Pseudomonas stutzeri RCH2 was isolated from chromium(VI) contaminated groundwater at the Department of Energy Hanford 100 Area, Benton County, WA (Han et al., 2010). Thirteen additional pseudomonas strains were isolated from Oak Ridge National Laboratory, Field Research Center, TN under conditions indicated in Supplementary Table 1. Liquid cultures were inoculated into 3-(N-morpholino)propanesulfonic acid (MOPS) buffered casein yeast magnesium broth (Mb-CYM): 10 g/L pancreatic digest of casein, 5 g/L Difco yeast extract, 1 g/L MgSO₄ heptahydrate, 10 mM MOPS. Mb-CYM agar was prepared with the addition of 1.5% w/v of agar. Unless otherwise noted, culture conditions were as follows. Strains were maintained as glycerol stocks; revived cultures were plated onto agar plates, incubated at 30°C for 24 h and stored at 4°C. Starter cultures for experiments were prepared from single colonies inoculated into liquid Mb-CYM broth and cultured aerobically with shaking at 30°C overnight. Uninoculated and unstreaked but incubated cultures/plates were used as negative controls for contamination. In figures and text, Pseudomonas stutzeri RCH2 is referred to as culture #1, while the 13 other strains are referred to as cultures #2–14, and uninoculated controls as culture #15 as indicated in Supplementary Table 1.

Rectangular petri dishes with ANSI standard dimensions were poured with agar to a height of 5 mm. Overnight cultures of the 14 effector strains (including RCH2) and uninoculated control broth (Supplementary Table 1) were diluted 1:50 in fresh Mb-MYM and then added to a 96-well plate. The plates were loaded onto a Hamilton Vantage Liquid Handling System equipped with a 96 pipetting head which was used to dip pipette tips into the liquid cultures and press/print them against the surface of the agar in a grid; a single colony of each unique strain was printed in a 3 × 5 grid (Figure 2A). The lidded-plate was sealed with Parafilm and incubated at 30°C for 2 days once visible colonies had formed of similar size (Supplementary Figure 1). Printing was repeated using only P. stutzeri RCH2 (the interaction strain) in all well positions of a 16 × 24 grid (ANSI standard dimensions of a 384 well plate), excluding the positions from the original print at time 0 (Figure 2A). Cultures were incubated for an additional 4 days prior to imaging using a digital camera. Image analysis was performed using ImageJ (Schneider et al., 2012). Briefly, the image was converted to 16-bit grayscale, corrected for uneven lighting using the FFT bandpass filter (filter large structures to 70 pixels and small structures to 5 pixels, 5% tolerance, autoscale and saturate after filtering), auto thresholded to black and white, and then the colonies were measured using the Analyze Particles tool (colonies missed by the automated selection were manually outlined and then measured). Measurements included area and position. Colony areas of surrounding (8 colonies in a square around the center interaction colony) and closest/farthest neighbors (sides/corners of the square) were compared with the colonies surrounding the control uninoculated position (#15) using Dunnett’s multiple comparison procedure using R version 3.6.2 (Hothorn et al., 2008; R Core Team, 2014).

Overnight culture of Pseudomonas sp. FW300-N1B4 (N1B4) was manually pipetted (10 μL) onto an agar plate and incubated at 30°C overnight. Overnight culture of P. stutzeri RCH2 was manually pipetted (10 μL) over the top of the N1B4 colony with care to not puncture the colony surface and to avoid allowing the droplet to spill over the sides. For time course analysis of macroscopic morphology, the rim of the petri dish was sealed with parafilm and placed on a black velvet cloth under a Leica M165FC microscope with a Planapo 1.0× objective lens. Images were acquired every 2 min for a 24 h period with a Leica DFC400 1.4 megapixel ccd sensor digital camera set at a 9 mm frame width. For microscopic colony interface analysis, after overnight growth of the overlay, the colony and underlying agar was cut out from the plate and fixed for 2 h with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Fixed samples were stained with 5 mM ruthenium red in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h and post-fixed with 1% osmium tetroxide for 1 h. Following staining, samples were dehydrated through a graded ethanol series (20, 40, 60, 80, 90, 100, 100, and 100%) followed by infiltration with an Embed-812 epoxy resin (Electron Microscopy Sciences):acetone series of 1:3 for 2 h, 2:3 for 2 h, and 100% resin overnight. Samples were then heat polymerized in 100% resin with N,N-dimethylbenzylamine accelerant for 2 h at 85°C. 100 nm sections were cut and sectioned using a Leica UC6 Ultra Microtome (Leica Microsystems Inc., Buffalo Grove, IL, United States) and then stained sequentially with 2% methanolic uranyl acetate and Reynolds’ lead citrate for 5 min each. Images were collected using a FEI Tecnai 12 transmission electron microscope (FEI Company, Hillsboro, OR, United States).

The construction of the P. stutzeri RCH2 DNA-barcoded transposon mutant was previously described (Wetmore et al., 2015). Overnight culture of Pseudomonas sp. FW300-N1B4 was manually pipetted (10 μL) in triplicate onto LB agar plates. The plates were incubated overnight and then 5 μL of the P. stutzeri RCH2 barcoded transposon mutant library (in LB media) starter culture at an OD₆₀₀ of 0.9 was spotted as overlay colonies on top of the three replicates of the underlay colonies. An aliquot of the starter culture was used for the initial RCH2 mutant abundances. Colonies were then carefully scraped and frozen for storage and subsequent analysis of the final P. stutzeri RCH2 mutant abundances. Gene fitness scores were calculated by comparing the initial and final mutant abundances as determined by deep sequencing of the DNA barcodes, as previously described (Wetmore et al., 2015).

The four mutants from the pooled fitness assay with the largest absolute differential fitness where fitness of RCH2 with N1B4 was less than fitness of RCH2 on agar alone were selected for constructing gene deletion mutant strains. Deletion mutants (Supplementary Table 2) for gamma-glutamyl phosphate reductase, OHCU decarboxylase, formyltetrahydrofolate deformylase, and glutamate 5-kinase were constructed by conjugation of unstable, marker-exchange plasmids into P. stutzeri RCH2, as previously described (Vaccaro et al., 2016) with modifications as follows. All plasmids and primers (IDT, Newark, NJ) used are listed in Supplementary Tables 3, 4, respectively. Briefly, deletion cassettes, containing kanamycin resistance gene (npt) flanked by chromosomal regions up and downstream of the gene to be deleted, were assembled using the “gene SOEing” technique (Horton et al., 1990). Cloned homologous regions were sequenced at the DNA core facility at the University of Missouri, Columbia, and compared with the published sequence for P. stutzeri RCH2 (Chakraborty et al., 2017). Cassettes and the template plasmid pM07704 were amplified by polymerase chain reaction (PCR) with Herculase II DNA polymerase (Stratagene). Marker-exchange plasmids were generated by ligation of the PCR products in α-select cells (Bioline) using the SLIC cloning method (Li and Elledge, 2007). The plasmids were isolated and transformed into E. coli strain WM3064, and then transferred to RCH2 via conjugation (Fels et al., 2013). The plasmids, containing up/downstream chromosomal regions flanking npt, allow for exchange of npt with the gene of interest in RCH2 via double homologous recombination. Exconjugates were selected on 50 μg/mL kanamycin solid medium, then screened for spectinomycin (100 μg/mL) sensitivity to ensure no single recombination isolates were selected. Deletion strains were confirmed by Southern blot analysis. One isolate for each deletion of interest was retained while the other isolates were discarded. All strains were frozen as early stationary phase cultures in 10% (v/v) glycerol. RCH2 mutants used for experiments were maintained in Mb-CYM broth or Mb-CYM agar with 50 μg/mL kanamycin sulfate; kanamycin was not included in subsequent cultures for metabolomics analysis.

Overnight cultures of RCH2 (wild-type and mutants) and N1B4 (500 μL) were centrifuged (5,000 × g × 3 min at RT) to collect cells, then washed 2 times in DPBS (resuspension in 500uL DPBS, centrifugation at 5,000 × g × 3 min at RT) with final resuspension adjusted to an OD (600 nm, 1 cm) of 0.5. Uninoculated control medium was washed and resuspended in a similar manner to account for carryover of nutrients from tube surfaces and residual volumes. Cultures (“underlays”) were manually spotted (2 μL) onto Mb-CYM agar plates in a 7 × 7 format and incubated overnight at 30°C. Fresh cultures were inoculated from agar stock plates into Mb-CYM. Overlay colonies were diluted and washed in the same manner as for the underlays and then carefully spotted on top of the underlay colonies. Images were taken using a digital camera of whole plates and through a LEICA M165 FC stereo microscope (2x objective) at 24 h (0 h of overlays), 48 h (24 h of overlays), 72 h (48 h of overlays). Morphological differences were visually evaluated.

Wild-type and mutant strains of P. stutzeri RCH2 were cultured in “spent” N1B4 medium (sterile filtered N1B4 culture supernatant) in liquid culture and then exometabolites were collected by extraction in methanol (Want et al., 2006; Baran et al., 2013; Sitnikov et al., 2016) for LCMS analysis. For collection of “spent” medium, overnight liquid cultures of RCH2 wild-type, RCH2 mutants, and N1B4 wild-type and uninoculated control medium were centrifuged (3,000 × g× 10 min) to pellet cells. Supernatants were sterile filtered (0.22 μm), supplemented with 1x Wolfe’s vitamins and minerals (ATCC) and stored at 4°C overnight. Additional overnight cultures were diluted, washed and adjusted as described above for mutant co-colony morphology analysis, except the final OD was adjusted to 0.12 in DPBS, 20 μL of which was added to 100 μL of spent medium in 96 well plates. Cultures were prepared in triplicates for all combinations of mutants and wild-type RCH2 on spent N1B4 and fresh Mb-CYM and for N1B4 on all combinations of spent mutants and wild-type RCH2 and fresh Mb-CYM; uninoculated controls were included for each spent medium and positive growth controls were included for each strain on fresh (non-spent) medium. Cultures were incubated for 20 h in a BioTek plate reader with OD readings taken every 30 min at 600 nm. Cross-feeding medium from the culture was then collected via centrifugation (3,000 × g for 5 min), transferred to a new plate that was sealed with heated foil (4titude), and frozen at −80°C. Holes were pierced in the foil with an 18 gauge needle and then supernatants were lyophilized to dryness. Dried material was resuspended in 200 uL of internal standard mix (15 μM 13C, 15N amino acid mix, 10 μg/mL 13C-mannitol, 13C-trehalose and 2 μM 15N4-inosine, 15N5-adenine, 13C4-15N2-uracil, 15N4-hypoxanthine, 13C4-15N2-thymine) in LCMS grade methanol, resealed, vortexed, sonicated in a room temperature water bath for 10 min, rechilled at −80°C for 5 min, and centrifuged 3,000 × g for 5 min to pellet insoluble material. Supernatants were filtered (0.22 μm, PVDF) using an Apricot positive pressure filtration device. Filtrates were then arrayed into 50 μL aliquots in a 384 well plate for LCMS analysis.

Extracts of polar metabolites were analyzed using hydrophilic interaction chromatography—mass spectrometry. Metabolites were retained and separated on an InfinityLab Poroshell 120 HILIC-Z column (Agilent, 683775-924, 2.7 μm, 150 × 2.1 mm) using an Agilent 1290 UHPLC. Samples, held at 4°C, were injected at 4 μL each; the column temperature was held at 40°C and flow rate was held at a constant 0.45 mL/min. Following injection, a gradient of mobile phase A (5 mM ammonium acetate, 0.2% acetic acid, 5 μM methylene di-phosphonic acid in water) and mobile phase B [5 mM ammonium acetate, 0.2% acetic acid in 95:5 (v/v) acetonitrile:water] was applied as follows: initial equilibration at 100% B for 1.0 min, linear decrease to 89% B over 10 min, linear decrease to 70% B over 4.75 min, linear decrease to 20% B over 0.5 min, hold at 20% B for 2.25 min, linear increase to 100% B over 0.1 min, re-equilibration at 100% B for 2.4 min. Eluted metabolites were subjected to mass spectrometry analysis on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) equipped with a HESI-II source probe using Full MS with Data Dependent tandem MS. Source settings were as follows: sheath gas at 55 (arbitrary units), aux gas flow at 20, sweep gas at 2, spray voltage at 3 | kV| spray, capillary temperature at 400C, and S-lens RF at 50. MS1 was set at 70,000 mass resolution, with automatic gain control target at 3.0E06 with a maximum allowed injection time of 100 ms, at a 70–1,050 m/z scan range. dd-MS2 was set at 17,500 mass resolution with automatic gain control target at 1.0E5 and a maximum allowed injection time of 50 ms, a 2 m/z isolation window and stepped normalized collision energies at 10, 20, and 30 (dimensionless units). All data was collected in centroid mode. The scan cycle included a single MS1 scan followed by sequential MS/MS of the top two most intense MS1 ions excluding any fragmented within the previous 10 s. Ions selected for fragmentation must meet a minimum AGC target threshold of 1.0E3 with absolute charge less than four. Each sample was analyzed in negative ionization mode. Sample were injected in randomized order with solvent blank injections between each; internal and external standards were used for quality control purposes and for retention time predictions of compounds from in an in-house standards library. Using custom python scripts and metabolite atlases (Bowen and Northen, 2010; Yao et al., 2015), mass-to-charge ratios, retention times and where possible spectra fragmentation patterns were used to confirm metabolite identification by comparison to metabolite standards analyzed using the same LC-MS/MS methods. Mutant and RCH2 cultures on N1B4 medium were compared with uninoculated control N1B4 spent medium using ANOVA and Tukey HSD in R.

As the first step of BIMA, a colony printing method was developed on an automated liquid handling system for the purpose of scalability and transferability between labs. Fourteen preprinted “effector” Pseudomonas strains (including RCH2) were evaluated for their effect on the growth of subsequently printed neighboring colonies of P. stutzeri RCH2 (Figures 2A,B). Pseudomonas strains 3–9, and 11 inhibited the growth of the four closest RCH2 colonies, located along the sides of the square of neighboring colonies (Figure 2C). Interestingly, for a subset of these (3, 4, 5), the more distant RCH2 colonies at the corners of the square of neighboring colonies had larger mean areas (Figure 2D). Pseudomonas strain 2 (Pseudomonas fluorescens FW300-N1B4) was the only strain that did not inhibit RCH2 (closest side colonies) and enhanced the growth of RCH2 (corner colonies). This strain “N1B4,” was selected for further analysis in co-culture with P. stutzeri RCH2.

Time lapse images of the RCH2 overlay on N1B4 were taken to evaluate the co-colony morphology. Highly wrinkled, rugose colony morphology had formed by 12 h after application of the RCH2 overlay (Figure 3A). The co-colony species interface was further examined using transmission electron microscopy (TEM) (Figure 3B). TEM after 2 h showed a stratification with a clear distinction between RCH2 on the surface and N1B4 underneath. Over the course of 24 h, the colonies became more mixed with infiltration of the N1B4 layer by RCH2. In both the co-culture and in isolate culture, RCH2 biofilm developed sacs containing groups of spherical cells at the air interface; whereas when spotted on the surface of N1B4, single, elongated RCH2 are observed at the N1B4 interface. The sacs became visible in the 24 h co-colony. The anaerobic co-colony culture developed a compact RCH2 layer with no visible EPS sacs.

A pooled mutant fitness assay was used to identify RCH2 genes essential for successful aerobic co-culture growth with N1B4; the anaerobic culture was not evaluated further since no morphological interaction was observed. The top four genes with the largest differential gene fitness between RCH2 on LB and RCH2 on N1B4, where fitness on N1B4 was negative included gamma-glutamyl phosphate reductase (proA), OHCU decarboxylase (uraD), formyltetrahydrofolate deformylase (purU), and glutamate 5-kinase (proB) (Figure 4A). To investigate these genes further, we constructed isogenic single-gene deletion strains for all four genes.

In liquid culture, while all mutants were capable of growth on fresh medium, only the ΔuraD deletion strain had similar growth to the wild-type when cultured on spent N1B4 medium (Figure 4B). Minimal to no growth was observed for the ΔproA and ΔproB deletion mutants, and delayed growth was observed for the ΔpurU deletion mutant. N1B4 had similar growth on the spent medium of RCH2 wild-type and mutants (not shown). The limited or delayed growth indicated these mutants were unable to acquire some required metabolite from the medium that was consumed by N1B4 in liquid culture or that growth may have been inhibited by something N1B4 was producing.

The biofilm interactions were analyzed by manual printing of the RCH2 wild-type and mutant strains on the top of an established N1B4 colony. The overlays were imaged at 24 and 48 h after overlay printing (Figure 4C). Wild-type RCH2, and ΔproA and ΔuraD deletion strains formed rugose co-colony biofilms after 24 h similar to the wild-type but with a smoother center for ΔproA, while the ΔpurU mutant had delayed rugose formation and the ΔproB mutant remained smooth up to 72 h of observation. All mutants formed rugose colonies after 24 h of overlay on control medium. On its own, the ΔpurD formed more tubular structures than the wild-type alone, similar to growth on N1B4 for both (Supplementary Figure 3).

To further evaluate why the four RCH2 genes identified in the mutant fitness assay were important to growth in the aerobic co-colony, spent medium from N1B4 was fed to each of the mutants described above and the wild-type RCH2 and then metabolomics was performed to check for altered metabolism in the mutants. Despite limited growth of three of the mutants on N1B4 spent medium (Figure 4B), all four RCH2 mutants appeared to be metabolically active with significant increases and decreases in metabolite abundances relative to uninoculated N1B4 spent medium. In many cases the changes were significantly different than the wild-type (Figure 5 and Supplementary Figures 4, 5). The P. stutzeri RCH2 wildtype consumed several N1B4 metabolites involved in one-carbon (C1) metabolism though consumption of methionine was reduced in P. stutzeri RCH2 strain JWST9066 (ΔpurU) (Supplementary Figure 5). P. stutzeri RCH2 strain JWST9063 (ΔuraD) accumulated allantoin, presumably from spontaneous conversion of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) to (R)-allantoin. P. stutzeri RCH2 strain JWST9060 (ΔproA) and P. stutzeri RCH2 strain JWST9069 (ΔproB), had reduced consumption of glutamate (precursor in proline synthesis) from N1B4, when compared to wild-type, and similar consumption of proline (Supplementary Figure 5).