Differential Regulation by Organic Compounds and Heavy Metals of Multiple Laccase Genes in the Aquatic Hyphomycete Clavariopsis aquatica

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete<jats:named-content content-type="genus-species">Clavariopsis aquatica</jats:named-content>) for the first time. Five putative laccase genes (<jats:italic>lcc1</jats:italic>to<jats:italic>lcc5</jats:italic>) identified in<jats:named-content content-type="genus-species">C. aquatica</jats:named-content>were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc.<jats:italic>lcc4</jats:italic>is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laccases of terrestrial fungi, none of the<jats:named-content content-type="genus-species">C. aquatica</jats:named-content>laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in<jats:named-content content-type="genus-species">C. aquatica</jats:named-content>, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 μM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats.</jats:p>

Identification of enzymes for lignocellulose degradation in the genome and transcriptome of the aquatic hyphomyceteClavariopsis aquatica

“…2-4 mm), were autoclaved (121 °C, 20 min) twice and suspended in a nitrogen-limited solution previously described for manganese peroxidase production in Stropharia rugosoannulata (Schlosser and Höfer, 2002). Control cultures were grown on liquid malt extract medium (1% malt extract, w/v; pH 5.6-5.8) (Solé et al, 2012). The flasks were inoculated with 5 mL of a mycelial suspension of C. aquatica prepared in sterile water (Junghanns et al, 2005).…”

“…Cultures were agitated at 120 rpm and incubated at 14 °C in the dark. Flasks were harvested after 7 days (trophophase/exponential growth phase) and 20 days (stationary growth phase) of cultivation (Junghanns et al, 2005;Solé et al, 2012). Liquid media were removed by stepwise centrifugation of the content of a flask in sterile 50-mL conical tubes at 7197 g and 4 °C for 10 min (Eppendorf centrifuge 5430R, rotor FA-35-6-30; Eppendorf, Hamburg, Germany).…”

“…Fungal growth phases and related starvation conditions are well known to play important roles in the regulation of enzymes involved in lignocellulose degradation (Delmas et al, 2012;Solé et al, 2012). Liquid culturing with milled plant material allows for clear temporal differentiation between exponential and stationary growth phase, and was applied in this study.…”

“…Besides general genome annotation, we specifically searched for gene families known to be involved in lignin degradation. We performed a blast search against our newly assembled C. aquatica genome to check for five previously described, partially sequenced laccase genes (Solé et al, 2012 Multiple Gene Set Activation (MGSA) analysis uses a Bayesian network approach to predict a probability of activation for sets of genes for each comparison (e.g., wheat straw -alder) based on differentially expressed genes (Bauer et al, 2011(Bauer et al, , 2010. We defined gene sets in three ways for the MGSA analysis using three different annotations: (1) all genes annotated with one GO term, (2) all genes assigned to one CAZy family, and (3) all genes assigned to one KEGG pathway based on assignment to KEGG orthology groups (see above).…”

Assessing the tolerance of immobilized laccase from a salt-tolerant strain of Trichoderma viride Pers NFCCI-2745 to heavy metal ions, detergents and copper chelating agents

“…Due to their low substrate specificity and strong oxidative abilities, laccases have a number of industrial applications such as textile dye bleaching, enzymatic conversion of chemical intermediates, biopulping, prevention of wine decolouration, the production of valuable compounds from lignin, soil bioremediation and biodegradation of environmental phenolic pollutants and removal of endocrine disruptors (Abadulla et al 2000;Cuoto and Herrera 2006;Divya et al 2013a, b, c;Fukuda et al 2001;Hemmat and Mazaheriassadi 2013;Mayer and Staples 2002;Nayanashree and Thippeswamy 2014;Rama et al 1998;Sole et al 2012;Tyagi et al 2014). The ideal laccases for industrial use would exhibit stability at high temperature and pH conditions (Niladevi et al 2008).…”