Identification of a Copper-Inducible Promoter for Use in Ectopic Expression in the Fungal Pathogen
            <i>Histoplasma capsulatum</i>

Son

et al. 2010

Appl Environ Microbiol

The ascomycete fungus Gibberella zeae is an important plant pathogen that causes fusarium head blight on small grains. Molecular studies of this fungus have been performed extensively to uncover the biological mechanisms related to pathogenicity, toxin production, and sexual reproduction. Molecular methods, such as targeted gene deletion, gene overexpression, and gene fusion to green fluorescent protein (GFP), are relatively easy to perform with this fungus; however, conditional expression systems have not been developed. The purpose of this study was to identify a promoter that could be induced by zearalenone (ZEA) for the development of a conditional expression system in G. zeae. Through microarray analysis, we isolated one zearalenone response gene (ZEAR) whose expression was increased more than 50 times after ZEA treatment. Northern blot analysis showed that the ZEAR transcript dramatically increased after 1 h of ZEA treatment. To determine the utility of the ZEAR promoter, called Pzear, in a conditional expression system, we transformed a Pzear::GFP fusion construct into G. zeae. Our data showed a ZEA concentrationdependent increase in GFP expression. We also replaced the promoter of G. zeae metE (GzmetE), an essential gene for methionine biosynthesis, with the Pzear promoter. The growth of the Pzear-GzmetE mutant on minimal medium was dependent on the ZEA concentration supplemented in the medium and showed that GzMetE expression was induced by ZEA. This study is the first report of an inducible promoter in G. zeae. Our system will be useful for the characterization of essential gene functions in this fungus through differential and ZEA-dependent gene expression. In addition, the Pzear promoter may be applicable as a biosensor for the detection of ZEA contamination in agricultural products.

Section: Vol 76 2010 Zearalenone-inducible Promoter 3091supporting

confidence: 66%

Section: Vol 76 2010 Zearalenone-inducible Promoter 3091mentioning

confidence: 59%

Development of a Conditional Gene Expression System Using a Zearalenone-Inducible Promoter for the Ascomycete Fungus Gibberella zeae

Son

et al. 2010

Appl Environ Microbiol

Proc. Natl. Acad. Sci. U.S.A.

“…Both Wor1 and Ryp1 bind to their own promoters, raising the possibility that Ryp1, like Wor1, may induce a self-sustaining feedback loop that promotes RYP1 transcript accumulation under appropriate conditions. Indeed, we observed that in particular transformants, ectopic expression of RYP1 under the control of a copper-inducible promoter (38) resulted in high-level induction of the endogenous RYP1 transcript (data not shown). However, this phenomenon was not consistently observed, suggesting that the switch between potential expression states may be more complex than a simple positive-feedback loop.…”

Section: Discussionmentioning

confidence: 93%

Temperature-induced switch to the pathogenic yeast form of Histoplasma capsulatum requires Ryp1, a conserved transcriptional regulator

Nguyen

Sil

2008

Self Cite

174

207

Histoplasma capsulatum, a fungal pathogen of humans, switches from a filamentous spore-forming mold in the soil to a pathogenic budding-yeast form in the human host. This morphologic switch, which is exhibited by H. capsulatum and a group of evolutionarily related fungal pathogens, is regulated by temperature. Using insertional mutagenesis, we identified a gene, RYP1 (required for yeast phase growth), which is required for yeast-form growth at 37°C. ryp1 mutants are constitutively filamentous irrespective of temperature. Ryp1 is a member of a family of fungal proteins that includes Wor1, a master transcriptional regulator of the whiteopaque transition required for mating in Candida albicans. Ryp1 associates with its own upstream regulatory region, consistent with a direct role in transcriptional control, and both the protein and its transcript accumulate to high levels in wild-type yeastphase cells. Microarray analysis demonstrated that Ryp1 is required for the expression of the vast majority of yeast-specific genes, including two genes linked to virulence. Thus, Ryp1 appears to be a critical transcriptional regulator of a temperature-regulated morphologic switch in H. capsulatum.fungal pathogenesis ͉ gene regulation ͉ morphology

“…Therefore, it may not contain sugar-regulated promoters, which are widely used for inducible gene expression in its relative, C. crescentus (18), and many other bacteria. Prompted by the successful application of heavy metal-inducible expression systems in other organisms (19)(20)(21)(22), we investigated whether this strategy also would be applicable to H. neptunium. In doing so, we specifically focused on copper and zinc, two heavy metals with moderate toxicity that also serve as essential trace elements (23,24).…”

Section: Identification Of Heavy Metal-inducible Promotersmentioning

confidence: 99%

Molecular Toolbox for Genetic Manipulation of the Stalked Budding Bacterium Hyphomonas neptunium

Jung

Eisheuer

Cserti

et al. 2015

Appl Environ Microbiol

The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. Bacteria are a phylogenetically diverse group of organisms whose study has provided important insights into the mechanisms that mediate the spatiotemporal organization of cells. However, most of our knowledge on bacterial cell biology so far has come from the analysis of only a few well-established model species, such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, which typically exhibit a rod-like morphology and divide by symmetric or asymmetric binary fission.To further our understanding of subcellular organization in bacteria, we have started to investigate the marine alphaproteobacterium Hyphomonas neptunium (1), a representative of the stalked budding bacteria (2, 3). Similar to other members of this polyphyletic bacterial group, H. neptunium is characterized by a unique mode of reproduction that involves the formation of buds at the tip of a stalk emanating from the mother cell body (Fig. 1A). During the budding process, the nascent daughter cell is equipped with a single polar flagellum at the pole opposite the stalk. Cytokinesis then gives rise to a motile swarmer cell, which initially is unable to replicate, and an immotile stalked cell, which immediately enters a new round of budding and cell division (4). At a defined time in the cell cycle, the swarmer cell undergoes a differentiation process during which it sheds the flagellum and establishes a stalk at the opposite pole. Subsequently, a bud emerges from the tip of the stalk, setting the stage for the next division event.H. neptunium was isolated from the harbor of Barcelona (Spain). Based on morphological criteria, it was originally described as Hyphomicrobium neptunium (1). Later, DNA-DNA hybridization experiments, 5S rRNA sequence analyses, and metabolic profiling revealed a close phylogenetic relationship to members of the genus Hyphomonas (5, 6). Interestingly, 16S rRNA-based phylogenetic studies identify H. neptunium as a member of the Rhodobacterales (7). However, 23S rR...