## 4 Discussion  
During the construction of the \(\Delta\)PMA1 mutant, extra point mutations were introduced in multiple sites downstream and upstream of the position of the PMA1 H+ ATPase gene, which resulted in a strain that accumulates melanin. A correct deletion of the Pma1 gene without the point mutations did not result in melanin accumulation, indicating that this  
Figure 9: Dye removal by FB2 and \(\Delta\)PMA1 cells. (**A**) Control of methylene blue solution. 90 ppm was the highest concentration to get 1.0 D.O; FB2 cells incubated by 24 and 48 h in the presence of different dye concentrations and \(\Delta\)PMA1 cells incubated at 24 and 48 h in the presence of methylene blue. (**B**) Typical view of the cells during biosorption of the dye. Bar = 20 \(\upmu\)m. (**C**) Graphical comparison of the residual absorbance after the incubation with the dye for 24 or 48 h.  
response is due to random point mutations. The pigment characterization involved TLC chromatography, UV-vis, and infrared spectra. The comparison of the samples with the commercial synthetic melanin indicated that the pigment found in _U. maydis_ was the same type. The chemical characteristics of the _U. maydis_ pigment agreed with other melanins described for microorganisms [30, 40]. In animals, bacteria, and fungi it is common to find melanin (DOPA-melanin) and its precursor can be either tyrosine or L-DOPA. Fungal melanin is synthetized in internal vesicles that look like the mammalian melanosomes and is then transported to the cell wall where the melanin crosslinks with polysaccharides [41, 42, 43]. We detected small spots of melanin in the cytoplasm of the \(\Delta\)PMA1 mutant, and cells became brown after 72 h of growth on solid media, suggesting that the production of melanin may be a response to the stress caused by the additional point mutation, and the excess of melanin is trapped in the cytosol. The content of melanin in fungi is variable [12]. In _U. maydis_, the saprophytic cells do not show melanogenesis. Normally, the spores are highly melanized, but once they become sporidia, the melanin is preferably located at the cell wall, presumably to protect the cell against environmental stresses. In this study, we measured the melanin content in wild-type FB2 and \(\Delta\)PMA1 strains cultured for 72 h and found a higher content of melanin in the \(\Delta\)PMA1 strain. To assess the basic behavior of the mutant in culture, cells were grown in YPD or MM media. Our results showed that the parent FB2 wild-type strain and \(\Delta\)PMA1 mutant displayed similar growth and small differences in the duplication time. These results suggest that the accumulation of melanin did not affect the cell division processes and vegetative growth. Furthermore, deletion of the \(pma1\) gene did not affect the glucose or oxygen consumption [31]. On the other hand, we noticed that the \(\Delta\)PMA1 mutant was stable after long periods of storage at \(-80\)\({}^{\circ}\)C.  
Cytosolic melanin accumulation was observed in the cells containing a mutation of the UmRrm 75 gene, which encodes a protein with three RNA recognition motifs with glycine-rich regions. UmRrm 75 mutant cells showed morphological alterations, decreased mating, and reduced virulence [17]. The additional phenotype was the accumulation of melanin in the cytosol. The accumulated pigment was melanin, passing this phenotype to the modification of pH by the present mutation. In our study, upon deletion of the PMA1 gene, the mutant showed an accumulation of melanin. The H+ ATPase is an enzyme located at the plasma membrane that pumps protons outside the cell and generates a proton electrochemical potential across the membrane [44, 45]. _U. maydis_ contains two H+ ATPases genes, PMA1 and PMA2, and both are expressed in the wild type cells growing in the YPD or MM media [46]. In contrast, in the yeast _Saccharomyces cerevisiae_, only one gene (PMA1) is expressed [45]. In a recent publication about the _U.