Regulation Of Filamentous Growth Research Articles

Comparative proteomics reveals the mechanism of cyclosporine production and mycelial growth in Tolypocladium inflatum affected by different carbon sources.

Cyclosporine A (CsA) is a secondary cyclopeptide metabolite produced by Tolypocladium inflatum that is widely used clinically as an immunosuppressant. CsA production and mycelial growth differed when T. inflatum was cultured in different carbon source media. During early fermentation, CsA was preferred to be produced in fructose medium, while the mycelium preferred to accumulate in sucrose medium. On the sixth day, the difference was most pronounced. In this study, high-throughput comparative proteomics methods were applied to analyze differences in protein expression of mycelial samples on day 6, revealing the proteins and mechanisms that positively regulate CsA production related to carbon metabolism. The differences included small molecule acid metabolism, lipid metabolism, organic catabolism, exocrine secretion, CsA substrate Bmt synthesis, and transcriptional regulation processes. The proteins involved in the regulation of mycelial growth related to carbon metabolism were also revealed and were associated with waste reoxidation processes or coenzyme metabolism, small molecule synthesis or metabolism, the stress response, genetic information or epigenetic changes, cell component assembly, cell wall integrity, membrane metabolism, vesicle transport, intramembrane localization, and the regulation of filamentous growth. This study provides a reliable reference for CsA production from high-efficiency fermentation. This study provides key information for obtaining more CsA high-yielding strains through metabolic engineering strategies.

Protein kinase A participates in hyphal and appressorial development by targeting Efg1-mediated transcription of a Rab GTPase in Setosphaeria turcica.

The cyclic adenosine monophosphate (cAMP) signalling pathway plays an important role in the regulation of the development and pathogenicity of filamentous fungi. cAMP‐dependent protein kinase A (PKA) is the conserved element downstream of cAMP, and its diverse mechanisms in multiple filamentous fungi are not well known yet. In the present study, gene knockout mutants of two catalytic subunits of PKA (PKA‐C) in Setosphaeria turcica were created to illustrate the regulatory mechanisms of PKA‐Cs on the development and pathogenicity of S. turcica. As a result, StPkaC2 was proved to be the main contributor of PKA activity in S. turcica. In addition, it was found that both StPkaC1 and StPkaC2 were necessary for conidiation and invasive growth, while only StPkaC2 played a negative role in the regulation of filamentous growth. We reveal that only StPkaC2 could interact with the transcription factor StEfg1, and it inhibited the transcription of StRAB1, a Rab GTPase homologue coding gene in S. turcica, whereas StPkaC1 could specifically interact with a transcriptional regulator StFlo8, which could rescue the transcriptional inhibition of StEfg1 on StRAB1. We also demonstrated that StRAB1 could positively influence the biosynthesis of chitin in hyphae, thus changing the filamentous growth. Our findings clarify that StPkaC2 participates in chitin biosynthesis to modulate mycelium development by targeting the Efg1‐mediated transcription of StRAB1, while StFlo8, interacting with StPkaC1, acts as a negative regulator during this process.

Evaluating the activity of the filamentous growth mitogen-activated protein kinase pathway in yeast.

Mitogen-activated protein kinase (MAPK) pathways are evolutionarily conserved signaling pathways that regulate diverse processes in eukaryotes. One such pathway regulates filamentous growth, a nutrient limitation response in budding yeast and other fungal species. This protocol describes three assays used to measure the activity of the filamentous growth pathway. First, western blotting for phosphorylated (activated) MAPKs (P∼MAPKs; Slt2p, Kss1p, Fus3p, and Hog1p) provides a measure of MAPK activity in yeast and other fungal species. Second, the PGU1 gene is a transcriptional target of the filamentous growth pathway. Cells that undergo filamentous growth secrete Pgu1p, an endopolygalacturonase that degrades the plant-specific polysaccharide pectin. We describe an assay that measures secreted pectinase activity, which reflects an active filamentous growth pathway. Finally, in yeast, two mucin-like glycoproteins, Msb2 and Flo11, regulate filamentous growth. Secretion of the processed and shed glycodomain of Msb2 is an indicator of MAPK activity. Flo11, the major adhesion molecule that controls filamentous growth and biofilm/mat formation, is also shed from cells. Detecting shed mucins with epitope-tagged versions of the proteins (secretion profiling) provides information about the regulation of filamentous growth across fungal species.

Regulation of Vacuolar H + -ATPase Activity by the Cdc42 Effector Ste20 in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc42 effector Ste20 plays a crucial role in the regulation of filamentous growth, a response to nutrient limitation. Using the split-ubiquitin technique, we found that Ste20 forms a complex with Vma13, an important regulatory subunit of vacuolar H(+)-ATPase (V-ATPase). This protein-protein interaction was confirmed by a pulldown assay and coimmunoprecipitation. We also demonstrate that Ste20 associates with vacuolar membranes and that Ste20 stimulates V-ATPase activity in isolated vacuolar membranes. This activation requires Ste20 kinase activity and does not depend on increased assembly of the V1 and V0 sectors of the V-ATPase, which is a major regulatory mechanism. Furthermore, loss of V-ATPase activity leads to a strong increase in invasive growth, possibly because these cells fail to store and mobilize nutrients efficiently in the vacuole in the absence of the vacuolar proton gradient. In contrast to the wild type, which grows in rather small, isolated colonies on solid medium during filamentation, hyperinvasive vma mutants form much bigger aggregates in which a large number of cells are tightly clustered together. Genetic data suggest that Ste20 and the protein kinase A catalytic subunit Tpk2 are both activated in the vma13Δ strain. We propose that during filamentous growth, Ste20 stimulates V-ATPase activity. This would sustain nutrient mobilization from vacuolar stores, which is beneficial for filamentous growth.

The Regulation of Filamentous Growth in Yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.

Coevolution of Cyclin Pcl5 and Its Substrate Gcn4

Gcn4, a transcription factor that plays a key role in the response of Saccharomyces cerevisiae to amino acid starvation, is regulated at both the levels of translation and of protein stability. Regulated degradation of Gcn4 depends on its phosphorylation by the cyclin-dependent kinase Pho85, in conjunction with the cyclin Pcl5. The pathogenic yeast Candida albicans contains a functional homolog of Gcn4, which is involved in amino acid metabolism, as well as in the regulation of filamentous growth in response to starvation. Here, we show that C. albicans Gcn4 (CaGcn4) is rapidly degraded and that this degradation depends on a Pho85 cyclin homolog, CaPcl5. The regulatory loop that includes Gcn4 and Pcl5 is conserved in C. albicans: like in S. cerevisiae, CaPcl5 is transcriptionally induced by CaGcn4 and is required for CaGcn4 degradation. However, the proteins have coevolved so that there is no cross-recognition between the proteins from the two species: phosphorylation-dependent degradation of CaGcn4 occurs only in the presence of CaPcl5, and S. cerevisiae Gcn4 (ScGcn4) requires ScPcl5 for its degradation. Phenotypic analysis of the Capcl5 mutant indicates that CaPcl5 also modulates the filamentous response of C. albicans in amino acid-rich media.

Colony shape as a genetic trait in the pattern-forming Bacillus mycoides

BackgroundBacillus mycoides Flügge, a Gram-positive, non-motile soil bacterium assigned to Bacillus cereus group, grows on agar as chains of cells linked end to end, forming radial filaments curving clock- or counter-clockwise (SIN or DX morphotypes). The molecular mechanism causing asymmetric curving is not known: our working hypothesis considers regulation of filamentous growth as the prerequisite for these morphotypes.ResultsSIN and DX strains isolated from the environment were classified as B. mycoides by biochemical and molecular biology tests. Growth on agar of different hardness and nutrient concentration did not abolish colony patterns, nor was conversion between SIN and DX morphotypes ever noticed. A number of morphotype mutants, all originating from one SIN strain, were obtained. Some lost turn direction becoming fluffy, others became round and compact. All mutants lost wild type tight aggregation in liquid culture. Growth on agar was followed by microscopy, exploring the process of colony formation and details of cell divisions. A region of the dcw (division cell wall) cluster, including ftsQ, ftsA, ftsZ and murC, was sequenced in DX and SIN strains as a basis for studying cell division. This confirmed the relatedness of DX and SIN strains to the B. cereus group.ConclusionsDX and SIN asymmetric morphotypes stem from a close but not identical genomic context. Asymmetry is established early during growth on agar. Wild type bacilli construct mostly uninterrupted filaments with cells dividing at the free ends: they "walk" longer distances compared to mutants, where enhanced frequency of cell separation produces new growing edges resulting in round compact colonies.

The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

In response to nitrogen starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. This dimorphic transition is regulated by the Galpha protein GPA2, by RAS2, and by elements of the pheromone-responsive MAP kinase cascade, yet the mechanisms by which nitrogen starvation is sensed remain unclear. We have found that MEP2, a high affinity ammonium permease, is required for pseudohyphal differentiation in response to ammonium limitation. In contrast, MEP1 and MEP3, which are lower affinity ammonium permeases, are not required for filamentous growth. Deltamep2 mutant strains had no defects in growth rates or ammonium uptake, even at limiting ammonium concentrations. The pseudohyphal defect of Deltamep2/Deltamep2 strains was suppressed by dominant active GPA2 or RAS2 mutations and by addition of exogenous cAMP, but was not suppressed by activated alleles of the MAP kinase pathway. Analysis of MEP1/MEP2 hybrid proteins identified a small intracellular loop of MEP2 involved in the pseudohyphal regulatory function. In addition, mutations in GLN3, URE2 and NPR1, which abrogate MEP2 expression or stability, also conferred pseudohyphal growth defects. We propose that MEP2 is an ammonium sensor, generating a signal to regulate filamentous growth in response to ammonium starvation.