# Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in _Candida albicans_
## 3 Results  
### Biotin Enhances Germ Tube Formation in both GPP and Lee's Medium  
The chemically defined media commonly used to achieve germ tube formation (GTF) in _C. albicans_ are remarkably diverse in their chemical composition. One feature which stands out is the concentration of biotin present, ranging from 20 \(\mathrm{\SIUnitSymbolMicro g}\)/L in GPP up to 200 \(\mathrm{\SIUnitSymbolMicro g}\)/L in RPMI 1640 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/L (4 \(\mathrm{\SIUnitSymbolMicro m}\)) in Lee's Medium ([PERSON] et al., 2016), even though [PERSON] and [PERSON] (2016) concluded that 1-5 \(\mathrm{\SIUnitSymbolMicro g}\)/L biotin was sufficient for maximal growth. Significantly, both yeast nitrogen base and yeast carbon base contain only \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Gene Abbreviation**} & \multicolumn{2}{c}{**Primer Sequences**} & \multirow{2}{*}{**Source**} \\  & **(Forward -F and Reverse -R)** & \\ \hline _BIO2_ & F: 5′-GCACACCCAAGATCATTGCCAA-3′ & This study \\ orf19.2593 & R: 5′-ACTGCCTCGTTCCTTCATG-3′ & \\ \hline _BIO4_ & F: 5′-AGTGCCTGAGATG-3′ & This study \\ orf19.2590 & R: 5′-TTAGAATGAGGATGTCGCGA-3′ & This study \\ \hline _BIO32_ & F: 5′-GTGCAGGATTATTTTGGGCAA-3′ & This study \\ orf19.3567 & R: 5′-TCCGCTATTGTCCCTTCCA-3′ & This study \\ \hline _BIO3_ & F: 5′-AAAATGGCCTGGAACACT-3′ & This study \\ orf19.2591 & R: 5′-GGCGAAACCCAAACACCTAAA-3′ & This study \\ \hline _BPL1_ & F: 5′-GTTGATGATGACGACGTGA-3′ & This study \\ orf19.7645 & R: 5′-GCCATTGTCAACGTCCACCTT-3′ & This study \\ \hline _CDC36_ & F: 5′-GACCGTCACATATAATCCACACACAC-3′ & [PERSON] et al. (2016) \\ R: 5′-TCAAAGAGGGCCTCCCACATTACTAT-3′ & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: List of primers used for gene expression. Amplification was done using _CDC36_ as the reference genes. 2 \(\mathrm{\SIUnitSymbolMicro}\)/L biotin (Difco Manual) as does the glucose synthetic dextrose medium commonly used for _S. cerevisiae_[31]. Thus, the concentration of biotin in [PERSON]'s medium is 500-fold higher than the 2 \(\mathrm{\SIUnitSymbolMicro}\)/L present in synthetic dextrose or yeast nitrogen base. Biotin (4 \(\mathrm{\SIUnitSymbolMicro}\)M) stimulated GTF in both GPP (Figure 2) and [PERSON]'s medium (Figure 3a). In [PERSON]'s medium we observed a dose-response relationship between % GTF and biotin (0 to 4 \(\mathrm{\SIUnitSymbolMicro}\)M) such that the high biotin grown cells initiated GTF earlier and by 2 h had formed ca. 10-fold more germ tubes (Figure 3a). This relationship was also observed in a modified [PERSON]'s medium from which L-pro, a known trigger for GTF, had been omitted (Figure 3b), and for [PERSON]'s medium in which the biotin precursors desthiobiotin and KAPA replaced biotin (Figure 3c). These data show that the high concentration of biotin in [PERSON]'s medium contributes to its ability to enhance GTF and that the presence of L-pro is not necessary for biotin to exert this activity. Figure 3: The germ tube formation (GTF) assays were conducted in [PERSON]’s media containing variable levels of biotin at 37 \({}^{\circ}\)C with rotary agitation at 220 rpm. Cultures were observed for 4 to 5 h. **(a)** Dose-response for biotin. Symbols:, no addition;, 10 nM biotin;, 100 nM biotin;, 400 nM biotin; and, 4 \(\mathrm{\SIUnitSymbolMicro}\)M biotin. **(b)** GTF assays in Lee’s Medium prepared without L-proline. Symbols:, no addition; and, 4 \(\mathrm{\SIUnitSymbolMicro}\)M biotin. **(c)** Desthiobiotin and KAPA also stimulate GTF., no addition;, 4 \(\mathrm{\SIUnitSymbolMicro}\)M biotin; and, 4 \(\mathrm{\SIUnitSymbolMicro}\)M KAPA. Values shown are the average of triplicate experiments \(\pm\) SD. Figure 2: The germ tube formation (GTF) assays were conducted in glucose-phosphate-proline (GPP) (pH 6.5) containing variable levels of biotin at 37 \({}^{\circ}\)C with rotary agitation at 220 rpm. Cultures were observed for 5 h, and plotted as, no addition;, 100 nM biotin;, 400 nM biotin; and, 1 \(\mathrm{\SIUnitSymbolMicro}\)M biotin. Values shown are the average of triplicate experiments \(\pm\) SD.