Background

Potassium is a nutrient element essential to every living organism. Potassium ions play an important role in regulating the osmotic pressure balance and ion balance in plant cells, maintaining the stability of the internal environment (Wu, 2003; Yin et al., 2006). In bacteria and eukaryotic cells, potassium is is an essential monovalent cation in living cells, which was an important component of the osmotic pressure of cells. It plays a crucial role in physiological processes, such as regulating osmotic pressure, regulating intracellular pH, various enzymes, membrane potential, transmission signals, and the so on (Epstein et al., 2003; Herrera et al., 2014). In order to ensure the normal operation of various metabolic activities in cells, Trk-like potassium absorption and translocation systems are prevalent in most prokaryotic, archaeal, eukaryotic, and filamentous fungi to maintain a relatively stable intracellular potassium concentration (Feng et al., 2009).

In recent years, studies on Trk genes have been reported in prokaryotic and eukaryotic microorganisms. In prokaryotic microorganisms, most bacteria usually suffered from a relatively complex growth environment, and a rapid and effective response to the range of external potassium ion mutations was necessary. Therefore, it is very important for the Trk gene family to regulate the potassium concentration. The Trk system has been reported in the prokaryotic microorganisms, such as Salmonella (Su et al., 2008), Escherichia coli (Schlösser et al., 1991; Harms et al., 2001), salinophilia (Guo et al., 2009), Vibrio parahaemolyticus (Cao et al., 2013), Vibrio alginolyticus (Nakamura et al., 1998a; 1998b), Vibrio vulnificus (Chen et al., 2004), and monosomonas long Halomonas (Kraegeloh et al., 2005; Kunte et al., 2005). Among of these, the Trk system of Escherichia coli is the most studied. By constructing E. coli mutants, the researchers found that Ec-TrkA can bind to NAD⁺ to regulate the K⁺ uptake activity of TrkH or TrkG, instead of the previously expected ATP. It was also found that the K⁺ uptake function of the cells did not change significantly regardless of the destruction of any protein in TrkH or TrkG. The found indicated that TrkH and TrkG could independently provide higher K⁺ transport capacity for organisms, which were related to the regulation of TrkA, that is, the system showed dependence on TrkA (Epstein et al., 2003; Su et al., 2008; Feng et al., 2009; Herrera et al., 2014). Two Trk gene family members, Va-TrkA and Va-TrkH, were also found in Vibrio alginolyticus. When the two Trk gene family members were expressed in E. coli △trkA and△trkH △trkG mutants, the mutants could resume growth in low K⁺ conditions (Nakamura et al., 1998a; 1998b). In addition, studies on fungal Trk systems have shown that most fungi contain at least one or two Trk family potassium transporters, such as Saccharomyces cerevisiae (Ariño et al., 2010; Bagriantsev et al., 2013; Herrera et al., 2014), Schizosaccharomyces pombe (Ariño et al., 2010), Kluyveromyces lactis (Prista et al., 2007), Ruthenium (Miranda et al., 2003), Cylindrotheca (Stříbný et al., 2012), Candida albicans (Rivetta et al., 2013) and so on.

Sc-Trk1 is the first member of the Trk gene family found in fungi, which was from Saccharomyces cerevisiae. Sc-Trk1 acts as a potassium transfer protein in the plasma membrane (Rodríguez-Navarro et al., 1984; Ramos et al., 1985). The study found that trkl-deficient strains of Saccharomyces cerevisiae still maintained a relatively low level of K⁺ uptake activity indicating that other members of the TRK gene family exist in Saccharomyces cerevisiae. Subsequent studies have found that ScTrk2 is also a potassium transporter and both play an important role in maintaining potassium balance in Saccharomyces cerevisiae (Chen et al., 2004; Alkhuder et al., 2010). It can be seen that there are TRK gene families in both prokaryotic and eukaryotic microorganisms to maintain osmotic balance in the organism.

Aspergillus niger is an important industrial fermentation microorganism and one of the edible safety strain identified by the US Food and Drug Administration. Furthermore, it is widely used in organic acids and industrial enzyme preparations. Aspergillus niger is always found in complex environment, such as food, plant products and soils, resulting to its salt and acid resistance. The regulation of osmotic pressure plays a crucial role in both high salt and low pH environments, but the genes and mechanisms associated with osmoregulation in Aspergillus niger have been rarely reported. In this study, bioinformatics methods were used to identify the entire Trk family members in Aspergillus niger, which provided the basis for the cloning of members of the TRK gene family. The analysis of their evolution, structure and protein interaction networks was of great significance for further study of their function and the osmotic regulation mechanism of Aspergillus niger.

1 Results

1.1 Identification of Aspergillus niger gene family members

Four TRK gene family members were identified in the Aspergillus niger genome, including AnTrk1(Accession number: EHA26772), AnTrk2 (NM_001181562), AnTrk3 (EHA26772) and AnTrk4 (NM_001181562). The encoded protein sequences are smaller than the Trk genes in yeast and the length of amino acid ranges from 617 to 826. The molecular weight ranges from 67.87 kD to 90.86 kD. Analysis of the domains revealed that the four members of the TRK gene family in Aspergillus niger and yeast were all contained a TrkH functional domain, but the size of the domains was different among the family. The TrkH functional domain is associated with cation transport. Therefore, it can be inferred from the above results that AnTrk1, AnTrk2, AnTrk3 and AnTrk4 are four members of the TRK gene family in Aspergillus niger (Table 1).

Table 1 The characteristics of identified TRK gene family in Aspergillus niger

1.2 Aspergillus niger TRK genes and yeast TRK genes alignment da/ds calculation

The evolutionary selection pressure is often expressed as da/ds. To analyze the evolutionary pressure of the TRK gene family in Aspergillus niger, the gene sequences and mRNA sequences of AnTrk1, AnTrk2, AnTrk3, and AnTrk4 were compared with Trk1 and Trk2, respectively. The results showed that AnTrk2 experienced a purification selection, while the other TRK gene family members in Aspergillus niger were positively selected (Table 2).

Table 2 The analysis of evolutionary selection pressure for TRK Note: S: Number of synonymous sites; N: Number of non-synonymous sites; dS: Synonymous substitution rate; dN: Non-synonymous substitution rate; dS/dN: Synonymous substitution rate/ Non-synonymous substitution rate

1.3 Phylogenetic Tree Construction of TRK Genes

Based on the multiple sequence alignment of the TRK genes, phylogenetic relationship was analyzed using the MEGA software. AnTrk1, AnTrk2, and Trk1, Trk2 in yeast were clustered together, while AnTrk3 and AnTrk4 were clustered together (Figure 1). This result shows that the phylogenetic relationship between AnTrk1, AnTrk2 and Trk1, Trk2 is closer, and we speculated that there is an intersection from their origins. AnTrk3 and AnTrk4 are relatively far apart, but they all belong to one big branch and were considered to be coordinative relationship. This also shows that there is a certain degree of phylogenetic relationship between them. The small branches appeared after large branches, which indicated that these subfamilies have undergone evolutionary losses and amplification events. Furthermore, the structure and function of genes are more disproportionate, revealing the diversity of gene expression and function in this gene family. Since the research of function of the TRK family genes in Aspergillus niger are limited, this result referred from the TRK genes in yeast which were studied more deeply can provide guidance for the functional studies and regulatory networks of the Aspergillus niger TRK family.

Figure 1 The phylogenetic tree construction of TRK gene

1.4 Motif Analysis of the TRK Gene Family in Aspergillus niger

Motif analysis of four TRK genes in Aspergillus niger and two TRK genes in yeast was performed by the MEME program. The result shows that AnTrk1, AnTrk2 and TRK genes in yeast all contained eight conserved motifs, but the positions of them are inconsistent (Figure 2A). On the other hand, AnTrk3 contains only six motifs and AnTrk4 contains only seven motifs. The results further confirmed the reliability of the phylogenetic analysis of the TRK genes. From the analysis of codon usage in each motif, it is obviously observed that the codons used by each motif are quite different among different genes (Figure 2B).

Figure 2 Motif analysis of TRK gene family in Aspergillus niger Note: A: 1~4 represents AnTrk1, AnTrk2, AnTrk3 and AnTrk4, 5~6 represents Trk1 and Trk2; B: Eight conserved functional amino acid residues in each P-loop are shown from top to bottom. Height of letter displays relative frequency of each amino acid residue; Abscissa and ordinate demonstrate the number of amino acid residues and relative frequency of each amino acid residue, respectively

1.5 Gene Structure Analysis of Gene Family

To further investigate the characteristics of the TRK genes, GSDS2.0 was used to analyze the intron, exon, and UTR regions of each gene (Figure 3). The result shows that the TRK genes in yeast do not contain intron, while the TRK gene family members in Aspergillus niger have intron structure in which AnTrk1 contain an intron and a UTR sequence is presented at the 5’ end. In addition, AnTrk2, AnTrk3 and AnTrk4 contained two, eight and seven introns, respectively.

Figure 3 The analysis of gene structure for TRK family Note: The coding sequences were represented in yellow. The upstream and downstream were represented in blue. The introns were represented by black gray lines

1.6 Protein interaction analysis

Analysis of protein interaction network for the TRK genes reveals that there is an interaction between TRK1 and TRK2, which forms a potassium transport system. This system can interact with the glucose transporters HXT3 and HXT1, the serine/threonine phosphatases PPZ1 and PPZ2, the phophoroyl cysteine decarboxylase SIS2, the K⁺/H⁺ antiporter KHA1 and the histone deacetylase RPD3 protein to regulate the osmotic balance of the cells (Figure 4). However, the function and mechanism of AnTRK3 and AnTRK4 have not been reported yet, and the interaction of the two members needs further experiments to identify.

Figure 4 Protein-protein interaction network of TRK

2 Discussion

Studies have shown that the TRK gene is expressed in plants as well as in prokaryotic and eukaryotic microorganisms. It plays an important role in maintaining the balance of intracellular and extracellular osmolarity in the regulation of potassium ion metabolism. Potassium ion is closely related to the metabolic activities of organisms and maintains the balance of potassium ion in the organism. Therefore, the importance of TRK genes becomes more prominent and is a hot spot in the current biological research. At present, some studies on the cloning and expression of TRK genes in microorganisms have been reported. Zhang et al. (2015) studied the cloning and expression of the TrkH gene in marine micro-organisms. Ma et al. (2001) studied the expression of brain-derived neurotrophic factor receptor trkB on NIH 3T3 cells. Guo et al. (2008) used homologous recombination to replace the TRK1 and TRK2 genes of Saccharomyces cerevisiae with new genes amplified by overlapping primers to obtain S. cerevisiae potassium-deficient strains, which is beneficial to the study of potassium ion gene function. With the completion of genome sequencing of various species, identification and analysis of gene family classification and relationship among members, sequence characteristics, evolutionary tree characteristics and gene function prediction from the genome level have become important issues in the field of life sciences.

To further understand the TRK genes in Aspergillus niger, the TRK gene family members of Aspergillus niger were identified and analyzed by bioinformatics methods, in which four TRK family members were identified. This study analyzed the motifs, the structure distribution, length of exons and introns, and the genetic relationship of the TRK gene family members systematically and comprehensively, which was beneficial to further understanding TRK functional characteristics. The Trk gene family includes four genes, TrkH, TrkG, TrkA and TrkE, of which TrkH is ubiquitous in E. coli and some intestinal bacteria (Wu, 2003). Schlösser et al. (1991) used gene cloning and expression methods to find that TrkA and TrkE are regulatory proteins, while TrkH and TrkG are transmembrane proteins. In this study, functional domains of the TRK genes of Aspergillus niger and yeast were analyzed by Pfam software. It was concluded that the TRK gene family members in Aspergillus niger all belong to TrkH, which is a transmembrane protein. This structure may be related to the secretion system of Aspergillus niger. The results provide theoretical basis for further understanding the function of TRK gene, researching the cation transport system and osmoregulation mechanism of Aspergillus niger. In addition, the results provide a reference for revealing the adaption of Aspergillus niger to the complex growth environment. These results also indicate that there are significant differences in the TRK gene family between yeast and Aspergillus niger. Therefore, it is speculated that there are other regulatory mechanisms in Aspergillus niger that balance the osmotic balance in vivo. In particular, the functions and mechanisms of AnTrk3 and AnTrk4 have not yet been determined and further experiments are needed to verify and analyze.

3 Materials and Methods

3.1 Identification of TRK Gene Family Members in Aspergillus niger

The whole genome sequence of the Aspergillus niger ATCC1015 strain (ACJE00000000) was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/) and constructed into the local BLAST database through the ncbi-blast-2.2.31+ formatdb program. Using the TRK gene family members TRK1 (NP_012406) and TRK2 (NP_012976) in yeast as reference sequences, the blast program was used to search for family members in Aspergillus niger with the E value of 1×10^-5. Both the identity and cover regions which exceed 50% were set as the filter criteria. After filtering, the domain analysis was finally performed using Pfam software to ensure that the screened sequences were non-redundant sequences.

3.2 Multiple sequence alignment and phylogenetic tree construction

The multiple sequence alignment of the identified TRK gene family members of Aspergillus niger was performed by the Muscle program. And then the phylogenetic tree was constructed using the maximum likelihood method and the neighbor-joining method of MEGA6.06 software (http://www.megasoftware.net/). The evolutionary tree was evaluated by the bootstrap method with a duplicate value of 1,000 (Peng et al., 2016).

3.3 Evolutionary pressure analysis

Evolutionary selection pressure analysis is always represented using non-synonymous substitution ratio and synonym substitution ratio (da/ds). If da/ds>1, it is considered to be a positive selection; if da/ds=1, it is considered to be a neutrality selection; if da/ds<1, it is considered to be a purification selection. The online software PAL2NAL (http://www.bork.embl.de/pal2nal/) was used to perform da/ds estimation of the TRK gene family members in Aspergillus niger and predict their evolutionary selection patterns.

3.4 Conservative motifs and gene structure analysis

Analysis of conserved motifs is performed using MEME software (http://memesuite.org/tools/meme) (Bailey et al., 2016). For the analysis of gene structure, the mRNA sequences and the corresponding genomic sequences were downloaded in the NCBI database, and the intron structure and distribution of each gene were analyzed by GSDS2.0 software (http://gsds.cbi.pku.edu.cn/).

3.5 Protein interaction network analysis

STRING is a database of protein-protein interactions through prediction and experimental validation, including direct physical interactions and indirect functional correlations. For the species included in the database, the interaction pairs of the target gene set can be directly extracted from the database to construct an interaction network. Since the database does not include information on Aspergillus niger, the BLAST software was used to compare the target genes with the proteins in the database to find homologous proteins. Then, an interaction network was constructed based on the interaction pairs of the homologous proteins. The constructed protein interaction network was imported into Cytoscape software for visualization (Ramos et al., 1985).

Authors' contributions

HB, LLL and LHR are the executors of the experimental design and experimental research of this study. They completed data analysis and wrote the first draft of the paper; DJ, ZYH, CF, HJZ and HJW participated in the experimental design and experimental results analysis; HB and ZB are the responsible persons of the project, guiding experimental design, data analysis, writing and revision of thesis. All authors have read and agreed with the final text.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (31171731), the National 863 Program (31460447), the Jiangxi Provincial Jiangxi Provincial Key Laboratory of 555 Engineering (3000039703), Jiangxi Provincial Key Laboratory of Bioprocessing, and the Collaborative Innovation Center for In Vitro Diagnostic Reagents and Instruments.

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