Laccases catalyze the oxidation of various aromatics, particularly phenolic and amine substrates, making them valuable in industrial applications. Laccases also play an important role in soil organic matter (SOM) turnover processes and the global carbon cycle due to their involvement in the synthesis and degradation of lignin as well as transformation of lignified substrates and humic substances. Laccases belong to the protein family of multicopper oxidases characterized by copper atoms in the active center. Laccases or laccase-like multicopper oxidases (LMCO) have been extensively studied especially in fungi. Recently, increasing evidence points to a wide occurrence of LMCO in bacteria. As bacterial communities are known to decompose pollutants and municipal wastes involving large quantities of phenolic substances and organic matter, it can be deduced that bacterial LMCO might also participate in lignin degradation and SOM cycling. Copper atoms not only constitute the catalytic center of laccases but can also confer copper resistance to bacteria. Consequently, copper ions have a well known use in isolating LMCO-producing bacteria. To gain deeper insights into the ecological role of bacteria with LMCO activity in soil, we used traditional plating methods and PCR-DGGE (denaturing gradient gel electrophoresis) technology to examine the community structure of LMCO-producing bacteria in forest soil of Liangshui nature reserve, Heilongjiang Province.
Soil samples were collected in three patterns of forests: Pinus koraiensis, Betula platyphylla and Picea dietrich. Soil samples were enriched in M9 basal medium and then screened in LB medium with gradient Cu²⁺. Ten bacteria strains were isolated and identified based on traditional plating methods of bacteria separation. The results showed that they all belonged to Bacillus sp., falling into four different species: Bacillus pumilus, Bacillus subtilis, Bacillus fusiformis and Bacillus amyloliquefaciens, among which no laccase-relative results have been reported on Bacillus fusiformis. To investigate the structural variation of LMCO-producing bacteria, 16S-rDNA-V3-fragment-based DGGE and clone sequence BLAST technologies were applied. The data based on DGGE plot indicated there was no distinct variation of LMCO-producing bacteria structure among soil samples of different forest patterns in the research district. The diversity of LMCO-producing bacteria in Pinus koraiensis forests was more abundant than that in Betula platyphylla and Picea dietrich. The results derived from BLAST revealed that Ralstonia sp., Enterobacter sp. and other uncultured species existed in soil samples in addition to Bacillus sp.. Band 3 was present in all soil samples and had a 100% homology to Ralstonia mannitolilytica. Ralstonia sp. had highly physical and biochemical similarities as well as sequence homology with Cupriavidus sp. which was a copper-philic bacteria community. Bands 2, 7 and 11 were commonly present except lane 8 and had a highly homology with Enterobacter ludwigii which had recently attracted interest in environment ecological research. Bands 5 and 9 had a highly homology with Bacillus circulans and Bacillus thuringiensis, respectively. Furthermore, Bands 1, 4, 6, 8 and 10 had highly homology with an uncultured bacterial clone which needed further investigation.