In deep‐sea hydrothermal vent environments, metal‐enriched fluids and sediments abound, making these habitats ideal to study metal resistance in prokaryotes. In this investigation, we employed transcriptomics and shotgun proteomics with scanning transmission electron microscopy and energy‐dispersive x‐ray spectroscopy (STEM‐EDX) to better understand mechanisms of tolerance for cadmium (Cd) and copper (Cu) at stress‐inducing concentrations in Nitratiruptor sp. SB155‐2 (phylum Campylobacterota). Transcriptomic profiles were remarkably different in the presence of these two metals, displaying 385 (19%) and 629 (31%) differentially transcribed genes (DTG) in the presence of Cd(II) and Cu(II), respectively, while only 7% of differentially transcribed (DT) genes were shared, with genes for non‐specific metal transporters and genes involved in oxidative stress‐response predominating. Transcriptomic and proteomic analyses confirmed that metal‐specific DT pathways under Cu(II) stress, including those involving sulfur, cysteine, and methionine, are likely required for high‐affinity efflux systems, while flagella formation and chemotaxis were over‐represented under Cd(II) stress. Consistent with these differences, STEM‐EDX analysis revealed that polyphosphate‐like granules (pPLG), the formation of CdS particles, and the periplasmic space are crucial for Cd(II) sequestration. Overall, this study provides new insights regarding metal‐specific adaptations of Campylobacterota to deep‐sea hydrothermal vent environments.

2016-07-01·The Journal of biological chemistry2区 · 生物学

Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability

2区 · 生物学

Article

作者: Inoue, Akira ; Ojima, Takao ; Anraku, Moe ; Nakagawa, Satoshi

Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of ϵ-Proteobacteria Nitratiruptor sp. SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 °C and around 6, respectively. A high concentration of NaCl (0.8-1.4 m) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 °C for 30 min. Heat stability was decreased in the presence of 5 mm DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.

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