Non-cognate immunity proteins provide broader defenses against interbacterial effectors in microbial communities
- Departments of Molecular and Cellular Biology, Harvard University, United States
- Department of Plant & Microbial Biology, University of California, Berkeley, United States
- Departments of Organismic and Evolutionary Biology, Harvard University, United States
- Division of Geological and Planetary Sciences, California Institute of Technology, United States
Figures
Figure 1 with 1 supplement
RdnE homologs act as DNA endonucleases and contain interchangeable domains.
(A) Cell viability (colony forming units [CFU] per mL) after protein production in swarms of P. mirabilis strain idrD*, which does not produce RdnE and RdnI. Cells produced GFPmut2, RdnE, or mutant variants in the predicted PD-(D/E)XK motif: D39A, E53A, K55A, or all. (B) In vitro DNA degradation assay for ProteusRdnE. Increasing concentrations of a negative control, ProteusRdnE-FLAG, or ProteusRdnED39A-FLAG were incubated with methylated or unmethylated lambda DNA (48,502 bp) and analyzed by gel electrophoresis. Plasmid DNA degradation is in Figure 1—figure supplement 1. (C) In vitro DNA degradation assay for domain deletions of ProteusRdnE. The first construct removed the first alpha helix without disturbing the catalytic residues, and the second construct contained the PD-(D/E)XK motif and removed region 2. Increasing concentrations were analyzed as in (B). (D) Multiple sequence alignment between P. mirabilis and R. dentocariosa RdnE sequences. The black bar marks the PD-(D/E)XK motif, and the gray bar marks the variable region 2 domain. Conserved residues are highlighted in dark blue. Secondary structure predictions identified using Ali2D (h for alpha helix, e for beta sheet); the catalytic residues (stars) are noted above the alignment. (E,F) In vitro DNA degradation assay and analysis as in (B). (E) Increasing concentrations of either a negative control, RothiaRdnE-FLAG, or RothiaRdnED39A-FLAG. (F) The PD-(D/E)XK motifs were swapped between the RothiaRdnE (orange bar) and the ProteusRdnE (green bar) sequences and compared to the wild-type RdnE proteins.
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Figure 1—source data 1
The full gels of the data in Figure 1B, C, E and F.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig1-data1-v1.zip
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Figure 1—source data 2
The individual, original gel scans for the data in Figure 1B, C, E and F.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig1-data2-v1.zip
Figure 1—figure supplement 1
RdnE, an endonuclease, is lethal in Escherichia coli and cuts plasmid DNA.
(A) Growth curve of E. coli cells overexpressing ProteusRdnE or variants with mutations in the PD-(D/E)K active site. Cells were grown at 37 °C for 16 hours. Optical density at 595 nm (OD595) was measured every half hour. A control strain expressing an empty vector was used as the negative control. (B) Micrographs of E. coli cells producing ProteusRdnE-FLAG or ProteusRdnED39A-FLAG, isolated during mid-logarithmic growth and imaged. DAPI was used to detect DNA within cells. Top, the empty vector as a negative control. Middle, E. coli producing ProteusRdnE from an inducible plasmid. Bottom, E. coli producing ProteusRdnED39A from an inducible plasmid. Left, phase; right, fluorescence. (C) Anti-FLAG western blot for ProteusRdnE-FLAG and ProteusRdnED39A-FLAG generated by in vitro translation. Protein levels were determined by comparison to a standard dilution of FLAG-BAP. A negative control (DHFR) without a FLAG tag was also produced with the in vitro translation reaction. A vertical orange line separates the membrane where the ladder was marked with a pencil after transfer (to the left) and the membrane after western blot detection (to the right). (D) In vitro DNase assay reactions on cut and uncut plasmid DNA. In vitro translation products of either a negative control (DHFR), ProteusRdnE-FLAG, or ProteusRdnED39A-FLAG were incubated with cut or uncut plasmid DNA and analyzed with gel electrophoresis.
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Figure 1—figure supplement 1—source data 1
The full gels of the data in Figure 1—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
It contains individual, original gel scans for the data in Figure 1—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
RdnI binds to and protects against RdnE in vivo and in vitro.
(A) Domain architecture for the idr locus in P. mirabilis strain BB2000. At the top are genes with Pfam domains listed below them. Gray boxes denote PAAR and Rhs domains in the N-terminal region of the full-length IdrD protein. (B) Micrographs of P. mirabilis strain idrD* cells carrying an empty vector, a vector for producing RdnE, or a vector for producing RdnE and RdnI. DNA was visualized by DAPI stain. Phase, left; fluorescence, right. (C) Swarm competition assay of wild-type P. mirabilis strain BB2000 (donor) competed against the vulnerable target, which is P. mirabilis strain ATCC29906 carrying an empty vector, a vector for producing RdnI-StrepII, or a vector for producing GFP, both under the fla promoter. Left: schematic of swarm competition assay where top left colony is BB2000, top right colony is ATCC29906 with its vector cargo, and bottom colony is a 1:1 mixture of BB2000 and ATCC29906 with its vector cargo. Gray boxes underneath indicate whether BB2000 (top) or ATCC29906 (bottom) dominate in the 1:1 mixture and white arrows point to a boundary line that forms between different strains. (D) Bacterial two-hybrid (BACTH) assay with RdnED39A-FLAG, RdnI-StrepII, and GFPmut2. The colorimetric change was discerned in the presence of the substrate X-gal and inducer IPTG. (E) An anti-FLAG batch co-immunoprecipitation of RdnED39A-FLAG and RdnI-StrepII. RdnED39A-FLAG or exogenous FLAG-BAP (soluble fraction) was incubated with anti-FLAG resin (FLAG flow through). RdnI-StrepII was then added to the resin (RdnI-StrepII flow through). Any proteins bound to resin were eluted with FLAG-peptide (Elution) and analyzed by anti-FLAG and anti-StrepII western blots.
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Figure 2—source data 1
It contains the full gels of the data in Figure 2E.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig2-data1-v1.zip
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Figure 2—source data 2
It contains the individual, original gel scans for the data in Figure 2E.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig2-data2-v1.zip
Figure 2—figure supplement 1
RdnI offers protection against and binds to RdnE.
(A) Viability assays of E. coli cells after production of RdnE, RdnI, or co-production of RdnE and RdnI within a cell. Cells were assayed for colony forming units per milliliter over a six-hour time course. (B) The Coomassie blue-stained gel for the anti-FLAG batch co-immunoprecipitation assay results shown in the main text, Figure 2E.
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Figure 2—figure supplement 1—source data 1
It contains the full gels of the data in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
It contains the individual, original gel scans for the data in Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig2-figsupp1-data2-v1.zip
Figure 3 with 2 supplements
RdnE and RdnI protein families share conserved residues and predicted structures.
(A) Gene neighborhoods for RdnE and RdnI homologs. Listed are gene neighborhoods, relevance, and niche, which we identified using IMG/M from the Joint Genomics Institute. Colors highlight conserved function/genes (not to scale). (Agr: Agriculture, Med: Medical, Env: Environmental), and the site of isolation. (B) Phylogenetic tree based on NCBI taxonomy. Scale is located below the graph. The colored circles represent phyla (green: Actinobacteriota; yellow: Firmicutes; blue: Bacteroidota; pink: Proteobacteria). (C) Unrooted maximum likelihood trees of the RdnE (left) and RdnI (right) homologs. Trees were created with RaxML (Kozlov et al., 2019), and the scale is annotated below. The colored circles represent phyla (same as in B). (D) Protein alignments overlaid with either predicted secondary structures (top) or conserved residues (bottom) of the RdnE and RdnI homologs. MUSCLE alignments (Edgar, 2004) are highlighted by secondary structures (red: alpha helices, light blue: beta sheets), or conserved residues (dark blue). White represents gaps in the protein alignment. The bars below mark the predicted conserved and variable domains. (E) Alignments of AlphaFold2 predictions for RdnE and RdnI sequences from P. mirabilis (green), R. dentocariosa (orange), P. jejuni (magenta), and P. ogarae (dark blue). Structures were generated using ColabFold (Mirdita et al., 2022) and aligned using PyMol.
Figure 3—figure supplement 1
RdnE and RdnI protein families show conserved structures.
(A) Diagram detailing the methodology used for identifying sequences homologous to RdnE and RdnI. Seven homologs of ProteusRdnE (orange) found using BLAST37 with their corresponding downstream genes (pink) were aligned and used as seed for a sequential search using HMMER search (Eddy, 2009) and the Ensembl database (Cunningham et al., 2022). Gene neighborhoods were analyzed for genomes with adjacent rdnE and rdnI genes. (B) Tanglegram (Scornavacca et al., 2011) of the RdnE and RdnI protein families from the 21 sequences. On the left is the maximum-likelihood tree for the RdnE protein family on the right is the maximum-likelihood tree for the RdnI protein family. Black lines match effector and immunity pairs from the same species. (C–F) MUSCLE alignment (Edgar, 2004) of RdnE (C and D) and RdnI (E and F) protein families highlighted with either predicted secondary structure predictions (C and E) or conserved residues (D and F). Secondary structures were predicted using Ali2D (Gabler et al., 2020; Zimmermann et al., 2018) and are shaded by confidence. Predicted α-helices in pink; β-strands in light blue. Conserved residues were highlighted (dark blue) using Jalview (Waterhouse et al., 2009). Black lines underneath mark the truncated variants of RdnI described in the main text (Figure 4).
Figure 3—figure supplement 2
AlphaFold2 predictions for RdnE and RdnI homologs.
(A) Confidence scores (pIDDT) for AlphaFold2 (Jumper et al., 2021; Mirdita et al., 2022) predictions for RdnE sequences from P. mirabilis, R. dentocariosa, P. jejuni, and P. ogarae. The confidence score (y-axis) for each residue (x-axis) are graphed for the five ranked models (rank 1: blue, rank 2: orange, rank 3: green, rank 4: red, rank 5: purple). (B) RdnE AlphaFold2 rank 1 models. Models were colored by confidence scores. Red indicates high confidence (90–100%) while blue indicates low confidence (30–50%). (C) Confidence scores for AlphaFold2 predictions of RdnI homologs from P. mirabilis, R. dentocariosa, P. jejuni, and P. ogarae. (D) We colored the AlphaFold2 rank 1 model, including both the BB2000 sequence and natural variant, by confidence scores where red is high confidence (90–100%) and blue is low confidence (30–50%).
Figure 4 with 3 supplements
The RdnI protein family can offer cross-protection due to an interchangeable conserved domain that is critical for function.
(A) Sequence logo of the RdnI’s conserved motif. Stars indicate the seven analyzed residues. (B) Swarm competition assay with ATCC29906 producing either RdnI-StrepII or RdnI7mut-StrepII, which contains mutations in all seven conserved residues. We used a sequence-optimized (SO) RdnI protein that had a higher GC% content and an identical amino acid sequence for ease of cloning. Left: schematic of swarm competition assay as in Figure 2. Gray boxes indicate which strain dominated over the other. White arrows point to the boundary formed between different strains. (C) BACTH assay of RdnED39A-FLAG with SO RdnI-StrepII or RdnI7mut-StrepII. GFPmut2 was used as a negative control. (D) Swarm competition assay with ATCC29906 expressing either the wild-type RdnI or a RdnI truncation. The three truncations were in the first alpha helix (amino acids 1–85), the second half of RdnI (amino acids 150–305), and the end of the protein (amino acids 235–305). (E) BACTH assay of RdnED39A-FLAG with wild-type RdnI and the three RdnI truncations. (F) Swarm competition assay with ATCC29906 expressing foreign RdnI proteins. (G) BACTH assay of RdnED39A-FLAG with each of the foreign RdnI proteins. GFPmut2 was used as a negative control. (H) Swarm competition assay with ATCC29906 producing SO RdnI with swapped conserved motifs. (I) BACTH assay of RdnED39A-FLAG with SO RdnI with swapped conserved motifs. Colored bars denote RdnI-StrepII proteins from P. mirabilis (green), R. dentocariosa (orange), P. jejuni (magenta), or P. ogarae (dark blue).
Figure 4—figure supplement 1
Single mutations in the RdnI conserved motif do not alter protective function.
Swarm competition assay (Wenren et al., 2013) with single residue mutations in the conserved motif of RdnI. P. mirabilis BB2000 (donor) was competed against the vulnerable P. mirabilis ATCC29906 expressing RdnI with single residue mutations in four of the seven conserved residues (highlighted in Figure 4A) in ProteusRdnI-StrepII (ProteusRdnIY197A-StrepII, ProteusRdnIH221A-StrepII, ProteusRdnIY244A-StrepII, ProteusRdnIY246A-StrepII). All constructs were made in a sequence-optimized RdnI.
Figure 4—figure supplement 2
RdnI protein levels are similar under constitutive fla promoter in P. mirabilis.
Swarm cell protein expression assay (Cardarelli et al., 2015) on P. mirabilis ATCC29906 cells expressing each of the four RdnI proteins under the constitutive fla promoter. Soluble fraction and whole cell extract samples were then run on SDS-Page gels and incubated with anti-StrepII antibodies (left) or Coomassie blue (right). 20 ng of GFP-StrepII (Iba Lifesciences, Gӧttingen Germany) was used as a positive control.
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Figure 4—figure supplement 2—source data 1
It contains the full gels of the data in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
It contains the individual, original gel scans for the data in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
Anti-FLAG co-IPs reveal mixed binding results for foreign immunity protein Anti-FLAG co-immunoprecipitation assay between ProteusRdnED39A-FLAG and the RdnI-StrepII proteins from P. mirabilis, R. dentocariosa, P. jejuni, or P. ogarae.
RdnED39A-FLAG was incubated with anti-FLAG resin (FLAG soluble fraction). RdnI-StrepII containing lysate was then added to the resin (RdnI-StrepII flow through). Proteins bound to resin were then eluted with FLAG-peptide (elution). The negative control was exogenous FLAG-BAP. Samples were incubated with either anti-FLAG antibodies (A), anti-StrepII antibodies (B), or were stained with Coomassie blue (C).
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Figure 4—figure supplement 3—source data 1
The full gels for the data in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
The individual, original gel scans for the data in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/90607/elife-90607-fig4-figsupp3-data2-v1.zip
Figure 5 with 2 supplements
The RdnI protein family has the potential for broader protection within oral and gut microbiomes.
(A) Methodology used to identify rdnE and rdnI genes in publicly available metagenomic data. Metagenomes were mapped against sequences with a stringency of 90%. ‘Coverage’ denotes the average depth of short reads mapping to a gene in a single sample. Colors represent rdnE and rdnI from P. mirabilis (green), R. dentocariosa (orange), P. jejuni (magenta), or P. ogarae (dark blue). (B) The experimentally tested rdnE gene sequences from different organisms (colors) are found in thousands of human-associated metagenomes. Each dot represents a single sample’s coverage of an individual rdnE gene, note log10-transormed y-axis. Only samples with >1 x coverage are shown. (C) Euler diagram showing the number of samples with co-occurring rdnE genes from different taxa (colors). (D) Kernel density plot of the ratio of rdnI to rdnE coverage. The ratio of rdnI to rdnE was defined as log10(I/E) where I and E are the mean nucleotide’s coverage for rdnI and rdnE, respectively. The distribution of ratios was summarized as a probability density function (PDF) for each taxon (color) in each environment (subpanel). Here, the y-axis (unitless) reflects the probability of observing a given ratio (x-axis) in that dataset. The colored numbers in the top right of each panel show the number of metagenomes above the detection limit for both rdnE and rdnI for each taxon. Dashed vertical lines represent the median ratio. (E) Skeleton-key model for immunity protein protection. Top, the current prevailing model for T6SS immunity proteins is that protection is defined by necessary and sufficient binding between cognate effectors (locks) and immunity proteins (keys). Bottom, a proposed, expanded model: multiple immunity proteins (skeleton-keys) can bind a single effector due to a flexible (promiscuous) binding site. Protection is a two-step process of binding and then neutralization.
Figure 5—figure supplement 1
Metagenomic analysis with a 70% stringency revealed similar patterns in RdnE and RdnI localization.
The same metagenomic analysis as described in Figure 5 was used but had a lower stringency (70% instead of 90% identity to the rdnE and rdnI sequences). (A) Each dot represents a single sample’s coverage of an individual rdnE gene, note log10-transormed y-axis. Only samples with >1 x coverage are shown. (B) Euler diagram showing the number of samples with co-occurring rdnE genes from different taxa (colors). (C) Kernel density plot of the ratio of rdnI to rdnE coverage. The ratio of rdnI to rdnE was defined as log10(I/E) where I and E are the mean nucleotide’s coverage for rdnI and rdnE, respectively. The distribution of ratios was summarized as a probability density function (PDF) for each taxon (color) in each environment (subpanel). Here, the y-axis (unitless) reflects the probability of observing a given ratio (x-axis) in that dataset. The colored numbers in the top right of each panel show the number of metagenomes above the detection limit for both rdnE and rdnI for each taxon. Dashed vertical lines represent the median ratio.
Figure 5—figure supplement 2
RdnE and RdnI sequences are found in metagenomic datasets.
(A) Heatmap of the log10-normalized coverage of rdnE and rdnI (rows) from the focal taxa for all metagenomes (columns) where any were detected. Metagenomes are sorted by decreasing rdnE coverage. (B) Span chart showing the difference in coverage between cognate rdnE (circles) to rdnI (crosses) for all taxa (colors) for metagenomes in which rdnE-rdnI from multiple taxa were detected.
Author response image 1
Tables
Table 1
RdnE and RdnI homolog species.
| Genus species strain | Phylum | Isolation Location | RdnE JGI unique ID | RdnI JGI unique ID |
|---|---|---|---|---|
| Acinetobacter baumannii BJAB0715 | Proteobacteria | Fresh water | 2562302616 | 2562302617 |
| Acinetobacter baumannii XH858 | Proteobacteria | Human sputum | 2686809281 | 2686809282 |
| Burkholderia sp. TSV86 | Proteobacteria | Water | 2766166119 | 2766166118 |
| Cellulophaga baltica 18 | Bacteroidota | Water | 2815949879 | 2815949878 |
| Chryseobacterium indologenes NBRC 14944 | Bacteroidota | Human trachea | 2565567985 | 2565567984 |
| Chryseobacterium populi CF314 | Bacteroidota | Soil rhizosphere | 2511231970 | 2511231971 |
| Chryseobacterium sp. IHB B 17019 | Bacteroidota | Soil undefined subtype | 2686963654 | 2686963655 |
| Cronobacter turicensis 564 | Proteobacteria | Human undefined subtype | 2532469359 | 2532469360 |
| Cronobacter turicensis z3032 | Proteobacteria | Human blood culture | 646327905 | 646327906 |
| Endozoicomonas numazuensis DSM 25634 | Proteobacteria | Marine sponge | 2574519540 | 2574519541 |
| Paenibacillus elgii M63 | Firmicutes | Hot spring | 2744846532 | 2744846531 |
| Paenibacillus sp. Aloe-11 | Firmicutes | Soil rhizosphere | 2549870597 | 2549870598 |
| Prevotella jejuni CD3:33 | Bacteroidota | Human intestine biopsy | 2804797915 | 2804797916 |
| Prevotella sp. C561 | Bacteroidota | Human respiratory tract | 2514485316 | 2514485315 |
| Prevotella sp. F0108 | Bacteroidota | Human oral cavity | 647936965 | 647936966 |
| Proteus mirabilis BB2000 | Proteobacteria | Human intestine biopsy | 2546214711 | 2546214712 |
| Pseudomonas ogarae F113 | Proteobacteria | Soil rhizosphere | 2511826458 | 2511826457 |
| Pseudomonas syringae pv. Coriandricola ICMP 12471 | Proteobacteria | Undefined | 2714543877 | 2714543878 |
| Rothia dentocariosa C6B | Actinobacteriota | Human oral cavity | 2611822673 | 2611822674 |
| Tannerella forsythia ATCC 43037 | Bacteroidota | Human oral cavity | 2512371376 | 2512371377 |
| Taylorella asinigenitalis MCE3 | Proteobacteria | Mammal reproductive system | 2511725471 | 2511725470 |
Table 2
List of strains used in this study.
| Strain | Strain Name | Description | Reference or Source |
|---|---|---|---|
| P. mirabilis BB2000 idrD*+pDS0062 | DS349 | BB2000 idrD::Tn5 (CmR) producing GFPmut2 under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0002 | DS104 | BB2000 idrD::Tn5 (CmR) producing RdnE under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0058 | DS344 | BB2000 idrD::Tn5 (CmR) producing RdnED39A under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0059 | DS345 | BB2000 idrD::Tn5 (CmR) producing RdnEE53A under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0060 | DS346 | BB2000 idrD::Tn5 (CmR) producing RdnEK55A under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0061 | DS347 | BB2000 idrD::Tn5 (CmR) producing RdnED39A E53A K55A under the aTc-inducible promoter | This study |
| E. coli MG1655 +pBBR1-NheI | DS068 | MG1655 carrying empty vector | This study |
| E. coli MG1655 +pDS0002 | DS151 | MG1655 producing RdnE under the aTc-inducible promoter | This study |
| E. coli MG1655 +pDS0058 | DS336 | MG1655 producing RdnED39A under the aTc-inducible promoter | This study |
| E. coli MG1655 +pDS0059 | DS337 | MG1655 producing RdnEE53A under the aTc-inducible promoter | This study |
| E. coli MG1655 +pDS0060 | DS338 | MG1655 producing RdnEK55A under the aTc-inducible promoter | This study |
| E. coli MG1655 +pDS0061 | DS339 | MG1655 producing RdnED39A E53A K55A under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 idrD*+pDS0003 | DS092 | BB2000 idrD::Tn5 (CmR) co-producing RdnE followed by RdnI under the aTc-inducible promoter | This study |
| E. coli MG1655 +pDS0003 | DS170 | MG1655 co-producing RdnE followed by RdnI under the aTc-inducible promoter | This study |
| P. mirabilis BB2000 +pBBR1-NheI | ANS1127 | BB2000 carrying empty vector | Wenren et al., 2013 |
| P. mirabilis ATCC29906 +pBBR1-NheI | ANS1280 | ATCC29906 carrying empty vector | This study |
| P. mirabilis ATCC29906 +pAK043 | AK0132 | ATCC29906 producing RdnI with a C-terminal Strep-II tag with the fla promoter | This study |
| P. mirabilis ATCC29906 +pLMW04-gfp | AK387 | ATCC29906 producing GFPmut2 with the fla promoter | This study |
| E. coli MG1655 +pDS0048 | DS248 | MG1655 producing RdnED39A with a C-terminal FLAG tag under an aTc-inducible promoter | This study |
| E. coli BL21(pLysS)DE3 +pAK023 | AK024 | BL21(pLysS)DE3 producing RdnI with a C-terminal Strep-II tag under the T7 promoter | This study |
| P. mirabilis ATCC29906 +pAK044 | AK0135 | ATCC29906 producing the RothiaRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK045 | AK0138 | ATCC29906 producing the PrevotellaRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK046 | AK0141 | ATCC29906 producing the PseudomonasRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| E. coli BL21(pLysS)DE3 +pAK058 | AK261 | BL21(pLysS)DE3 producing RothiaRdnI with a C-terminal Strep-II tag under the T7 promoter | This study |
| E. coli BL21(pLysS)DE3 +pAK059 | AK262 | BL21(pLysS)DE3 producing PrevotellaRdnI with a C-terminal Strep-II tag under the T7 promoter | This study |
| E. coli BL21(pLysS)DE3 +pAK060 | AK263 | BL21(pLysS)DE3 producing PseudomonasRdnI with a C-terminal Strep-II tag under the T7 promoter | This study |
| P. mirabilis ATCC29906 +pAK063 | AK318 | ATCC29906 producing the recoded ProteusRdnI sequence with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK065 | AK320 | ATCC29906 producing the RothiaRdnI sequence (aa195-271) inserted between aa192-266 in the recoded ProteusRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK066 | AK321 | ATCC29906 producing the PrevotellaRdnI sequence (aa170-245) inserted between aa192-266 in the recoded ProteusRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK067 | AK322 | ATCC29906 producing the PseudomonasRdnI sequence (aa181-255) inserted between aa192-266 in the recoded ProteusRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK086 | AK381 | ATCC29906 producing the recoded ProteusRdnI sequence (aa192-266) inserted between aa170-245 in the PrevotellaRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK087 | AK382 | ATCC29906 producing the recoded ProteusRdnI sequence (aa192-266) inserted between aa181-255 in the PseudomonasRdnI with a C-terminal Strep-II tag under the fla promoter | This study |
| P. mirabilis ATCC29906 +pAK064 | AK319 | ATCC producing the recoded ProteusRdnI sequence with seven alanine mutations (Y197A, H221A, S235A, P244A, E246A, R254A, K258A) and a C-terminal Strep-II tag under the fla promoter | This study |
| OneShot OmniMax 2 T1R Competent Cells | E. coli strain for cloning | Thermo Fisher Scientific, Waltham, MA | |
| MFDpir | Mu-free E. coli mating strain to introduce plasmids into P. mirabilis | Ferrières et al., 2010 |
Table 3
Plasmids used in this study.
| Plasmid Name | Description | Cloning Method or Source |
|---|---|---|
| pBBR1-NheI | empty vector with pBBR1 origin. | Gibbs et al., 2008 |
| pLMW04-gfp | GFPmut2 with a constitutive fla promoter, (pBBR1 origin, Kan resistance). | Wenren et al., 2013 |
| pDS0002 | rdnE with the anhydrotetracycline (aTc)-inducible promoter, Ptet, (pBBR1 origin, Kan resistance) | gDS0005 was recombined into amplified pAS1054 by SliCE |
| pDS0062 | gfpmut2 with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | restriction digest using amplified gfpmut2 and pDS0002 |
| pDS0048 | rdnED39A-FLAG with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | gDS0025 was recombined into pDS0002 using restriction digest |
| pDS0058 | rdnED39A with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | pDS0048 was recombined into pDS0002 using restriction digest |
| pDS0059 | rdnEE53A with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | gDS0026 was recombined into pDS0002 using restriction digest |
| pDS0060 | rdnEK55A with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | gDS0027 was recombined into pDS0002 using restriction digest |
| pDS0061 | rdnED39A, E53A, K55A with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | gDS0028 was recombined into pDS0002 using restriction digest |
| pDS0034 | rdnE-FLAG with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | gDS0023 was recombined into amplified pDS0002 using SliCE |
| pDS0003 | rdnE-rdnI with the aTc-inducible promoter, (pBBR1 origin, Kan resistance) | Amplified rdnE-rdnI from pAS1059 was recombined into pDS0002 using SOE PCR |
| pAK023 | rdnI-StrepII with the T7 promoter, (pUC, Amp resistance) | gAK001 and pDS0003 were recombined into pET17b vector using SOE PCR |
| pAK043 | rdnI-StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | rdnI-StrepII amplified from pAK023 was recombined into pLMW04 using restriction digest |
| pAK070 | T25-linker-rdnED39A-FLAG with the IPTG-inducible lac promoter, (p15A, Kan resistance) | rdnED39A-FLAG tag amplified from pDS0048 was recombined into pKT25 using restriction digest |
| pAK071 | T25-linker-rdnI with the lac promoter, (p15A, Kan resistance) | rdnI-Strep-II amplified from pAK043 was recombined into pKT25 using restriction digest |
| pAK074 | T18-linker-rdnED39A-FLAG with the lac promoter, (Col E1 origin, Amp resistance) | rdnED39A-FLAG tag amplified from pDS0048 was recombined into pUT18C using restriction digest |
| pAK075 | T18-linker-rdnI-StrepII with the lac promoter, (Col E1 origin, Amp resistance) | rdnI-Strep-II tag amplified from pAK043 was recombined into pUT18C using restriction digest |
| pAK076 | T25-gfpmut2 with the lac promoter, (p15A origin, Kan resistance) | Amplified gfpmut2 was recombined into pKT25 using restriction digest |
| pAK077 | T18-gfpmut2 with the lac promoter, (Col E1 origin, Amp resistance) | Amplified gfpmut2 was recombined into pUT18C using restriction digest |
| pAK044 | RothiardnI-StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | gAK003 was recombined into pAK043 using restriction digest |
| pAK045 | PrevotellardnI-StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | gAK004 was recombined into pAK043 using restriction digest |
| pAK046 | PseduomonasrdnI-StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | gAK005 was recombined into pAK043 using restriction digest |
| pAK079 | T18-RothiardnI-StrepII with the lac promoter, (pUC, Amp resistance) | gAK003 was recombined into pUT18C using restriction digest |
| pAK081 | T18-PrevotellardnI-StrepII with the lac promoter, (pUC, Amp resistance) | gAK004 was recombined into pUT18C using restriction digest |
| pAK083 | T18-PseudomonasrdnI-StrepII with the lac promoter, (pUC, Amp resistance) | gAK005 was recombined into pUT18C using restriction digest |
| pAK058 | RothiardnI-StrepII with the T7 promoter, (pUC, Amp resistance) | gAK003 was recombined into pAK023 using restriction digest |
| pAK059 | PrevotellardnI-StrepII with the T7 promoter, (pUC, Amp resistance) | gAK004 was recombined into pAK023 using restriction digest |
| pAK060 | PseudomonasrdnI-StrepII with the T7 promoter, (pUC, Amp resistance) | gAK005 was recombined into pAK023 using restriction digest |
| pAK063 | Sequence optimized rdnI-StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | gAK024 was recombined into pAK043 using restriction digest |
| pAK065 | Pm/RdrdnI-StrepII (Proteus rdnI with Rothia conserved motif insert) with the fla promoter, (pBBR1 origin, Kan resistance) | gAK026 was recombined into pAK043 using restriction digest |
| pAK066 | Pm/PjrdnI-StrepII (Proteus rdnI with Prevotella conserved motif insert) with the fla promoter, (pBBR1 origin, Kan resistance) | gAK028 was recombined into pAK043 using restriction digest |
| pAK067 | Pm/PordnI-StrepII (Proteus rdnI with Pseudomonas conserved motif insert) with the fla promoter, (pBBR1 origin, Kan resistance) | gAK027 was recombined into pAK043 using restriction digest |
| pAK086 | Pj/PmrdnI-StrepII (Prevotella rdnI with Proteus conserved motif insert) with the fla promoter, (pBBR1 origin, Kan resistance) | gAK029 was recombined into pAK043 using restriction digest |
| pAK087 | Pf/PmrdnI-StrepII (Pseudomonas rdnI with Proteus conserved motif insert) with the fla promoter, (pBBR1 origin, Kan resistance) | gAK030 was recombined into pAK043 using restriction digest |
| pAK092 | T18-linker-Sequence optimized ProteusrdnI with a C-terminal StrepII with the lac promoter (Col E1 origin, Amp resistance) | gAK024 was recombined into pAK075 using restriction digest |
| pAK093 | T18-linker-Sequence optimized Pm/RardnI (Proteus rdnI with Rothia conserved motif insert) with a C-terminal StrepII with the lac promoter (Col E1 origin, Amp resistance) | gAK026 was recombined into pAK075 using restriction digest |
| pAK094 | T18-linker-Sequence optimized Pm/PjrdnI (Proteus rdnI with Prevotella conserved motif insert) with the lac promoter (Col E1 origin, Amp resistance) | gAK028 was recombined into pAK075 using restriction digest |
| pAK095 | T18-linker-Sequence optimized Pm/PfrdnI (Proteus rdnI with Pseudomonas conserved motif insert) with the lac promoter (Col E1 origin, Amp resistance) | gAK027 was recombined into pAK075 using restriction digest |
| pAK096 | T18-linker- Pj/PmrdnI (Prevotella rdnI with Proteus conserved motif insert) with the lac promoter (Col E1 origin, Amp resistance) | gAK029 was recombined into pAK075 using restriction digest |
| pAK097 | T18-linker-Pf/PmrdnI (Pseudomonas rdnI with Proteus conserved motif insert) with a C-terminal StrepII with the lac promoter (Col E1 origin, Amp resistance) | gAK030 was recombined into pAK075 using restriction digest |
| pAK064 | Sequence optimized rdnI7mut -StrepII with the fla promoter, (pBBR1 origin, Kan resistance) | gAK025 was recombined into pAK043 using restriction digest |
| pAK085 | T18-linker-Sequence optimized rdnI7mut-StrepII with the lac promoter (Col E1 origin, Amp resistance) | gAK025 was recombined into pAK075 using restriction digest |
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Non-cognate immunity proteins provide broader defenses against interbacterial effectors in microbial communities
eLife 12:RP90607.
https://doi.org/10.7554/eLife.90607.3
