The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His 6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His 6 -YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His 6 -YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3-phosphatases. However, the purified protein lacked both 5-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3 blocking groups of DNA generated during both spore dormancy and germination.During unpredicted periods of dormancy Bacillus subtilis spores are constantly exposed to environmental conditions that have the potential to cause several types of DNA damage. Therefore, the existence of spore-specific protecting mechanisms would seem to be fundamental for spore survival. One of the factors intricately involved in protecting spore DNA from several types of damage, such as oxidative stress, UV-C irradiation, and desiccation, is the presence of ␣/␤ type small acid-soluble proteins (reviewed in references 16, 28, and 27). Although ␣/␤ type small acid-soluble proteins protect spore DNA from several stresses, they confer protection neither to base alkylation (29) nor to UV-induced DNA strand break formation (30). Thus, while the physiological state of the B. subtilis spores prevents or dramatically slows DNA damage during the long periods of dormancy, it is clear that spores do accumulate potentially lethal and mutagenic DNA lesions such as the spore photoproduct, strand breaks, cyclobutane pyrimidine dimers, chemically altered bases and apurinic/apyridiminic (AP) sites which could affect transcription and replication processes during germination (16,26,29). To remove these potentially deleterious DNA damages and alteratio...

The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/ apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the E G regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the G regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.Endogenous and environmental factors such as reactive oxygen species, UV light, and chemical carcinogens alter the chemical structure of DNA bases, producing lesions that are substrates for a myriad of DNA glycosylases of the base excision repair (BER) pathway (27). The apurinic/apyrimidinic (AP) sites generated not only by the action of DNA glycosylases but also by the spontaneous depurination and depyrimidination of DNA (29, 30) are inherently toxic and highly mutagenic and thus should be rapidly processed and eliminated (31). The first catalytic event during the repair of AP sites is carried out by AP endonucleases, which cleave the DNA backbone immediately 5Ј of an AP site, generating a 5Ј deoxyribose-phosphate group and a 3Ј deoxyribose-hydroxyl group. AP endonucleases have been classified into two families, namely, ExoIII and type IV AP endonucleases (3, 13), and these enzymes have been conserved across the species of the three domains of life (23).Dormant spores of Bacillus subtilis are more resistant than their vegetatively growing counterparts to several chemical substances, including acids, bases, alkylating agents, and oxidizing agents (reviewed in references 40, 41, and 58). The existence of core coats, the low permeability of spores to hydrophilic compounds, and the protection of spore DNA from damage by its saturation with ␣/␤-type small acid-soluble proteins (SASPs) account for this resistance (reviewed in references 40, 56, and 58). It has been demonstrated that ␣/␤-type SASPs slow DNA depurination-depyrimidination, as well as hydroxyl radical-induced DNA backbone cleavage, thus contributing to spore resistance to heat and oxidizing agents (reviewed in references 40 and 58). ␣/␤-...

We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting) with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70) was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells.In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.Key words: sporotrichosis -Sporothrix schenckii -cell wall -glycoproteins -2D-immunoblotting analysisThe dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America, whose prevalence has significantly increased during the last few years mainly in immunocompromised patients (Callens et al. 2006, Lopes-Bezerra et al. 2006). The S. schenckii cell wall is composed of alkali-soluble and insoluble glucans, which are found in both morphological phases of the fungus (Previato et al. 1979, Lopes-Bezerra et al. 2006. Despite significant progress in the knowledge of structural polysaccharides, little is known regarding the identity and characteristics of cell wall proteins. Thus far, a peptide-rhamnomannan and peptide-rhamnogalactan have been isolated and characterized from wall preparations of yeast-like cells (Lloyd & Bitoon 1971, Nakamura 1976, Lopes-Bezerra et al. 2006). In addition, there are some reports dealing with the role of a cell wall glycoprotein of 70 kDa in adhesion of S. schenckii to host tissues and extracellular matrix components as well as fungal pathogenesis (Nascimento et al. 2008, Ruiz-Baca et al. 2009, Teixeira et al. 2009).2D-immunoblotting analysis has become one of the most frequently used tools to search antigens present in fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus (Pitarch et al. 2002, Gautam et al. 2007, Young et al. 2009). This approach was used here to further advance in the identification of antigenic proteins present in the cell wall of both morphologies of S. schenckii. Antibodies raised against the whole fungal cell allowed the identification of morphology-specific cell wall glycoproteins as well as glycoproteins present in both fungal phases.Organism and culture conditions -S. schenckii ATCC 58251 was used in this study. For mycelia propagation, 2-l Erlenmeyer flasks containing 600 mL of YPG medium (0.3% yeast extract, 1% peptone and 2% glucose, pH 4.5) were inoculated with 1 x 10 6 conidia mL -1 and incubated for 24 h at 28ºC with shaking (120 rpm). To obtain the yeast-like form, the pH of the medium was adjusted to 7.2 and the flasks were shaken for six days at 37ºC. Hyphae were collected by filtration in a Büchner filter and yeast-like cells were harvested by centrifugation at...

One of the processes most used in biotechnology today for handling industrial liquid wastes is biological wastewater treatment. The efficiency and quality of its operation depends on the composition and activity of the microbial community that is present. The application of traditional and molecular techniques has provided a glimpse into the "black box" and has given information to improve the wastewater treatment process. However, bleach pulp and paper mill effluents require a better understanding of the active bacterial population. For the study of these microorganisms, molecular techniques have been used for more than 15 years. However, there has been a lack of knowledge of the physiological requirements and relations with the environment, which seems to be very difficult to obtain involving profile on the diversity. Nowadays, highthroughput sequencing technology is a promising method that makes it possible to identify the entire profile of microbial communities. In combination with fingerprint methods, this approach allows the identification and analysis of the whole biodiversity of microbial communities. In this review, several identification techniques will be discussed.

Introduction The participation of non-Saccharomyces yeasts in fermentation processes is of great importance due to their participation in the formation of esters and superior alcohols, which confer characteristic aromas to beverages such as wine and mescal. The aim The aim of this study was identify and evaluate the potential aroma production of yeast native of Agave fermentation by the mescal production in Durango, Mexico. Isolated yeasts were molecularly identified by 5.8s ribosomal gene; the potential production of aromas was carried out in fermentations with the addition of L-phenylalanine and evaluated after 24 h of fermentation. Extraction and quantification of aromatic compounds by headspace solid-phase micro-extraction (HS-SPME) and gas chromatograph mass spectrometry (GC-MS). Results The isolated non-Saccharomyces yeasts could be classified into six different genera Saccharomyces cerevisiae, Clavispora lusitaniae, Torulaspora delbrueckii, Kluyveromyces dobzhanskii, Kluyveromyces marxianus, and Kluyveromyces sp. All probed strains presented a potential aroma production (ethyl acetate, isoamyl acetate, isoamyl alcohol, benzaldehyde, 2-phenylethyl butyrate, and phenylethyl propionate), particularly 2-phenylethanol and 2-phenylethylacetate; the levels found in the Kluyveromyces marxianus ITD0211 yeast have the highest 2-phenylethylacetate production at 203 mg/L and Kluyveromyces marxianus ITD0090 with a production of 2-phenylethanol at 1024 mg/L. Conclusion Non-Saccharomyces yeasts were isolated from the mescal fermentation in Durango; the Kluyveromyces genus is the most predominant. For the production of aromas, highlighting two strains of Kluyveromyces marxianus produces competitive quantities of compounds of great biotechnological interest such as 2-phenylethanol and 2-phenylethylacetate, without resorting to the genetic modification of yeasts or the optimization of the culture medium.