Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa

Abstract

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall.

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea

“…Transcripts of this gene were also highly abundant during growth on xylan. GH72 enzymes are known to be involved in elongation and remodelling of the β-1,3-glucan of the fungal cell wall [ 53 ]. Thus, MalCi_235.14 is not likely to be involved in plant biomass degradation, but may be important for hyphal growth during an abundance of nutrients.…”

Stress Reshapes the Physiological Response of Halophile Fungi to Salinity

“…In this fungus, gel4 is an essential gene [60], whereas ∆gel2 mutants have reduced growth, and gel1 deletion has no evident effect on morphology [61]. In Neurospora crassa, individual deletions of gel genes do not influence stress sensitivity [59], indicating that there is higher redundancy in their function than in A. fumigatus. Noticeably, if the function of GEL proteins is abolished-with a triple mutation in N. crassa [59] or a single mutation of the GEL homolog gene gas1 in S. cerevisiae [62]-the chitin content in the cell wall is increased.…”

“…In Neurospora crassa, individual deletions of gel genes do not influence stress sensitivity [59], indicating that there is higher redundancy in their function than in A. fumigatus. Noticeably, if the function of GEL proteins is abolished-with a triple mutation in N. crassa [59] or a single mutation of the GEL homolog gene gas1 in S. cerevisiae [62]-the chitin content in the cell wall is increased. This could be a compensatory mechanism to restore the rigidity of the cell wall, indicating a balancing effect between β(1,3)-glucan crosslinking and chitin production.…”

“…Their function is to cleave the newly synthesized linear β(1,3)-glucan and transfer it to another β(1,3)-glucan molecule, resulting in elongation or shortening of the glucan fibers. GEL proteins also regulate the cross-linking of proteins into the cell wall, therefore allowing the correct assembly of cell wall structures [59]. In A. fumigatus, seven gel genes have been identified, of which only three (gel1, gel2, and gel4) are expressed during mycelial growth [50,60].…”

The PHR Family: The Role of Extracellular Transglycosylases in Shaping Candida albicans Cells

“…Cell reshaping occurs during the complex morphological transitions a fungus typically undergoes in its life cycle (yeast/hyphal growth, spore or conidiophore formation) and requires the expression of different paralogs. Well-known examples of a dynamic interplay among paralogs during the fungal life cycle were described for the GAS genes of S. cerevisiae , the gas+ genes of Schizosaccharomyces pombe , and the GEL s of Neurospora crassa and of A. fumigatus [ 28 , 29 , 30 , 31 , 32 ]. Gene redundancy occurs not only in microorganisms that underwent whole genome duplication, e.g., S. cerevisiae , but other species as well.…”