RESEARCH ARTICLE Jiang et al., Microbiology 2017;163:1–15 DOI 10.1099/mic.0.000427 The function of the three phosphoribosyl pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans Ping Jiang,1† Wen-fan Wei,1† Guo-wei Zhong,2 Xiao-gang Zhou,1 Wei-ran Qiao,1 Reinhard Fisher3 and Ling Lu1,* Abstract Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-a-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity. INTRODUCTION in Charcot-Marie-Tooth disease type 5 (or Rosenberg Chutorian Syndrome), Arts syndrome, PRS-I Phosphoribosyl pyrophosphate synthetase (Prs – EC: superactivity and X-linked nonsyndromic sensorineural 2.7.6.1) catalyses the reaction of ATP to form 5-phosphori- deafness [10–12, 14–16]. In prokaryotes, Prs genes have bosyl-a-1-pyrophosphate (PRPP) and AMP [1–5]. It has been found and sequenced from a variety of bacteria; for been identified that the Prs reaction product, PRPP, is example, the genome of Mycobacterium tuberculosis, which involved in many different pathways, such as the pentose is the main pathogen of tuberculosis, contains a single Prs- phosphate pathway and the de novo and salvage purine and encoding gene, Mt-prs (Rv1017c) [6, 17, 18]. Because PRPP pyrimidine nucleotide pathways, and in the biosynthesis of is required for the cell wall biosynthetic precursor decapre- nucleotide coenzymes, histidine and tryptophan [2, 6–8]. nol-1-monophosphoarabinose to maintain cell integrity, Therefore, this information indicates the central importance this Mt-prs gene has been used as a potential drug target. In of Prs enzymes in cellular metabolism. contrast, several isoforms of Prs have been cloned and Prs genes and their products are of interest not only for described in plants, such as Arabidopsis thaliana and Spina- their importance to our understanding of biochemistry but cia oleracea, which have five and four Prs genes, respec- also for their medical significance. In Homo sapiens, there tively, and are located in different cellular organelles [3, 19, are three Prs genes (PRPS1, PRPS2 and PRPS1L1) whose 20]. Similar to the eukaryotic system, most fungi also pos- superactivity is connected to gouty arthritis and whose sess multiple Prs genes. For instance, in the filamentous diminished activity is associated with various neuropathies hemiascomycete Ashbya gossypii, PRPP synthetase encoded [9–13]. In addition, the missense mutations of PRPS1 result by four genes (Aer083cp, Agl080cp, Agr371cp and Adr314cp) Received 21 June 2016; Accepted 11 January 2017 Author affiliations: 1Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Microbiology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, PR China; 2Department of Hygiene Analysis and Detection, School of Public Health, Nanjing Medical University, Nanjing 211166, PR China; 3Department of Microbiology, Karlsruhe Institute of Technology (KIT) - South Campus Institute for Applied Biosciences, Karlsruhe, Germany. *Correspondence: Ling Lu, [email protected] Keywords: phosphoribosyl pyrophosphate synthetase; Aspergillus nidulans; hyphal growth; conidiation. Abbreviations: PRPP, 5-phosphoribosyl-a-1-pyrophosphate; RT-PCR, reverse transcription PCR; SIN, septum initiation network; aa, amino acids. †These authors contributed equally to this work. Eight supplementary figures are available with the online Supplementary Material. 000427 ã 2017 © Nanjing Normal University 2016 1 Jiang et al., Microbiology 2017;163:1–15 plays important roles in cell growth and riboflavin produc- Constructions of gene replacement strains tion [21]. In comparison, the single cell yeast Saccharomyces A strain containing the AnprsB-null mutation was created cerevisiae is equipped with a set of five unlinked Prs genes by double joint PCR [39]. The Aspergillus fumigatus pyrG – (ScPRS1 ScPRS5). Deletion of any single or specific double gene in plasmid pXDRFP4 and the Asp. nidulans pyroA combinations of ScPRS genes did not cause a lethal pheno- gene in plasmid pQa-pyro were used as selectable nutri- type, indicating that some of the ScPRS genes are redundant. tional markers for fungal transformation, respectively. The Moreover, genetic analysis and enzyme activity detection linearized DNA fragment 1 which included a sequence of suggest that there existed more than one minimal functional about 686 bp that corresponded to the regions immediately unit capable of synthesizing phosphoribosyl pyrophosphate upstream of the AnprsB start codon was amplified with the – in S. cerevisiae [20, 22 28]. These results also suggest that primers DAnprsB-p1 and DAnprsB-p3 (Table 2). Linearized Prs may have their own working paradigms in different DNA fragment 2 including a sequence of about 541 bp that species. corresponded to the regions immediately downstream of In our previous study, we found that there are three putative the AnprsB stop codon was amplified with primers D D annotated genes (AnprsA, AnprsB and AnprsC) encoding AnprsB-p4 and AnprsB-p6 (Table 2). Lastly, purified lin- PRPP synthetase in Aspergillus nidulans, according to the earized DNA fragments 1 and 2 plus the pyrG gene or pyroA Aspergillus genome database (www.aspgd.org/). Moreover, gene were mixed and used in a fusion PCR with primers D D we identified the AnPrs family as a suppressor of the AnprsB-p2 and AnprsB-p5. The final fusion PCR prod- septum initiation network (SIN) and that it acts antagonisti- ucts were purified and used to transform Asp. nidulans cally against SepH, a main component of SIN, so the down- strains TN02A7. A diagnostic PCR assay was performed to regulation of the AnPrs family can bypass the requirements identify the deletion of the AnprsB gene by primers D ¢ ¢ for SIN in septum formation and conidiation [29]. How- AnprsB-p1 and Diag-AfpyrG-3 (Diag-AnpyroA-3 ). The ever, knowledge about how AnPrs family members work AnprsA and AnprsC deletion transformants with the marker together and contribute to total PRPP synthetase activity is of AnpyrG and AnpyroA were constructed by using a similar limited. In this study, we used constructed deletion and con- strategy and transformed to TN02A7. To further identify auxotrophic requirements of AnprsB and AnprsC, conidia ditional strains for three Anprs genes to identify that AnprsB D D and AnprsC are auxotrophic genes, i.e. either deletion of from the AnprsB and AnprsC heterokaryotic transform- AnprsB or AnprsC confers auxotrophic requirement for the ants selected by AnpyroA were inoculated into the selection downstream product of PRPP. In comparison, AnprsA is a medium MMDRUU supplemented with the predicted non-auxotrophic gene. Moreover, we found that these three downstream products (uracil/uridine, adenine, guanine, his- genes have different expression profiles at different develop- tidine, tryptophan and AMP). Oligonucleotides used in this mental stages. The important functions of AnprsB and study are listed in Table 2. AnprsC are not irreplaceable but are mainly due to the con- Tagging of AnPrsA, AnPrsB and AnPrsC with GFP tribution of Prs proteins to PRPP synthetase activity. To generate an alcA(p)-gfp-AnprsB fusion construct, a 909 bp fragment of AnprsB was amplified from TN02A7 genomic DNA with primer AnprsB-5¢ (NotI site included) METHODS and primer AnprsB-3¢ (XbaI site included) (Table 2). The 909 bp amplified DNA fragment was cloned into the corre- Strains, media, culture conditions, plasmids and ¢ transformation sponding sites of pLB01, yielding pLB-AnprsB-5 [40]. This plasmid was transformed into TN02A7. Homologous AlistofAsp. nidulans strains used in this study is provided in recombination of this plasmid into the AnprsB locus should supporting Table 1. The media YAG (yeast + agar + glucose result in an N-terminal GFP fusion of the entire AnprsB media), YUU (YAG + uridine + uracil), MMPGRUU (MM + gene under control of the alcA promoter and a fragment of pyridoxine + glycerol + riboflavin + uridine + uracil), AnprsB under its own promoter. The transformants, which MMPDR (MM + pyridoxine + glucose + riboflavin), were able to form the normal colony under the derepressing MMPDRUU (MM + pyridoxine + glucose + riboflavin + uri- condition but could not grow