Isolation, Storage, and Identification of Bacterial Isolates
Fourteen cryopreserved bacterial isolates were revived using LB broth. Isolates were previously obtained by Dr. Cuellar-Gempeler from greenhouse and field collections of Darlingtonia fluid and maintained in cryovials using 2% DMSO. Stored isolates were thawed and 1mL aliquot was transferred to a microcentrifuge tube, and sample was centrifuged at 100xg for 10 minutes. DMSO supernatant was removed, and bacteria pellet was mixed into LB broth which was incubated for 48 hours at 30⁰C. Broth was streaked onto LB plates to confirm purity.
To perform colony PCR, a needle was used to remove bacteria from a single colony and diluted in tube with 20 µL ultrapure water. PCR cocktail consisted of 25 µL DreamTaq PCR Mastermix, 23 µL Nuclease-Free Water, 0.5 µL forward primer (27F), 0.5 µL reverse primer (1492R) per sample. 49 µL of PCR cocktail was combined with 1 µL of diluted bacterial sample. Samples were run in the thermocycler with settings: 35 cycles, 95⁰C 4 minutes, 94⁰C 1 minute, 58⁰C 1 minute, 72⁰C 7 minutes, 4⁰C upon completion of 35 cycles. Samples were submitted to Eurofins for Sanger sequencing.
Cultured Community
Once isolates were acclimated to lab condition, they were streaked on LB agar media to confirm purity and then plated to quantify abundance. Samples were diluted with LB broth to 107 cells/mL. They were then inoculated into duplicate monocultures by adding 5mL of 107 cell/mL culture to 40 mL LB broth for a total of 28 samples, plus two media controls, and incubated at room temperature for 48 hours. Five groups each of 2, 3, 4, and 5 mixed cultures were randomly assembled in equal amounts. For mixed cultures with 2 species, 2.5mL of each species was added, for mixed cultures with 3 species, 1.67 mL of each species was added. For mixed cultures with 4 species, 1.25mL of each species was added. For mixed cultures with 5 species, 1mL of each species was added. This equated to adding a total of 5mL of 107 cells/mL culture to 40mL LB broth for a total of 20 samples, plus two media controls; these were incubated at room temperature for 48 hours. Bacterial species and composition of groups are detailed in Table 1 and Table 2 respectively.
Table 1
Bacterial isolate identities.
Isolate Number | Scientific Name | Percent Identity | Acquisition Number |
1 | Bacillus sp. WYT007 | 98.30% | JQ807855.1 |
2 | Curtobacterium sp. b213 4 | 90.75% | KF733315.1 |
3 | Leucobacter sp. 1 | 98.14% | MH671536.1 |
4 | Staphylococcus equorum 1 | 87.03% | KC513844.1 |
5 | Acinetobacter sp. | 99.18% | MF462948.1 |
6 | Leucobacter sp. 4 | 93.75% | MH671536.1 |
7 | Arthrobacter gandavensis 1 | 85.32% | MT539758.1 |
8 | Leucobacter sp. 2 | 98.14% | MH671536.1 |
9 | Bacillus pumilus | 81.56% | AJ494732.1 |
10 | Curtobacterium sp. b213 3 | 90.40% | KF733315.1 |
11 | Staphylococcus equorum 2 | 95.05% | JX134628.1 |
12 | Curtobacterium sp. b213 2 | 90.29% | KF733315.1 |
13 | Curtobacterium sp. b213 1 | 92.32% | KF733315.1 |
14 | Leucobacter sp. 3 | 99.15% | MH671536.1 |
Table 2
Composition of mixed culture communities.
Group Name | Bacteria Included (By isolate number) |
Mix 1 | 7, 13 |
Mix 2 | 4, 7 |
Mix 3 | 8, 9 |
Mix 4 | 11, 14 |
Mix 5 | 3, 9 |
Mix 6 | 8, 10, 13 |
Mix 7 | 9, 12, 14 |
Mix 8 | 3, 6, 13 |
Mix 9 | 5, 7, 14 |
Mix 10 | 10, 12, 14 |
Mix 11 | 3, 4, 6, 8 |
Mix 12 | 1, 6, 11, 14 |
Mix 13 | 1, 3, 6, 11 |
Mix 14 | 1, 5, 11,14 |
Mix 15 | 7, 10, 11, 12 |
Mix 16 | 1, 7, 8, 10, 11 |
Mix 17 | 3, 8, 10, 13, 14 |
Mix 18 | 1, 6, 9, 10, 13 |
Mix 19 | 5, 8, 12, 13, 14 |
Mix 20 | 5, 7, 11, 13, 14 |
Because Bacillus sp. WYT007 did not produce single colonies upon plating (it formed filamentous mats), counts for this species have been converted from a percentage of plate coverage into colony counts by taking that percent of coverage and multiplying it by the maximum species abundance counted, that being of Curtobacterium sp. b213 2 at an abundance of 1.2×1011 cells/mL, as they visually looked the most similar at max coverage.
Fruit Fly Preparation for Degradation Experiments
Drosophilia fruit flies were purchased from Arcata Pet Supply in Arcata, CA. Culture was then frozen over night at -20⁰ C to sacrifice flies. Flies were then collected and dried for 48 hours at 60⁰ C and autoclaved to sterilize. These were then transferred into sterile 1.5mL cages, produced by drilling 24 holes into a 1.5mL centrifuge tube to allow for flow of fluid and bacteria, for a total of 12-15mg of flies per 1.5mL cage. Exact mass per cage was recorded by recording each cage without flies and with flies.
Degradation Experiment
The cultured community and serial dilution community were subjected to the following degradation experiment. Following acclimation of the communities, samples were plated to calculate cell density at the start of the degradation. Fruit fly cages were placed in each sample and samples were loosely capped to allow for airflow. Samples were then incubated at room temperature for 11 days in still conditions to simulate the still conditions in which Darlingtonia leaves live. Samples were inverted once daily to encourage flow of bacteria in and out of the fruit fly cages for the duration of the degradation experiment. At the end of the 11 days, 1.5 mL fluid was collected for each solubilized protein, protease, and chitinase quantification, and 10 mL was collected for nitrate and ammonia quantification. Samples were plated to quantify end of experiment densities.
Relevant Measures of Specific Nitrogen Function Assays
Following degradation, samples were collected for measurement of specific nitrogen metabolic functions and frozen at -20⁰C until measurement. Degradation rate, enzymatic activity of protease and chitinase, solubilized protein, nitrate, and ammonia were measured. Degradation rate was measured by drying fly cages at 60⁰ C for 3 days and subtracting from pre-degradation mass to calculate fly mass loss. Chitinase activity was quantified using the Chitinase Microplate Assay Kit from MyBioSource (catalog #MBS8243204) and read on Spectra iMax at 585 nm. Protease activity was quantified using the Protease Assay Kit from ThermoScientific (catalog #23263) and read on the Spectra iMax at 450 nm. Solubilized protein was quantified using a Bradford assay and reading on a Nanodrop 1000 at 595 nm. Solubilized protein and enzymatic activity of chitinase and protease were measured in duplicate to verify accuracy of spectrophotometric quantification. Ammonia and nitrate was quantified using the Orion™ High Performance Ammonia Electrode and calibrated using standards from USAbluebook.
Bioinformatics and Data Analysis
Sanger sequences were read using the package sangerseqR (version 1.30.1) [29]. Reverse sequences were reversed using the function reverseComplement from the package Biostrings (version 2.60.2) [30]. Low quality bases were trimmed and sequences were merged using the package sangeranalyseR (version 0.1.0) [31]. Files were saved using the function write.dna from the package ape (version 5.5) [32]. DNA sequences were then matched to previously isolated strains in the NCBI database using BLAST.
We calculated degradation and growth rates from the monocultures to establish relationships between these parameters. To calculate bacterial growth rates at the start of degradation, we subtracted bacterial cell concentration upon inoculation from bacterial cell concentration at the start of the 11 day degradation experiment. To calculate end of degradation growth, we subtracted cell concentration at the start of the degradation experiment from cell concentration at the end of the experiment. Significance of correlation was tested using a linear model with a significance test for linear regression using the function lm. All analyses and plots were performed and produced using the R statistical environment (version 4.1.1, R Core Team, 2020). Relationships were plotted and visualized using the ggplot2 package (version 3.3.6) [33]. P-values were adjusted using the Benjamini and Hochberg method; degradation rate was the dependent variable and specific nitrogen functions were the explanatory variables [34].
To establish the biodiversity-function relationship in the mixed community experiments, we used a linear regression model with diversity metrics as the explanatory variables and function metrics as the dependent variable. We used function lm which included a significance of correlation test.
To evaluate the relationship between the cultured communities and complementarity and selection, we used the partitionBEFsp package (version 1.0) [35]. Monoculture and coculture plate counts were used and functions calculate_DRY and classic_partition were used to calculate complementarity and selection values. These values were plotted using a linear regression model and function lm was used to measure significance of correlation.