# Genetic, Physiological, and Industrial Aspects of the
## 3 _S. bacillaris_ Physiology  
_S. bacillaris_ grows as ellipsoid to elongated (2.2-3.0 mm \(\times\) 3.0-5.2 mm) cells, which divide by multilateral budding [9] (Figure 2). Indigenous strains of this yeast species have been isolated worldwide, from grapes and grape musts, using the general yeast growth media YPD-agar (Figure 3a), the differential media WL-nutrient-agar (Figure 3b), and the selective media YPD agar, supplemented with cycloheximide (Figure 3d-f) and/or lysine-agar [20; 48]. In standard YPD-agar media, _S. bacillaris_ form small, white, creamy, shiny colonies (Figure 3a), while similar colonies, but green with a white peripheral halo, develop in WL-nutrient agar media (Figure 3b,c), which enables it to be differentiated from other non-_Saccharomyces_ species (Figure 3c). Enological species of the _Hansenispora_ genera (i.e., _H. oputtiae_, _H. osmophila_, _H. uvarum_, and _H. vineae_) also form green colonies in WL-nutrient agar, although these are larger and flat (see Figure 3c). _S. bacillaris_ can ferment glucose, sucrose, fructose, and raffinose, but not galactose, maltose, and lactose [9]. It shows a marked preference for fructose over glucose when both sugars are present simultaneously [7; 10; 25; 50; 51]. This trtotophilic character is also associated with other yeast species found in high sugar environments (e.g., _Candida apicola_, _Candida magnoline_, _Candida versatilis_, _S. bombicola_, _Zygosaccharomyces bailii_, and _Zygosaccharomyces rouxii_) [10; 52; 30]. Genetic evidence indicates that the trtotophilic character of members of the W/S clade is dependent on a specific, low affinity, high capacity fructose transporter named \"Frz1\" [52]. In silico analyses of _S. bacillaris_ draft genome sequences showed the presence of two distinct _FFZ1_ genes at a distance of approximately 4 kb from each other [52]. These transporters (i.e., Ffz1a and Ffz1b) proved to enable growth on fructose and mannose when expressed as sole hexose transporters in a _S. cerevisiae_ hxt-null mutant strain [52]. Kinetic parameters of these two transporters revealed that they are not functionally identical: Ffz1a more closely resembles the Ffz1 transporter from _Z. rouxii_, \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Restriction Enzyme**} & \multicolumn{3}{c}{**Fragment Size (bp)**} \\ \cline{2-4}  & _S. bacillaris_ & _C. stellata_ & _S. bombicola_ \\ \hline Uncut & 460 & 468 & 467 \\ _Cf0_I & \(56+103+105+196\) & \(56+200+212\) & \(39+56+172+200\) \\ _Hae_III & 460 & 468 & \(54+87+138+326\) \\ _Hint_I & 225 + 235 & 229 + 239 & \(8+227+232\) \\ _DraI_ & 309 + 115 + 36 & 119 + 349 & \(143+324\) \\ _MbI_ & 22 + 145 + 293 & \(22+135+149+162\) & \(22+137+138+170\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Digestion pattern of _S. bacillaris_ ITS-5.8S region. Figure 2: _S. bacillaris_ cell morphology. Cellular morphology of _C. zemplinina_ (syn., _S. bacillaris_) strain CBS 9494 as observed under differential interference contrast (DIC) microscopy (**a**) or fluorescence (i.e., calcofluor-white staining) microscopy, which highlights cell walls and budding scars (**b**). Images were obtained using a Leica DMI8 inverted microscope and 63\(\times\) objective. which seems to be indispensable for fructophily [53], while Ffz1b supports weaker growth on fructose and supports growth on mannose [52]. An interesting finding concerning fermentation in members of the W/S clade is the apparent absence of a typical pyruvate decarboxylase (PDC) enzyme [30]. In fact, decarboxylation of pyruvate to acetaldehyde is a key step in the alcoholic fermentative pathway, catalyzed by Pdcl in _S. cerevisiae_[54; 55; 56]. Although orthologs of this gene appear to be absent in W/S-clade genomes, a modification of specificity in the enzyme Aro10, allowing this enzyme to accept pyruvate in addition to phenylpyruvate as a substrate, appears to be involved in the remodeling of alcoholic fermentation in W/S clade yeasts [30].