Journal: Cell

Article Title: A chemical radar allows bacteria to detect and kill predators.

doi: 10.1016/j.cell.2025.02.033

Figure Lengend Snippet: Figure 1. Phylogenetic and phenotypic analyses (A) Phylogenetic tree based on the whole genomes of all sequenced Pseudomonas strains with complete assembly level deposited in the NCBI. The strains SZ47 and SZ57 (red) belong to the plant pathogen complex P. syringae. (B) Fruiting bodies of the amoeba and bacterial predator Polysphondylium pallidum. (C and D) Amoebal plaque assays for P. syringae SZ47 and SZ57 and mutants thereof in combination with P. pallidum. SZ47 can kill the predator P. pallidum, whereas SZ57 is eaten by the amoeba. The edibility status underneath indicates whether the bacterium is edible or inedible by the amoeba. The bacterium Klebsiella aerogenes served as an edible control. In the case of edible strains, numerous amoebal fruiting bodies were visible on the plates resulting in the appearance of cloud-like structures on the plates. The amoebal isolate is phylogenetically closely related to the axenic laboratory strain P. pallidum PN500 (Figure S1B). Initial screens were thus performed with P. pallidum PN500. Edibility tests involving P. syringae SZ47 and SZ57 strains were then confirmed with both the axenic and the isolated amoebal strains. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies 6x-His Tag Monoclonal Antibody Thermo Fischer Cat#MA1-21315; RRID: AB_557403 Goat anti-Mouse IgG (H+L) Secondary Antibody, AP Thermo Fischer Cat#31320; RRID: AB_228304 Bacterial and virus strains Pseudomonas syringae SZ47 Zhang et al.25 N/A Pseudomonas syringae SZ57 Zhang et al.25 N/A Klebsiella aerogenes Tindall et al.51 N/A Escherichia coli Top10 ThermoFisher Cat#C404010 Escherichia coli S17-1lpir Fisher Scientific Cat#NC1526716 Escherichia coli BL21 (DE3) ThermoFisher Cat#C600003 Escherichia coli K12 Bachmann52 N/A Pseudomonas protegens pf-5 Clardy Lab N/A Pseudomonas sp. SZ63a Zhang et al.25 N/A Paenibacillus sp. SZ31 Zhang et al.25 N/A Pseudomonas syringae DSM 1241 DSMZ Cat#DSM 1241 Pseudomonas syringae DSM 50274 DSMZ Cat#DSM 50274 Pseudomonas syringae DSM 50255 DSMZ Cat#DSM 50255 Pseudomonas syringae DSM 50252 DSMZ Cat#DSM 50252 Pseudomonas syringae DSM 1242 DSMZ Cat#DSM 1242 Pseudomonas syringae DSM 1856 DSMZ Cat#DSM 1856 Pseudomonas syringae DSM 50256 DSMZ Cat#DSM 50256 Pseudomonas syringae DSM 50272 DSMZ Cat#DSM 50272 Pseudomonas syringae DSM 50293 DSMZ Cat#DSM 50293 Pseudomonas syringae DSM 50277 DSMZ Cat#DSM 50277 Pseudomonas syringae DSM 50281 DSMZ Cat#DSM 50281 Pseudomonas syringae IMET10777 JMRC-HKI N/A Pseudomonas syringae IMET10891 JMRC-HKI N/A Pseudomonas syringae DSM10604 JMRC-HKI N/A Pseudomonas syringae CECT7752 JMRC-HKI N/A Pseudomonas syringae DSM 5176 DSMZ Cat#DSM 5176 Pseudomonas syringae DSM 50292 DSMZ Cat#DSM 50292 Pseudomonas syringae pv. tomato DC3000 Höfte lab N/A Candida auris JMRC-HKI N/A Candida krusei JMRC-HKI N/A Candida zeylanoides JMRC-HKI N/A Saccharomyces cerevisiae JMRC-HKI N/A Aspergillus nidulans JMRC-HKI N/A Aspergillus oryzae JMRC-HKI N/A Aspergillus niger JMRC-HKI N/A Polysphondylium pallidum WS320 Winckler lab N/A Polysphondylium pallidum H168 Winckler lab N/A Polysphondylium asymmetricum OH567 Winckler lab N/A Dictyostelium purpureum QSPU1 Winckler lab N/A Dictyostelium citrinum OH594 Winckler lab N/A (Continued on next page) Cell 188, 1–10.e1–e12, May 1, 2025 e1

Techniques: Control, Isolation

Journal: Cell

Article Title: A chemical radar allows bacteria to detect and kill predators.

doi: 10.1016/j.cell.2025.02.033

Figure Lengend Snippet: Figure 2. Metabolite profiles and biosynthetic scheme (A) HPLC profile of extracts of microorganisms cultured on solid medium (detection at l = 210 nm). P. syringae SZ47 and SZ57 both produce the syringafactin lipopeptides 1. In combination with their predator P. pallidum, SZ57 produces deacylated products 2 and 3 whereas SZ47 produces pyrofactins (6). (B) Pyrofactin biosynthesis requires the action of both Pseudomonas and P. pallidum strains; CraA is an annotated amidase and CraC is an annotated cyclase. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies 6x-His Tag Monoclonal Antibody Thermo Fischer Cat#MA1-21315; RRID: AB_557403 Goat anti-Mouse IgG (H+L) Secondary Antibody, AP Thermo Fischer Cat#31320; RRID: AB_228304 Bacterial and virus strains Pseudomonas syringae SZ47 Zhang et al.25 N/A Pseudomonas syringae SZ57 Zhang et al.25 N/A Klebsiella aerogenes Tindall et al.51 N/A Escherichia coli Top10 ThermoFisher Cat#C404010 Escherichia coli S17-1lpir Fisher Scientific Cat#NC1526716 Escherichia coli BL21 (DE3) ThermoFisher Cat#C600003 Escherichia coli K12 Bachmann52 N/A Pseudomonas protegens pf-5 Clardy Lab N/A Pseudomonas sp. SZ63a Zhang et al.25 N/A Paenibacillus sp. SZ31 Zhang et al.25 N/A Pseudomonas syringae DSM 1241 DSMZ Cat#DSM 1241 Pseudomonas syringae DSM 50274 DSMZ Cat#DSM 50274 Pseudomonas syringae DSM 50255 DSMZ Cat#DSM 50255 Pseudomonas syringae DSM 50252 DSMZ Cat#DSM 50252 Pseudomonas syringae DSM 1242 DSMZ Cat#DSM 1242 Pseudomonas syringae DSM 1856 DSMZ Cat#DSM 1856 Pseudomonas syringae DSM 50256 DSMZ Cat#DSM 50256 Pseudomonas syringae DSM 50272 DSMZ Cat#DSM 50272 Pseudomonas syringae DSM 50293 DSMZ Cat#DSM 50293 Pseudomonas syringae DSM 50277 DSMZ Cat#DSM 50277 Pseudomonas syringae DSM 50281 DSMZ Cat#DSM 50281 Pseudomonas syringae IMET10777 JMRC-HKI N/A Pseudomonas syringae IMET10891 JMRC-HKI N/A Pseudomonas syringae DSM10604 JMRC-HKI N/A Pseudomonas syringae CECT7752 JMRC-HKI N/A Pseudomonas syringae DSM 5176 DSMZ Cat#DSM 5176 Pseudomonas syringae DSM 50292 DSMZ Cat#DSM 50292 Pseudomonas syringae pv. tomato DC3000 Höfte lab N/A Candida auris JMRC-HKI N/A Candida krusei JMRC-HKI N/A Candida zeylanoides JMRC-HKI N/A Saccharomyces cerevisiae JMRC-HKI N/A Aspergillus nidulans JMRC-HKI N/A Aspergillus oryzae JMRC-HKI N/A Aspergillus niger JMRC-HKI N/A Polysphondylium pallidum WS320 Winckler lab N/A Polysphondylium pallidum H168 Winckler lab N/A Polysphondylium asymmetricum OH567 Winckler lab N/A Dictyostelium purpureum QSPU1 Winckler lab N/A Dictyostelium citrinum OH594 Winckler lab N/A (Continued on next page) Cell 188, 1–10.e1–e12, May 1, 2025 e1

Techniques: Cell Culture

Journal: Cell

Article Title: A chemical radar allows bacteria to detect and kill predators.

doi: 10.1016/j.cell.2025.02.033

Figure Lengend Snippet: Figure 5. 1H-15N-TROSY-HSQC spectrum of the CraR protein, RT- qPCR analysis, and MALDI imaging of P. syringae SZ47 and P. pallidum (A) 1H-15N-TROSY-HSQC spectrum of 15N-labeled CraR protein in the absence (black) or presence of equal concentrations of either octapeptide 2A (orange) or heptapeptide 3A (blue). Chemical-shift perturbations show that both ligands bind CraR, but that 3A has a lower affinity. (B) RT-qPCR experiments. CraA, C, and R gene expression in SZ47 was increased upon addition of octapeptide 2A. Expression levels are normalized with respect to entries 1, 3, 5, 7, and 9 (left to right). CraC gene expression levels in SZ47DcraR and SZ57::craR were downregulated and upregulated, respectively, upon treatment with octapeptide 2A. Expression level of craC in octapeptide-treated SZ57 or SZ47 was arbitrarily set to 1. The respective rpoD genes were used for internal normalization. The data are means ± SD (n = 3) using the Student’s t test (*p < 0.05, **p < 0.01, and ***p < 0.001). (C) MALDI imaging of cultures of SZ47 (blue circle) with P. pallidum (orange circle) on solid medium, showing the spatial distribution of degradation products of syringafactin A. The color scale on the bottom indicates the relative intensity of the respective ions detected. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies 6x-His Tag Monoclonal Antibody Thermo Fischer Cat#MA1-21315; RRID: AB_557403 Goat anti-Mouse IgG (H+L) Secondary Antibody, AP Thermo Fischer Cat#31320; RRID: AB_228304 Bacterial and virus strains Pseudomonas syringae SZ47 Zhang et al.25 N/A Pseudomonas syringae SZ57 Zhang et al.25 N/A Klebsiella aerogenes Tindall et al.51 N/A Escherichia coli Top10 ThermoFisher Cat#C404010 Escherichia coli S17-1lpir Fisher Scientific Cat#NC1526716 Escherichia coli BL21 (DE3) ThermoFisher Cat#C600003 Escherichia coli K12 Bachmann52 N/A Pseudomonas protegens pf-5 Clardy Lab N/A Pseudomonas sp. SZ63a Zhang et al.25 N/A Paenibacillus sp. SZ31 Zhang et al.25 N/A Pseudomonas syringae DSM 1241 DSMZ Cat#DSM 1241 Pseudomonas syringae DSM 50274 DSMZ Cat#DSM 50274 Pseudomonas syringae DSM 50255 DSMZ Cat#DSM 50255 Pseudomonas syringae DSM 50252 DSMZ Cat#DSM 50252 Pseudomonas syringae DSM 1242 DSMZ Cat#DSM 1242 Pseudomonas syringae DSM 1856 DSMZ Cat#DSM 1856 Pseudomonas syringae DSM 50256 DSMZ Cat#DSM 50256 Pseudomonas syringae DSM 50272 DSMZ Cat#DSM 50272 Pseudomonas syringae DSM 50293 DSMZ Cat#DSM 50293 Pseudomonas syringae DSM 50277 DSMZ Cat#DSM 50277 Pseudomonas syringae DSM 50281 DSMZ Cat#DSM 50281 Pseudomonas syringae IMET10777 JMRC-HKI N/A Pseudomonas syringae IMET10891 JMRC-HKI N/A Pseudomonas syringae DSM10604 JMRC-HKI N/A Pseudomonas syringae CECT7752 JMRC-HKI N/A Pseudomonas syringae DSM 5176 DSMZ Cat#DSM 5176 Pseudomonas syringae DSM 50292 DSMZ Cat#DSM 50292 Pseudomonas syringae pv. tomato DC3000 Höfte lab N/A Candida auris JMRC-HKI N/A Candida krusei JMRC-HKI N/A Candida zeylanoides JMRC-HKI N/A Saccharomyces cerevisiae JMRC-HKI N/A Aspergillus nidulans JMRC-HKI N/A Aspergillus oryzae JMRC-HKI N/A Aspergillus niger JMRC-HKI N/A Polysphondylium pallidum WS320 Winckler lab N/A Polysphondylium pallidum H168 Winckler lab N/A Polysphondylium asymmetricum OH567 Winckler lab N/A Dictyostelium purpureum QSPU1 Winckler lab N/A Dictyostelium citrinum OH594 Winckler lab N/A (Continued on next page) Cell 188, 1–10.e1–e12, May 1, 2025 e1

Techniques: Quantitative RT-PCR, Imaging, Labeling, Gene Expression, Expressing