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.

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.

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 - lac Z fusion suggested that yqfS may be an additional member of the Eσ ^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 σ ^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.

Review of Molecular Techniques for the Identification of Bacterial Communities in Biological Effluent Treatment Facilities at Pulp and Paper Mills

ObjectiveThe main aim of the current study was to investigate whether SKA2 gene expression in the postmortem brain of rs7208505 genotype are altered in suicide victims from a Mexican population.MethodsIn this study, we report a genetic analysis of expression levels of the SKA2 gene in the prefrontal cortex of the postmortem brain of suicidal subjects (n = 22) compared to subjects who died of causes other than suicide (n = 22) in a Mexican population using RT-qPCR assays. Additionally, we genotyped the rs7208505 polymorphism in suicide victims (n = 98) and controls (n = 88) and we evaluate the association of genotypes for the SNP rs7208505 with expression level of SKA2.ResultsThe results showed that the expression of the SKA2 gene was significantly higher in suicide victims compared to control subjects (p = 0.044). Interestingly, we observed a greater proportion of allele A of the rs7208505 in suicide victims than controls. Even though there was no association between the SNP with suicide in the study population we found a significative association of the expression level from SKA2 with the allele A of the rs7208505 and suicide.ConclusionThe evidence suggests that the expression of SKA2 in the prefrontal cortex may be a critical factor in the etiology of suicidal behavior.

Aerated lagoons are a main unit operation for wastewater treatment in the paper industry. Many such operations involve inoculation with bacterial formulations in which in situ effectiveness has not been proven; this can be translated into low efficiency in treatment and unnecessary investments. Lack of knowledge of bacterial biodiversity present in a lagoon limits the capacity to exploit the maximum degradation. To overcome such problems, various methods to identify and study these microorganisms have been developed. In this study, a PCR-DGGE analysis was performed to estimate the bacterial diversity and to verify the presence of bacteria present in a commercial inoculum in two aerated lagoons of a pulp and paper effluent. Phylogenetic affiliation of predominant member’s correspondent to γ- and β-proteobacteria and Firmicutes were found. The dominant bacteria present in lagoon 2 belonged to the following genus Microbacterium sp. Rhodocyclaceae sp., Eubacterium sp. and B. subtilis. In lagoon 1 the dominant genus included Microbacterium sp., Rhodocyclaceae sp. Tepidimonas sp., Acetanaerobacterium sp., and Flavobacteria sp. The two characterized lagoons were not similar to the commercial inoculum. In addition, non-dominant bacteria (less relative intensity) were composed mostly of bacteria of the commercial inoculum.