Control of reactive oxygen species (ROS) production through histidine kinases in Aspergillus nidulans under different growth conditions

Sensor histidine kinases (HKs) are important factors that control cellular growth in response to environmental conditions. The expression of 15 HKs from Aspergillus nidulans was analyzed by quantitative real-time PCR under vegetative, asexual, and sexual growth conditions. Most HKs were highly expressed during asexual growth. All HK gene-disrupted strains produced reactive oxygen species (ROS). Three HKs are involved in the control of ROS: HysA was the most abundant under the restricted oxygen condition, NikA is involved in fungicide sensing, and FphA inhibits sexual development in response to red light. Phosphotransfer signal transduction via HysA is essential for ROS production control.


Introduction
His-Asp phosphorelay signal transduction helps cells adapt to environmental changes and is common among bacterial and some eukaryotic cells. Sensor histidine kinases (HKs) recognize external signals, autophosphorylate on their own histidine residues, transfer phosphoryl groups to their own aspartic acid residues, and subsequently transfer the phosphate signals to histidine-containing phosphor transmitter (HPt). Finally, response regulators (RRs) receive the phosphate signals on their aspartic acid residues and regulate gene expressions directly or by controlling the downstream signal transduction pathways. Aspergillus nidulans is a model filamentous fungus that contains 15 HKs, 1 HPt, and 4 RRs [ 1 ]. Several HKs have been studied in attempts to characterize the roles of His-Asp phosphorelay systems in A. nidulans . TcsA is important for the formation of conidia during asexual development [ 2 ]. Meanwhile, TcsB is an ortholog of Sln1, which is an osmosensor in Saccharomyces cerevisiae [ 3 ]. NikA  Table S1 lists the primers used for PCR. Error bars represent the standard deviations of at least 3 independent experiments. ABPU1 strain including ligD gene deletion to induce efficient homologous recombination [ 10 ]. ΔnapA was a kind gift from Dr. T. Mizuno [ 11 ]. A set of 14 HK deletion strains was purchased from Fungal Genetics Stock Center [ 12 ].

Construction of a hysA deletion and alcA promoter control strain
A hysA -deletion plasmid, pKK001, was constructed and introduced into ABPU1 by protoplast transformation. The wild-type hysA gene was connected to an alcA promoter to create the plasmid, pTM003, which was introduced into the hysA -deletion strain. The details of plasmid construction are described in the Supplementary methods .

Total RNA preparation
Total RNA was prepared, and relative transcription levels were determined by quantitative real-time PCR as described in the Supplementary methods .

Nitro-blue tetrazolium staining
A. nidulans cultivation and nitro-blue tetrazolium (NBT) staining were performed as described previously [ 9 ] with some modifications. To observe germling growth, conidia were inoculated in liquid culture on cover glasses and cultivated for 12 h at 37 • C. To observe sporulating hyphae, each strain was grown beneath cover glass crossed with square holes on agar plates for 2 days at 37 • C. A. nidulans strains growing along the cover glass were drifted and sunk into NBT solution (0.1% Nitro Blue Tetrazolium [Wako], 100 mM Sodium phosphate buffer [pH 7.0]). After 4 h in the dark, blue-colored precipitate, which is the reduction product of NBT by superoxide anion, was observed under a microscope (OLYMPUS BX51).

HysA protein purification
The construction of expression plasmids, cultivation for protein induction, and purification of recombinant HysA HR and HysA H are described in the Supplementary methods .

In vitro autophosphorylation experiment
The in vitro autophosphorylation reaction of purified HysA proteins was carried out according to the method of Azuma et al. [ 13 ]. SDS, 6% glycerol, and 0.02% bromophenol blue). Samples were subjected to SDS-PAGE. The gel was dried and analyzed with an imaging scanner (BAS-2500, Fuji Film, Tokyo, Japan).

Expression levels of HK proteins in A. nidulans
To determine the roles of unknown signaling proteins in the cell development of A. nidulans , we analyzed the expression levels of all HK proteins at different stages of cell growth ( Fig. 1 ) [ 14 ]. Only a few HKs were expressed during vegetative growth ( Fig. 1 A), whereas   most HKs were expressed with the progression of asexual development, even though their overall expression levels were low ( Fig. 1 B).
These results indicate His-Asp phosphorelay signal transduction in A. nidulans mainly occurs during asexual development. However, under the restricted oxygen conditions by sealing the plates with tape (the induction of sexual development) ( Fig. 1 C), only 1 HK, originally called HK8-2, was markedly expressed; this implies a functionally important role of HK8-2 in response to low oxygen. Herein, we refer to HK8-2 as HysA ( hy poxia expressed s ensor protein A ) and continued to analyze its functions along with other characteristic HKs.

Construction of the hysA-deletion strain ( ΔhysA)
To determine the function of hysA in A. nidulans , the entire hysA region in the A. nidulans genome was replaced with auxotrophic marker, argB (see Supplementary methods ). The deletion was performed in wild-type ABPU1, and confirmed by PCR and Southern blotting (data not shown). Functional growth defects of A. nidulans due to deletion were investigated by comparison with BPU1, which was constructed by introducing wild-type argB into the ABPU1 strain. However, there were no differences between the BPU1 and ΔhysA strains with respect to growth even under the restricted oxygen condition (data not shown).

ROS production in HK gene-deletion strains
Since ROS are important signals for cell growth in A. nidulans like all organisms [ 9 ], we determined whether differences in NBT staining were detected using microscopy in ΔhysA , including differences in other HK gene disruptants. After 12 h of incubation at 37 • C in minimal medium liquid culture, cells were stained with NBT, which reacts with superoxide anion, one of ROS. Wild-type BPU1 produced ROS at the tip of growing hyphae ( Fig. 2 A) as reported previously [ 9 ]. PhkA and PhkB are thought to play roles in oxidative stress responses in A. nidulans because they have functional domains similar to those of Phk1-3, which are histidine kinases that function in response to oxidative stresses in Schizosaccharomyces pombe [ 1 ]. However, both ΔphkA and ΔphkB exhibited the same sensitivity to oxidative stress as the wild-type ( Fig. 2 D) and almost the same level of NBT staining as the wild-type ( Fig. 2 A). On the other hand, ΔnikA [ 4 , 5 ] and ΔhysA exhibited abnormal NBT staining at various parts of growing hyphae, indicating both NikA and HysA are involved in the control of ROS production ( Fig. 2 A).
Approximately 50 growing cells were observed for each strain and categorized into 8 different NBT staining patterns ( Fig. 2 B, left 1-8, black representing NBT-stained areas). The resultant distribution of ROS production is summarized in Fig. 2 B (right graphs). It indicates that ΔnikA and ΔhysA produce ROS at any parts of cells during the germling growth.
A. nidulans also developed conidiophores after hyphal growth on minimal medium agar plate. Wild-type BPU1 minimally produced ROS in some conidia and aerial hyphae ( Fig. 2 C). The ΔnikA strain exhibited poor formation of conidia as described previously [ 4 , 5 ] but still produced ROS at several parts of the hyphae. ΔhysA strongly produced ROS at both developed conidiophores and aerial hyphae ( Fig. 2 C).
The oxidative stress sensitivity of cells is thought to be linked to cellular ROS production. However, in the present study, the ΔhysA strain exhibited the same sensitivity as the wild-type but growth patterns different from those of ΔnikA on the oxidative stress plates To investigate the effects of other HKs on ROS production, we obtained 14 HK gene-deletion strains listed in the Fungal Genetics Stock Center (FGSC) except ΔphkA , which was not included in the list [ 12 ]. In addition, we observed specific ROS production in ΔfphA strain ( Fig. S1 ).
These are the first findings indicating HKs are involved in ROS production as directly observed by NBT staining.

ROS production in napA gene-disrupted strain
As NapA is a transcription factor that controls several genes for oxidative stress response, the deletion strain ( ΔnapA ) exhibited sensitivity to oxidizing regents [ 11 ]. Curiously, the ΔnapA strain exhibited ROS production similar to that of ΔhysA during the germling ( Fig.   2 A and B) but not at the conidiophore development ( Fig. 2 C). These results indicate HysA and NapA play different roles in ROS production during the development of A. nidulans .
To determine whether the involvement of HysA in ROS production is dependent on the presence of oxygen in growing cells, we cultivated ΔhysA under the restricted oxygen condition and observed the resultant ROS production. ROS levels were reduced mostly in the hyphae ( Fig. 2 E) but not at the tips in ΔhysA . It must be mentioned that other HK gene-disrupted strains and the ΔnapA strain including the wiltype did not exhibit different ROS production when oxygen was restricted, indicating they maintain their characteristic ROS production. Since ROS are mainly generated through the respiratory reactions at mitochondria under the presence of oxygen, HysA might be involved in the regulation of mitochondria. These results of NBT staining are summarized in Table 1 .

Functional importance of the His-Asp phosphorelay system in the control of ROS production
To test whether His-Asp phosphorelay signal transduction via HysA is essential for controlling ROS production in A. nidulans , we constructed a strain in which hysA expression was controlled under the alcA promoter [ 15 ] by introducing the alcA(p)::hysA fusion at the pyroA locus in the ΔhysA strain (OPHysA). Histidine residue at position 566 (His566) of HysA is supposed to be an autophosphorylation site based on the homology among HKs, and aspartic acid residue at position 1134 (Asp1134) is a phosphate-receiving site. HysA HQ and HysA DN mutant strains were also constructed by means of 2 alcA(p)::hysA fusions: alcA(p)::hysAHQ , which included 1 point mutation (His566 → Glu), and alcA(p)::hysADN , which included another mutation (Asp1134 → Asn). These 3 strains were cultivated in minimal medium liquid culture and stained by NBT, and ROS production was observed by microscopy ( Fig. 3 ). OPHysA produced ROS only on the tips of hyphae like the wild-type. However, HysA HQ and HysA DN exhibited the same abnormal ROS production as ΔhysA . In this experiment, we cultivated all strains in the non-inducing condition of alcA promoter including glucose, because the original expression of hysA promoter was similar to that of alcA promoter in this condition (data not shown). The induction condition of alcA promoter also resulted in similar NBT staining.

Autophosphorylation of purified HysA protein in vitro
Since it was unclear how HKs transmit phosphate signals in A. nidulans , we first examined the autophosphorylation activity of purified HysA protein in vitro . Plasmids were constructed in order to induce HysA HR and HysA H, i.e., the entire wild-type HysA protein and the HysA protein lacking the RR domain ( Fig. 4 A). Other plasmids were also prepared in order to introduce an amino acid change at the autophosphorylation site (i.e., His566) in HysA HR and H. The mutant proteins were purified in the same manner as the wild-type proteins ( Fig. 4 B). The phosphorylation reaction mixture included 2 mM DTT ( Fig. 4 C, lanes 1-4) or 10 mM GSH ( Fig. 4 C, lanes 5-8). 32 P-labeled protein bands were detected for HysA HR and HysA H ( Fig. 4 C, lanes  1, 3, 5, and 7) but not for the HysA HR (HQ) or H (HQ) mutant proteins ( Fig. 4 C, lanes 2, 4, 6, and 8). These in vitro results indicate HysA autophosphorylates its histidine residue at position 566. The autophosphorylation activities of HysA HR and HysA H were not detected without any reducing reagent ( Fig. 4 C, lanes 9 and 10). Because HysA controls ROS production, it is possible some redox conditions affect HysA activity.

Evidence of phosphotransfer from HysA to YpdA in vitro
YpdA is a histidine-containing phosphate transmitter in A. nidulans . YpdA (HQ) includes an amino acid change at His85 and does not receive phosphate signals from any histidine kinases or upstream sensor proteins [ 13 ]. We examined the in vitro phosphate signal transmission from HysA to YpdA in detail ( Fig. 5 ). When purified HysA HR was mixed with YpdA ( Fig. 5 A, HysA HR + YpdA), a radiolabeled protein band of YpdA was observed, and it was confirmed by the lack of a protein band for YpdA (HQ) at the same position as YpdA ( Fig. 5 A, HysA HR + YpdA(HQ)). HysA H does not include the C-terminal RR domain of HysA HR. Therefore, HysA H exhibits autophosphorylation activity ( Fig. 4 C, lanes 1 and 5) but does not transfer its phosphate signal to YpdA ( Fig. 5 B, HysA H + YpdA). On the other hand, HysA HR (HQ) includes an amino acid change at a histidine residue for autophosphorylation but still has the ability to receive the phosphate signal at the aspartic acid residue in the C-terminal RR domain. As expected, HysA HR (HQ) did not phosphorylate YpdA (data not shown). However, a radiolabeled protein band of YpdA was detected when it was mixed with both HysA H and HysA HR (HQ) ( Fig. 5 B, HysA H + HR (HQ) + YpdA), indicating HysA H can transfer a phosphate signal to YpdA via the RR domain of HysA HR (HQ). These in vitro results collectively indicate phosphotransfer including wild-type HysA occurs from His (i.e., the HK domain of HysA) to Asp (i.e., the RR domain of HysA) and then to His (i.e., YpdA).

Conclusions
ROS are generated during cell growth in the presence of oxygen in all organisms. It makes sense that His-Asp signal transduction is involved in ROS production, because it should be an important system that controls cell growth and development. The lack of a direct link between ROS production and oxidative sensitivity was evidenced by the HK deletion mutant strains. Therefore, HysA might be a key HK controlling ROS production in response to redox conditions. Further studies are required to elucidate signal transduction after HysA-YpdA as well as the genes under the control of HysA.