Filamentous fungi produce high levels of polysaccharide-degrading enzymes and are frequently used for the production of industrial enzymes. Because of the high secretory capacity for enzymes, filamentous fungi are effective hosts for the production of foreign proteins. Genetic studies with Aspergillus nidulans have shown pathway-specific regulatory systems that control a set of genes that must be expressed to catabolize particular substrates. Besides the pathway-specific regulation, wide domain regulatory systems exist that affect a great many individual genes in different pathways. A molecular analysis of various regulated systems has confirmed the formal models derived from purely genetic data. In general, many genes are subject to more than one regulatory system. In this article, we describe two transcriptional activators, AmyR and XlnR, and an enhancer, Hap complex, in view of their regulatory roles in the expression of the amylolytic and (hemi-)cellulolytic genes mainly in aspergilli. The amyR gene has been isolated as a transcriptional activator involved in the expression of amylolytic genes from A. oryzae, A. niger, and A. nidulans, and the xlnR gene, which has been isolated from A. niger and A. oryzae, activates the expression of xylanolytic genes as well as some cellulolytic genes in aspergilli. Both AmyR and XlnR have a typical zinc binuclear cluster DNA-binding domain at their N-terminal regions. Hap complex, a CCAAT-binding complex, enhances the overall promoter activity and increases the expression levels of many fungal genes, including the Taka-amylase A gene. Hap complex comprises three subunits, HapB, HapC, and HapE, in A. nidulans and A. oryzae as well as higher eukaryotes, whereas HAP complex in Saccharomyces cerevisiae and Kluyveromyces lactis has the additional subunit, Hap4p, which is responsible for the transcriptional activation. Hap complex is suggested to enhance transcription by remodeling the chromatin structure. The regulation of gene expression in filamentous fungi of industrial interest could follow basically the same general principles as those discovered in A. nidulans. The knowledge of regulation of gene expression in combination with traditional genetic techniques is expected to be increasingly utilized for strain breeding. Furthermore, this knowledge provides a basis for the rational application of transcriptional regulators for biotechnological processes in filamentous fungi.
In screening for resistance to tannic acid, mutants of Saccharomyces cerevisiae with an altered cell wall composition were recently isolated. Here we show that these mutants were all respiratory deficient. Cytoplasmic petite mutants isolated after ethidium bromide mutagenesis were resistant to tannic acid and had cell wall characteristics similar to the mutants isolated by screening for tannic acid resistance as shown by the lower sensitivity to zymolyase, a cell wall hydrolyzing enzyme, and by a changed sensitivity to calcofluor white, a molecule interfering with the cell wall assembly. Reintroducing active mitochondria to a tannic-acid-resistant mutant reduced the tannic acid resistance and zymolyase resistance to the wild-type level, showing that a mitochondrial mutation was responsible for the changes in cell wall composition and in tannic acid sensitivity.
To envisage the roles of MexXY- and MexAB-multidrug efflux pumps in the intrinsic multidrug resistance of wild-type strain Pseudomonas aeruginosa PAO1, we constructed mutants lacking either individual or both efflux pumps. A mutant lacking MexXY showed increased susceptibility to aminoglycosides, erythromycin, and tetracycline, but not to β-lactams, chloramphenicol, or quinolones. A mutant lacking MexAB showed increased susceptibility to β-lactams, chloramphenicol, and nalidixic acid, but not to aminoglycosides, erythromycin, tetracycline, or fluoroquinolones. A mutant lacking both MexXY and MexAB showed an increased susceptibility to all antimicrobial agents tested compared with the wild type. Very similar results were obtained with a mutant lacking MexAB-OprM and a mutant lacking both MexXY and MexAB-OprM. Thus it is clear that OprM is essential not only for the function of MexAB, but also for the function of MexXY. Furthermore, we found that each pump compensated to some extent for the lack of another pump with respect to the common substrates (tetracycline, quinolones, and cefpirome). The introduction of a plasmid carrying the mexXY genes into P. aeruginosa PAO1 cells increased the resistance to fluoroquinolones. This suggests that the mexXY genes could be involved in acquired resistance to fluoroquinolones in P. aeruginosa PAO1.
By the application of a bioassay based on cresson seedlings, two phytotoxic compounds were isolated by thin-layer chromatography from the culture fluid of a Calonectria morganii isolate. The structure of both compounds was elucidated by ESI/MS and NMR spectroscopy. According to the Chemical Abstracts database, they were identified as chaetoglobosin A and 19-O-acetylchaetoglobosin A, mycotoxins originally described for Chaetomium globosum.
The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg+), intermediate (Bvgi), and modulated (Bvg+mod) phenotypes. Potential vaccine antigens can be expressed by Bvg+ strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg+B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg+ strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvgi phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg+ strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.