Worldwide prevalence of class 2 integrases outside the clinical setting is associated with human impact.

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (< or =98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised approximately 70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction ( approximately 40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (< or =98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3-4 M KCl. However, specific activity values were two-fold higher in the presence of 3-4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.