1. Microbiology and Infectious Disease

Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism

  1. See-Yeun Ting
  2. Kaitlyn D LaCourse
  3. Hannah E Ledvina
  4. Rutan Zhang
  5. Matthew C Radey
  6. Hemantha D Kulasekara
  7. Rahul Somavanshi
  8. Savannah K Bertolli
  9. Larry A Gallagher
  10. Jennifer Kim
  11. Kelsi M Penewit
  12. Stephen J Salipante
  13. Libin Xu
  14. S Brook Peterson
  15. Joseph D Mougous  Is a corresponding author
  1. Department of Microbiology, University of Washington School of Medicine, United States
  2. Department of Medicinal Chemistry, University of Washington School of Pharmacy, United States
  3. Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, United States
  4. Department of Biochemistry, University of Washington School of Medicine, United States
  5. Howard Hughes Medical Institute, University of Washington, United States
https://doi.org/10.7554/eLife.74658
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Cite this article
  1. See-Yeun Ting
  2. Kaitlyn D LaCourse
  3. Hannah E Ledvina
  4. Rutan Zhang
  5. Matthew C Radey
  6. Hemantha D Kulasekara
  7. Rahul Somavanshi
  8. Savannah K Bertolli
  9. Larry A Gallagher
  10. Jennifer Kim
  11. Kelsi M Penewit
  12. Stephen J Salipante
  13. Libin Xu
  14. S Brook Peterson
  15. Joseph D Mougous
(2022)
Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism
eLife 11:e74658.
https://doi.org/10.7554/eLife.74658
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5 figures, 5 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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Multiple pathways under Gac/Rsm control contribute to P. aeruginosa defense against antagonism.

(A) Transposon library sequencing-based comparison of the fitness contribution of individual P. aeruginosa genes during growth competition with wild-type B. thai versus B. thai ∆T6S. Genes under Gac/Rsm control (blue) and those encoding core Gac/Rsm regulatory factors (purple, labeled) are indicated. (B) Overview of the Gac/Rsm pathway (SD, Shine–Dalgarno). The rsmY and rsmZ genes encode small RNA molecules that sequester the translational regulator RsmA. Hybrid sensor kinase PA1611 is here renamed RetS-regulating sensor kinase A (RskA). (C) Left: P. aeruginosa gene clusters hit (greater than threefold in replicate screens; Figure 1—source data 1) in this study. Numbers below genes indicate transposon insertion ratio (B. thai ∆T6S/wild-type) for screen in (A) followed by the replicate screen (Figure 1—figure supplement 1); light toned genes were hit in only one replicate. Asterisk indicates genes for which insertion was undetected in libraries obtained from B. thai wild-type competition. Right: zoom-in of boxed region of (A) with genes colored corresponding to clusters at left. (D, E) Recovery of P. aeruginosa cells with the indicated genotypes following growth competition against B. thai wild-type (D) or ∆T6S (E). For interbacterial competition assays, the mean ± SD of biological duplicates and associated technical duplicates is shown: *p<0.05 using unpaired two-tailed Student’s t-test.

Figure 1—source data 1

Transposon sequencing-based analysis of P. aeruginosa fitness determinants during antagonism by B. thai compared to B. thai with an inactive T6SS (∆icmF).

https://cdn.elifesciences.org/articles/74658/elife-74658-fig1-data1-v2.docx
Figure 1—figure supplement 1
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Multiple pathways under Gac/Rsm control contribute to P. aeruginosa defense against antagonism.

(A) Growth of wild-type P. aeruginosa in co-culture with the indicated B. thai strains. (B) Transposon library sequencing-based comparison of the fitness contribution of individual P. aeruginosa genes during growth competition with wild-type B. thai versus B. thai ∆T6S. Genes under Gac/Rsm control (blue) and those encoding core Gac/Rsm regulatory factors (purple, labeled) are indicated. This experiment is a biological replicate of that in Figure 1A. (C) P. aeruginosa gene clusters hit (Figure 1—source data 1) in this study. (D) Zoom-in of boxed region of (B) with genes colored corresponding to clusters at left. The interbacterial competition assay represents two biological replicates with two associated technical replicates.

Figure 1—figure supplement 2
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Antagonism resistance clusters possess functionally related genes and are in diverse bacteria.

(A) Schematic depicting the arc1 gene cluster of P. aeruginosa. Predicted enzymatic activity of Arc1A, B, D, F is indicated (right) and placed onto a phospholipid biosynthesis pathway (bottom). (B) Depiction of arc2 genes from the indicated species. Genes encoding the predicted MoxR-like ATPase and von Willebrand factor (VWF) domain protein are indicated below the respective genes. Orthologs are colored to highlight synteny. (C) Depiction of arc3 genes from the indicated species. S. dynata exemplifies an instance of translational fusion of arc3A,B. Domains of unknown function constituting the two proteins are indicated below.

Figure 1—figure supplement 3
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Interbacterial competition assays measure the contribution of arc1-3 to P. aeruginosa fitness during antagonism by B. thai.

(A) Recovery of P. aeruginosa strains bearing the indicated single or double deletions within arc1-3 following growth competition against wild-type B. thai. (B) Recovery of P. aeruginosa cells with the indicated genotypes following growth competition against wild-type B. thai. The mean ± SD of biological duplicates and associated technical duplicates is shown: n.s. corresponds to p>0.05 by unpaired two-tailed Student’s t-test. The fold difference in growth yield between mutants and the parental strain is indicated above.

Figure 2 with 3 supplements
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Arc pathways can provide toxin-specific antagonism defense that eclipses that of other cellular factors.

(A) Recovery of B. thai cells following growth competition against a relative abundance of P. aeruginosa containing the indicated gene deletions, colored according to Figure 1C. (B) Relative survival of P. aeruginosa Arc1-3-inactivated mutants following growth in competition with an excess of the indicated B. thai strains (mutant CFU/parental CFU × 100 for each B. thai competitor). The loss of ColA or Tle3 activity in B. thai increases the relative survival of P. aeruginosa lacking Arc2 or Arc3 activity, respectively. (C, D) Transposon library sequencing-based comparison of the fitness contribution of individual P. aeruginosa genes during growth competition with wild-type B. thai versus B. thai lacking Tle3 (C) or ColA (D) activity, colored according to Figure 1C. Values in parentheses correspond to the number of genes hit (greater than threefold change in transposon insertion frequency) within each cluster compared to the number within each cluster hit in the initial screens (B. thai wild-type versus B. thai ∆T6S) followed by the average transposon insertion frequency ratio (B. thai mutant/B. thai wild-type) of the genes in the depicted screen. (E) Fitness contribution of each P. aeruginosa Arc pathway and the H1-T6SS to defense against Tle3 versus ColA. For interbacterial competition assays, the mean ± SD of biological duplicates and two technical replicates is shown: *p<0.05 using unpaired two-tailed Student’s t-test.

Figure 2—source data 1

Transposon sequencing-based analysis of P. aeruginosa fitness determinants during antagonism by B. thai compared to B. thai tle3S264A.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-data1-v2.docx
Figure 2—source data 2

Transposon sequencing-based analysis of P. aeruginosa fitness determinants during antagonism by B. thai compared to B. thai ∆colA.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-data2-v2.docx
Figure 2—figure supplement 1
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Arc1-3 do not contribute to the survival of P. aeruginosa exposed to common environmental stresses.

Survival of the indicated P. aeruginosa strains during exposure to hydrogen peroxide (50 mM, 30 min) (A), high salinity (3 M NaCl, 20 hr) (B), high temperature (55°C, 30 min) (C), or detergent (0.5% [w/v] SDS) (D). The mean ± SD of a representative biological replicate with three technical replicates is shown.

Figure 2—figure supplement 2
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Arc1-3 are subject to tight regulation by the Gac/Rsm signaling pathway.

Anti-VSV-G immunoblot analysis was used to probe the levels of the indicated VSV–G-tagged (–V) Arc proteins encoded at their respective native genomic loci in P. aeruginosa wild-type, ∆retS, or ∆gacS backgrounds. The asterisk denotes a nonspecific VSV-G antibody-reactive band. The arrow highlights the position of Arc2G–V. Blot images are representative of at least two biological replicates. See Figure 2—figure supplement 2—source data 1–6.

Figure 2—figure supplement 2—source data 1

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc1A–V.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data1-v2.pdf
Figure 2—figure supplement 2—source data 2

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc1A–V RpoB control.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data2-v2.pdf
Figure 2—figure supplement 2—source data 3

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc2G–V.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data3-v2.pdf
Figure 2—figure supplement 2—source data 4

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc2G–V RpoB control.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data4-v2.pdf
Figure 2—figure supplement 2—source data 5

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc3A–V.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data5-v2.pdf
Figure 2—figure supplement 2—source data 6

Original uncropped image and original uncropped image with relevant bands indicated for Figure 2—figure supplement 2, Arc3A–V RpoB control.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig2-figsupp2-data6-v2.pdf
Figure 2—figure supplement 3
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The loci encoding Tle1 and Tle3 are duplicated in B. thai, and Tle3 induces potent self-intoxication.

(A) Schematic depicting the tle1/tli1 (top) and tle3/tli3 (bottom) loci in the genome B. thai E264 strain. Predicted catalytic serine residues mutagenized in this study are indicated by red asterisks. (B) Recovery of B. thai strain sensitized to Tle3 intoxication (∆tle3∆tli3) following growth competition against B. thai wild-type or a strain in which both orthologs of Tle3 contain an inactivating point mutation. The mean ± SD of two biological replicates with two technical replicates is shown.

Figure 3 with 1 supplement
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Arc3B is a predicted massive polytopic membrane protein with functionally complementing family members that occur widely in Gram-negative and -positive bacterial phyla.

(A) Schematic depiction of Arc3A and Arc3B based on bioinformatic predictions. (B) Phylogeny of bacterial phyla with representative arc genes depicted and colored as in (A). Membrane-associated regions (grayed) and the number of predicted transmembrane segments are indicated. (C) Recovery of P. aeruginosa strains bearing the indicated mutations and containing a control chromosomal insertion (–) or insertion constructs expressing Arc3B proteins derived from assorted bacteria (P. aer, P. aeruginosa; P. pro, P. protegens; B. cen, B. cenocepacia; P. syr, P. syringae; C. rod, Citrobacter rodentium) following growth competition against B. thai wild-type. (D) Recovery of the indicated P. protegens strains following growth competition against B. thai wild-type or a strain lacking Tle3 activity. For interbacterial competition assays, the mean ± SD of biological duplicates and two or three associated technical replicates is shown: *p<0.05 using unpaired two-tailed Student’s t-test.

Figure 3—figure supplement 1
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EstA and Arc3B inactivation does not significantly impact P. aeruginosa fitness in pairwise growth competition experiments with B. thai.

Recovery of the indicated P. aeruginosa strains following growth in competition with B. thai wild-type or a strain lacking Tle3 activity (tle3S264A). Means ± SD of biological duplicates with three technical replicates s shown.

Figure 4 with 3 supplements
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Arc3 prohibits Tle3-catalyzed lysophospholipid accumulation by a mechanism distinct from known pathways.

(A–D) Mass spectrometric (A) and radiographic thin layer chromatography (TLC) (B) analysis of major phospholipid and lysophospholipid species within lipid extracts derived from the indicated mixtures of P. aeruginosa and B. thai strains. P. aeruginosa were grown in 32PO42- prior to incubation with B. thai. Strains were allowed to interact for 1 hr before phospholipids were harvested. Radiolabeled molecules of interest within biological triplicate experiments resolved by TLC were quantified by densitometry (C, D) (E) Mass spectrometric analysis of major phospholipid and lysophospholipid species within lipid extracts derived from mixtures containing B. thai lacking Tle3-specific immunity factors competing with the indicated B. thai strains. (F) Recovery of the indicated P. aeruginosa strains following growth competition against B. thai wild-type or a strain lacking Tle3 activity. (G) Radiographic TLC analysis of products extracted after incubation of P. aeruginosa-derived spheroplasts with purified radiolabeled lysophosphatidylethanolamine (LPE). For interbacterial competition assays, the mean ± SD of biological replicates and associated technical replicates is shown: *p<0.05 using unpaired two-tailed Student’s t-test. TLC images shown are representative of at least two biological replicates. See also Figure 4—source data 1–3.

Figure 4—source data 1

Original uncropped image and original uncropped image with relevant spots indicated for Figure 4B, left TLC.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig4-data1-v2.pdf
Figure 4—source data 2

Original uncropped image and original uncropped image with relevant spots indicated for Figure 4B, right TLC.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig4-data2-v2.pdf
Figure 4—source data 3

Original uncropped image and original uncropped image with relevant spots indicated for Figure 4G, right TLC.

https://cdn.elifesciences.org/articles/74658/elife-74658-fig4-data3-v2.pdf
Figure 4—figure supplement 1
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Tle3 intoxication does not impact free fatty acids nor intact parent phospholipids in P. aeruginosa.

Mass spectrometric analysis of free fatty acid (A) and parent phospholipid species (B) within lipid extracts derived from the indicated mixtures. Data represent the mean ± SD of biological duplicates with two technical replicates.

Figure 4—figure supplement 2
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Arc3B genes are encoded adjacent to aas genes in diverse bacteria.

Schematic depicting arc3B and aas loci in the indicated bacterial species.

Figure 4—figure supplement 3
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Overexpression of aas fails to complement the competitive defect associated with Arc3A inactivation.

Recovery of the indicated P. aeruginosa strains carrying empty pPSV39 (control) or pPSV39-aas grown in competition with an excess of B. thai with Isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction of expression from the plasmid. Data represent mean and standard deviation from technical duplicates from one biological replicate representative of two conducted.

Author response image 1
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Inactivation of Arc3A does not detectably impact P. aeruginosa growth in competition with assorted phospholipase producing species.

(A) Recovery of P. aeruginosa with the indicated genotypes following growth in competition with an excess of wild-type or Tle1-inactivated B. thai. (B and C) Recovery of P. aeruginosa (B) or competitive index obtained (C; final donor:recipicient ratio divided by initial donor:recipienct ratio) following growth in competition with the indicated organisms.

Tables

Appendix 1—table 1
Mass spectrometric analysis of P. aeruginosa Arc3A and Arc3B immunoprecipitation samples.
Locus ID*Peptide counts of sample (%)†Fold change (VSV-G/Ctrl ‡)
Arc3B-VSV-G sample
PA5113 (Arc3B)5.86> 200
PA43851.622.29
PA50161.450.82
PA00901.170.72
PA37291.121.30
PA47611.120.71
PA5114 (Arc3A)1.12N.D. §
PA52391.061.10
PA38611.001.10
PA42691.001.10
PA10920.950.78
PA01410.890.71
PA09630.841.27
PA29760.840.83
PA42600.840.69
PA42650.840.61
PA42700.841.04
PA18030.780.74
PA21510.780.77
PA39500.781.19
PA30010.731.10
PA36560.730.80
PA51730.730.76
PA55540.731.20
PA55560.732.05
Arc3A-VSV-G sample
PA55542.924.81
PA5113 (Arc3B)2.22> 200
PA5114 (Arc3A)1.44N.D.
PA47511.3012.87
PA55561.263.55
PA37291.101.28
PA50161.100.62
PA44290.99N.D.
PA49420.966.36
PA24930.922.60
PA31600.90N.D.
PA42460.870.79
PA00900.850.53
PA15520.83N.D.
PA00770.815.33
PA24940.81ND
PA29760.720.71
PA06590.70N.D.
PA47610.700.44
PA49410.652.57
PA42650.630.46
PA36560.610.67
PA37940.61N.D.
PA21510.560.55
PA38210.56N.D.
  1. *

    Proteins containing at least two peptides identified in a given immunoprecipitation sample are included. Arc3A and Arc3B are bolded.

  2. †

    Value corresponds to the abundance (peptide counts) of the protein within the total immunoprecipitation sample. Only the 25 most abundant proteins in each sample are shown.

  3. ‡

    Immunoprecipitation from a P. aeruginosa strain lacking a VSV-G-tagged protein served as the control sample (Ctrl).

  4. §

    The protein was not detected in the control sample.

Appendix 1—table 2
Internal standards and parameters used in lipidomic analysis.
Internal standards used
Compound formulaCompound NameAlternative name
C20H41O9PLysoPG(14:0)
C24H49O9PLysoPG(18:0)
C34H67O10PPG(14:0/14:0)
C46H91O10PPG(20:0/20:0), i.e. diphytanoyl PG
C19H40O7PNLysoPE(14:0)
C23H48O7PNLysoPE(18:0)
C29H58O8PNPE(12:0/12:0)
C45H90O8PNPE(20:0/20:0), i.e. diphytanoyl PE
C66H120O17P2CL(57:4)14:1(3)–15:1 CA
C70H134O17P2CL(61:1)15:0(3)–16:1 CA
C89H166O17P2CL(80:4)22:1(3)–14:1 CA
Mass spectrometry global parameters on Waters Xevo TQS mass spectrometer
ParameterValue
Cycle timeAutomatic
Source temperature (°C)150
Desolvation temperature (°C)250
Cone gas flow (L/h)150
Desolvation gas fow (L/h)650
Collision gas flow (mL/min)0.14
Nebuliser gas (Bar)7
LM 1 Resolution2.8
HM 1 Resolution14.8
Mass spectral acquisition and data processing parameters
Acquisition parameters
CompoundFunctionm/z rangeStart/end time (min)Scan duration (s)Ionization modeCone voltage (V)Collision energy (V)Total scan time (min)
PGNeutral Loss of 189.04680–8801.6–52ES+3083.4
LPE, PENeutral Loss of 141.02422–9221.6–55ES+40203.4
LPGParents of 152.9420–5705.2–100.75ES-30384.8
CL, MLCLParents of 153865–1,5721.6–304.7ES-407528.4
Data processing parameters
SubtractionSmooth meanCentroid mid peak width at half height (all top 50%)
1, 40, 0.012 × 0.42
1, 40, 0.012 × 0.42
1, 40, 0.011 × 0.65
1, 40, 0.012 × 0.65
Appendix 1—table 3
Transposon sequencing data summary.
Growth experimentMutant pool pre-competitionPool with WT B. thai - rep 1Pool with δt6s B. thai - rep 1
fastq file nametn_mutant_pool.fastqpool_v_wt_rep1.fastqpool_v_deltaT6_rep1.fastq
Total reads515,4345,788,7286,452,899
Trimmed (valid Tn prefix)515,2935,786,4276,450,594
Mapped350,9234,666,5975,283,645
Mapped to TA sites349,9154,634,9425,241,672
TA_sites94,40494,40494,404
TAs_hit50,34857,64460,331
Max reads per TA site3249,5195,886
Mean reads per hit TA site6.980.486.9
Pool with WT B.thai - rep 2Pool with ΔT6S B.thai - rep 2Pool with tle3S264A B. thaiPool with ΔcolA B. thai
pool_v_wt_rep2.fastqpool_v_deltaT6_rep2.fastqpool_v_tle3.fastqpool_v_colA.fastq
411,515720,1333,226,9783,130,545
411,401719,8953,225,8163,129,574
266,466481,4663,024,0472,938,318
265,661479,7692,997,4662,922,995
94,40494,40494,40494,404
46,50053,14859,14558,101
6694473,4843,568
5.79.050.750.3
Appendix 2—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Pseudomonas aeruginosa)PAO1Stover et al., 2000
Strain, strain background (P. aeruginosa)PAO1 ∆pa2541This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa2541∆pa2536This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa4323This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa4323∆pa4320This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114∆pa5113This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa2541, Tn7::AraE::pa2541This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa4323, Tn7::AraE::pa4323This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114, Tn7::AraE::pa5114This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa2541∆pa4323∆pa5114This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆gacS (∆pa0928)LeRoux et al., 2015a
Strain, strain background (P. aeruginosa)PAO1 ∆icmF1 (∆pa0077)Silverman et al., 2011
Strain, strain background (P. aeruginosa)PAO1 ∆pa5112This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114,Tn7::AraE::emptyThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114,Tn7::AraE:: pfl_rs01995This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114,Tn7::AraE:: i35_rs27920This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114, Tn7::AraE::pspto_rs26695This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114, Tn7::AraE::ta05_rs00690This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa3267This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆pa5114∆pa3267This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS (∆pa4856)Mougous et al., 2006
Strain, strain background (P. aeruginosa)PAO1 ∆retS ∆pa3267This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS ∆pa5114This studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆rpoS (∆pa3622)Jørgensen et al., 1999
Strain, strain background (P. aeruginosa)PAO1 ∆vacJ (∆pa2800)Munguia et al., 2017
Strain, strain background (P. aeruginosa)PAO1 pa2541_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS, pa2541_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆gacS, pa2541_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 pa4323_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS, pa4323_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆gacS, pa4323_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 pa5114_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS, pa5114_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆gacS, pa5114_vsv-gThis studySee Materials and methods
Strain, strain background (P. aeruginosa)PAO1 ∆retS, pa5113_vsv-gThis studySee Materials and methods
Strain, strain background (Burkholderia thailandensis)E264 (ATCC 700388)Yu et al., 2006
Strain, strain background (B. thailandensis)E264 ∆icmF1 (∆BTH_I2954)LeRoux et al., 2015a
Strain, strain background (B. thailandensis)E264 tle3S264A(BTH_II0090S264A + BTH_I3226S264A)This studySee Materials and methods
Strain, strain background (B. thailandensis)E264 tle1S267A(BTH_I2698S267A + BTH_I2701S267A)This studySee Materials and methods
Strain, strain background (B. thailandensis)E264 ∆colA (∆BTH_I2691)This studySee Materials and methods
Strain, strain background (B. thailandensis)E264 ∆tae2 (∆BTH_I0068)Russell et al., 2012
Strain, strain background (B. thailandensis)E264 Tn7::Tp-PS12-mCherryLeRoux et al., 2012
Strain, strain background (B. thailandensis)E264 tle3 sensitized strain (∆BTH_I3225-8, ∆BTH_II0089-94)This studySee Materials and methods
Strain, strain background (B. thailandensis)E264 Tn7::Tp-PS12-mCherry tle3 sensitized strain (∆BTH_I3225-8, ∆BTH_II0089-94)This studySee Materials and methods
Strain, strain background (Pseudomonas protegens)pf-5 (ATCC BAA-477)Paulsen et al., 2005
Strain, strain background (P. protegens)Pseudomonas protegens pf-5 ∆pfl_rs01995This studySee Materials and methods
Strain, strain background (Escherichia coli)DH5αThermo Fisher ScientificCat# 18258012
Strain, strain background (E. coli)SM10Biomedal Lifescience
Cat# BS-3303
Strain, strain background (E. coli)UE54MG1655 lpp-2Δara714 rcsF::mini-Tn10 cam pgsA::FRT-kan-FRTHarvat et al., 2005
Recombinant DNA reagentpEXG2-∆pa2541 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa2536 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa4323 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa4320 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa5114 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa5114/pa5113 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa3267 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa5113 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pa5112 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-∆pfl_rs01995 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpEXG2-pa2541_vsv-g (plasmid)This studyConstruct to introduce VsvG tag, see Materials and methods
Recombinant DNA reagentpEXG2-pa4323_vsv-g (plasmid)This studyConstruct to introduce VsvG tag, see Materials and methods
Recombinant DNA reagentpEXG2-pa5114_vsv-g (plasmid)This studyConstruct to introduce VsvG tag, see Materials and methods
Recombinant DNA reagentpEXG2-pa5113_vsv-g (plasmid)This studyConstruct to introduce VsvG tag, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE (plasmid)Hoang et al., 2000Arabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-pa2541 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-pa4323 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-pa5114 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-pfl_rs01995 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-i35_rs27920 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-pspto_rs26695 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpUC18-Tn7t-pBAD-araE-ta05_rs00690 (plasmid)This studyArabinose-inducible expression system, see Materials and methods
Recombinant DNA reagentpTNS3 (plasmid)Choi et al., 2008
Recombinant DNA reagentpJRC115-BTH_II0090S264A (plasmid)This studyPoint mutation construct, see Materials and methods
Recombinant DNA reagentpJRC115-BTH_I3226S264A (plasmid)This studyPoint mutation construct, see Materials and methods
Recombinant DNA reagentpJRC115-BTH_I2698S267A (plasmid)This studyPoint mutation construct, see Materials and methods
Recombinant DNA reagentpJRC115-BTH_I2701S267A (plasmid)This studyPoint mutation construct, see Materials and methods
Recombinant DNA reagentpJRC115-∆BTH_I2691 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpJRC115-∆BTH_I3225-8 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpJRC115-∆BTH_II0089-94 (plasmid)This studyDeletion construct, see Materials and methods
Recombinant DNA reagentpUC18T-miniTn7T-Tp-PS12-mCherry (plasmid)LeRoux et al., 2012Construct for mCherry expression
Software, algorithmSeqmagickMatsen Group, 2020, https://github.com/fhcrc/seqmagick
Software, algorithmTRANSIT TPPhttps://transit.readthedocs.io/en/latest/tpp.html
AntibodyAnti-VSV-G (rabbit polyclonal)MilliporeSigmaCat# V4888-200UGWestern blot (1:5000 dilution)
AntibodyAnti-rabbit IgG HRP conjugated (goat)MilliporeSigmaCat# A6154-1MLWestern blot (1:5000 dilution)
AntibodyAnti-ribosome polymerase β (mouse monoclonal)BioLegendCat# 663903; RRID: AB_2564524Western blot (1:1000 dilution)
AntibodyAnti-mouse IgG HRP conjugated (sheep)MilliporeSigmaCat# AC111PWestern blot (1:4000 dilution)
Appendix 3—table 1
Oligonucleotides used in this study.
Oligonucleotides 5′–3′Source
pEXG2_∆PA2541_F1CAAGCTTCTGCAGGTCGACTCTAGAAGGTGGAACCGGACCTGAAGIntegrated DNA Technology
pEXG2_∆PA2541_R1CTCAGGCCTGGGAAATCATGTCAGCCAGTCCIntegrated DNA Technology
pEXG2_∆PA2541_F2CATGATTTCCCAGGCCTGAGAGGGAACGIntegrated DNA Technology
pEXG2_∆PA2541_R2TAAGGTACCGAATTCGAGCTCGTATGACCCAGGCGGTCGIntegrated DNA Technology
pEXG2_∆PA4323_F1AGCTCGAGCCCGGGGATCCTCTAGAGACGCAGAACCCGATCGAGIntegrated DNA Technology
pEXG2_∆PA4323_R1AAAGCACGCCCGATGGCTTCATACGCGGIntegrated DNA Technology
pEXG2_∆PA4323_F2GAAGCCATCGGGCGTGCTTTGACGGATCIntegrated DNA Technology
pEXG2_∆PA4323_R2GGAAGCATAAATGTAAAGCAAGCTTGAGCGATGCCGAGCTGGAIntegrated DNA Technology
pEXG2_∆PA5114_F1CAAGCTTCTGCAGGTCGACTCTAGAGCCGCATGCCCTTGACCTIntegrated DNA Technology
pEXG2_∆PA5114_R1CGACCTCGGCAATCCATTGCATGCGTCGAATCIntegrated DNA Technology
pEXG2_∆PA5114_F2GCAATGGATTGCCGAGGTCGCATCGGAGIntegrated DNA Technology
pEXG2_∆PA5114_R2AATTCGAGCTCACCAGATCATCGGCGCCGIntegrated DNA Technology
pEXG2_∆PA2536_F1CAAGCTTCTGCAGGTCGACTCTAGACTCGGCCTGGCCCGAGCIntegrated DNA Technology
pEXG2_∆PA2536_R1CAGGCGAACCTCAGGGCGTTTCGTTCATCTCAGGCCTCCIntegrated DNA Technology
pEXG2_∆PA2536_F2GGAGGCCTGAGATGAACGAAACGCCCTGAGGTTCGCCTGIntegrated DNA Technology
pEXG2_∆PA2536_R2TAAGGTACCGAATTCGAGCTCCACATCGGTCTCAGCGAAGCIntegrated DNA Technology
pEXG2_∆PA4320_F1AGCTCGAGCCCGGGGATCCTCTAGATGTTCGGCTTCTACATCATGAACIntegrated DNA Technology
pEXG2_∆PA4320_R1TGCGCCAGCCCACGCTGGCGTCAGTCAGIntegrated DNA Technology
pEXG2_∆PA4320_F2CGCCAGCGTGGGCTGGCGCAGCCTTTTCIntegrated DNA Technology
pEXG2_∆PA4320_R2GGAAGCATAAATGTAAAGCAAGCTTGTCGGCGGTTTCCTCGCTIntegrated DNA Technology
pEXG2_∆PA5114/PA5113_F1CAAGCTTCTGCAGGTCGACTCTAGAGCCGCATGCCCTTGACCTIntegrated DNA Technology
pEXG2_∆PA5114/PA5113_R1TGTTCGGCGAAATCCATTGCATGCGTCGAATCIntegrated DNA Technology
pEXG2_∆PA5114/PA5113_F2GCAATGGATTTCGCCGAACAAGCCATGAGIntegrated DNA Technology
pEXG2_∆PA5114/PA5113_R2TAAGGTACCGAATTCGAGCTCAACAGCCAGACCACGATGTAGCIntegrated DNA Technology
pEXG2_∆PA5113_F1CAAGCTTCTGCAGGTCGACTCTAGATTGCTAGGGGTGCTGGCGIntegrated DNA Technology
pEXG2_∆PA5113_R1ATGGCTTGTTGGAGCGCGTCATGGCTGCIntegrated DNA Technology
pEXG2_∆PA5113_F2GACGCGCTCCAACAAGCCATGAGCCGGTTCIntegrated DNA Technology
pEXG2_∆PA5113_R2TAAGGTACCGAATTCGAGCTCAACAGCCAGACCACGATGTAGIntegrated DNA Technology
pEXG2_∆PA5112_F1CAAGCTTCTGCAGGTCGACTCTAGATACCGCTGGCAGTTGCCGIntegrated DNA Technology
pEXG2_∆PA5112_R1AGAAGTCCAGCGCCATTCTGATCATTCTCTTACTCIntegrated DNA Technology
pEXG2_∆PA5112_F2CAGAATGGCGCTGGACTTCTGAAACGGCGGCIntegrated DNA Technology
pEXG2_∆PA5112_R2TAAGGTACCGAATTCGAGCTCCGCGCAACCGCCGGTTGGIntegrated DNA Technology
pEXG2_∆PA3267_F1CAAGCTTCTGCAGGTCGACTCTAGATCTACATCGACTTCGACGIntegrated DNA Technology
pEXG2_∆PA3267_R1ATCAGCGAGCTTGGGTCATCGTCCTTGTTACIntegrated DNA Technology
pEXG2_∆PA3267_F2GATGACCCAAGCTCGCTGATCGATCCGCIntegrated DNA Technology
pEXG2_∆PA3267_R2TAAGGTACCGAATTCGAGCTCTTCGCCGGCCTGTTCGAAGIntegrated DNA Technology
pEXG2_∆PFL_RS01995_F1CAAGCTTCTGCAGGTCGACTCTAGAAACTACAACGTCAGCCTGIntegrated DNA Technology
pEXG2_∆PFL_RS01995_R1TTCATTCAACGGTTTGCCTATCCATTGCATGTGTCGIntegrated DNA Technology
pEXG2_∆PFL_RS01995_F2CGACACATGCAATGGATAGGCAAACCGTTGAATGAAIntegrated DNA Technology
pEXG2_∆PFL_RS01995_R2TAAGGTACCGAATTCGAGCTCCTGCGACCACACCAGCGIntegrated DNA Technology
pEXG2_PA2541_VSVG_F1CAAGCTTCTGCAGGTCGACTCTAGAGCGCTGATACGGACCATGCIntegrated DNA Technology
pEXG2_PA2541_VSVG_R1TTTTCCTAATCTATTCATTTCAATATCTGTATAGGCCTGGCCGTCGCTGCCCCGIntegrated DNA Technology
pEXG2_PA2541_VSVG_F2TATACAGATATTGAAATGAATAGATTAGGAAAATGAGAGGGAACGGGCGAACIntegrated DNA Technology
pEXG2_PA2541_VSVG_R2TAAGGTACCGAATTCGAGCTCCTCCTGCGGCGCACGATGIntegrated DNA Technology
pEXG2_PA4323_VSVG_F1CAAGCTTCTGCAGGTCGACTCTAGATGGAGTTCCACCAGTTGCGCGIntegrated DNA Technology
pEXG2_PA4323_VSVG_R1TGATCCGTCATTTTCCTAATCTATTCATTTCAATATCTGTATAAAGCACGCCGGCGCGCTIntegrated DNA Technology
pEXG2_PA4323_VSVG_F2TATACAGATATTGAAATGAATAGATTAGGAAAATGACGGATCAGACCGACTGIntegrated DNA Technology
pEXG2_PA4323_VSVG_R2TAAGGTACCGAATTCGAGCTCAAGGTACACTTCTCCGCCIntegrated DNA Technology
pEXG2_PA5114_VSVG_F1CAAGCTTCTGCAGGTCGACTCTAGACTGGAGCTGCGCTACCTGTTCGIntegrated DNA Technology
pEXG2_PA5114_VSVG_R1TAATCTATTCATTTCAATATCTGTATATGGCTGCTCGGCCTCCGAIntegrated DNA Technology
pEXG2_PA5114_VSVG_F2GATATTGAAATGAATAGATTAGGAAAATCGGAGGCCGAGCAGCCAIntegrated DNA Technology
pEXG2_PA5114_VSVG_R2TAAGGTACCGAATTCGAGCTCGCTCGGACCAGATCATCGGCIntegrated DNA Technology
pEXG2_PA5113_VSVG_F1CAAGCTTCTGCAGGTCGACTCTAGACTGCGTCTGGCCTGGCCGIntegrated DNA Technology
pEXG2_PA5113_VSVG_R1TTTTCCTAATCTATTCATTTCAATATCTGTATATGGCTTGTTCGGCGAGGAACIntegrated DNA Technology
pEXG2_PA5113_VSVG_F2TATACAGATATTGAAATGAATAGATTAGGAAAATGAGCCGGTTCCGCGCTATGIntegrated DNA Technology
pEXG2_PA5113_VSVG_R1TAAGGTACCGAATTCGAGCTCGTCGGGCAACAGCCAGACIntegrated DNA Technology
pUC18_PA2541_FAGC GAATTCGAGCTCGGTACCACGGGAGGAAAG ATGATTTCCGTCTATCAACTCIntegrated DNA Technology
pUC18_PA2541_RCTCATCCGCCAAAACAGCCAAGCTTTCAGGCCTGGCCGTCGCIntegrated DNA Technology
pUC18_PA4323_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGAAGCCATCGCGCGCCCTGCTGGIntegrated DNA Technology
pUC18_PA4323_RCTCATCCGCCAAAACAGCCAAGCTTTCAAAGCACGCCGGCGCGCTTCCAGIntegrated DNA Technology
pUC18_PA5114_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGCAATGGATTTTCATGCTGGIntegrated DNA Technology
pUC18_PA5114_RCTCATCCGCCAAAACAGCCAAGCTTTCATGGCTGCTCGGCCTCIntegrated DNA Technology
pUC18_pfl_rs01995_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGCAATGGATATTCATGCTGCIntegrated DNA Technology
pUC18_pfl_rs01995_RCTCATCCGCCAAAACAGCCAAGCTTTCACGATGACACTCCTTCATTCIntegrated DNA Technology
pUC18_i35_rs27920_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGAACTGGGCATTCGCCGIntegrated DNA Technology
pUC18_i35_rs27920_RCTCATCCGCCAAAACAGCCAAGCTTTCATGGCTGGCCGTCCTGIntegrated DNA Technology
pUC18_pspto_rs26695_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGCTTTGGATTTGTCTGGTAGIntegrated DNA Technology
pUC18_pspto_rs26695_RCTCATCCGCCAAAACAGCCAAGCTTTCATGGCGTTTCAGGCGCTGIntegrated DNA Technology
pUC18_ta05_rs00690_FAGCGAATTCGAGCTCGGTACCACGGGAGGAAAGATGGACGACCTTTTAATCCTGIntegrated DNA Technology
pUC18_ta05_rs00690_RCTCATCCGCCAAAACAGCCAAGCTTTCATTTGTTTTCTCCAGCTTTGIntegrated DNA Technology
pJRC115_ BTH_II0090S264A_F1TAAAACGACGGCCAGTGCCAAGCTTCGACGGATCAGCGTTTCAAGCTGCIntegrated DNA Technology
pJRC115_ BTH_II0090S264A_R1ACCTTGGGCATGACCCATCACCGTGATCGTTTCGTGIntegrated DNA Technology
pJRC115_ BTH_II0090S264A_F2GGTCATGCCCAAGGTACGATCATCACGCTGCTCGIntegrated DNA Technology
pJRC115_ BTH_II0090S264A_R2GCTCGGTACCCGGGGATCCTCTAGACGGCTCGGCACGATGCGCIntegrated DNA Technology
pJRC115_ BTH_I3226S264A_F1TAAAACGACGGCCAGTGCCAAGCTTCGACGGATCAGCGTTTCAAGCTGCIntegrated DNA Technology
pJRC115_ BTH_I3226S264A_R1ACCTTGGGCATGACCCATCACCGTGATCGTTTCGTGIntegrated DNA Technology
pJRC115_ BTH_I3226S264A_F2GGTCATGCCCAAGGTACGATCATCACGCTGCTCGIntegrated DNA Technology
pJRC115_ BTH_I3226S264A_R2GCTCGGTACCCGGGGATCCTCTAGACGGCTCGGCACGATGCGCIntegrated DNA Technology
pJRC115_ BTH_I2698S267A_F1TCAATCAGTATCTAGAGGGACACCTTTCTCAAGCGAAATCIntegrated DNA Technology
pJRC115_ BTH_I2698S267A_R1GCCGCGCGCGAAACCAAACACATAAAGGCGAATGCGIntegrated DNA Technology
pJRC115_ BTH_I2698S267A_F2GGTTTCGCGCGCGGCGCAGCGGAAGCTCGCACGTTTTCIntegrated DNA Technology
pJRC115_ BTH_I2698S267A_R2TGTTAAGCTAGAATTCCATCAAACCCGCTGTCCCATGCTCIntegrated DNA Technology
pJRC115_ BTH_I2701S267A_F1TAAAACGACGGCCAGTGCCAAGCTTGAAAGACGAGCAGGACGCGIntegrated DNA Technology
pJRC115_ BTH_I2701S267A_R1ACCCCGCGCGAAACCGAATACGTATAGCCGAATGCGIntegrated DNA Technology
pJRC115_ BTH_I2701S267A_F2GGTTTCGCGCGGGGTGCAGCGGAGGCTCGCACIntegrated DNA Technology
pJRC115_ BTH_I2701S267A_R2GCTCGGTACCCGGGGATCCTCTAGACTCGACCTCCAGCAGATCGIntegrated DNA Technology
pJRC115_ ∆BTH_I2691_F1TAAAACGACGGCCAGTGCCAAGCTTATTTCAAGCGCGGCCAGTCIntegrated DNA Technology
pJRC115_ ∆BTH_I2691_R1ATCTATGCGAGCTTCCCGCCATTTTTATTCCIntegrated DNA Technology
pJRC115_ ∆BTH_I2691_F2GGCGGGAAGCTCGCATAGATGAGTGATGIntegrated DNA Technology
pJRC115_ ∆BTH_I2691_R2GCTCGGTACCCGGGGATCCTCTAGATTCTGTCAATACTTAAAATACAATTTTCIntegrated DNA Technology
pJRC115_ ∆BTH_II0089-94_F1TAAAACGACGGCCAGTGCCAAGCTTGAATCAGTGCATCGCTGTACIntegrated DNA Technology
pJRC115_ ∆BTH_II0089-94_R1ATGTTTTGCTTAGCGTATCGTTCAAATTGGIntegrated DNA Technology
pJRC115_ ∆BTH_II0089-94_F2CGATACGCTAAGCAAAACATGAGATTATTGAAGAGIntegrated DNA Technology
pJRC115_ ∆BTH_II0089-94_R2GCTCGGTACCCGGGGATCCTCTAGATATGCAACGCATTGCCGAAACIntegrated DNA Technology
pJRC115_ ∆BTH_I3225-8_F1TAAAACGACGGCCAGTGCCAAGCTTCCGGTCAATATACCACCATCIntegrated DNA Technology
pJRC115_ ∆BTH_I3225-8_R1ATGTGCGCCCCGTATCGTTCAAATTGGTCACIntegrated DNA Technology
pJRC115_ ∆BTH_I3225-8_F2GAACGATACGGGGCGCACATAATAAGACTTGIntegrated DNA Technology
pJRC115_ ∆BTH_I3225-8_R2GCTCGGTACCCGGGGATCCTCTAGAATTTGCTGTTTCTGCTGATGIntegrated DNA Technology
PCR_1AGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGGGGGGGGGGGGGGIntegrated DNA Technology
PCR_1BTCATCGGCTCGTATAATGTGTGGIntegrated DNA Technology
PCR_2ACAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGIntegrated DNA Technology
PCR_2BCAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGIntegrated DNA Technology
PCR_2CAATGATACGGCGACCACCGAGATCTACACCTAGAGACCGGGGACTTATCAGCCAACCTGTTAIntegrated DNA Technology
Seq_primerCTAGAGACCGGGGACTTATCAGCCAACIntegrated DNA Technology
  

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  1. See-Yeun Ting
  2. Kaitlyn D LaCourse
  3. Hannah E Ledvina
  4. Rutan Zhang
  5. Matthew C Radey
  6. Hemantha D Kulasekara
  7. Rahul Somavanshi
  8. Savannah K Bertolli
  9. Larry A Gallagher
  10. Jennifer Kim
  11. Kelsi M Penewit
  12. Stephen J Salipante
  13. Libin Xu
  14. S Brook Peterson
  15. Joseph D Mougous
(2022)
Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism
eLife 11:e74658.
https://doi.org/10.7554/eLife.74658