Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system

Levin, Ana M.; Vries, Ronald P. de; Wösten, Han A. B.

doi:10.1016/j.mimet.2007.01.003

Cited by 28 publications

(46 citation statements)

References 6 publications

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“…To assess the complexity of the profile of secreted proteins, 6-day-old maltose-and xylose-grown sandwiched colonies were transferred to a ring plate (9). This polycarbonate disc with ring-shaped wells (see Materials and Methods) was filled with fresh medium.…”

Section: Resultsmentioning

confidence: 99%

“…Colonies were grown as a sandwiched culture (24) in 9-cm petri dishes in a 0.2-mm thin layer of 1.25% agarose in between two perforated polycarbonate membranes (diameter, 76 mm; pore size, 0.1 m; Osmonics, GE Water Technologies, Trevose, PA) placed on top of solidified (1.5% agar) minimal medium (7) with 25 mM maltose or xylose as a carbon source. Sandwiched cultures were inoculated with 1.5 l of spore suspension (10 8 spores l Ϫ1 ) and were either transferred or not transferred after 6 days of growth to fresh solid medium or to liquid minimal medium contained in a ring plate (9). A ring plate consists of a polycarbonate disc (9 cm in diameter, 1.2 cm thick) with six ring-shaped wells.…”

Section: Methodsmentioning

confidence: 99%

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Spatial Differentiation in the Vegetative Mycelium of Aspergillus niger

et al. 2007

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Fungal mycelia are exposed to heterogenic substrates. The substrate in the central part of the colony has been (partly) degraded, whereas it is still unexplored at the periphery of the mycelium. We here assessed whether substrate heterogeneity is a main determinant of spatial gene expression in colonies of Aspergillus niger. This question was addressed by analyzing whole-genome gene expression in five concentric zones of 7-day-old maltose-and xylose-grown colonies. Expression profiles at the periphery and the center were clearly different. More than 25% of the active genes showed twofold differences in expression between the inner and outermost zones of the colony. Moreover, 9% of the genes were expressed in only one of the five concentric zones, showing that a considerable part of the genome is active in a restricted part of the colony only. Statistical analysis of expression profiles of colonies that had either been or not been transferred to fresh xylosecontaining medium showed that differential expression in a colony is due to the heterogeneity of the medium (e.g., genes involved in secretion, genes encoding proteases, and genes involved in xylose metabolism) as well as to medium-independent mechanisms (e.g., genes involved in nitrate metabolism and genes involved in cell wall synthesis and modification). Thus, we conclude that the mycelia of 7-day-old colonies of A. niger are highly differentiated. This conclusion is also indicated by the fact that distinct zones of the colony grow and secrete proteins, even after transfer to fresh medium.Filamentous fungi fulfill an essential role in global carbon cycling by degrading organic material. The organic material is colonized by a mycelium that consists of interconnected hyphae. These hyphae are exposed to a heterogenic substrate. The periphery of the colony is exposed to unexplored organic material, whereas its center is surrounded with a substrate that has been (partly) utilized. Little is known of how this heterogeneity affects cellular processes and gene expression in different parts of the mycelium. However, previous studies suggest that the vegetative colonizing mycelium is highly differentiated (11,21,24). Growth of Aspergillus niger and Phanerochaete chrysosporium mainly takes place in specific zones at the periphery and the center of the colony (11, 24). These zones also secrete proteins. For instance, glucoamylase is secreted at the periphery of an A. niger colony (24), whereas lignin peroxidase is secreted in the central growth zone of P. chrysosporium (11). Interestingly, the hyphae that release the peroxidase are morphologically distinct from the secreting hyphae at the periphery of the vegetative mycelium. The high degree of differentiation in fungal vegetative mycelia is also indicated by the fact that only a subset of growing hyphae at the periphery of an A. niger colony secrete glucoamylase (21, 24).Previous studies have shown that certain genes are differentially expressed in central and peripheral zones of the vegetative mycelium (11,18,21,25)....

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Spatial Differentiation in the Vegetative Mycelium of Aspergillus niger

et al. 2007

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“…Protein secretion on solid medium was investigated using the ring-plate culturing system adapted from Levin et al (2007) and modified as follows. Cells were cultured essentially as described above for the study of transcriptional fusions.…”

Section: Methodsmentioning

confidence: 99%

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

et al. 2011

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Although classical type II secretion systems (T2SSs) are widely present in Gram-negative bacteria, atypical T2SSs can be found in some species. In Pseudomonas aeruginosa, in addition to the classical T2SS Xcp, it was reported that two genes, xphA and xqhA, located outside the xcp locus were organized in an operon (PaQa) which encodes the orphan PaQa subunit. This subunit is able to associate with other components of the classical Xcp machinery to form a functional hybrid T2SS. In the present study, using a transcriptional lacZ fusion, we found that the PaQa operon was more efficiently expressed (i) on solid LB agar than in liquid LB medium, (ii) at 25 °C than at 37 °C and (iii) at an early stage of growth. These results suggested an adaptation of the hybrid system to particular environmental conditions. Transposon mutagenesis led to the finding that vfr and fimV genes are required for optimal expression of the orphan PaQa operon in the defined growth conditions used. Using an original culturing device designed to monitor secretion on solid medium, the ring-plate system, we found that T2SS-dependent secretion of exoproteins, namely the elastase LasB, was affected in a fimV deletion mutant. Our findings led to the discovery of an interplay between FimV and the global regulator Vfr triggering the modulation of the level of Vfr and consequently the modulation of T2SS-dependent secretion on solid medium.

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“…The mycelium of 7-day-old maltose-grown sandwiched colonies of N402 and the DfluG strain were divided in five concentric zones (Levin et al 2007a, Levin et al 2007b. Gene expression was assessed in the central zone 1, the intermediate zone 3, and the outer zone 5 using RNAseq.…”

Section: Rna Profiles In Zones Of Dflug Coloniesmentioning

confidence: 99%

“…niger forms a mycelium, also known as colony, that consists of a network of interconnected hyphae. Zones in colonies of A. niger are heterogeneous with respect to growth (Wösten et al 1991), RNA composition (de Bekker et al 2011;Levin et al 2007a), secretion (Levin et al 2007b;Wösten et al 1991;Krijgsheld et al 2012) and sporulation (Krijgsheld et al 2013b). For instance, secretion of GlaA (Wösten et al 1991) and expression of its encoding gene glaA (Levin et al 2007a) mainly take part at the periphery of the colony.…”

Section: Introductionmentioning

confidence: 99%

FluG affects secretion in colonies of Aspergillus niger

Wang

Krijgsheld

Hulsman

et al. 2014

Antonie van Leeuwenhoek

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Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3 0 end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the DfluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown DfluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion.Keywords Fungus Á Conidiogenesis Á Asexual development Á Aspergillus fluG Á Secretion Á Heterogeneity Electronic supplementary material The online version of this article

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Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system

Cited by 28 publications

References 6 publications

Spatial Differentiation in the Vegetative Mycelium of Aspergillus niger

Spatial Differentiation in the Vegetative Mycelium of Aspergillus niger

Role of fimV in type II secretion system-dependent protein secretion of Pseudomonas aeruginosa on solid medium

FluG affects secretion in colonies of Aspergillus niger

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