2 results in diminished fluffy colony morphology while overexpression of AQY2 causes strong agar invasion and adherence to plastic surfaces. Hyper‐osmotic stress inhibits morphological developments including the above characteristics as well as AQY2 expression through the osmoregulatory Hog1 mitogen‐activated protein kinase. Moreover, two pathways known to control morphological developments are involved in regulation of AQY2 expression: the protein kinase A pathway derepresses AQY2 expression through the Sfl1 repressor, and the filamentous growth Kss1 mitogen‐activated protein kinase pathway represses AQY2 expression in a Kss1 activity‐independent manner. The AQY2 expression pattern resembles in many ways that of MUC1/FLO11, which encodes a cell surface glycoprotein required for morphological developments. Our observations suggest a potential link between aquaporins and cell surface properties, and relate to the proposed role of mammalian aquaporins in tumour cell migration and invasion.