Vegetation Leachate During Arctic Thaw Enhances Soil Microbial Phosphorus

Leachate from litter and vegetation penetrates permafrost surface soils during thaw before being exported to aquatic systems. We know this leachate is critical to ecosystem function downstream and hypothesized that thaw leachate inputs would also drive terrestrial microbial activity and nutrient uptake. However, we recognized two potential endpoint scenarios: vegetation leachate is an important source of C for microbes in thawing soil; or vegetation leachate is irrelevant next to the large background C, N, and P pools in thaw soil solution. We assessed these potential outcomes by making vegetation leachate from frozen vegetation and litter in four Arctic ecosystems that have a variety of litter quality and soil C, N, and P contents; one of these ecosystems included a disturbance recovery chronosequence that allowed us to test our second hypothesis that thaw leachate response would be enhanced in disturbed ecosystems. We added water or vegetation leachate to intact, frozen, winter soil cores and incubated the cores through thaw. We measured soil respiration throughout, and soil solution and microbial biomass C, N, and P pools and gross N mineralization immediately after a thaw incubation (−10 to 2°C) lasting 6 days. Vegetation leachate varied strongly by ecosystem in C, N, and P quantity and stoichiometry. Regardless, all vegetated ecosystems responded to leachate additions at thaw with an increase in the microbial biomass phosphate flush and an increase in soil solution carbon and nitrogen, implying a selective microbial uptake of phosphate from plant and litter leachate at thaw. This response to leachate additions was absent in recently disturbed, exposed mineral soil but otherwise did not differ between disturbed and undisturbed ecosystems. The selective uptake of P by microbes implies either thaw microbial P limitation or thaw microbial P uptake opportunism, and that spring thaw is an important time for P retention in several Arctic ecosystems

Proximity assays for sensitive quantification of proteins

Proximity assays are immunohistochemical tools that utilise two or more DNA-tagged aptamers or antibodies binding in close proximity to the same protein or protein complex. Amplification by PCR or isothermal methods and hybridisation of a labelled probe to its DNA target generates a signal that enables sensitive and robust detection of proteins, protein modifications or protein–protein interactions. Assays can be carried out in homogeneous or solid phase formats and in situ assays can visualise single protein molecules or complexes with high spatial accuracy. These properties highlight the potential of proximity assays in research, diagnostic, pharmacological and many other applications that require sensitive, specific and accurate assessments of protein expression

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7- amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly- Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 mM21?sec21) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 mM21?sec21). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCAhydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule