Studys On Molecular Mechanism Of Penicillium Funiculosum Acidic Resistance

In nature, living organisms can be found over a wide range of extreme environments, such as high temperature, acidity/alkalniry, salinity, pressure and radiation. The organisms that thrive in extreme environments are extremophiles. The mechanisms by which different microorganisms adapt to extreme environments provide a unique perspective on the fundamental characteristics of biological processes present in most species. Acidophiles are organisms which are able to grow down to pH1.0and are able to actively grow at pH<4.0. However, their adaptability mechanism to high H+concentrations is unclear.The filamentous fungus was identified as Penicillium funiculosum X33. The fungus has the widest range of pH values from pH0.6to pH13for growth in life. An Agrobacterium tumefaciens-mediated transformation system of P.funiculosum X33was established and the transformation procedure was optimized. The transformation efficiency of ATMT on P. funiculosum is100-200mutants per106conidia.We constructed a T-DNA insertional mutant library of P.funiculosum with9300transformants using this transformation system. PCR and Southern blot analysis showed that the T-DNA was integrated into the genome via random recombination and about87.7%tansfromants contained a single copy T-DNA. Screening and analysis of the phenotypic mutants from T-DNA insertional library of P. funiculosum, several strains of mutant morphological abnormalities were found.The mutant M4was used for isolation of disrupted gene. Using TAIL-PCR, a novel gene was cloned and named PfMFS gene (KF815490). The PfMFS gene showed88%identity to Penicillium marneffei ATCC18224conserved hypothetical protein (XP002144911). It contained an open reading frame of1656bp encoding a putative551-amino-acid protein. The calculated molecular weight and pi of the encoded putative mature protein were59.5kDa and6.08, respectively. BLASTP analysis showed the encoded putative protein belongs to the Major Facilitator Superfamily (MFS). These search results indicate that the putative protein is a novel MFS transporter, but whether this is of functional significance is unclear.Sequence analysis of the genomic DNA franking T-DNA showed that the T-DNA insert site in the mutant M4was located in the promoter region of PfMFS gene at position-372. To further confirm the function of the PfMFS in P. funiculosum acid-tolerance, PfMFS was disrupted by homologous recombination. The PfMFS gene replacement vector pROK2/PfMFS was used for fungal transformation. Southern blotting and PCR were used to confirm disruption of PfMFS in P. funiculosum transformants. These data further demonstrate that PfMFS was required for P. funiculosum acid-tolerance.To complement the PfMFS mutant, we transformed the disrupted mutant M7spores with the complementation vector p3SR2/PfMFS. The expression of PfMFS was then confirmed by RT-PCR and Northern blot analysis. The data indicated that disrupted PfMFS is responsible for the phynotype observed in the two mutants, M4and M7.Protoplasts of P. funiculosum were prepared for the determination of the cellular pH at pH1.0in P. funiculosum wild type and PfMFS disruption mutant M7using highly fluorescent BCECF that is particularly sensitive to pH. The wild-type strain was found to show higher intracellular pH than the PfMFS disruption mutant in response to pH1.0. These results show that PfMFS is important for cellular pH homeostasis.In order to further investigate biological functions of PfMFS, it was selected for heterologous expression. Results show that the transformant PfMFS-22yeast cells had a higher growth biomass than the control yeast cells at pH2.0, but both the PfMFS-22yeast cells and the control yeast cells showed no growth at pH1.0and had almost same growth at pH3.0These results also suggest that the P. funiculosum PfMFS gene has the ability of acid resistance, especially at low pH.The PfMFS mutant M7was not sensitive to NaCl, while the wild type exhibited a significant increase in growth in the presence of NaCl. In the measurement of cell suspension pH, pH in WT cell suspension exhibited a significant decrease in the presence of Na+. In contrast, pH in M7cell suspension showed a little decrease in the presence of Na+. These results demonstrate that Na+was related to the PfMFS transporter of P. funiculosum, suggesting that the PfMFS transporter might be a Na+-dependent MFS transporter.To examine the cellular distribution of PfMFS, we constructed the plasmid pROK II-GFP-PJMFS used for subcellular location. A. tumefaciens strain EHA105contained the pROK II-GFP-PfMFS vector was used to infect onion epidermal cells. In fluorescence microscope, localization of the PfMFS-GFP fusion protein was visualized exclusively in the cytomembrane. These results indicate that the PfMFS transporter was a cytomembrane protein.