Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications

Laccase activity is proportional to the abundance of bacterial laccase-like genes in soil from subtropical arable land

Laccase enzymes produced by both soil bacteria and fungi play important roles in refractory organic matter turnover in terrestrial ecosystems. We investigated the abundance and diversity of fungal laccase genes and bacterial laccase-like genes in soil from subtropical arable lands, and identified which microbial group was associated with laccase activity. Compared with fungal laccase genes, the bacterial laccase-like genes had greater abundance, richness and Shannon-Wiener diversity. More importantly, laccase activity can be explained almost exclusively by the bacterial laccase-like genes, and their abundance had significant linear relationship with laccase activity. Thus, bacterial laccase-like gene has great potential to be used as a sensitive indicator of laccase enzyme for refractory organic matter turnover in subtropical arable lands.</p

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2\u2032-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75\ua0\ub0C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions

Pitfalls and Rewards of Setting Up a Liquid Biopsy Approach for the Detection of Driver Mutations in Circulating Tumor DNAs: Our Institutional Experience

We describe our institutional experience of developing a liquid biopsy approach using circulating tumor DNA (ctDNA) analysis for personalized medicine in cancer patients, focusing on the hurdles encountered during the multistep process in order to benefit other investigators wishing to set up this type of study in their institution. Blood samples were collected at the time of cancer surgery from 209 patients with one of nine different cancer types. Extracted tumor DNA and circulating cell-free DNA were sequenced using cancer-specific panels and the Illumina MiSeq machine. Almost half of the pairs investigated were uninformative, mostly because there was no trackable pathogenic mutation detected in the original tumor. The pairs with interpretable data corresponded to 107 patients. Analysis of 48 gene sequences common to both panels was performed and revealed that about 40% of these pairs contained at least one driver mutation detected in the DNA extracted from plasma. Here, we describe the choice of our overall approach, the selection of the cancer panels, and the difficulties encountered during the multistep process, including the use of several tumor types and in the data analysis. We also describe some case reports using longitudinal samples, illustrating the potential advantages and rewards in performing ctDNA sequencing to monitor tumor burden or guide treatment for cancer patients