We report here that in addition to a cytoplasmic copper-zinc-containing superoxide dismutase (SOD) and a mitochondrial manganese-containing SOD, Candida albicans expresses a third SOD gene (SOD3). The deduced amino acid sequence contains all of the motifs found in previously characterized manganese-containing SODs, except the presence of a mitochondrial transit peptide. Recombinant Sod3p expressed and purified from Escherichia coli is a homotetramer with a subunit mass of 25.4 kDa. Mass absorption spectrometry detected the presence of both iron and manganese in purified Sod3p but, as determined by metal replacement experiments, the enzyme displays activity only when bound to manganese. Overexpression of SOD3 was shown to rescue the hypersensitivity to redox cycling agents of a Saccharomyces cerevisiae mutant lacking the cytoplasmic copper-zinc-containing SOD. Northern blot analyses showed that the transcription of SOD3 is induced neither by the transition from the yeast to the mycelial form of C. albicans nor by drug-induced oxidative stress. In continuous cultures, the expression of SOD3 was strongly stimulated upon the entry and during the stationary phase, concomitantly with the repression of SOD1. We conclude that Sod3p is an atypical cytosolic manganese-containing superoxide dismutase that is involved in the protection of C. albicans against reactive oxygen species during the stationary phase.

The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal ؊ strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal ؉ strain grew and produced acid more rapidly than the Gal ؊ wild-type strain.Streptococcus thermophilus is widely used in yoghurt fermentation and cheese making. Specific S. thermophilus strains may also have the potential to improve human health as probiotics. For instance, they may interfere with the adhesion of indigenous cariogenic bacteria to teeth (9) and may prolong the useful life of indwelling voice prostheses by preventing colonization by Candida spp. (8).The ability of S. thermophilus to rapidly take up sugars from the environment is a prerequisite for these applications. Unlike several other lactic acid bacteria, S. thermophilus only uses a few sugars, with most strains showing a marked preference for saccharose and lactose, while glucose and fructose are more slowly fermented, if they are fermented at all (15,19). S. thermophilus takes up lactose via the membrane protein LacS, a member of the glycoside-pentoside-hexuronide:cation symporter family (29). Inside the cell, the disaccharide is hydrolyzed into glucose and galactose by the enzyme ␤-galactosidase. While all strains metabolize glucose via the EmbdenMeyerhof-Parnas pathway, most are unabl...

In Streptococcus thermophilus, a lactic acid bacterium widely used by the dairy industry, lactose is transported via a secondary symporter-type transport system consisting of a single membrane protein, LacS, that belongs to the glycoside-pentoside-hexuronide:cation symporter family (34), a subgroup of the major facilitator superfamily (39). In most bacteria that use this mode of transport, internalized lactose is hydrolyzed by ␤-galactosidase into glucose and galactose, which are metabolized via the Embden-Meyerhof-Parnas and Leloir pathways, respectively (15,20). However, most strains of S. thermophilus are unable to metabolize galactose due to insufficient expression levels of the galactokinase-encoding galK gene (29,48,49) and release the hexose into the external medium (19). The galactose expulsion phenomenon is closely associated with S. thermophilus LacS, which is able to catalyze two modes of transport: a ⌬p-driven lactose uptake in symport with protons and a lactose-galactose exchange (32-34). The exchange mode is stimulated by phosphorylation of a histidine residue at the C-terminal end of LacS. The phosphate donor has been identified as HPr(HisϳP) (16), and the target histidine is part of a hydrophilic domain homologous to IIA proteins (35). Both HPr and IIA proteins are components of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS).The PTS sequentially catalyzes the transport and PEP-dependent phosphorylation of mono-and disaccharides in a group translocation process involving the non-sugar-specific proteins enzyme I (EI) and HPr and sugar-specific EII proteins or domains called IIA, IIB, IIC, and IID (36,40). Sugar transport by the PTS is initiated by phosphorylation of HPr on a histidine residue at position 15 (His 15 ) by EI at the expense of PEP to generate HPr(HisϳP). The phosphate molecule is then sequentially transferred to the IIA and IIB domains or proteins. Sugar substrates of the PTS pass through the membrane by pores made up of IIC or IIC/IID proteins and are phosphorylated by phospho-IIBs. In addition to its pivotal role in sugar transport and its involvement in the control of S. thermophilus LacS, the HPr(HisϳP) of gram-positive bacteria is also involved, via phosphotransfer reactions, in the regulation of gene transcription and enzyme activity (7,42).HPrs of gram-positive bacteria can also be phosphorylated on a serine residue at position 46 (Ser 46 ) by an ATP-dependent

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(HisϳP), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(HisϳP) or HPr(Ser-P)(HisϳP). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.Streptococcus salivarius is the predominant bacterial species among the pioneer microorganisms that colonize the mouth (19). Acquisition of and competition for nutrients, particularly sugars, which serve as the major energy source for oral streptococci, constitute vital ecological determinants for the survival of oral bacteria. S. salivarius is able to metabolize a broad variety of sugars that can be grouped into two categories, non-PTS sugars, which are taken up by transport systems energized by proton motive force or ATP, and PTS sugars, which are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (4,35,38). The PTS uses phosphoenolpyruvate (PEP) in a group translocation process to phosphorylate incoming mono-and disaccharides via a phosphoryl-transfer cascade involving the non-sugar-specific proteins, Enzyme I (EI) and HPr, and a family of sugar-specific enzyme II complexes (EII) (27). In gram-positive bacteria, the PTS controls sugar metabolism by regulating transporter activities and gene transcription via the protein HPr (6, 29). This protein can be phosphorylated by EI at the expense of PEP on a histidine at position 15, generating HPr(HisϳP), and by a ATP-dependent protein kinase/phosphorylase, called HPrK/P, on a serine at position 46, generating HPr(Ser-P) (6,8,29). Both HPr(HisϳP) and HPr(Ser-P) possess regulatory functions. HPr(HisϳP) accomplishes its regulatory functions by reversibly phosphorylating its targets, and HPr(Ser-P) accomplishes its regulatory functions by protein-protein interactions (7,13,31,42). In addition to the aforementioned phosphorylated forms of HPr, rapidly growing streptococcal cells contain substantial amounts of the doubly phosphorylated form HPr(Ser-P)(HisϳP), whose functions remain unclear (33,36,37).Lactose (milk sugar) is a disacc...

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