Bacterial strains, plasmids, and growth conditions

Bacterial strains used are listed in Supplementary Table 1. A. xylosoxidans MN00152 and Escherichia coli were grown in lysogeny broth (LB) at 37 °C unless otherwise specified. When necessary, growth media were supplemented with gentamicin at 20 μg/ml (E. coli) or 100 μg/ml (A. xlyosoxidans), ampicillin at 100 μg/ml (E. coli) or 300 μg/ml (A. xylosoxidans), carbenicillin (300 μg/ml), tetracycline (300 μg/ml), or chloramphenicol (30 μg/ml). E. coli strain β2155 was supplemented with 360 μM diaminopimelic acid (DAP).

Transposon mutagenesis

The transposon delivery vector pTnTet containing the hyperactive mariner transposon21 was introduced by transformation into the E. coli donor bacterial strain β2155.53 A. xylosoxidans MN001 and E. coli β2155 (carrying pTnTet) were grown overnight in LB and LB containing chloramphenicol (30 mg/ml) and 360 μM DAP, respectively, at 37 °C. Cells were combined in a donor-to-recipient ratio of 5:1, centrifuged at 4000 × g for 5 min, resuspended in 200 μL fresh LB-DAP and spot-plated (10 μL) onto LB agar. Mating proceeded for 8 h at 37 °C, at which point cells were harvested and resuspended in 1 mL of LB. Cell suspensions were diluted 1:5, and 100 μL aliquots were plated on LB-tetracycline agar and allowed to grow for 48 h at 37 °C. Colonies were randomly selected for downstream biofilm assays.

Biofilm microtiter plate assay

To identify determinants of biofilm development, transposon mutants were screened using a modified crystal violet (CV) assay approach.22 Briefly, individual mutants were transferred to single wells of a 96-well microtiter plate containing 200 μL LB per well, and incubated while shaking at 37 °C. Following 24 h of growth, 2 μL was transferred to 198 μL ABT medium [15 mM (NH 4 ) 2 SO 4 , 40 mM Na 2 HPO 4 , 20 mM KH 2 PO 4 , 50 mM NaCl, 1 mM MgCl 2 , 0.1 mM CaCl 2 and 0.01 mM FeCl 3 supplemented with 0.5% casamino acids and 0.5% glucose]25 in a new microtiter plate, and grown for an additional 24 h at 37 °C in a humidified chamber. Plates were first measured spectrophotometrically (OD 600 ) to determine culture cell density. Supernatants were discarded, and plates were washed 3X with ultrapure water. Plates were dried in a biosafety hood for 2.5 h and stained with 200 μL of 0.1% CV for 20 minutes. Plates were then washed 5X to remove excess stain, air-dried for 4 h, and 200 μL 30% acetic acid was added to each well. Following a 15 min incubation, CV absorbance was measured spectrophotometrically (OD 560 ) and normalized to culture density. Wells exhibiting less than 50% absorbance than the wildtype (MN001) were considered putative hits (134 total) and were subjected to a secondary screen using the same protocol (n = 4 for each mutant). Transposon mutants showing a significant reduction in biofilm growth (31 total, as determined by a Mann-Whitney U test) were stored for further characterization.

Biofilm growth of MN001, its ΔechA derivative and complemented strain (see below) were also tested using the same microtiter assay. In these experiments, growth medium was supplemented with 2% ethanol to drive expression of the alcA promoter in pBMB4 (below). When indicated, cis-2-decenoic acid (F13807D, Carbosynth) was also added at a concentration of 310 nM.35

Arbitrary PCR and mutant sequencing

An arbitrary-PCR-based approach22 was used to identify sequences flanking transposon insertion sites. The PCR method involved two rounds of reactions, with the first using a primer unique for the mariner transposon and one degenerate primer (pair 1, Table S2).54 The second round included nested primers (pair 2) unique to the transposon and 5’ end of the arbitrary primer for amplification of PCR products obtained in the first round. PCR products were sequenced at the University of Minnesota Genomics Center (UMGC) and were mapped to the A. xylosoxidans MN001 genome (Accession#PRJNA288995).

Construction and complementation of an echA deletion mutant

To generate a deletion vector compatible with A. xylosoxidans, pSMV855 was first digested using ApaI. A tetracycline resistance cassette was then amplified from pEX18tc31 using primer pair 3 (Supplementary Table 2) and digested with ApaI. Vector and insert (1:3 ratio) were then ligated using T4 ligase, transformed into E. coli UQ950 and selected for on LB agar containing tetracycline (15 μg/mL). Colonies were screened using primers M13F and TetR (pair 4) to confirm insertion orientation. One plasmid, pBMB1, was selected for further use.

To generate an in-frame, unmarked deletion of Axylo1_0405 (echA), ~1 kb sequences flanking echA were PCR amplified using primer pairs 5 and 6 (Supplementary Table 2). These flanking regions were combined and cloned into pBMB1 digested with SpeI and XhoI using Gibson assembly, resulting in pBMB2 (Supplementary Fig. 1). This plasmid was then chemically transformed into UQ950, and positive ligations were screened by PCR. pBMB2 was then transformed into E. coli strain WM3064 and mobilized into A. xylosoxidans MN001 by conjugation. Recombinants were selected for on LB-tetracycline agar and double recombinants were selected for on LB agar containing 6% sucrose.

Complementation was achieved via exogenous expression of echA (Ax_0405) from pBBR1MCS-5.55 To do so, an alcohol-inducible promoter, alcA32 was first amplified from pGGA008 using primers alcA_F and alcA_R (Supplementary Table 2) before digestion with restriction enzymes HindIII and BamHI. Ligation into similarly digested pBBR1MCS-5 yielded pBMB3 (pBBR1MCS-5::alcA). echA was then amplified from A. xylosoxidans MN001 genomic DNA using primers echA_F and echA_R, and digested with BamHI and SacI before ligation into pBMB3 using T4 ligase. The resulting vector, pBMB4 (pBBR1MCS-5::alcAechA; Supplementary Fig. 1), was transformed into E. coli UQ950. This vector was then introduced into A. xylosoxidans MN001 via conjugation with an E. coli donor strain WM3064, and transconjugants were selected on LB agar containing 300 μg/mL gentamicin sulfate. All constructs and positive transformants were verified by Sanger sequencing.

Attachment assay

A modified attachment assay36 was used to assess early attachment of A. xylosoxidans to a polystyrene substratum. Briefly, MN001 and its ΔechA derivative were grown for 18 h at 37 °C in ABT followed by dilution 1:100 into fresh medium. Cells were then grown to mid-log phase (OD 600 = 0.6) before dilution in ABT to an OD 600 of 0.1. 200 μl of each culture was added to an 8-chamber coverslip slide (Ibidi, #80824) and incubated at 37 °C for 1 h. Following incubation, slides were rinsed twice with 200 μl of PBS to remove unattached biomass, and attached cells were stained using SYTO 9 (Invitrogen) in PBS according the manufacturer’s protocol. Substrata were imaged using an Olympus IX83 inverted fluorescence microscope with a transmitted Koehler illuminator and a 40X objective lens (Olympus). Four images per strain per biological replicate (n = 4) were captured on a Hamamatsu ORCA camera, and post-acquisition analysis was performed using FIJI software56 by calculating the integrated density of SYTO 9.

Colony biofilm assay

MN001 and ΔechA were grown overnight in LB medium, diluted 1:1000, and 10 μL was spotted on nutrient agar containing 1% tryptone, 1% agar, 20 μg/mL Coomassie Brilliant Blue, and 40 μg/mL Congo red.36 Plates were incubated at 37 °C for 6 days and monitored daily for colony morphology.

Scanning electron microscopy

Overnight cultures were diluted 1:10 in fresh LB medium and were added to 48-well microtiter plates containing autoclaved Aclar fluoropolymer film (Electron Microscopy Sciences, Hatfield, PA). Biofilms were grown for 48 h at 37 °C, shaking at 50 rpm, and prepared for SEM using cationic dye stabilization methods.57,58 Briefly, Aclar membranes containing biofilm growth were washed three times in 0.2 M sodium cacodylate buffer, and submerged in primary fixative (0.15 M sodium cacodylate buffer, pH 7.4, 2% paraformaldehyde, 2% glutaraldehyde, 4% sucrose, 0.15% alcian blue 8 GX) for 22 h. Samples were washed three more times prior to a 90 minute treatment with secondary fixative (1% osmium tetroxide, 1.5% potassium ferrocyanide, 0.135M sodium cacodylate, pH 7.4). After three final washes, biofilms were chemically dehydrated in a graded ethanol series (25, 50, 70, 85, 95 [2×] and 100% [2×]) before CO 2 -based critical point drying. Aclar membranes were attached to SEM specimen mounts using carbon conductive adhesive tape and sputter coated with ~ 5 nm iridium using the Leica ACE 600 magnetron-based system. Biofilms were imaged using a Hitachi S-4700 field emission SEM with an operating voltage of 2 kV.

Antibiotic challenge

Biofilm antimicrobial susceptibility testing was performed using a chamber slide assay.59 Briefly, MN001 and ΔechA were grown in LB overnight, diluted 1:1000 and grown to an OD 600 of 0.6 before dilution to an OD 600 of 0.5. In total 200 μL of each culture was then added to each well of an Ibidi 8-chamber coverslip slide and incubated at 37 °C in a humidified chamber. After 24 h, medium was replaced with fresh LB and incubated for an additional 24 h. Media was gently aspirated from each well, replaced with LB containing either tobramycin or levofloxacin (0, 10, 100, and 1000 μg/mL), and incubated at 37 °C for 6 h. Cells were washed in sterile PBS to remove unattached biomass, stained for 15 minutes using the BacLight Live/Dead viability assay (Life Technologies, #L7012) and visualized by fluorescent imaging as described above. Integrated density for SYTO 9 and propidium iodide (PI) for each image was determined using FIJI,56 and percentage of dead biomass was determined by (average integrative density of PI)/(average integrated density of PI+ integrated density of SYTO 9).59 Data were generated using four biological replicates (n = 4).

Prior publication

This work was previously published as a preprint.60

Reporting Summary

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