Sa-Ouk Kang

Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is required for the protection of Candida albicans against oxidative stresses and the expression of its full virulence

Copper- and zinc-containing superoxide dismutase (Cu/ZnSOD) is suspected to be one of the anti-oxidant enzymes and virulence determinants active in some pathogenic micro-organisms. To elucidate the role of Cu/ZnSOD in the major human fungal pathogen Candida albicans, its gene, designated SOD1, was disrupted by the URA-blaster technique. The resulting sod1/sod1 mutant showed delayed hyphal growth on Spider medium but could still form hyphae on other solid media or in liquid media, particularly in response to serum. Moreover, the sod1/sod1 mutant was more sensitive to menadione, a redox-cycling agent, than the isogenic wild-type strain, although it still showed an adaptive oxidative stress response. Furthermore, the sod1/sod1 mutant cells exhibited slow growth in minimal medium when compared to the wild-type cells, but their growth was restored by the addition of lysine to the medium. Interestingly, C. albicans cells lacking Cu/ZnSOD showed increased susceptibility to macrophage attack and had attenuated virulence in mice. Thus, these results suggest that Cu/ZnSOD is required for the protection of C. albicans against oxidative stresses and for the full virulence of the organism to be expressed.

Crystal structures of human DJ-1 and Escherichia coli Hsp31, which share an evolutionarily conserved domain.

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinsons disease is discussed.

A reducing system of the superoxide sensor SoxR in Escherichia coli

The soxRS regulon functions in protecting Escherichia coli cells against superoxide and nitric oxide. When SoxR is activated by oxidation of its [2Fe–2S] cluster, it increases the synthesis of SoxS, which then activates its target gene expression. How the oxidized SoxR returns to and is maintained in its reduced state has been under question. To identity genes that constitute the SoxR‐reducing system, we screened an E.coli mutant library carrying a chromosomal soxSp::lacZ fusion, for constitutive mutants. Mutations mapped to two loci: the rsxABCDGE operon (named for reducer of SoxR) that is highly homologous to the rnfABCDGE operon in Rhodobacter capsulatus involved in transferring electrons to nitrogenase, and the rseC gene in the rpoE–rseABC operon. In‐frame deletion of each open reading frame in the rsxABCDGE operon produced a similar constitutive phenotype. The double mutation of rsx and rseC suggested that rsxABCDGE and rseC gene products act together in the same pathway in reducing SoxR. Electron paramagnetic resonance analysis of SoxR and measurement of re‐reduction kinetics support the proposal that rsx and rseC gene products constitute a reducing system for SoxR.

The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc.

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state‐dependent interaction of a membrane‐bound protein with a repressor. The dephospho‐form of enzyme IICBGlc, but not its phospho‐form, interacts directly with Mlc and induces transcription of Mlc‐regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc‐regulated genes is caused by dephosphorylation of the membrane‐bound transporter enzyme IICBGlc, which directly recruits Mlc to derepress its regulon.

D‐Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae

D‐Arabinono‐1,4‐lactone oxidase catalysing the final step of D‐erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59 493 Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L‐gulono‐1,4‐lactone oxidase from rat and L‐galactono‐1,4‐lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8 kb in size. In the alo1 mutants, D‐erythroascorbic acid and the activity of D‐arabinono‐1,4‐lactone oxidase could not be detected. The intracellular concentration of D‐erythroascorbic acid and the enzyme activity increased up to 6.9‐fold and 7.3‐fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.

A Proposal To Reclassify Nocardia pinensis Blackall et al. as Skermania piniformis gen. nov., comb. nov.

The type strain of Nocardia pinensis was the subject of chemotaxonomic and 16S ribosomal DNA sequencing studies. The resultant nucleotide sequence was aligned with the sequences of representatives of the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella, and phylogenetic trees were generated by using the Fitch-Margoliash, maximum-parsimony, maximum-likelihood, and neighbor-joining methods. It was evident from the phylogenetic analyses that N. pinensis represents a distinct phyletic line that is most closely associated with the Gordona clade. This genealogical evidence, together with chemotaxonomic and phenotypic data derived from this and previous studies, indicates that N. pinensis merits generic status within the family Nocardiaceae. Therefore, we propose that N. pinensis Blackall et al. 1989 be reclassified as Skermania piniformis gen. nov., comb. nov. The type strain of Skermania piniformis cleaved an array of conjugated substrates based on the fluorophores 7-amino-4-methylcoumarin and 4-methylumbelliferone.

Streptomyces seoulensis sp. nov.

The taxonomic position of an actinomycete strain isolated from Korean soil was examined by a polyphasic approach. The isolate, designated IMSNU-1, was clearly assigned to the genus Streptomyces on the basis of morphological and chemotaxonomic data. The test strain was the subject of a probabilistic identification study using the identification matrices generated by Langham et al. (J. Gen. Microbiol. 135:121-133, 1989) and found to be marginally close to clusters 19 and 39. An almost complete 16S rRNA gene (rDNA) sequence was obtained for the test strain and compared with those of representative streptomycetes. 16S rDNA sequence data not only support the strains membership in the genus Streptomyces but also provide strong evidence that our isolate is genealogically distant from representatives of clusters 19 and 39, forming a separate phyletic line in a clade encompassed by streptomycetes. It is therefore proposed from the polyphasic evidence that strain IMSNU-1 be classified in the genus Streptomyces as Streptomyces seoulensis sp. nov.

Protective roles of mitochondrial manganese-containing superoxide dismutase against various stresses in Candida albicans

Candida albicans contains copper‐ and zinc‐containing superoxide dismutase but also two manganese‐containing superoxide dismutases (MnSODs), one in the cytosol and the other in the mitochondria. Among these, the SOD2 gene encoding mitochondrial MnSOD was disrupted and overexpressed to investigate its roles in C. albicans. The null mutant lacking mitochondrial MnSOD was more sensitive than wild‐type cells to various stresses, such as redox‐cycling agents, heating, ethanol, high concentration of sodium or potassium and 99.9% O2. Interestingly, the sod2/sod2 mutant was rather more resistant to lithium and diamide than the wild‐type, whereas overexpression of SOD2 increased susceptibility of C. albicans to these compounds. The inverse effect of mitochondrial MnSOD on lithium toxicity was relieved when the sod2/sod2 and SOD2‐overexpressing cells were grown on the synthetic dextrose medium containing sulphur compounds such as methionine, cysteine, glutathione or sulphite, indicating that mitochondrial MnSOD may affect lithium toxicity through sulphur metabolism. Moreover, disruption or overexpression of SOD2 increased or decreased glutathione reductase activity and cyanide‐resistant respiration by alternative oxidase, respectively. Taken together, these findings suggest that mitochondrial MnSOD is important for stress responses, lithium toxicity and cyanide‐resistant respiration of C. albicans. Copyright

D-Arabinose dehydrogenase and its gene from Saccharomyces cerevisiae.

D-Arabinose dehydrogenase was purified 843-fold from the cytosolic fraction of Saccharomyces cerevisiae with a recovery of 9%. The purified enzyme gave two bands with a molecular mass of 40 and 39 kDa on SDS-PAGE. The native enzyme had a molecular mass of 74 kDa as estimated by Sephacryl S-200 chromatography. Therefore, this enzyme was considered to be a heterodimer. The purified enzyme exhibited maximum activity at pH 10.0 and around 30 degrees C. The enzyme catalysed the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+. The apparent Km values at pH 10.0 with 50 microM NADP+ for D-arabinose, L-xylose, L-fucose, and L-galactose were 161, 24, 98 and 180 mM, respectively. The pH profile of Vmax and kcat/Km showed one ionisable groups around pH 8.3. D-Erythroascorbic acid was formed in vitro from D-arabinose by D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase. The N-terminal amino acid sequence of the heavy subunit was Ser-Thr-Glu-Asn-Ile-Val-Glu-Asn-Met-Leu-His-Pro-Lys-Thr-. The N-terminus of the light subunit was blocked. The obtained peptide sequence was identical to the translational product of an unknown open reading frame, YBR149W, in chromosome II of S. cerevisiae. When compared with the translational product of this open reading frame, the peptide sequence was identical to the amino acid sequences of residues 7 to 20. The first six amino acids of this open reading frame were lost in protein sequence, which may be modified post-translationally. The heavy subunit was composed of 344 amino acid residues and its deduced amino acid sequence contained the motifs I, II, and III of aldo-keto reductase and also leucine zipper motif. This enzyme is the first heterodimeric protein of aldo-keto reductase family. In the deletion mutant of this gene, D-arabinose dehydrogenase activity and D-erythroascorbic acid were not detected.

Ssn6, an important factor of morphological conversion and virulence in Candida albicans

Candida albicans , the major fungal pathogen in humans, undergoes morphological conversion from yeasts to filamentous growth forms depending upon various environmental conditions. Here, we have identified a C. albicans gene, namely SSN6 , encoding a putative global transcriptional co‐repressor that is highly homologous to the Saccharomyces cerevisiae Ssn6. The isolated C. albicans SSN6 complemented the pleiotropic phenotypes of S. cerevisiae ssn6 mutation, and its expression levels declined significantly in response to a strong true hyphal inducer, serum. The mutant lacking C. albicans Ssn6 displayed a stubby pseudohyphal growth pattern, derepressed filament‐specific genes in response to elevated temperature 37°C and failed to develop true hyphae, extensive filamentation and virulence. Such morphological defects of ssn6 / ssn6 mutant were not rescued by overexpression of Tup1, Cph1 or Efg1. Moreover, epistatic analysis showed that, as far as cell morphology was concerned, Ssn6 was epistatic to Tup1 at the higher temperature but that, at the lower temperature, the ssn6 / ssn6 tup1 / tup1 double mutant grew in a stubby form of pseudohyphae distinct from the phenotypes of either single mutant. Furthermore, overexpression of SSN6 in C. albicans led to enhanced filamentous growth and attenuated virulence. These findings suggest that Ssn6 may function as an activator as well as a repressor of filamentous growth and be a target for candidacidal drugs, as its excess or deficiency resulted in impaired virulence.