Sucrase

The evaluation was performed on the Sanjiang Plain (47°35′N, 133°31′E) in northeastern China. The mean month‐long temperature spans from −21.6°C in January to 21.5°C in July, with a yearly mean temperature of 1.9°C. The average yearly precipitation is approximately 560 mm, where 80% precipitates from May to October. Eight vegetation types along a forest succession gradient in a degraded wetland were selected for this study: original natural wetland (NW), wetland edge (EW), shrub‐invaded wetland (IW), shrub‐dominant wetland (DW), young‐Betula forest (YB), mature‐Betula forest (MB), Populus and Betula mixed forest (PB), and conifer forest (CF) (Table 1, Figure 1). These eight types can be divided into two main groups, that is, an aquatic group including permanent or seasonal wetland (NW, EW, IW, and DW), and a dryland group (YB, MB, PB, and CF) (Table 1).
Three plots (10 m × 10 m) were established in each vegetation type, and the distance between any two plots was >50 m. At each plot, we identified all plant species and calculated the plant's Shannon diversity index. Soil samples (0–20 cm depth) were taken on October 15, 2016. Soil samples were collected using a sterile soil drill from 5 randomly selected locations inside each plot and were pooled to obtain a mixed soil specimen (approximately 1 kg of fresh soil) for each plot. Each soil sample was split into 2 subsamples, one of them was kept at −80°C for DNA analysis and the second one was air dried for soil physico‐chemical analyses, with soil moisture content (Mc) measured gravimetrically and soil pH quantified with a pH meter following the soil being mixed using water (1:5 w/v) for 30 min. The total organic carbon (TC) and total nitrogen (TN) concentrations were measured with an elemental analyzer (VarioEL III; Elementar Analysen systeme GmbH, Langensel bold, Germany). Soil sample was performed digestion and extraction through H₂SO₄‐HClO₄, 0.5 M NaHCO_3, and 2.0 M KCl in succession, followed by assay with a continuous flow analytical system (SAN⁺⁺, Skalar Analytical, the Netherlands). The catalase activity was tested using the method presented by Aebi (1984), the urease activity was established with the method detailed by Kandeler and Gerber (1988), the sucrase activity was assayed by ammonium molybdate colorimetry (Guan et al., 1984), and the acid phosphataseactivity was determined using ρ‐nitrophenyl phosphate following a method described by Eivazi and Tabatabai (1977).

Seven strains were selected for genomic sequencing based upon their characterized and distinctive biological control properties and their isolation from different habitats (bulk or rhizosphere soil or aerial plant surfaces) (Table 1). The seven strains and three previously-sequenced strains (Pf-5, Pf0-1, and SBW25) evaluated in this study exhibited the conserved phenotypes of the P. fluorescens group: positive for fluorescence under UV light, arginine dihydrolase activity, and oxidase activity; and negative for growth at 41°C and induction of a hypersensitive response on tobacco, determined through standard methods [138] (Table S16). The ten strains were subjected to a panel of biochemical and biological assays (nitrate reduction, levan sucrase production, potato soft rot, gelatinase activity, and catabolic spectra) [138] , [139] to assign each to a biovar of P. fluorescens or to a species of Pseudomonas[129] (link) (Table 1 and Table S16). Strains A506, 30-84, SS101, and BG33R are rifampicin-resistant (100 µg/ml) derivatives of field isolates; previously, spontaneous mutants with resistance to rifampicin were selected to facilitate tracking of these strains in field studies. Strain A506 is known to have a single nucleotide insertion in rpoS, which causes a frameshift resulting in a truncated form of the stationary-phase sigma factor RpoS [140] (link). During the course of this work, we discovered that strain Pf0-1 has a mutation in gacA, which encodes a component of the GacA/GacS global regulatory system in Pseudomonas spp. [61] (link). We sequenced gacA and gacS from the strain Pf0-1 in our collection and confirmed that the sequences are identical to those in the published genome of Pf0-1 [32] (link). It is not possible to know whether the mutations in A506 and Pf0-1 were present in the strains prior to isolation or if they developed in the laboratory during storage, but all strains have been maintained as frozen stocks (−80°C) throughout this study and for many years preceding.

Lucifer Yellow (450 Da) added to the epithelial channel of the Intestine Chip to assess intestinal barrier permeability. The concentration of dye that diffused through the membrane into endothelial channel was measured in the effluent, and apparent paracellular permeability (P_app) was calculated using the following formula: $${\mathit{P}}_{\mathit{app}}=\frac{{\mathit{V}}_{\mathit{\text{rec}}}\cdot \mathit{d}{\mathit{C}}_{\mathit{rec}}}{\mathit{A}\cdot \mathit{dt}\cdot {\mathit{C}}_{\mathit{don},\mathit{t}=0}}$$ where V_rec is volume receiver (endothelial compartment), C_rec concentration receiver, A the seeded area and C_don concentration donor (epithelial compartment).
To measure sucrase activity, upper and lower chamber of the Intestine Chip were perfused PBS with Ca²/Mg² for 1 h to remove any residual glucose. 30 mM sucrose reaction buffer was prepared in PBS with Ca²/Mg²; the NaCl concentration in the PBS was reduced to 120 mM to adjust for osmolarity in the presence of 30 mM sucrose, and the sucrose was replaced with 30 mM mannitol in control samples, as described previously^{46 (link)}. Sucrose or mannitol reaction buffer was then introduced in the upper microfluidic channel and PBS with Ca²/Mg² into the lower channel. Glucose levels were measured using an Amplex Red Glucose assay kit (Thermo Fisher); total protein content in epithelial cell lysates was determined using a Pierce BCA Protein Assay (Thermo Fisher). Glucose concentrations were determined from a standard curve and sucrase activity expressed as units per gram of protein; 1 unit (1 U) = activity that hydrolyzes 1 μmol substrate min⁻¹ at 37 °C. To assess MUC2 production, the luminal effluent was collected over night and mucin 2 content was measured using a Human Mucin 2/MUC2 ELISA Kit (LSBio).

Five-micrometer consecutive sections of jejunal and ileal segments were prepared for morphological observations after staining with hematoxylin-eosin (HE). Ten representative and well-oriented villi and the associated crypt of each sample were selected for morphological observations using a Leica DMi8 optical microscope (Leica Corp., Weztlar, Germany). Villus height (VH) was ascertained by measuring distance from the apex of the villus until the junction of the villi and crypt [17 (link)]. Crypt depth (CD) was defined as the depth between the villus and the basal membrane. Accordingly, the villus height to crypt depth ratio (VCR) was calculated. Moreover, ten intact and neat rows of intestinal villi stained with periodic acid Schiff (PAS) were selected to image goblet cells (GC). The number of GC was quantified by counting the number of stained goblet cells per 100 μm length of villi and presented as the means per ten villi. The activities of mucosal sucrase, maltase, alkaline phosphatase, and Na⁺-K⁺ ATPase were determined colorimetrically via commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. These indices were normalized by the total protein contents of the intestinal mucosa, which were quantified using bicinchoninic acid (BCA) protein assay kits (CWBiotech Co. Ltd., Beijing, China). Specific principles and operating procedure were inquired through the instruction manual of the kits.

Air-dried bulk soil samples were used to measure organic matter, pH, total and available nitrogen (N), phosphorus (P), and potassium (K), and catalase and sucrase activity. The soil organic matter was measured using the K₂Cr₂O₇ (Walkley-Blach) method. The pH value was determined by the glass electrode method; total N by the Kjeldahl method; total P and available P by the molybdenum antimony resistance colorimetric method; total K by the NaOH melting-flaming luminosity method; available N by diffusion; and available K was determined in ammonium acetate (NH₄OAc) extract by flame photometry (Zhang et al., 2014 (link)). Sucrase activity was measured using the 3,5-dinitrosalicylic acid colorimetric method, and catalase activity was determined by the KMnO₄ titration method (Ge et al., 2018 (link)).

Soil pH was determined using a pH meter (PHS-25, Shanghai, China) at a constant soil/water ratio of 1:2.5. The available nitrogen (AN) concentration in the soil samples was determined using the DigiPREP TKN System (KJELTEC 8400, Foss, Denmark). A UV–Vis spectrophotometer (UH5300, North Points Ruili) was utilized for assessing the available phosphorus (AP) concentration in the soil. The available potassium (AK) in the soil was quantified using an inductively coupled plasma (ICP) spectrometer (Spectro Analytical Instruments, Kleve, Germany). The K₂Cr₂O₇-H₂SO₄ oxidation approach was adopted to assess the organic matter (OM) content in the soil.
Soil enzymes associated with nitrogen, phosphorus, and carbon degradation, including peroxidase (POD), sucrase (SC), and urease (UE), were evaluated for their activity. The soil UE activity was determined as described by Bao (2000) (Bao, 2000 ) using urea as the substrate. The spectrophotometric approach was adopted for the soil POD quantification in a 96-well microplate, using L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate (Bach et al., 2013 (link)). The soil SC activity was evaluated by determining the glucose discharge from an 8% sucrose solution following 24 h of incubation at 37°C (Chen et al., 2010 (link)).