Diversity of Ascomycete Laccase Gene Sequences in a Southeastern US Salt Marsh

Abstract

The diversity of ascomycete laccase sequences was surveyed in a southeastern US salt marsh using a degenerate primer set designed around copper binding sites conserved in fungal laccases. This gene was targeted for diversity analysis because of its potential function in lignin degradation in the salt marsh ecosystem and because few studies have assessed functional gene diversity in natural fungal communities. Laccase sequences were amplified from genomic DNA extracted from 24 isolates (representing 10 ascomycete species) cultured from decaying blades of Spartina alterniflora, and from DNA extracted directly from the decaying blades. Among the ascomycete isolates, 21 yielded a PCR product of expected size (900 bp) that was tentatively identified as laccase based on sequence similarities to previously published laccase sequences from related organisms. Overall, 13 distinct sequence types, containing 39 distinct sequences, were identified among the isolates, with several species yielding multiple distinct laccase types. PCR amplifications from early and late decay blades of S. alterniflora yielded seven laccase types. Of these, five were composed of sequences >96% similar at the amino acid level to sequences from three cultured ascomycetes previously found to be dominant members of the fungal communities on decaying S. alterniflora blades. Two of the laccase types from the natural-decay clone library were novel and did not match any of the sequences obtained from the cultured ascomycetes. The 39 distinct sequences and 15 distinct laccase sequence types retrieved from the S. alterniflora decay system demonstrate high sequence diversity of this functional gene in a natural fungal community.

On the Diversity of the Laccase Gene: A Phylogenetic Perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and Other Related Taxa

“…Amplification of the Copper-Oxidase Domain of a Laccase Gene PCR amplification was carried out using the degenerate primers (Lac2for 5 0 GGIACI WIITGGTAYCAYWSICA3 0 and Lac3rev 5 0 CCRTGIWKRTGIAWIGGRTGIGG3 0 ) capable of amplifying a 900 bp laccase gene fragment from ascomycetes and basidiomycetes (Lyons et al 2003). Ambiguous bases are defined as follows: R, A/G; W, A/T; Y, C/T; S, C/G; K, T/G; I, inosine.…”

Isolation of Fungal Cellobiohydrolase I Genes from Sporocarps and Forest Soils by PCR

“…Moreover, testing this hypothesis has been difficult, because determining the function of fungal species typically requires isolating organisms and examining their effects on defined substrates in pure culture (7,15,(30)(31)(32), which has well-documented limitations (2). The recent development of degenerate primer sets for both basidiomycete (22) and ascomycete laccase genes (25) provides cultivation-independent tools for assessing the genetic diversity and activity of a "lignindegrading guild" within fungal communities. To our knowledge, a complementary tool to examine the "cellulolytic guild" is still lacking, despite an increasing knowledge of cellulase gene structure (4,6,14,33).…”

Biochemical and Molecular Analysis of Laccase Enzyme in Saprobic Fungus; Sordaria fimicola

“…3) and this amino acid change may have impact on laccase enzyme activity of strains N6 and N7 as evident in biochemical assay performed for laccase enzyme. The sequence variations in laccase genes and their proteins were reported earlier in Ascomycetes fungi (Lyons et al, 2003). The laccase genes amplified and sequenced from Ganoderma (Galhaup et al, 2002).…”