# Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in _Candida albicans_
## 3 Results
### Biotin Auxotrophy and Carbon Dioxide  
_C. albicans_ is a biotin auxotroph [16; 17] and biotin is added routinely to all defined growth media for _C. albicans_[1]. We found that biotin, desthiobiotin, and KAPA were equivalent in fulfilling the biotin auxotrophy but pimelic acid was not (data not shown). Additionally, some vitamin requirements in bacteria and fungi are temperature dependent [39]. However, we found that the biotin auxotrophy in _C. albicans_ is not temperature dependent; biotin was required for growth at all temperatures from 20 to 42 \({}^{\circ}\)C (Figure 8). Figure 8: Lack of temperature dependence for the biotin auxotrophy. _C. albicans_ SC5314 were grown for two cycles in GPP without biotin at 30 \({}^{\circ}\)C for 30 h and then washed three times. The graph shows cultures grown into GPP for another 30 h with or without the addition of biotin. Cultures were observed at different temperature, and plotted as (closed symbol—no addition of biotin): \(\blacklozenge\), 25 \({}^{\circ}\)C; \(\blacksquare\), 30 \({}^{\circ}\)C; \(\blacklozenge\), 37 \({}^{\circ}\)C; \(\blacklozenge\), 42 \({}^{\circ}\)C; (open symbol—with 100 nM biotin): \(\bigcirc\), 25 \({}^{\circ}\)C; \(\square\), 30 \({}^{\circ}\)C; \(\bigtriangleup\), 37 \({}^{\circ}\)C; \(\bigtriangledown\), 42 \({}^{\circ}\)C. After two washes, dry weights of the cells were determined. Values shown are the average of triplicate experiments \(\pm\) SD. Figure 7: Relative expression of _C. albicans_ predicted biosynthesis genes in synthetic complete (SC) medium under biotin starvation (no added biotin) relative to growth with 2 ug/L biotin (set as “1”) and normalized for the housekeeping gene _CDC36_. The experiment was done in three biological replicates and displayed as the mean \(\pm\) SE. Significant upregulation of genes are marked by asterisk* (_p_ < 0.05). (**a**) _BIO2, BIO3, BIO4_, and _BIO32_. (**b**) Biotin protein ligase, _BPL1_. We also examined the possible influence of 5% CO\({}_{2}\) on biotin-dependent cell yields. This idea was tested because all biotin-containing enzymes have CO\({}_{2}\) as either their substrate or product [40]. GS agar plates containing four levels of biotin (0-4 uM) along with YPD control plates were incubated under four conditions (30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C both with and without 5% CO\({}_{2}\)) and colony diameter and dry weight were recorded after 5 days (Table 4). On these defined agar plates, the colonies were barely visible without the addition of biotin (Table 4 and Figure 9). However, with biotin the colony diameters and colony weights were only slightly smaller than those observed on the YPD plates (Table 4 and Figure 9). Significantly, it did not matter whether the biotin supplemented plates contained 5, 50, or 4000 nM biotin; the colony diameters and mass were not statistically different (Figure 9). High biotin (4 uM) did not induce hyphal growth in these cases because the GS agar plates start at pH 5.6 and become more acidic as growth proceeds. Furthermore, 5% CO\({}_{2}\) did not increase the colony diameter or mass except in one case (37 \({}^{\circ}\)C with 5% CO\({}_{2}\)) where the colony diameters with biotin (7 \(\pm\) 0.3 mm) were twice as large (Table 4) because the colonies were predominantly filamentous and had spread. The filamentous nature of the colonies was determined by both microscopy and the wrinkled appearance of the colonies (Figure 9). Wrinkled colonies were observed for the 37 \({}^{\circ}\)C/CO\({}_{2}\) grown cells regardless of whether the biotin supplemented plates contained 5, 50, or 4000 nM biotin (Figure 9) because 5%-10% CO\({}_{2}\) also triggers GTF [41; 42]. Finally, the short spikes seen emerging from the creamy, predominantly yeast colonies on YPD at 30 \({}^{\circ}\)C/CO\({}_{2}\) (Figure 9) were also filamentous when observed by microscopy. Their presence likely results from the fact that the colony interior is anaerobic and anaerobically hyphal growth is expected at 25 \({}^{\circ}\)C and 30 \({}^{\circ}\)C as well as at 37 \({}^{\circ}\)C [7].