Self-Identity Barcodes Encoded by Six Expansive Polymorphic Toxin Families Discriminate Kin in Myxobacteria

Self-identity barcodes encoded by six expansive polymorphic toxin families discriminate kin in myxobacteria

Christopher N. Vassalloa,1 and Daniel Walla,2

aDepartment of Molecular Biology, University of Wyoming, Laramie, WY 82071

Edited by Christine Jacobs-Wagner, Yale University, West Haven, CT, and approved October 24, 2019 (received for review July 20, 2019) Myxobacteria are an example of how single-cell individuals can dynamic OM proteins, and, when 2 compatible cells touch, transition into multicellular life by an aggregation strategy. For these multiple receptor complexes from each cell coalesce into distinct and all organisms that consist of social groups of cells, discrimination foci that bridge the boundary between the 2 cells. This transient against, and exclusion of, nonself is critical. In myxobacteria, TraA is interaction culminates in an apparent membrane fusion and bi- a polymorphic cell surface receptor that identifies kin by homotypic directional transfer of proteins and lipids before cells separate by binding, and in so doing exchanges outer membrane (OM) proteins gliding motility (5–7). This striking and robust behavior is and lipids between cells with compatible receptors. However, TraA thought to help rejuvenate and maintain homeostasis of the cell variability alone is not sufficient to discriminate against all cells, as envelope in a population that ages or encounters insults in traA allele diversity is not necessarily high among local strains. To constantly fluctuating environments (8, 9). increase discrimination ability, myxobacteria include polymorphic In nutrient-rich soils, myxobacteria populations are numerous OM lipoprotein toxins called SitA in their delivered cargo, which and diverse (10, 11). Local strains compete with each other and poison recipient cells that lack the cognate, allele-specific SitI immu- must establish and maintain a group identity by recognizing and nity protein. We previously characterized 3 SitAI toxin/immunity cooperating with kin while excluding nonkin. TraA serves as one pairs that belong to 2 families. Here, we discover 4 additional SitA self-recognition determinant by binding to cells with matching families. Each family is unique in sequence, but share the character- receptors (2, 12). Sequence polymorphisms within the TraA istic features of SitA: OM-associated toxins delivered by TraA. We MICROBIOLOGY variable domain, which determines recognition specificity, is high, demonstrate that, within a SitA family, C-terminal nuclease domains are polymorphic and often modular. Remarkably, sitA loci are strik- and prior studies with a limited allele set experimentally de- > termined or predicted >60 distinct TraA recognition groups (3). ingly numerous and diverse, with most genomes possessing 30 and Myxococcus up to 83 distinct sitAI loci. Interestingly, all SitA protein families are However, analysis of TraA allele variation between xanthus serially transferred between cells, allowing a SitA inhibitor cell to strains that are colocalized in the soil revealed that some poison multiple targets, including cells that never made direct con- divergent strains are in fact compatible for OME (2, 13). In other tact. The expansive suites of sitAI loci thus serve as identify barcodes words, TraA is not always sufficient to discriminate between clonal to exquisitely discriminate against nonself to ensure populations are cells and competitors. This suggests that myxobacteria have ad- genetically homogenous to conduct cooperative behaviors. ditional mechanisms to identify clonemates. Indeed, to increase

kin recognition | polymorphic toxins | outer membrane exchange | Significance myxobacteria Social organisms that share resources must identify their kin ulticellular organisms or groups of social cells need to to avoid exploitation by nonself competitors; however, un- Midentify clonal cells to coordinate specific behaviors and derlying mechanisms to explain discrimination are lacking. allow resources to be directed toward them. Central to under- Myxobacteria, which aggregate into tissue-like groups, use a standing these fundamental processes is identifying the proteins 2-step self-identification mechanism in which cells interact by a involved in self/nonself-recognition and the mechanisms indi- highly variable cell surface receptor that catalyzes cellular viduals use to discriminate against nonkin to form cohesive and cargo exchange. This cargo includes polymorphic toxins that harmonious populations. Myxobacteria represent tractable model poison nonclonal cells, which lack specific immunity genes. systems to study how kin recognition evolves and functions at a Here, we identified 6 unique families of toxins that are strik- molecular level. Myxobacterial cells typically live in social groups ingly numerous in myxobacterial genomes. Together, arrays of in the soil, where they move and feed on prey microbes. When toxins form what we describe as self-identity barcodes that nutrients are depleted, they undergo a synchronized, cooperative exquisitely distinguish clonal cooperators from nonself. This work highlights how selfish and discriminating genes, which developmental program culminating in the formation of a multi- expand in vast combinations in bacterial genomes, help to di- cellular fruiting body that harbors dormant spores. Cooperating versify and insulate social groups. with kin cells while excluding incompatible individuals is impera-

tive for them to maintain a viable social network. During vege- Author contributions: C.N.V. and D.W. designed research; C.N.V. performed research; tative growth, cells maintain close contacts as they move past one C.N.V. analyzed data; and C.N.V. and D.W. wrote the paper. another by gliding motility. Upon each physical contact, cells The authors declare no competing interest. monitor the identity of their neighbors by homotypic interactions This article is a PNAS Direct Submission. of a highly polymorphic cell surface receptor called TraA, along Published under the PNAS license. – with its partner protein TraB (1 3). When neighboring cells have 1Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, identical or matching TraA receptors, they exchange large MA 02142. amounts of cell envelope material in a process called outer 2To whom correspondence may be addressed. Email: [email protected]. membrane exchange (OME). OME can be directly visualized This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. microscopically by rapid and efficient cell-to-cell transfer of 1073/pnas.1912556116/-/DCSupplemental. outer-membrane (OM) fluorescent reporters (4, 5). TraA/B are

www.pnas.org/cgi/doi/10.1073/pnas.1912556116 PNAS Latest Articles | 1of11 Downloaded by guest on September 24, 2021 specificity of OME beyond TraA–TraA interactions, there is a modules that share no sequence similarity. The escort domains are second authentication or discrimination step. OM-localized poly- thought to facilitate CT toxin delivery into the cytoplasm following morphic toxins are included among the wide array of cell envelope delivery to the OM of the target cell. sitA3 is similar to the sitA1/2 cargo that is delivered during OME (14). genes in that it encodes a lipoprotein toxin delivered by OME, Polymorphic toxin/immunity pairs are ubiquitous in microbial contains a modular CT toxin domain, and is associated with a genomes and provide a means to exclude nonkin from clonal downstream sitI3 immunity gene. Interestingly, however, SitA3 populations (1, 15). Toxins typically consist of a domain that shares no sequence homology with SitA1 and SitA2. Each of the 3 facilitates delivery of a C-terminal (CT) toxin domain, which sitA loci have an overlapping homologous upstream open reading causes growth inhibition or death of a susceptible cell that re- frame (ORF) called sitB, whose gene product enhances the ability ceives it. Immunity genes, almost always encoded next to the of SitA to kill target cells, but is not essential for SitA function toxin, provide allele-specific protection from the toxic activity. (14). SitB contains a signal sequence, and is predicted to form a These systems can diversify by amino acid changes in residues β-barrel in the OM, but has no clear homology to any character- involved in the molecular recognition between the toxin and the ized domains. Since SitA1/2 and SitA3 are not homologous, it immunity proteins, resulting in polymorphisms and the forma- raises the possibility that there are other SitA proteins that share tion of new toxin/immunity specificity pairs (16). As microbial similar function and delivery mechanism but do not necessarily strains diversify, so too do their toxin repertoires, and horizontal share sequence homology. Here, we describe the discovery and gene transfer (HGT) plays a major role in toxin/immunity dis- characterization of 4 additional families of SitAI that belong to the semination and diversification between populations (15, 17, 18). overarching SitA class of proteins. We demonstrate that SitAI4, Further, toxins involved in interstrain warfare often have a SitAI5, SitAI6, and SitAI7 are OME-dependent toxin families that modular architecture in that diverse toxin domains are found at are strikingly numerous and diverse within myxobacterial genomes the C terminus of a particular delivery domain and appear to be predicted to contain functional TraAB proteins. Remarkably, mixed and matched by recombination between various delivery some myxobacterial genomes contain >80 total sitAI loci. Within systems (15). In consequence, organisms can encode an array of each family, CT toxin domains and SitI immunity proteins are unique toxin/immunity pairs that together facilitate intergenomic highly diverse and are often modular. Many of these toxin and conflict. A number of functionally diverse delivery mechanisms are immunity domains are previously uncharacterized, but, also, many known, such as the type-6 secretion system (T6SS) and contact- are conserved in other polymorphic toxin delivery systems, such as dependent inhibition (CDI), which deliver their toxic domains via CDI and RHS (30), found in diverse taxa. Our discovery greatly a needle-like secretion machine or a large, extended filament expands the known myxobacterial toxic SitA arsenal that con- structure, respectively (19–21). Additionally, new polymorphic tributes to kin discrimination following the mutual decision of toxins and delivery systems continue to be discovered (14, 22–27). partner cells to engage in OME. We suggest that the plethora of We recently described a polymorphic toxin system in myxobacteria polymorphic SitA toxins act as self-identify barcodes to help se- that is delivered by OME between cells with compatible TraA quester the cooperative behavior of OME to clonal cells. receptors (Fig. 1A). These proteins, called SitA, are lipoproteins that reside in the OM and contain CT nuclease domains Results (14). Once delivered to the target cell OM by OME, SitA Identification of Six Families of SitAI Proteins. The previously char- must traverse the cell envelope, by an unknown mechanism, acterized sitA gene families (sitA1/2 and sitA3) are each typically to reach the cytoplasm. There, the CT nuclease causes target cell associated with an overlapping upstream sitB gene. Although these death. Target cells that express the allele-specific, cognate im- SitA families have negligible homology to each other, their up- munity protein, called SitI, are protected (14). sitA and sitI genes stream SitBs are homologous. To investigate whether other sitBAI are always adjacent in myxobacterial genomes. SitA, or swarm loci reside in myxobacterial genomes, we performed BLAST inhibition toxin A, is so named because it was discovered as an analysis and determined that there is another family of genes as- effector that prevented outward swarming in a recipient strain sociated with sitB homologs. This family, designated SitA4, also during a 2-strain coculture (7, 28). That is, nonmotile M. xanthus contains a lipoprotein signal sequence, an AHH nuclease CT mutants that express SitA inhibit the outward swarming of sus- domain found in other toxin systems, and a downstream putative ceptible motile cells that lack the corresponding SitI (14). Swarm immunity gene. Its central escort domain, however, shared no inhibition is therefore caused by poisoning of the motile strain homology with SitA1/2 or SitA3. This suggested that SitA4 may be before outward swarming occurs. A ΔtraA mutation in either the a new, independent family of sitA genes that may share a similar nonmotile inhibitor or the motile target strain blocks toxin function. transfer, and therefore swarm expansion is restored to the motile To determine if myxobacteria encode other potential OME- strain. Thus, SitA transfer is traA-dependent. Furthermore, the dependent toxins, we used various HHM profiles (Pfams [31]) of SitA proteins are polymorphic, and some myxobacteria encode known toxin or immunity domains (15, 32) as queries to probe multiple sitA alleles per genome. The requirement that a SitA publically available Myxococcales genomes for remote homology. recipient have a compatible traA allele suggests that SitA func- Further, we searched for putative toxins that contain lipopro- tions as a kin discrimination factor. Therefore, sitAI loci act as a tein signal sequences, a hallmark of OME cargo. These efforts verification step to ensure OME is occurring among clonal cells returned multiple conserved hypothetical gene families with sim- (14). Unique among delivery systems, SitA transfer is infectious ilarity to conserved nucleases and immunity domains or otherwise within a population because the toxins can be serially transferred conspicuous gene pairs. We further narrowed our search by fo- between cells by multiple OME events. Consequently, a single cusing on gene families that were well-conserved in a subset of SitA-producing cell can kill many target cells as the toxin pool myxobacteria yet had obvious polymorphic regions when aligned rapidly spreads throughout the population (14). by sequence homology. Many of these resulting gene candidates In our prior study, 3 sitAI loci were found in the domesticated encode N-terminal lipoboxes within their signal sequence. Since lab strain (SitA1, SitA2, and SitA3) (14). Interestingly, all 3 loci OME delivers cell envelope proteins, we focused our attention on reside in similar relative locations on 3 tandem but divergent only those containing signal sequences. From the NCBI (33) and repeats of ∼100-kb prophage-like elements (∼300 kb total) (14, IMG databases (34), the ORF start codon was frequently incor- 28). Historically named “Mx-alpha,” these defective prophage rectly assigned by automated algorithms, and therefore many signal produce nonvirulent transducing particles, suggesting that HGT sequences were only found by manual curation for the correct start plays a role in acquiring new sitAI loci (14, 29). SitA1 and SitA2 codon. In total, these efforts returned 3 additional families of po- share homologous central “escort” domains, but have CT nuclease tential SitAI toxin/immunity pairs that we predicted function like

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1912556116 Vassallo and Wall Downloaded by guest on September 24, 2021 A SitA exchange by OME B TraA/B Fusion complex IM OM

OM IM SitA

C SitA escort domain pairwise identity D

SitA4

SitA5

SitA6 MICROBIOLOGY

SitA3 SitA7 SitA1/2 Percent identity

0 20 40 60 80 100

Fig. 1. Four newly discovered SitA families. (A) Model for exchange of SitA by OME. Two adjacent cells with compatible TraA receptors form a TraAB OM fusion junction. Subsequently, transient membrane fusion allows the passive diffusion of SitA toxins between cells. SitA then traverses the cell envelope to access the cytoplasm, where they either act as nucleases or are inactivated by their allele-specific SitI immunity protein. (B) Overview of the domain organization of SitBAI and SitAI families. SitA families are classified into 2 groups by whether they have a modular toxic CTD (red dashed boxes) or a single polymorphic toxin domain (solid red boxes). Colors correspond to unique escort domains belonging to each family. Invariant lipobox cysteine (“C”) residues or alternative residues (in SitA4) are shown. Genes not to scale; Fig. 5 provides more detail. (C) Pairwise percent identity matrix of Myxococcaceae proteins from each SitA family. SitA proteins belonging to one family share homology with each other but not with proteins of a different SitA family. (D) Locations of sitA loci on the lab strain M. xanthus chromosome and the pMF1 plasmid from M. fulvus 124B02. DK101 is a parent strain of DK1622 that contains a ∼200-kb region of Mx-alpha that was spontaneously lost during the construction of DK1622, which harbors 2 additional sitA alleles (Inset). Numbers correspond to MXAN locus tags. Colors indicate SitA family based on the color scheme in B. Each sitA gene contains a cognate downstream sitI gene (not pictured). *MXAN_1054 has a frame-shift mutation.

other SitAs. However, these families were never found with an this gene pair is the only annotated feature on one strand of the upstream sitB gene. Hereafter, we refer to these gene families plasmid DNA. The other strand codes for the remaining 21 as SitA5, SitA6, and SitA7, representing the order in which they predicted ORFs (37). were discovered. An overview of the characteristic gene and We previously showed that the SitA1/2 and SitA3 families domain architecture of the predicted SitAI families is shown in have modular CT domains, meaning diverse and distinct toxin Fig. 1B.Wedefinea“family” of SitA as a group of genes that domain modules are found at the C terminus, following the share conservation in their central escort domain (colored conserved escort domain (Fig. 1B). By comparing genes within domains in Fig. 1B). Importantly, the escort domain of a SitA the newly discovered families, we found that SitA5 proteins are family does not share significant homology with the escort also modular. SitA4, 6, and 7 proteins are not modular; instead, domain of another family (Fig. 1C), again suggesting that there they are polymorphic over the length of the proteins. A more may be multiple SitA gene families that share a similar func- detailed analysis of domain architecture and conserved domains tion, but no sequence homology. Each family had multiple of the 6 SitA families is described later. Our analysis brings the representative genes in myxobacterial genomes. For example, total number of putative SitA families to 6, with SitA1 and SitA2 the parent of the lab strain M. xanthus DK1622 has 25 total constituting a single family, “SitA1/2,” based on their homologous sitAI loci, which represent all 6 families (Fig. 1D). Additionally, escort domains. we found a sitAI6 cassette on the only known replicating plasmid in myxobacteria, called pMF1 (35), harbored within strain The Six Families of SitAI Are OM-Associated Toxins Delivered by OME. Myxococcus fulvus 124B02 (Fig. 1D), a loci that was previously To experimentally probe the function of the 4 putative SitA toxin shown to be a toxin/immunity pair (Fig. 1D) (36). Interestingly, families, we cloned representative sitAI cassettes from M. fulvus

Vassallo and Wall PNAS Latest Articles | 3of11 Downloaded by guest on September 24, 2021 Mf HW-1 (sitA ) into the M. xanthus chromosome. Importantly, A Competition assay each of these gene cassettes was divergent from those found in M. xanthus 10 sitA– (mock inhibitor) the genome. For example, the closest homolog of SitA5 from M. fulvus in M. xanthus was 44.4% identical. Each of 8 Target these constructed inhibitor strains were then competed with a WT 6 WT (“target”) strain, which lacked the corresponding sitAI cas- ΔtraA Target cell target CFU 4 sette, but contained identical traA alleles, in a one-to-one co-

10 2 morphology culture on solid agar growth media. CFUs of the target strain were

Log 0 determined following 0, 6, and 24 h of coincubation. In all cases, 01224 the CFUs of the target strain were dramatically lowered compared hr to mock-inhibitor control at the 6- and 24-h time points (Fig. 2A). In order to test if these proteins were dependent on OME for Mx1 Mf1 10 sitBAI1 10 sitAI3 delivery, we repeated the CFU competitions using an isogenic ΔtraA traA 8 8 mutant as the target strain. As mentioned, a mutation 6 6 in one or both strains abolishes transfer because OME is a mutual decision where both cells must have compatible TraA receptors. 4 4 ΔtraA 2 Importantly, targets were all immune from antagonism, as 2 their CFU output was similar to the control (Fig. 2A). Taken together, these data suggest that, like SitA1/2 and 3, the additional 0 24 0 24 families of SitA are toxins delivered by OME. Prior work showed that SitA antagonism leads to morpho- 10 sitBAI4Mf1 10 sitAI5Mf1 logical changes in the target cells (14). To observe target cells 8 8 during coincubation, we competed the above inhibitor strains 6 6 with a fluorescently labeled target strain on agarose pads. 4 4 Along with causing cell lysis, each coincubation resulted in dra- 2 2 matic filamentation, enlargement, and lysis of the fluorescent target cells (Fig. 2A). Mean cell lengths of control cells were μ = n = 0 24 0 24 6.05 m(SD 1.38, 50), whereas the mean cell length of SitA- poisoned cells was 14.38 μm(SD= 4.58, n = 200). These results further suggest that delivery of the newly discovered SitA pro- sitA61Mf1 10 sitAI7Mf1 10 teins is toxic to recipient cells that lack the matching SitI 8 8 immunity protein. 6 6 As noted earlier, one outcome of SitA-mediated antagonism is 4 4 the ability of a nonmotile, SitA-producing strain to inhibit the 2 2 outward swarming of a SitA-susceptible motile strain when the 2 strains are cultured together on an agar surface. To test whether, 0 24 0 24 like SitA1/2 and 3, the newly described SitA families caused a swarm inhibition phenotype, we expressed the various SitAI loci from M. fulvus in a nonmotile M. xanthus strain. Resulting co- B Motile cultures between the nonmotile inhibitor and susceptible motile targets that lacked immunity showed swarm inhibition of the motile strain that was dependent on TraAB (Fig. 2B and SI Ap- pendix,Fig.S1A). SitA6 inhibitors exhibited the weakest antago- – sit sit1+ nistic phenotype. It was therefore necessary to increase the + nonmotile to motile cell ratio to 5:1 to achieve swarm inhibition in sit1 sit3+ elitom sit3+ these mixtures. All other assays were conducted at a 1:1 ratio. sit4+ SitI proteins have predicted immunity functions, and we pre- sit4+ viously showed that SitI1, SitI2, and SitI3 selectively protect target

noN sit5+ sit5+ cells from death by their cognate SitA (14). To probe the function of SitI4 to SitI7, we repeated the swarm inhibition assay using sit6+ sit6+ motile target strains that expressed the same SitAI operon as the sit7+ sit7+ nonmotile inhibitor strain. Expression of the operon resulted in relief of swarm inhibition and hence protected the motile strain B SI Appendix A Swarm inhibition (antagonism) from toxicity (Fig. 2 and ,Fig.S1 ). To test for specificity between the various SitAI systems, we then competed Relief of swarm inhibition (no antagonism) all combinations (except as noted later) of nonmotile and motile Fig. 2. Expression of heterologous sitAI cassettes results in traA-dependent strains together. Unlike the previous assay, these combinations target cell death. (A) CFU time course of target cells from agar plates when can involve reciprocal antagonism. Therefore, we gave the non- competed against SitAI-expressing inhibitors. Open circles and dashed lines motile strain a starting advantage with a cell ratio of 8:1. The indicate that target cells are ΔtraA; closed circles and solid lines indicate that motile strain in these combinations escaped swarm inhibition only + target cells are traA . Adjacent image shows the morphology (filamentation) sitAI + + when both strains expressed the same cassette. Otherwise, of fluorescently labeled traA target cells after coincubation with SitA in- when strains expressed different cassettes, there was reciprocal an- μ hibitor cells. (Scale bar, 5 m.) (B)(Left) Results of swarm inhibition coculture tagonism and complete swarm inhibition (Fig. 2B and SI Appendix, experiments in which a nonmotile inhibitor strain expresses a unique sitAI + + cassette and susceptible motile target cells are traA , ΔtraA,orSitI.Strains were mixed at a 1:1 ratio with the exception of SitA6Mf1 inhibitors, which were mixed at 5:1. (Right) Results of swarm inhibition cocultures of SitAI-expressing killed by the opposing motile strain and never caused swarm inhibition. SI nonmotile and SitAI-expressing motile strains at an 8:1 cell ratio. SitAI6Mf1- Appendix, Fig. S1, provides micrographs of these results. All strain, plasmid, expressing nonmotile inhibitors were excluded because they were always and primer details are given in SI Appendix, Tables S1–S3.

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.1912556116 Vassallo and Wall Downloaded by guest on September 24, 2021 Fig. S1B). The nonmotile SitAI6 inhibitor was excluded from this in its signal sequence. This resulting strain also reduced target assay because the SitA6Mf1 toxins were too weak and thus the strain CFUs in a TraAB-dependent manner (SI Appendix,Fig. motile strain easily outcompetes and escapes swarm inhibition, S2), suggesting that SitA4 proteins that are not lipidated are even at the 8:1 starting ratio. Together, these experiments dem- nevertheless transferred. Further experiments are needed to test onstrate that the newly described SitAI cassettes code for toxic the subcellular localization of SitA4 proteins and whether they are proteins delivered by OME and that expression of these cassettes soluble periplasmic proteins that are delivered by OME. In ad- Mf confer gene-specific immunity in target strains. dition, sitAI4 2 does not have a cognate sitB gene upstream. As mentioned earlier, a sitAI6 cassette was found on the 18.6-kb Therefore, like SitA1 and SitA3, a cognate SitB4 protein is not autonomously replicating plasmid pMF1 from M. fulvus 124B02 required for SitA4 function (14). Results from competition ex- (aka pMF1.20 and pMF1.19 genes). We cloned and recombined periments with 3 additional heterologous sitAI cassettes are also this operon into the genome of M. xanthus and found that this shown in the SI Appendix,Fig.S2. resulting strain also reduces CFUs of a target strain in a TraA- dependent manner (SI Appendix,Fig.S2). Although the pMF1.20 Serial Transfer of SitA Toxins. We previously showed that the was noted previously to be a toxin family distributed in myxobacteria unique delivery system of OME led to serial transfer of SitA1 (36), we suggest adopting the name SitAI6 based on these proteins (14). In other words, cells infected with SitA proteins in their having a similar function and delivery mechanism as other SitA OM by OME act as carriers that spread the toxin to neighboring family members. cells by subsequent OME events. In this way, toxin-producing Interestingly, although all other SitA proteins have an invari- cells poison target cells that they never made direct contact ant cysteine residue in their lipobox and a sorting sequence that with. We therefore tested whether this attribute applies to the indicates OM localization, many sitA4 alleles do not, revealing that families of SitA described here. To test this, we used a 3-strain SitA4 proteins are not strictly lipoproteins. For example, 3 of coculture composed of an isogenic inhibitor and target strain 5 sitA4 genes in M. fulvus HW-1 do not have a cysteine in their that have incompatible traA alleles and a third-party intermedi- Mf signal sequence. Nevertheless, SitA4 1 is functional and TraA- ary strain that expresses both traA alleles (Fig. 3). Therefore, for dependent (Fig. 2). To this point, OME has been observed for the inhibitor strain to deliver SitA to the target strain, OME OM-localized proteins and lipids, including those that contain a must first occur with the traA merodiploid intermediary that acts type I signal sequence and hence are not lipoproteins (4, 38). To a conduit for SitA delivery to the target. Importantly, we pre- confirm whether other SitA4 proteins that do not have a lipobox viously demonstrated that TraA itself does not transfer since it is are functional, we cloned and expressed a separate sitAI4 locus apparently anchored to the cell envelope (5, 14), and therefore

Mf MICROBIOLOGY from M. fulvus HW-1 (sitAI4 2), whose SitA also lacks a cysteine serial transfer cannot be explained by exchange of TraA mediating

Experimental design Target fitness Target morphology

Inhibitor (TraAMf) SitA– ns

SitA4 ** ) DK1622

No OME SitA5 * , TraA Mf Intermediary (TraA SitA6 *

SitA7 *

Target (TraADK1622) 0.01 0.1 1.0 Competitive index

+ Fig. 3. New SitA families are serially transferred. Experimental design: cell types are labeled where the SitAI inhibitor cell expresses an incompatible traA allele with the target cell and therefore cannot engage in OME. The intermediary cell expresses both traA alleles and thus can engage in OME with both strains to serve as a conduit for serial transfer. Intermediary cell is also susceptible to the inhibitor cell toxin. Target fitness: competitive index of target (green data points) and intermediary cells (red data points) in the 2-strain and 3-strain cocultures when the inhibitor cells express one of the SitA toxin families or is − an isogenic SitA control. Competitive index is a measure of the target-to-inhibitor cell ratio at 24 h normalized to the ratio at 0 h as enumerated by fluorescent microscopy. Starting ratios are 1:1 and 1:1:1 with the exception of SitA6 inhibitors, which were mixed at 3:1 and 3:1:1 ratios. Area between vertical dashed lines indicates experiments in which target cells were not killed and were not filamentous as observed by the target morphology panel. Error bars indicate SE, and significance indicators show results from a 2-tailed nonparametric t test. ns, not significant (*P < 0.05, **P < 0.01). Target morphology: concurrent micrographs of the quantified experiments show that, in the 3-strain coculture, green fluorescent target cells are either eliminated or show filamentation and morphological defects (arrows). In the 2-strain cocultures, green cells appear normal. (Scale bar, 10 μm.)

Vassallo and Wall PNAS Latest Articles | 5of11 Downloaded by guest on September 24, 2021 direct delivery between producer and target. As a control, for each Total sitA genes TraAB absent SitA inhibitor strain, we competed the inhibitor and target without SitA1/2 158 TraAB present SitA3 200 Unknown the intermediary strain and found that there was no antagonism SitA4 236 and no morphological changes to the target cells because OME SitA5 599 SitA6 236 cannot occur between these stains (Fig. 3). In contrast, when the SitA7 153 intermediary strain was included, the competitive index of the target cells drops dramatically, and target cells become filamentous and lyse (Fig. 3). Therefore, as was shown with SitA1, the newly characterized SitAs were all serially transferred, leading to target cell death dependent on the presence of the intermediary strain.

Phylogenetic Distribution of SitAI Proteins. To determine the distribution of SitA toxins, we used representative SitA proteins as

queries to BLAST the NCBI nonredundant database. We col- Myxococcales lected sequences that aligned over the conserved central domain of the query SitA and organized the sequences based on phylogeny. We found that SitA proteins were restricted to the Cystobacterineae suborder of the Myxococcales and were not found outside of myxobacteria (Fig. 4). Further, genomes that are predicted to contain functional traAB genes all contained a collection of sitA genes (Fig. 4, green branches [3]). No sitA genes were found in suborders of Myxococcales that do not have TraAB homologs (Fig. 4, red branches). The cooccurrence of TraAB and

SitA further supports that SitAs are delivered by OME and Histogram scale: 5 genes function as a kin discrimination determinant to differentiate OME partners. Distant TraAB homologs are found in clades outside of Fig. 4. sitA genes are numerous in myxobacteria that contain functional the Cystobacterineae (Fig. 4, yellow branches), but OME has not TraAB proteins. Tree depicting the NCBI taxonomic organization of sequenced been demonstrated with these strains or TraAB homologs (3). Myxococcales genomes and the distribution of TraAB and SitA. Species that Consistent with this, no sitA genes were found in these genomes, have TraAB homologs are depicted with green branches, whereas genera that do not have TraAB or have distant TraAB homologs that may not function in suggesting that these distant TraAB homologs may have a dif- OME are depicted with red and yellow branches, respectively. Further detail is ferent function from OME. Interestingly, the majority of genomes provided in the text. Number and family designation of all sitA alleles are contain sequences for a striking number of distinct sitA genes, with depicted by colored histograms adjacent to the species names. Scale bar in- the average number of loci per genome being 38 and the number dicates a histogram bar length of 5 genes per genome. SI Appendix,TableS4, of genes per genome ranging from 13 in Corallococcus sp. provides corresponding numbers of genes per genome. Total number of loci in H22C18031205 to 83 in Cystobacter fuscus DSM 52655 (Fig. 4 and 41 SitA-containing genomes is shown at upper left. SI Appendix,TableS4).

Sequence Analysis of SitAI Toxin and Immunity Domains. As noted Table S5). Interestingly, 3 toxin/immunity domain modules were earlier, the SitA1/2 and SitA3 family toxins contain a conserved shared between SitA3 and SitA5 families (Fig. 5B), suggesting escort domain and a polymorphic and modular CT toxin domain. that recombination occurred between sitA3 and sitA5 loci and/or When distinct SitAs do share a modular CT toxin domain, they these domains were acquired independently from common ancestral contain polymorphisms. To characterize all of the domains sequences. within SitAI families, we undertook a comprehensive analysis Next, we used HHpred (39) as a sensitive search tool to find of their domain architectures using the described collection of homology to conserved domains in the PDB (40), Pfam (31), and Myxococcales sequences. First, we aligned the sequences of each NCBI CDD (41) databases for each of the CTD and immunity family and looked for modularity at the C terminus. We found protein domains. Most of the toxin domains had remote ho- that, in addition to SitA1/2 and SitA3 families, SitA5 contains a mology to conserved nuclease domains (Fig. 5B). Since HHpred modular C-terminal domain (CTD), as conservation clearly drops could detect remote homology between SitA-CT and conserved following the conserved escort domain (Fig. 5A). SitA4, SitA6, domains, direct sequence similarity was not always conserved, and SitA7 were not modular. For example, unlike the modular and often sequences were only partially aligned over the SitA CT SitAs, all SitA4 sequences align at their CT but are enriched in query. Therefore, although some SitA CT and immunity domains substitutions along the length of the protein. It is therefore likely returned hits to conserved domain superfamilies, they appear to that polymorphisms at the CT domains are responsible for the belong to more distinct subclasses of these domains. For this specificity of interaction between these SitAs and their cognate reason, although some CT/immunity domains have the same SitI proteins. To illustrate these fundamental differences between name, they are, in fact, phylogenetically very distinct (Fig. 5B and modular and nonmodular toxins, alignments of representative al- SI Appendix,TableS5). In addition, a number of the SitA CT and leles of SitA4 and SitA5 are shown in Fig. 5A. Also of note in this SitI domains did not return hits and were thus uncharacterized. To figure, between the signal sequence and the conserved escort determine if these uncharacterized CT module domains and their domain, we typically found a nonconserved region that is pre- corresponding immunity proteins were found in other organisms, dicted to be intrinsically disordered. we used BLAST to search against sequences outside of the − To characterize the toxin and immunity domains of each Myxococcales order (E < 10 05 over 80- to 200-residue cutoff). modular SitA family, we clustered their sequences by similarity Interestingly, nearly all of the CTD and immunity sequences were and found they formed distinct groups with <20% amino acid found adjacent to one another in other genomes. Further, these identity between clusters (Fig. 5B). Therefore, each cluster is CTD sequences were always found as CTD toxin modules to considered an independent toxin module. Consistent with this, characterized toxin systems such as RHS, CdiA, WXG, MafB, and each member of a CTD module cluster is associated with a filamentous hemagglutinin (FH) in diverse taxa (Fig. 5B). Our similar but distinct downstream immunity protein (SI Appendix, sequence searches also returned other uncharacterized toxins that

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1912556116 Vassallo and Wall Downloaded by guest on September 24, 2021 had toxin processing sites such as HINT, PsrW, and pretoxin-TG domains. SitAI1/2 and SitA7 CTDs were the only cases in which the toxin and immunity domains were exclusively found in myxobacteria, but these SitAs are numerous and were experimentally charac- A Modular and polymorphic terized (14). Many toxin CTDs and their corresponding immunity Conservation 10 proteins were only found once or twice in SitA sequences, but 0 numerous homologous domains were found at the CT of other SS Disorder Conserved Toxin Module toxin systems in organisms quite distant from myxobacteria Escort Domain (SI Appendix,TableS5). This implies that these are in fact functional domains conserved in a wide range of phylogenies, despite

Conservation Polymorphic being either rare in SitA proteins or simply underrepresented in 10 0 publically available myxobacterial genomes. A number of the domains we identified were listed as domains of unknown function SS Disorder Conserved Escort Domain Toxin (DUF), and this study thus provides insight into their function. The conserved escort domains did not show strong similarity to B = 200 residues Toxin domain Other toxin systems any conserved domains, with the exception of SitA6, which was prediction with this domain classified as TIGR02269 (PF14412), previously recorded as a M. xanthus Uncharacterized None family with 9 paralogous lipoproteins in , all of which Colicin-DNase(1) WXG, FH, RHS we classified here as SitA6 family members (SI Appendix,Table

SitA1/2 BrnT toxin(1) RHS S5). DUF2380 also describes a group of paralogous lipoproteins EndoU endonuclease RHS, MafB with AHH nuclease domains (SitA4 and 6 and some SitA5 HNH endonuclease RHS modules; Fig. 5B and SI Appendix,TableS5). Uncharacterized RHS Uncharacterized RHS Variation of sitA Genes between Related Genomes Establishes Self- CdiA-CT(1) RHS, FH, CDI Identity Barcodes. The finding that myxobacterial genomes pos- Uncharacterized RHS sess high numbers of sitA genes suggests that any 2 myxobacteria Uncharacterized WXG that employ this system are unlikely to possess immunity to one SitA3 Uncharacterized RHS ’ Ntox47 RHS, MafB another s full complement of toxins. As a result, 2 strains or EndoU endonuclease RHS species that are compatible for OME would likely inhibit one MICROBIOLOGY DUF2380 RHS, MafB another upon contact. To explore this concept, we conducted Tox-Rease-5 CDI, FH whole-genome comparisons of sitA genes between M. xanthus AHH nuclease(1) RHS, FH, MafB DK1622 and a related strain, Myxococcus virescens DSM 2260. CdiA-CT(2) FH, CDI We chose these 2 genomes because their traA alleles are highly AHH nuclease(2) FH, RHS similar (97.36% amino acid identity) and are predicted to be Nup96 RHS compatible for OME (3). Despite having different species des- Colicin-DNase(2) FH, RHS ignations, these strains are in fact closely related, with 99.93% Ntox43 FH, RHS identity at the 16S rRNA locus. We used progressiveMauve (42) CdiA-CT(3) FH, CDI, RHS to align the 2 genomes and looked at sitAI loci variation. Twelve GIY-YIG nuclease FH, RHS sitAI Uncharacterized FH, RHS loci were shared between the 2 strains, meaning that they tRNA synth binding RHS are found on aligned regions with perfect gene synteny (Fig. 6). HNH endonuclease 5 RHS Ten of these 12 loci were very similar in sequence, ranging from

SitA5 CdiA-CT tRNase WXG 93 to 99% amino acid identity. At one such matching locus, sitA6 Colicin D FH, RHS of DK1622 had acquired a frame-shift mutation, but the down- BrnT toxin(2) MafB, RHS stream sitI was intact and 97% identical to its SitI ortholog in M. Uncharacterized RHS virescens. In contrast, 2 sitAI5 loci were perfectly colocalized Tox-SHH RHS between the 2 strains but had a significant number of substitu- Tox-ART-HYD1 RHS, WXG tions enriched in the CTD of SitA and in SitI (example shown in PIN 11 (1) RHS Fig. 6). Beyond the orthologous matching loci, there were 15 PIN 11 (2) RHS sitAI EndoU endonuclease RHS individual operon pairs that were located on genomic is- HNH 4 nuclease RHS lands present in one genome but not the other. Together, these Ntox28 WXG, FH, RHS results suggest that a small proportion of sitAI loci diversify much SitA4 AHH nuclease(3) RHS faster than others, and that incompatibilities between strains are SitA6 AHH nuclease(4) RHS mostly derived from horizontal acquisition of new loci and gene SitA7 HNHc endonuclease None loss rather than diversification at existing loci. Importantly, this analysis indicates that, if populations of these Fig. 5. Domain organization and bioinformatic prediction of SitAI func- 2 strains were to interact, OME between them will result in the tions. (A) Domain organization of representative sequences of a modular M. xanthus and polymorphic SitA family (SitA5) and a polymorphic family (SitA4). Note transfer of 20 unique toxins: 8 delivered to and 12 that the SitA5 family has a nonconserved CTD, i.e., different domain mod- delivered to M. virescens (Fig. 6B). Based on the present and ules compared to the conserved CTD of SitA4. Conservation scores are based prior work (14), subsequent cell death and boundary formation on an algorithm that takes into account conservation of physicochemical between swarms will ensue, establishing barriers between these properties of individual residues (52). Proteins not shown to scale. (B) competitors and blocking further OME. Therefore, the sitAI loci Functional prediction of all uniquely clustering toxin domains found in in these genomes together constitute a self-identity code that Myxococcales sitA alleles by HHpred. The top relevant domain hit is shown, distinguishes social groups. with preference given to Pfam domains deposited in comprehensive domain To investigate the extent and diversity of sitAI loci between analyses (15, 32). Search criteria are described in Materials and Methods, and genomes of related myxobacteria, we performed a similar anal- additional details, including HHpred probability score, domain size, and SitI sitAI functional predictions, are provided in SI Appendix,TableS5. Domains are ysis of operons in a recently deposited group of genomes from shown to scale. Domains followed by a number are to distinguish those that the genus Corallococcus (43). First, TraA sequences were collected, share a common domain prediction despite having very little sequence similarity. and each isolate was parsed into predicted TraA recognition

Vassallo and Wall PNAS Latest Articles | 7of11 Downloaded by guest on September 24, 2021 xanthus virescens 13031 Compatible 1899 10971 traAB 1296 4478 0119 Frame-shift 0485 0848 Legend 1231/12551 101408 4323 105331 DK1622 chromosome 4844 M. virescens contigs 6511 102579 7256 115177 Mxα Colinear blocks 2 7453 106332 Aligned regions 106179 Location (Mb) 0242 10656 Mxα1 0253 101787 101634 10542 101579 Examples of co-localized SitA 1544 2496 103339 sitA5 sitI5 5843 104488 6330 102752 6448 102643 7411 0598.5 108142 6560

Mx Mv

Fig. 6. Comparative analysis of sitA genes from Myxococcus strains that are compatible for OME. (A) Whole-genome alignment depicts conserved and unique SitA toxins between 2 related Myxococcus genomes. Aligned regions are shown in green with red boundaries (“Legend”). Bars represent sitAI loci on the chromosomes. Bars spanning both genomes are colocalized sitA loci in regions of gene synteny. Inner-facing bars are unique M. virescens loci, whereas outer-facing bars are unique DK1622 loci. Amino acid percent identity between colocalized SitA alleles is shown. Inside the circular plot is an amino acid substitution analysis of one example of colocalized and conserved alleles (Lower) between the 2 genomes and colocalized alleles that have diverged at the SitA-CTD and SitI (Upper). Vertical lines indicate substitutions. (B) Total full-length sitA genes between the 2 genomes. MXAN and SAMN4488504 locus tag numbers are shown. Alleles that are predicted to be shared by both genomes (≥93% amino acid identity), i.e., reciprocal immunity, are placed side by side and marked with an open green circle. Genes unique to one genome are marked with a red circle. 1Although MXAN_1231 is an unmatched allele, its sitI is nearly identical to MXAN_1255 and so is not considered a unique allele, as M. virescens has a matching sitI downstream of SAMN4488504_101408. 2MXAN_1054 has acquired a frame-shift mutation, even though its SitI remains intact. The schematic (Bottom Right) is an overview of the total sitA loci from both genomes, whose presence in either genome is indicated by a black fill underneath the color-coded legend. Note that sitA1 and 2 are in the DK101 draft genome (Fig. 1), which is identical to DK1622 but contains 2 additional Mx-alpha regions that are not in the DK1622 genome and are not pictured here.

groups based on the sequence of the region that determines kin absence (no line) of each of the 145 SitA groups in each genome specificity, i.e., the variable domain (VD), using criteria from a (Fig. 7). Of the 14 possible interactions among these 6 isolates, prior study (3). Of the 27 alleles, we predicted 15 distinct TraA the range of unique or discriminating SitA toxins that could be recognition groups (SI Appendix, Fig. S3), which is illustrated in a transferred by OME is 29 to 76. These data highlight the precise maximum likelihood tree based on the amino acid sequence of and extensive nonself-discrimination power that these expansive their VD with the lab strain M. xanthus DK1622 serving as the SitA families enable when 2 TraA-compatible cells interact. The outgroup (Fig. 7). The largest predicted recognition group was outcome from such OME encounters determines whether indi- composed of 7 Corallococcus isolates, for 6 of which genomic viduals are poisoned or remain viable to undergo cooperative sequence was publically available (Fig. 7). We then examined the interactions as validated clonemates. SitA repertoires contained in this group. By comparing only the variable CT toxin domains of each SitA sequence, we clustered Discussion them into unique groups based on a stringent cutoff of >95% In-depth analysis of myxobacterial genomes revealed 6 families amino acid identity. This analysis concluded that the 224 total of SitA toxins that share a similar function but are not related by SitA-CT sequences belonged to 145 distinct specificity groups sequence. Interestingly, however, 3 families can be associated (Fig. 7). To visually represent the social compatibility of these with a homologous sitB gene. This suggests that SitBAI1/2, strains, we created SitA barcodes that displayed the sequence SitBAI3, and SitBAI4 share a common origin and subsequently analysis with vertical black lines that represent the presence or diverged. The sitA5, 6, and 7 genes are never associated with sitB

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1912556116 Vassallo and Wall Downloaded by guest on September 24, 2021 TraA recognition groups genomes, probably owing to cooperative social fitness gains OME plays, for example in membrane homeostasis and repair (5, 9). 205: P A SitA identity Our results show that the newly discovered SitA families of barcodes proteins, and, by extension, other classes of cargo proteins, can be serially transferred between cells. A model for the mechanism Scale 0.1 of serial transfer was described in a prior study (14). Previously, we suspected that SitB may be required for serial transfer be- AB038B cause we showed that ΔsitB inhibitor cells could poison intermediary cells in a serial transfer assay, but not secondary target cells (14). However, here we demonstrated serial transfer of SitA without a cognate SitB protein, suggesting that SitB is not always * AB018 required for serial transfer, but instead enhances SitA delivery to primary cells, which in turn enhances serial transfer. Finally, although our serial transfer assay was artificially designed by con- traA AB004 structing a merodiploid strain, serial transfer in nature nevertheless likely occurs between 3 or more distinct genotypes ** that all contain compatible traA alleles for OME. Importantly, we found that sitAI genes are expansive in CA041A myxobacteria, with several genomes possessing >50 loci. While there is inherent selective pressure to retain immunity genes to any toxin to avoid poisoning by neighboring clonal cells, the AB049A reason to retain so many toxins is less clear. For example, in some bacterial toxin systems, arrays of immunity genes remain Bootstrap intact despite not retaining their cognate toxins (15). However, 207 999 from our analysis, in nearly all cases, complete sitAI operons are AB047A retained despite there being multiple genes of any one family present in their genomes. In fact, all 11 heterologous sitAI cassettes that we cloned into M. xanthus were functional. We suggest that one major advantage of containing a vast array of toxins MICROBIOLOGY Barcode legend is to allow stringent discrimination against OME partners. A 145 second reason for many sitAI loci is that it provides a competitive total advantage over related cells. For instance, if an individual cell within a clonal population gains a novel sitAI by HGT, that cell can now efficiently poison and outcompete its siblings through sitAI Fig. 7. SitA loci define molecular barcodes that discriminate kin between cells OME. Similarly, there is selective pressure to retain cas- in a TraA recognition group. TraA and SitA analysis within the Corallococcus settes because, if a cell loses a cassette, it would be eliminated by genus from Fig. 4. Maximum likelihood tree depicts the relationship between its siblings. Such HGT and exploitation schemes were likely re- TraA variable domains from 27 genomes. Colored shape backdrops indicate peated many times over during evolution, such that some strains the predicted recognition groups of traA alleles based on their sequence acquired and retained a large arsenal of sitAI loci. identity and the presence of proline or alanine at relative position 205 as The large array of sitAI cassettes that cells harbor likely results described (3). The largest recognition group is denoted by the dashed outline, in differential expression of these loci in response to environ- excluding the allele with 205P. The 6 available genomes from this group (black mental and developmental cues. A previous study on SitA1 and bars, white text) encode 224 total sitAI loci, which clustered into 145 distinct SitA2 supports the notion that these toxins are constitutively groups. Barcode diagrams show the presence (black line) or absence (white line) of each SitA group for the 6 genomes. The barcode legend (Bottom) expressed during swarming on solid agar surfaces (28), but future shows the total sitA loci used to generate the barcodes (color-coded cells studies should examine the regulation and natural expression separated by black borders). The thickness of each cell is proportional to the levels of the SitA protein families in more detail. Although ex- number of strains that encode the corresponding locus. *MCy1075 belongs to pression levels of certain SitA toxins are high enough to display a recognition group 13 (SI Appendix,Fig.S3), but genomic sequence for sitA strong killing phenotype (28), it is possible that endogenous ex- analysis was not available. **AB043A was not included in recognition group 13 pression levels at other loci are not, and therefore low levels of because its VD percent identity fell below the 90% threshold in 4 of 8 cases multiple toxins may act in concert to kill target cells by multiple with other family members. mechanisms of action. Genome sequences of myxobacterial isolates that were colo- cated in the soil have demonstrated that many strains that live in and thus may have evolved in a convergent fashion to adopt a close proximity possess identical traA alleles, but nevertheless are similar function. We classified SitA proteins into distinct families incompatible and antagonize one another upon physical contact defined by their conserved escort domains. Based on prior studies (13, 47, 48). Although it is beyond the scope of this work, we of bacteriocins (44) and CdiA (45) toxins, we hypothesize that found that these environmental isolates have different repertoires these SitA escort domains facilitate entry of the toxin module into of sitAI loci (14) despite their genomes being very similar and the target cell cytoplasm. Because these escort domains are unique containing identical traA alleles. These observations suggest that to each family, we further hypothesize that they exploit distinct the plethora of SitA toxins contained in these cells play a key role cellular proteins to gain cytoplasmic entry after they are delivered in kin discrimination. Future studies from our lab will investigate to the OM by OME. Our future work will examine this question. this hypothesis. Myxobacteria also possess other antagonistic, Possessing an arsenal of SitA toxins with different cytoplasmic polymorphic kin discrimination systems such as the T6SS (49, 50). entry pathways is advantageous for this system, as it is unlikely that A fundamental difference of these systems is that SitA delivery a target cell will evolve resistance by blocking entry to a divergent requires matching sequence identity at another locus, traA,whereas panel of toxins without mutations to TraA or TraB (46). In this the T6SS can indiscriminately inject toxins into neighboring cells. regard, it is interesting to note that we found no evidence of de- Therefore, in the case of SitA, discrimination does not function at generate TraAB mutant sequences from >100 myxobacterial a broad level of interstrain or interspecies antagonism, but instead

Vassallo and Wall PNAS Latest Articles | 9of11 Downloaded by guest on September 24, 2021 functions to establish a narrow relatedness threshold to guard recognition groups, of which 13 are new and supplement the 42 against OME with related but nonkin genotypes. recognition groups previously demonstrated or predicted from Our comparative genomic analysis of M. xanthus and M. virescens the suborder Cystobacterineae (3). Although the complete col- provides clues to how SitA proteins were acquired and diversified. lection of Cystobacterineae isolates that exist in nature obviously For instance, we found that sitA loci that are exclusively found in contains many more than 55 TraA recognition groups, there is one strain were all present on either small genetic islands, where nevertheless a finite number of specificities one receptor family sitAI makes up the majority of the island, or on islands that also can offer, which is clearly much smaller than the number of com- contain mobile genes, e.g., prophage. This implies that these sitAI patible social groups found in nature (13). However, SitA diversity loci were acquired by HGT and, in some cases, are components of adds another layer, with increased resolution and specificity, to prophage elements. Similarly, a sitAI locus was found on the only discriminate nonkin among natural isolates. Indeed, our analysis of known natural myxobacterial plasmid, again suggesting a role in the one TraA recognition group, consisting of 6 genotypes, revealed a selfish element expansion and retention. Our previous study found large constellation of diverse sitAI loci (145 distinct groups pre- that SitA1, 2, and 3 all reside on Mx-alpha prophages located in the dicted). This small sampling of genomes highlights that, within the same chromosomal region of the lab strain (14). In contrast, the 4 Cystobacterineae suborder, there is a vast pool of sitAI loci with families of sitAI loci described here are found at diverse positions unique specificities. Further, these loci can be mixed and matched around the genome, including associations with different and in different genomes in astronomically large possibilities of combi- smaller types of prophage elements. These findings suggest that the nations to serve as exquisitely specific kin discrimination barcodes. global diversity of SitA families arises, at least in part, from dif- In turn, discrimination by a suite of sitAI loci protects against ex- ferent phage elements that carry them. In contrast, there are also ploitation of OME by nonclonemates. Based on the findings that sitAI loci positioned in the same chromosomal location in M. xanthus sitAI loci are numerous and frequently found on genomic islands, and M. virescens strains, and these loci have retained high pairwise it is likely that SitAI diversity has played a major role in the genetic sequence identity. This is despite significant divergence between isolation and diversification of myxobacteria social groups (48). these strains, including the fact that they were isolated from different geographic locations (M. xanthus DK1622, Iowa [28]; M. virescens Methods and Methods DSM 2260, Ontario, Canada [51]). These 10 sitAI loci are thus di- Strains, plasmids, and primers used in this study are described in SI Appendix, versifying slowly, whereas there were only 2 colocalized sitAI loci Tables S1–S3, respectively. Bacterial growth conditions, cloning, strain con- with lower sequence identity, suggesting they diversified rapidly (Fig. struction, competition experiments, and sequence analysis are described in SI 6). Taken together, this suggests that SitAI repertoire, and thus Appendix, Material and Methods. identity barcode, of any one strain is primarily changed by the acquisition of new loci by HGT as opposed to diversifying se- Data Availability. All data and protocols are described in this manuscript, SI lection at existing loci. Appendix, or references therein. Strains, plasmids, and other reagents or information are available upon request. To gain a more comprehensive grasp of the role SitAI proteins play in kin discrimination, we analyzed a relatively large group of ACKNOWLEDGMENTS. We thank Wei Hu and Yue-Zhong Li for M. fulvus Corallococcus strains (43). To initiate this analysis, we predicted 124B02 and the pMF1 plasmid. This work was supported by National Institutes that these Corallococcus strains contained 15 distinct TraA of Health Grant GM101449 (to D.W.).

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