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Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium.

ABSTRACT: The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

SUBMITTER: Guo C

PROVIDER: S-EPMC8069351 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Benefiting from the rapid developments and wide applications of high-throughput sequencing, great advancements have been made in investigating microbiota, which are highly diverse and play key roles in both element cycling and the energy flow of ecosystems. There have been inherent limitations of amplicon sequencing that could introduce uncertainty and raise concerns about the accuracy and reproducibility of this technology. However, studies focusing on the reproducibility of amplicon sequencing are limited, especially in characterizing microbial communities in deep-sea sediments. To evaluate reproducibility, 118 deep-sea sediment samples were used for 16S rRNA gene sequencing in technical replicates (repeated measurements of the same sample) that demonstrate the variability of amplicon sequencing. The average occurrence-based overlaps were 35.98% and 27.02% between two and among three technical replicates, respectively, whereas their abundance-based overlaps reached 84.88% and 83.16%, respectively. Although variations of alpha and beta diversity indices were found between/among technical replicates, alpha diversity indices were similar across samples, and the average beta diversity indices were much smaller for technical replicates than among samples. Moreover, clustering methods (i.e., operational taxonomic units [OTUs] and amplicon sequence variants [ASVs]) were shown to have little impact on the alpha and beta diversity patterns of microbial communities. Taken together, although there are variations between/among technical replicates, amplicon sequencing is still a powerful tool with which to reveal diversity patterns of microbiota in deep-sea sediments. IMPORTANCE The reproducibility of amplicon sequencing is vital for whether the diversities of microbial communities could be accurately estimated. Thus, reproducibility influences the drawing of sound ecological conclusions. Nevertheless, few studies have focused on the reproducibility of microbial communities that are characterized by amplicon sequencing, and studies focusing on microbiota in deep-sea sediments have been especially lacking. In this study, we evaluated the reproducibility of amplicon sequencing targeting microbiota in deep-sea sediments of cold seep. Our results revealed that there were variations between/among technical replicates and that amplicon sequencing was still a powerful tool with which to characterize the diversities of microbial communities in deep-sea sediments. This study provides valuable guidelines for the reproducibility evaluation of future work in experimental design and interpretation.

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Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium.

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