# Biotin Auxotrophy and Biotin Enhanced Germ Tube Formation in _Candida albicans_
## 1 Introduction  
_Candida albicans_ (_C. albicans_) is an important fungal pathogen of humans. As such, there has been intense interest in the virulence factors which make _C. albicans_ such a versatile pathogen, and of these virulence factors the one which has garnished the greatest attention is the ability to interconvert between the yeast and hyphal morphologies. In his monumental work on Candida and candidiasis, [PERSON] [1] listed 34 chemical and environmental factors which favored filamentous forms and 25 factors which favored yeast/blastospore forms or suppressed hypha formation. Media commonly used to produce _C. albicans_ hyphae in vitro include: (i) [PERSON]'s medium which contains eight amino acids [2]; (ii) serum [1,2]; (iii) buffered _N_-acetyl-D-glucosamine [3,4]; (iv) L-proline [5]; and (v) glucose-phosphate-proline (GPP) which may also have 2.5 mM N-acetyl-D-glucosamine (mGPP) [6], all at \(\geq\)37 \({}^{\circ}\)C. In addition, anaerobic growth gave hyphae at all temperatures tested from 25 \({}^{\circ}\)C to 37 \({}^{\circ}\)C [7]. Current research has focused on the signal transduction mechanisms and transcription factors which connect these external triggers with the induction of hyphae-associated genes [8]. [PERSON] [8] distinguished nine positive and negative environmental cues. Farnesol and bacterial signaling molecules in the 3-oxo-homoserine lactone family were negative cues while the seven positive cues included: (i) growth in an embedded matrix or microaerophilic environment; (ii) low nitrogen; (iii) serum; (iv) \(>\)5% CO\({}_{2}\); (v) L-methionine; (vi) _N_-acetylglucosamine; and (vii) a neutral pH or the absence of an acidic pH. However, there are still many unanswered questions regarding the identity of the hyphal triggers, like which component(s) of [PERSON]'s medium and serum actually induce hypha formation. These questions persist despite evidence that for serum both glucose [9] and bacterial peptidoglycan-like components [10] are part of the answer. Thus, we believe that some important hyphal triggers are still unknown or at least under-appreciated. Throughout, we will distinguish between a hyphal trigger, which by itself can initiate hyphal development, and an enhancer, which improves the effectiveness of a hyphal trigger. _C. albicans_ is a biotin auxotroph [1] and our previous work on biotinylated histones in _C. albicans_[11] showed that the cell yields for _C. albicans_ in a biotin-free glucose-phosphate-proline (GPP) medium through three growth cycles (ca. 20 buddings) were ca. 60%, 40%, and 20%, respectively, of those achieved in a biotin replete medium. The presence of an internal biotin reservoir, possibly biotinylated histones, was indicated by the same decreasing cell yields being observed with and without added neutravidin in the biotin-free medium. Neutravidin is a strong biotin-binding protein. The present paper studies the role of added biotin in germ tube formation at 37 \({}^{\circ}\)C. The interactions of biotin with yeasts date back to the discovery of biotin by [PERSON] and [PERSON] [12]. They identified biotin as a growth factor obtained from eggs and yeast which promoted the growth of all baker's and distiller's yeasts tested, as well as a large number of other yeasts. Subsequently it was shown that _S. cerevisiae_ needs biotin or the biotin analogs D-desthiobiotin, biocytin, or biotin-D-sulphoxide [13]. Biotin biosynthesis is a variable trait in _S. cerevisiae_ in that many strains only contain a partial biosynthetic pathway. Presumably the pathway was lost in a distant ancestor and then rebuilt by horizontal gene transfer, gene duplication, and neofunctionalization [14]. However, the last four steps are generally conserved: pimeloyl-CoA to 7-keto-8-aminopelargonic acid (KAPA) to 7,8-diaminopelargonic acid (DAPA) to desthiobiotin to biotin (Figure 1). 7-keto-8-amino-pelargonic acid and 7,8-diamino-pelargonic acid are also called 8-amino-7-oxononanoate and 7,8-diaminononanoate respectively. Thus, [PERSON] et al. [15] found that _S.