Key drivers of microbial abundance, activity, and diversity in karst spring waters across an altitudinal gradient in Slovenia.

In aquatic ecosystems, the biomass, activity and composition of microorganisms are determined to a large extent by local and regional environmental conditions. While karst aquifers are an important source for drinking water, the ecology of microbial communities in karst waters has hardly been studied. This study examined the regional variations and seasonal dynamics of microbial communities in pristine karst spring waters of Slovenia (Central Europe). Fifteen springs distributed across 5 eco-regions exhibiting a strong altitudinal gradient were sampled 4 times a year. Evaluation of the microbial communities included quantification of prokaryotic biomass via total cell counts and microbial activity estimated via measurements of electron transport system activity. The taxonomic structure of the bacterial communities was analysed by terminal restriction fragment length polymorphism fingerprinting. Biological measures were complemented by a set of physico-chemical parameters, including dissolved organic carbon, nutrients, major ions, temperature, electrical conductivity, pH, and dissolved oxygen. Bacterial community structure differed significantly with seasons and eco-regions, with the latter causing greater variation. While the overall power of the environmental variable tested was a moderate factor (15%) in explaining the variability in microbial community structure, catchment altitude was a key determinant. Prokaryotic cell density and microbial activity in spring water decreased with an increase in catchment altitude and were significantly positively correlated. For a better understanding of karst ecosystems and the ecosystem service of water purification, future investigation should address karst microbial communities at a higher phylogenetic and functional resolution

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