RdnE homologs act as DNA endonucleases and contain interchangeable domains.

A) Cell viability (colony forming units 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 Supplemental Figure 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. Black bar show the PD-(D/E)XK motif and the grey bar marks the variable region 2 domain. Conserved residues are highlighted in dark blue. Secondary structure predictions identified using Ali2D55, 56 (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) and the ProteusRdnE (green) sequences and compared to the wild-type RdnE proteins.

RdnI binds to and protects against RdnE in vivo and in vitro.

A) Domain architecture for the idr locus in P. mirabilis strain BB200018. 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 assay18 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. 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) dominated in the 1:1 mixture and white arrows point to the 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 Xgal 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.

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 RaxML60, 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 alignments52 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 ColabFold32 and aligned using PyMol. The P. mirabilis RdnI sequence is a natural variant with two residue substitutions (V101E and I222M) as compared to BB2000 (supplemental figure 6).

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).

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 >1x 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 immunity proteins is that protection is defined by necessary and sufficient binding between cognate effectors (locks) and immunity proteins (keys). Bottom, our proposed skeleton-key model for protection is that 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.

List of strains used in this study.

Plasmids used in this study.

RdnE and RdnI homolog species.