A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in Escherichia coli and the purified enzymes exhibited temperature optima of between 40–50°C. Substrate-profiling studies revealed that the characterised enzymes were ‘true’ carboxylesterases based on their preferences for short to medium chain length p-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches.

Marion Island is a Sub-Antarctic island made up of distinct ecological habitats based on soil physiochemical, plant cover and physical characteristics. The microbial diversity and ecological determinants in this harsh Sub-Antarctic environment are largely uncharacterized. Actinobacteria have diverse ecological functions related to soil and plant functioning. This study was aimed at characterizing the diversity and community structures of the dominant actinobacteria in the distinct habitats and to identify their determinant soil and plant characteristics. Using the 16S rRNA gene, the denaturing gradient gel electrophoresis patterns and clone library diversity were correlated with the soil and plant characteristics. Multivariate statistical methods were also used to identify determinant soil and plant characteristics. Salinity and pH were the most important soil determinants, and a number of important site-specific plant species may have been important. The Coastal Fellfield Habitat was dominated by sequences of the suborders Micrococcineae (44%) andPropionibacterineae (18%), with salinity identified as the principal determinant. The Cotula Herbfield Habitat was dominated by Frankineae (37%) and Streptosporangineae (38%), which were correlated with organic nutrient concentrations. The Wet Mire Habitat was dominated by Acidimicrobineae (61%), with moisture and organic carbon content as principal components. Culture-dependent studies were complementary to culture-independent studies with the majority of actinobacteria isolated not identified in 16S rRNA gene clone libraries. This study demonstrates how the soil physiochemical characteristics and plant species independently determine the community structures of the dominant actinobacteria in distinct ecological habitats. These factors subsequently influence their ecological adaptation, roles and functions.

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