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 membranetype 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 His 552 residue or the phosphorelay site Asp 989 residue had been replaced failed to complement the sln1-ts mutant. In addition, introduction of the tcsB cDNA into an sln1⌬ sho1⌬ 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 (tcsB⌬) and examined its phenotype. However, unexpectedly, the tcsB⌬ 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.Living cells are equipped with mechanisms for sensing environmental stimuli, such as osmotic stress, oxidative stress, and hormones, which allow adaptation to the stimuli through a variety of cellular responses triggered by the sensing and subsequent signaling systems. Two-component signaling systems, which involve a phosphorelay from the histidine of the sensor kinase to the aspartic acid of the response regulator, are widespread in bacteria (23 CaNIK1, and CaSLN1 [19]). Some of the histidine kinases, including yeast SLN1, plant ATHK1, and fungal NIK1 and tcsA, are involved in osmoregulation. In Saccharomyces cerevisiae, Sln1p consists of an extracellular sensor, a kinase, and a response regulator domain in a single polypeptide and is thus a transmembrane hybrid-type histidine kinase (22). Under low-osmolarity conditions, a specific histidine residue within the histidine kinase domain is autophosphorylated. The phosphate moiety of the histidine kinase is transferred to an aspartic acid residue within the response regulator domain and then via a phosphorelay is transferred to the downstream proteins Ypd1p and Ssk1p, shutting off the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) cascade (24). Histidine kinase activity and phosphorylation of Sln1p are essential for growth at low osm...

In VitroAnalysis of His-Asp Phosphorelays inAspergillus nidulans: The First Direct Biochemical Evidence for the Existence of His-Asp Phosphotransfer Systems in Filamentous Fungi

“…15) Indeed, TcsB can suppress the lethality of the S. cerevisiae sln1-ts mutant, indicating that TcsB is orthologous to the wellcharacterized S. cerevisiae osmotic sensor Sln1p, and that TcsB functions as an osmosensor histidine kinase. The tcsB deletion strain, however, did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media, suggesting that A. nidulans has a more complex and robust osmoregulatory system than S. cerevisiae.…”

“…The tcsB deletion strain, however, did not exhibit a detectable phenotype for either hyphal development or morphology on standard or stress media, suggesting that A. nidulans has a more complex and robust osmoregulatory system than S. cerevisiae. 15) On the other hand, based on the analogy with S. pombe, 20,21) PhkA, the homolog of Phk1/2, and PhkB, the homolog of Phk3, are candidate sensors for the hydroxyl peroxide signal. We have constructed deletion mutants of these genes.…”

“…E. coli BL21(DE3) cells transformed with various His-tagged constructs were grown in 100 ml of LuriaBertani broth with 50 mg/ml of ampicillin at 37 C. When the OD 600 reached 0.5, the culture was shifted to 15 C. Then IPTG was added to a final concentration of 1 mM and the cultures were shaken for an additional 24 h at 15 C. Cells were resuspended in 2 ml of buffer S (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, and 1 mM DTT). Bacteria were lysed by sonication, and the lysates were clarified by centrifugation at 7;000 Â g for 15 min at 4 C. The supernatant were applied to 0.25 ml of Ni-NTA Agarose (Qiagen, Hilden, Germany), and washed with 10 ml of buffer S. His-tagged proteins were eluted with buffer S containing 100 mM imidazole.…”

“…To date, only four histidine kinases have been reported: TcsA (gene ID, AN5296.3), 14) TcsB (AN1800.3), 15) FphA (AN9008.3), 16) and NikA (AN4479.3). 13) TcsA and FphA have been characterized with reference to asexual and/or sexual development processes, whereas TcsB appears to be orthologous to the well-characterized Saccharomyces cerevisiae osmotic sensor Sln1p.…”

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

“…To address this issue, it is necessary to characterize the physiological roles of these 15 HKs one by one. So far, only three of them have been characterized to some extent, namely, TcsA (gene ID, AN5296.3), 5) TcsB (AN1800.3), 6) and FphA (AN9008.3). 7) TcsA and FphA were characterized with reference to asexual and/or sexual development processes, whereas TcsB appears to be orthologous to the well-characterized Saccharomyces cerevisiae osmotic sensor Sln1p.…”