(A) Genomic arrangement of T6SS chaperone-effector-immunity genes for characterized effector-associated gene family members (eag; shown in purple), which encode DUF1795 domain-containing chaperones. …
(A) Phylogenetic distribution of 975 prePAAR-containing proteins identified in the UniProtKB database using the N-terminus of Tse6 (Tse6NT) as a search query (see Materials and Methods). The TM …
(A) Genomic context of two prePAAR-containing effector-immunity pairs from P. protegens Pf-5. RhsA is a class I effector (shown in green) and Tne2 is a class II effector (shown in blue). Shading is …
(A) Western blot of supernatant (sup) and cell fractions of the indicated P. protegens Pf-5 strains grown to OD 0.8. An Hcp (PFL_6089)-specific antibody was used to assess T6SS activity. (B) …
(A) Domain architecture of P. protegens RhsA and a truncated variant lacking its prePAAR and TMD-containing N-terminus (RhsA∆NT). (B) EagR1 interacts with the N-terminus of RhsA. His6-tagged RhsA or …
(A and B) Growth competition assays between the indicated P. protegens donor strains and either Tne2 (A) or RhsA (B) susceptible recipients. (C) Western blot of lysate and pull-down elution …
(A) An X-ray crystal structure of the Eag chaperone SciW bound to the N-terminus of Salmonella Typhimurium class I prePAAR effector Rhs1 (Rhs1NT, residues 8–57 are modeled) shown in two views …
Unprocessed micrographs (A, C, E, G) and representative 2-D class averages (B, D, F, H) of negatively stained VgrG1 (A, B), RhsA∆NT (C, D), EagR1-RhsA complex (E, F) and EagR1-RhsA-VgrG1 complex (G, …
(A) Alignment of Eag chaperones that interact with class I (SciW, EagR1) or class II (EagT6 and EagT2) prePAAR effectors plotted with secondary structure elements. (B) Residues making intimate …
(A) Structural comparison of apo-SciW and apo-EagT6. Two views are shown related by an ~90° rotation. Each chaperone is colored by chain as in Figure 4. (B) Conserved surface residues as determined …
(A) Western blot analysis of Tse6 from cell fractions of the indicated P. aeruginosa strains. Low-molecular-weight band indicates Tse6 alone whereas high-molecular-weight band indicates Tse6-VgrG1a …
(A) Surface representation of structural models of the PAAR domain from each of the indicated prePAAR effector proteins (purple) overlaid with a ribbon representation of the c1882 PAAR protein …
Phylogenetic distribution of 564 orphan PAAR sequences (blue) and 1765 split PAAR (green) sequences. The scale bar indicates the substitutions per base.
(A) PAAR proteins exist with or without prePAAR domains. Those that lack prePAAR (orphan), can interact with VgrG and form a functional T6SS spike complex without any additional factors. By …
SciW (native) | SciW (Iodide) | SciW-Rhs11-59 | EagT6-Tse61-61 | |
---|---|---|---|---|
Data Collection | ||||
Wavelength (Å) | 1.5418 | 1.5418 | 0.97895 | 1.5418 |
Space group | P212121 | P212121 | P3121 | P32 |
Cell dimensions | ||||
a, b, c (Å) | 55.27 75.1 76.6 | 55.6 75.3 76.4 | 105.3 105.3 248.4 | 68.9 68.9 173.1 |
α, β, γ (°) | 90 90 90 | 90 90 90 | 90 90 120 | 90 90 120 |
Resolution (Å) | 29.03–1.75 | 19.63–2.21 | 91.20–1.90 | 28.22–2.55 |
(1.82–1.75) | (2.33–2.21) | (1.98–1.90) | (2.65–2.55) | |
Unique reflections | 32309 (3162)* | 29933 (4888) | 126298 (12473) | 29267 (2832) |
CC(1/2) | 99.8 (89.1) | 99.6 (81.4) | 99.9 (53.9) | 99.6 (52.8) |
Rmerge (%)† | 6.2 (91.3) | 6.1 (44.7) | 5.7 (34.6) | 15.5 (179.8) |
I/σI | 14.2 (1.9) | 8.0 (1.8) | 11.6 (1.26) | 7.27 (0.92) |
Completeness (%) | 99.5 (98.8) | 96.0 (97.9) | 99.9 (99.9) | 99.3 (96.9) |
Redundancy | 7.0 (6.8) | 2.0 (1.9) | 9.9 (9.7) | 4.9 (4.8) |
Refinement | ||||
Rwork/Rfree (%)‡ | 19.8/22.6 | 18.7/21.4 | 22.9/26.6 | |
Average B-factors (Å2) | 46.1 | 42.9 | 71.7 | |
Protein | 45.1 | 42.5 | 72.1 | |
Ligands | 60.8 | 123.4 | ||
Water | 53.9 | 42.2 | 59.3 | |
No. atoms | ||||
Protein | 2331 | 10492 | 7827 | |
Ligands | 10 | 60 | ||
Water | 256 | 1119 | 248 | |
Rms deviations | ||||
Bond lengths (Å) | 0.003 | 0.005 | 0.004 | |
Bond angles (°) | 0.67 | 0.68 | 0.73 | |
Ramachandran plot (%)§ | ||||
Total favored | 99.65 | 99.24 | 98.26 | |
Total allowed | 0.35 | 0.68 | 1.74 | |
PDB code | 6XRB | 6XRR | 6XRF |
*Values in parentheses correspond to the highest resolution shell.
†Rmerge = Σ Σ |I(k) - < I > |/ Σ I(k) where I(k) and <I > represent the diffraction intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements.
‡Rwork = Σ ||Fobs| - k|Fcalc||/|Fobs| where Fobs and Fcalc are the observed and calculated structure factors, respectively. Rfree is the sum extended over a subset of reflections excluded from all stages of the refinement.
§As calculated using MOLPROBITY (Chen et al., 2010).
List of prePAAR motif-containing proteins identified in the UniProtKB Database .
The document contains two separate sheets. List A corresponds to 2054 prePAAR-containing sequences that were identified through an iterative search of the UniprotKB using Tse6NT. List B corresponds to 975 sequences collected following filtering of list A (see Materials and Methods for details).
List of prePAAR motif-containing proteins from assembled genomes of all species belonging to the genera Burkholderia, Escherichia, Enterobacter, Pseudomonas, Salmonella, Serratia, Shigella, and Yersinia.
The document contains two separate sheets. List C corresponds to 6101 prePAAR-containing sequences that were identified through an iterative search of the UniprotKB using Tse6NT. List D corresponds to 1166 sequences collected following filtering of list C (see Materials and methods for details).
Summary of the number of genomes and effector sequences used in our informatics analyses.
This document contains three separate sheets. The ‘UniprotKB-effectors’ sheet shows the quantity of initial prePAAR-containing sequences that were identified in our search and the number of sequences that were used following filtering and removal of redundancy (plotted in the cladogram in Figure 1—figure supplement 1A). The numbers in bold indicate the number of sequences in Supplementary file 1. The ‘eight genera - genomes’ sheet corresponds to the number of genomes from the eight genera (Burkholderia, Escherichia, Enterobacter, Pseudomonas, Salmonella, Serratia, Shigella, and Yersinia) that contained one prePAAR-containing sequence and the number that remained following filtering and removal of redundancy. The ‘8-genera – effectors’ sheet corresponds to initial and final numbers of prePAAR-containing sequences that were identified in the eight genera listed above. The final number of sequences in this sheet were used to construct the cladogram in Figure 1E. The numbers in bold indicate the numbers of sequences in the lists in Supplementary file 2.
Strains used in this study.
Plasmids used in this study.