Co-option and Detoxification of a Phage Lysin for Housekeeping Function

Temperate phages constitute a potentially beneficial genetic reservoir for bacterial innovation despite being selfish entities encoding an infection cycle inherently at odds with bacterial fitness. These phages integrate their genomes into the bacterial host during infection, donating new, but deleterious, genetic material: the phage genome encodes toxic genes, such as lysins, that kill the bacterium during the phage infection cycle. Remarkably, some bacteria have exploited the destructive properties of phage genes for their own benefit by co-opting them as toxins for functions related to bacterial warfare, virulence, and secretion. However, do toxic phage genes ever become raw material for functional innovation? Here we report on a toxic phage gene whose product has lost its toxicity and has become a domain of a core cellular factor, SpmX, throughout the bacterial order Caulobacterales. Using a combination of phylogenetics, bioinformatics, structural biology, cell biology, and biochemistry, we have investigated the origin and function of SpmX and determined that its occurrence is the result of the detoxification of a phage peptidoglycan hydrolase gene. We show that the retained, attenuated activity of the phage-derived domain plays an important role in proper cell morphology and developmental regulation in representatives of this large bacterial clade. To our knowledge, this is the first observation of phage gene domestication in which a toxic phage gene has been co-opted for a housekeeping function.

prophages contain genes that benefit the host, promoting retention of prophages in many 47 bacterial lineages, even after mutations have inactivated the prophages and they no longer are 48 capable of producing phage [7][8][9]. Accumulation of host-specific beneficial mutations in 49 prophages has been referred to as "domestication." Many so-called domesticated segments of 50 inactivated prophages unexpectedly contain lytic and phage structural genes, which would 51 intuitively be of little use or even detrimental to the bacterial host [7]. These genes are often used 52 by the bacteria as weapons against competing bacteria and eukaryotic hosts [10][11][12][13][14]. In contrast, 53 we have identified an instance in which a toxic phage gene has not been repurposed as a weapon, 54 but has rather been incorporated into a bacterial housekeeping factor, spmX. Here, we report that 55 SpmX resulted from an ancient domestication event at the root of the alphaproteobacterial order 56 Caulobacterales, in which co-option and detoxification of a toxic phage gene gave rise to a novel 57 bacterial gene with roles in developmental regulation and morphogenesis. 58 SpmX was first identified as a developmental regulator in the model organism 59 Caulobacter crescentus [15]. Like most members of Caulobacterales, stalked C. crescentus cells 60 divide asymmetrically to produce a "mother" cell with a polar stalk and a motile "daughter" or 61 "swarmer" cell with a polar flagellum. The Caulobacter developmental cycle depends on strict 62 coordination of cell growth, chromosome replication and segregation, and division by various 63 regulatory proteins that differ in localization and timing [16]. This network depends on 64 regulatory phospho-signaling factors localized and regulated by polar scaffolds. SpmX is one 65 scaffold that localizes at the old pole during the swarmer-to-stalked cell transition and recruits 66 and potentially activates the histidine kinase DivJ [15]. Intriguingly, SpmX is required for stalk 67 synthesis initiation and elongation in the closely related Asticcacaulis species A. excentricus and 68 A. biprosthecum [17]. Therefore, this gene appears to have evolved multiple roles within this 69 family of dimorphic, stalked bacteria. 70 Perplexingly, SpmX contains an N-terminal putative phage muramidase domain that is 71 generally toxic to bacteria. This enzymatic domain is typically used by phage to lyse bacteria and 72 release infectious phage particles during the lytic cycle. As a part of SpmX, this domain is 73 critical for SpmX's role in both developmental regulation and stalk biogenesis. In particular, the 74 muramidase domain is necessary for proper localization of SpmX in both C. crescentus [15,18] 75 and the Asticcacaulis genus [17]. Various studies have shown that SpmX localizes with the polar 76 scaffold PopZ in C. crescentus [19,20], and this interaction occurs entirely through the 77 muramidase domain [18]. A previous study was unable to measure enzymatic activity from 78 purified C. crescentus SpmX muramidase domain, and consequently concluded that the domain 79 was repurposed for protein interactions and oligomeric assembly [18]. However, given the 80 remarkable sequence similarity of the SpmX muramidase domain to functional phage lysozymes, 81 including the canonical catalytic glutamate, it seems unlikely that its enzymatic activity is 82 entirely lost. Why would this domain be so highly conserved if its new function were merely for 83 non-essential protein-protein interactions? 84 To better characterize the SpmX muramidase domain and the constraints underlying its 85 conservation, we performed an in-depth bioinformatics study of more than 60 available SpmX 86 genes together with structural determination, biochemical analysis, and comparative cell biology 87 between Caulobacter and Asticcacaulis. We show that SpmX arose prior to the diversification of 88 Caulobacterales, a large class of stalked bacteria. We establish that the SpmX muramidase 89 domain is a close relative of GH24 (T4-like) autolysin/endolysins that have been laterally 90 exchanged throughout Proteobacteria via prophages. We find that the SpmX domain is still an 91 active muramidase, albeit with attenuated ancestral phage activity, consistent with its remodeled 92 domain in a bacterial protein that is no longer within prophage and is under direct cellular 139 control. It likely fused with the intermediate and TM domains in a common ancestor of 140 Parvularculales and Caulobacterales. The vertical transmission of SpmX and strong sequence 141 conservation of the muramidase domain suggests an important function in members of 142 Caulobacterales.

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The SpmX muramidase domain retains the canonical GH24 motif but contains mutations 145 in the catalytic cleft known to inactivate phage lysozymes. To determine if critical enzymatic 146 residues in SpmX muramidase were conserved, we compared the amino acid sequences of SpmX 147 and other lysozymes. By definition, lysozymes catalyze the hydrolysis of β1,4-linked glycosidic 148 bonds in peptidoglycan and chitin [25]. This superfamily includes at least seven distinct groups 149 that are unrelated by sequence similarity but share a common fold in which the catalytic Glu and 150 the beta-hairpin motif in the N-terminal lobe form the catalytic cleft by packing against the C-151 terminal lobe ( Fig. 2A) [26]. This beta-hairpin, or GH motif, contains family-specific residues 152 critical for enzyme activity in all members of the lysozyme superfamily [26]. 153 We compared the GH motif of SpmX to lysozymes from the T4L and endolysin/autolysin 154 classes with varying degrees of relatedness. Figure 2B shows the amino acid conservation in the 155 GH motif of T4L-like, autolysin/endolysin, closely related non-SpmX muramidase, and SpmX 156 muramidase protein sequences. Because the autolysin/endolysin class and the closest non-SpmX 157 relatives are likely to be active phage enzymes, highly conserved residues shared by these groups 158 with T4L delineate positions that are evolutionarily constrained for phage lysozyme activity and 159 stability. For example, D10 is not conserved outside of T4L-like enzymes because the 160 autolysin/endolysin class does not have a salt bridge between D10 and the C-terminal lobe [27]. 161 On the other hand, all of the putative phage sequences (Fig. 2B(i-iii)) conserve the "catalytic 162 triad" of T4 lysozymes: the catalytic residue E11 and active site residues D20 and T26. While 163 the exact roles of D20 and T26 are not clear, they are posited as being critical for effective 164 catalysis [26][27][28][29]. Position D20 is very sensitive to mutation, with only substitutions D20C/A 165 retaining the hydrolytic activity of T4L or P22 phage lysozymes [30]; these substitutions are 166 tellingly well represented amongst the putative phage sequences. Remarkably, SpmX 167 muramidase domains demonstrate strong conservation of residues required for T4L stability and 168 creating the GH fold, but low conservation of residues associated with catalysis, with the 169 exception of the main catalytic residue, E11 ( Fig. 2B(iv)). The majority of SpmX genes contain 170 the mutation D20L/R, both of which reduced T4L activity to less than 3% of WT in previous 171 studies [27] and which are distinctly unrepresented in the other phage muramidases. Moreover, 172 the T26 position no longer appears to be under constraint in SpmX. The conservation of the GH 173 motif coupled with the apparent inactivation of the catalytic triad across all SpmX genes suggests 174 that muramidase function may not remain fully active.

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The SpmX muramidase domain has a wider, more dynamic catalytic cleft than related 177 phage lysins. Obtaining the structure of the SpmX muramidase domain (residues 1-150) from 178 Asticcacaulis excentricus (SpmX-Mur-Ae) allowed us to directly visualize the effect of the D20L 179 and T26X mutations on the catalytic cleft. We solved the X-ray crystal structure by molecular 180 replacement using the atomic coordinates of phage P22 lysozyme (PDB 2ANX) to 1.9 Å 181 resolution (Rcryst of 21.1%, Rfree of 25.5%,) ( Table S2). SpmX-Mur-Ae exhibited the expected 182 structural similarity with the phage P22 lysozyme (root mean square deviation (rmsd) of 1.7 Å 183 and 40% identity over 141 aligned Cα atoms, Dali Z-score of 21.5). It therefore shares the 184 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  characteristic structure of T4 lysozymes, with the predicted catalytic glutamate occurring at the  185  C-terminal end of the first alpha-helix and situated in the catalytic cleft formed between the N-186 and C-terminal lobes (Fig. 3A). The active conformation of the distantly related SAR endolysin 187 protein R21 (PDB 3HDE) from bacteriophage P21 (Fig. S2) is included in the structural 188 alignment in Figure 3A to emphasize the ways in which the SpmX muramidase domain deviates 189 from phage lysozymes: SpmX-Mur-Ae contains an extended beta-hairpin in the C-terminal lobe 190 and, more importantly, the canonical GH beta-hairpin in the N-terminal lobe of SpmX-Mur-Ae 191 splays away from the catalytic cleft relative to the GH beta-hairpins of the phage lysozymes.

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There is additional evidence for the flexibility of the GH beta-hairpin in the various 193 conformations observed in the crystallography data, as among the three molecules of  Mur-Ae in the asymmetric unit, this region exhibited the most conformational differences. The 195 overlay of the three chains in Figure 3B illustrates how the orientation of the GH beta-hairpin is 196 tilted by about 16° between chains A and B; moreover, chain A appears to have lost some of its 197 beta-strand secondary structure. That SpmX-Mur-Ae is capable of both conformations suggests a 198 heightened flexibility in this region compared to other T4L-like lysozymes, which may reduce 199 the ability of the enzyme to coordinate the hydrolysis of peptidoglycan in the catalytic cleft.

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Sequence comparisons of the GH motif in Figure 2B show that SpmX muramidase 201 domains have lost a highly conserved tyrosine residue at position 18. Although T4L enzymatic 202 activity is not sensitive to mutation at this position [27], it is invariant across all the phage 203 lysozyme classes we analyzed. Visualization of Y18 in the P22 lysozyme structure (Fig. 3C) 204 shows that it interacts with R14 at the base of the beta-hairpin, possibly a critical interaction for 205 coordinating the beta-hairpin with the catalytic glutamate. In SpmX-Mur-Ae, Y18S still appears 206 to make hydrogen-bonding contact with R14; however, most SpmX muramidase domains have 207 non-polar residues at position 18 ( Fig. 2B(iv)), which may reduce coordination between the 208 catalytic glutamate and the GH beta hairpin. It has been previously shown that the Y18 position 209 is a hot-spot for compensatory mutations that restore activity to inactive catalytic mutants [31], 210 and it is intriguing to imagine that mutations at this position in SpmX muramidase are the result 211 of a remodeled catalytic cleft. 212 Figure 3 shows the catalytic clefts of both P22 lysozyme (D) and SpmX-Mur-Ae (E). In 213 P22 lysozyme, the E11 carbonyl, D20 carboxyl, and T26 hydroxyl groups point into the aqueous 214 catalytic cleft. In SpmX-Mur-Ae, the cleft is slightly reorganized, with the S-methyl thioether of 215 T26M still within 20 Å of E11 and potentially capable of interacting with peptidoglycan. In 216 about two thirds of the SpmX genes, position 26 has either a valine or an isoleucine, which have 217 no polar moieties to contribute to the cleft ( Fig. 2B(iv) (P22Lyso-D20L) (Fig. 4A). The activity curves demonstrate that both SpmX-Mur-Cc and 234 P22Lyso-D20L exhibit similarly attenuated hydrolytic activity in comparison to P22Lyso: both 235 reached maximal levels of RBB release near enzyme concentrations of 15 µM while P22Lyso 236 reached the same levels near 5 µM. Mutants in which the catalytic glutamate was replaced with 237 alanine (SpmX-Mur-Cc-E11A and P22Lyso-E11A) did not exhibit activity (Fig. S4A). Restoring 238 the ancestral D20 (SpmX-Mur-Cc-L20D) did not increase SpmX activity in vitro (Fig. S4B). 239 These data indicate that the "inactivating" substitution D20L attenuates enzymatic activity 240 whereas mutating the catalytic glutamate abolishes it altogether. 241 We were surprised by the activity of P22Lyso-D20L and SpmX-Mur given the reported 242 inhibition of lytic activity of T4L with D20 mutations in phage plaque assays [27]. We were 243 interested in the biological implications of this in vitro activity, and whether the attenuated 244 mutant was active in the periplasmic environment. We therefore designed an experimental 245 system to test the activity of P22Lyso and SpmX-Mur-Cc mutants in the periplasmic space of E. 246 coli. In this system, we expressed the various muramidase constructs with N-terminal PelB 247 leader sequences (pET22b) for periplasmic expression in Lemo21(DE3) cells, which allow 248 tunable expression of toxic products (see Methods). Lemo21(DE3) cells carrying P22Lyso lysed 249 even in the absence of induction (Fig. 4B). In contrast, Lemo21(D32) cells carrying P22Lyso-250 D20L lysed only after induction, and strains carrying SpmX-Mur-Cc and P22Lyso-E11A never 251 lysed ( Fig. 4C and S4C) despite equivalent periplasmic expression levels (Fig. S4D). Different 252 growth conditions and media did not affect the viability of cells expressing SpmX-Mur-Cc ( Fig.  253  S4C). Moreover, SpmX-Mur-Cc was active on sacculi isolated from Lemo21(DE3), eliminating 254 the possibility that it cannot cleave E. coli peptidoglycan (Fig. S4E) with eGFP gene fusions (Fig. 5). We determined the effects of the E11A mutation on cellular 272 morphology, as the ∆spmX strains in all three species have morphological phenotypes ( Fig.  273  5ABCii). In C. crescentus, ∆spmX cells have a characteristic elongated morphology resulting 274 from failed division cycles and often grow stalks prematurely from daughter cells that fail to 275 divide completely (Fig. 5Aii) [15]. In Asticaccaulis, ∆spmX cells lack stalks and do not appear to 276 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; have other developmental phenotypes (Fig. 5BCii) [17]. In C. crescentus, the E11A mutant 277 population contained both WT-like cells and cells exhibiting the division defect, but with less 278 severity than in ∆spmX (Fig. 5Aiii). In both Asticcacaulis species, the E11A mutants still grew 279 stalks (Fig. 5BCiii). Nevertheless, the A. biprosthecum E11A mutant exhibited a significant loss 280 of bilateral stalks (3.5 fold reduction) and an increase in the frequency of cells with a single stalk 281 (Fig. S5D). 282 We used GFP fusions of WT and mutant SpmX to monitor changes in SpmX 283 localization. In all species, WT SpmX localizes at the future position of the stalk, either at the 284 pole as in C. crescentus, or at sub-polar or bilateral positions in Asticcacalis, and is retained at 285 this position (Fig. 5ABCiii). Both C. crescentus and A. biprosthecum spmX E11A mutants 286 exhibited an increase in mislocalized SpmX throughout the cell body compared to WT (Fig.  287  5ABiv). Quantification of the fluorescence data indicated that while the overall mean 288 fluorescence of the cells were the same as WT, the SpmX foci were significantly less intense in 289 the mutants (Fig. S5AB). We also observed a 3X increase in SpmX E11A in the stalks of A. 290 biprosthecum compared to WT (Fig. S5B). Although no difference in focal fluorescence 291 intensity was observed in A. excentricus spmX E11A mutant cells, more cells had a second 292 SpmX focus at the tip of the stalk than WT cells (Fig. 5iv, S5C delocalization phenotypes as E11A (Fig. 5BCiiv). Western blots of cells expressing WT SpmX-298 eGFP and SpmX mutants indicated that the delocalization was not due to clipping (Fig. S5E).

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Overall, these data show that eliminating the catalytic glutamate affects SpmX localization and 300 function. Although it is not clear whether the E11A mutation specifically affects the potential 301 hydrolytic activity or the peptidoglycan-binding ability, the similar phenotype from mutating a 302 predicted peptidoglycan-interacting residue (N105R) underscores the importance of SpmX-303 peptidoglycan interactions. That the catalytic mutant has an intermediate morphological 304 phenotype in C. crescentus and one Asticcacaulis species indicates that the muramidase domain 305 may coordinate SpmX functions similarly in the two genera and that this function may rely on its 306 enzymatic activity. 307 308 Replacing the muramidase domain, or removing it, interferes with native SpmX protein 309 levels in vivo. In an effort to understand the role of the highly conserved muramidase fold in 310 SpmX function, we made chimeras wherein P22 lysozyme replaced the muramidase domain of 311 SpmX in C. crescentus (P22Lyso-SpmX). While similarly made constructs with SpmX and the 312 SpmX-E11A mutant exhibited the previously determined morphological and delocalization 313 phenotypes ( Fig. 6Aii-iii), replacing the SpmX muramidase domain with P22 lysozyme 314 phenocopied the parent ∆spmX strain. However, we were unable to detect any sfGFP-fusion 315 products in these chimeras by Western blot (Fig. 6B). We confirmed that P22Lyso-SpmX had 316 been correctly inserted in these strains by sequencing, suggesting that the chimeras were 317 expressed but degraded quickly in C. crescentus. Therefore the phenotype of this chimera is due 318 to loss of SpmX and not the addition of the P22 lysozyme domain. Inactivation of P22Lyso 319 (E11A) did not change the outcome, suggesting that the toxicity of the phage muramidase was 320 not driving degradation of SpmX. Deletion of the muramidase domain from the spmX locus in all 321 three species also resulted in strains that failed to produce detectable amounts of ∆mur-SpmX-322 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; sfGFP by Western blot (Fig. S5E). These results suggest that the SpmX muramidase domain is 323 necessary to produce and/or maintain WT levels of SpmX in all three species, and that phage 324 muramidase P22Lyso, with high sequence similarity (51%) and structural homology (RMSD 1.7 325 Å), is not sufficient to replace it. 326 Given that P22Lyso and SpmX muramidase are fairly distant relatives despite high 327 sequence conservation, we tested the ability of other SpmX muramidase domains to replace that 328 of C. crescentus. First, we attempted to restore the phage active site D20 (L28D in SpmX 329 numbering) in the native SpmX gene, but this had no evident effect on SpmX localization or cell 330 morphology (Fig. 6iv). Previously, the muramidase domains of C. crescentus and Asticcacaulis 331 were shown to be interchangeable [17], so we extended the sequence distance to SpmX 332 muramidases from the next closest relative B. subvibrioides, which has D20R in the catalytic 333 cleft, and the most distant relative P. bermudensis, which shares the D20L mutation. We found 334 that the muramidase domain from B. subvibrioides supported the WT phenotype in C. crescentus 335 (Fig. 6v), but that the SpmX muramidase domain from P. bermudensis did not. We were 336 surprised to see no evidence of delocalization in the B. subvibrioides SpmX chimera because the 337 L20R mutant of SpmX in C. crescentus showed some delocalization (Fig. 6vi) prophage exist in the literature. We have investigated the origin and function of a taxonomically 354 restricted gene from Caulobacterales, spmX, and determined that its occurrence is the result of 355 the fusion and domestication of a peptidoglycan hydrolase gene commonly found in prophage.

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Despite the previously demonstrated role of SpmX as a scaffold in developmental regulation and 357 morphology, the muramidase domain retains high sequence similarity to phage lysozymes, 358 which are toxic to bacteria. The active cleft contains mutations that have attenuated the toxic 359 activity of the domain, presumably making it available for genetic innovation and bacterial use. 360 We show here that the domain remains enzymatically active on peptidoglycan and that reducing 361 or eliminating this activity alters the function of the full-length protein in vivo. Thus, the SpmX 362 gene represents a bacterial gene innovation, specific to the Caulobacterales order and with 363 housekeeping function, that originally arose from a prophage gene with antibacterial activity. 364 Previously, it was suggested that the SpmX muramidase domain functions only in 365 protein-protein and self-oligomerizing interactions in SpmX's role as a developmental regulator 366 and scaffold in C. crescentus [18]. This conclusion was based on the lack of detectable activity 367 from the purified domain and the inability of the catalytic E11R (E19R in SpmX numbering) 368 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; mutant to self-oligomerize. The conservation of the GH fold and the catalytic glutamate clearly 369 suggests that the activity of SpmX is important for its domesticated function, and, importantly, 370 we were able to observe muramidase activity in vitro with purified protein. It is highly likely that 371 the E11R mutation greatly destabilizes the structure of the muramidase domain. We found that 372 the E11A mutant eluted in multiple fractions during purification, indicating decreased 373 conformational stability. Moreover, the E11R mutant exactly phenocopies the ∆mur-SpmX 374 phenotype in vivo in that the protein product was no longer detectable in the cells expressing the 375 gene [18]. In this sense, the effect of the E11R mutation is similar to using a distantly related 376 muramidase domain (like P22Lyso) or deleting portions of the muramidase domain entirely. 377 These data indicate that the muramidase domain plays an unanticipated role in maintaining stable 378 levels of SpmX protein across all tested species: without the muramidase domain, the protein is 379 misfolded or misprocessed and is therefore quickly degraded. 380 Inactivating the SpmX muramidase domain resulted in developmental defects in C. 381 crescentus and significant loss of the bilateral stalk morphology in A. biprosthecum. It is curious 382 that inactivating the enzymatic domain did not yield a null phenotype or complete delocalization. 383 Since SpmX-E11A and N105R still bind to sacculi in vitro, it is possible that enough 384 peptidoglycan-interactions are maintained in the mutants for partial SpmX function. It is also 385 possible that SpmX recruits another protein with redundant enzymatic activity that cannot be 386 recruited in the ∆spmX mutant, as it is already known that SpmX interacts with targeting factors 387 via its C-terminal domains in Asticcacaulis [17] and possibly via its transmembrane segments 388 with DivJ [15]. Finally, it is hard to distinguish whether there is a direct relationship between 389 catalytic activity and peptidoglycan binding, and therefore localization, or if cleavage of 390 peptidoglycan by SpmX could indirectly impact SpmX localization. The multiple domains and 391 pleiotropic effects of SpmX make it difficult to assess the effects of an individual domain on its 392 in vivo function. However, our data support a model in which the muramidase domain of SpmX 393 is still active, and this activity is used to localize SpmX directly or indirectly.

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SpmX emerges in the genomic record at the root of Caulobacterales with the attenuating 395 D20L mutation already in the muramidase domain (Fig. 1B). The D20L mutation is therefore 396 ancestral and potentially the initial step in the co-option of the domain. The conservation of 397 D20L throughout most of Caulobacterales suggests evolutionary constraint on this position 398 despite no observable phenotype from the SpmX-Mur-Cc L20D reversion mutation in vivo or in 399 vitro. There are several modifications to the active cleft, including the loss of selection on the 400 third catalytic triad position, T26, and the invariant residue Y18. This pair is interesting in that 401 Y18 was identified as a hotspot for spontaneous second site revertants of T26 mutants in T4L 402 [31]. It is possible that the scatter we see at these two positions are compensatory mutations 403 retaining attenuated activity, although a clear history of covariation is not clear. In two groups, 404 SpmX has diverged from the ancestral D20L mutation: the marine genera Oceanicaulis and 405 Maricaulis (D20R/G), and the freshwater/soil genus Brevundimonas (D20R) (Fig. 1B, S1). 406 Interestingly, D20R is covariant with residue N105 (Fig. S1), which is a peptidoglycan-407 interacting residue in T4 lysozyme [33]. The covariance of peptidoglycan-interacting residues in 408 these diverging genera further underscores the importance of this domain in peptidoglycan 409 interactions, rather than just protein-protein interactions. 410 The SpmX gene has arisen recently enough to see the hallmarks of novel gene emergence 411 and adaptation in a constrained bacterial clade. The gene either arose from a fusion event in the 412 bacterial genome, or the original phage gene contained the transmembrane segments.

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Domestication of the muramidase appears concomitant with the occurrence of the fused SpmX 414 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; domains in the extant genetic record. Maintenance of the muramidase domain since the 415 emergence of SpmX and its activity in current living members of Caulobacterales suggest that its 416 activity was selected for in the ancestral protein and still implicated in its modern function. 417 Given the variability of the intermediate domain throughout Caulobacterales (Fig. 1A), this 418 domain is under few constraints and appears to have undergone multiple independent events of 419 elaboration and reduction in this order. This region of charged residues and prolines drives 420 SpmX self-oligomerization in vitro [18], and may also facilitate other protein interactions. infection. In one strange case, a phage lysozyme gene has been co-opted in bivalve genomes, 444 which apparently still use the gene for its antibacterial properties [52]. 445 The domestication of the muramidase domain in SpmX is distinct from the above cases 446 of "guns for hire" because the phage gene has been incorporated into a novel bacterial gene with 447 new function in basic cellular processes. The SpmX muramidase domain, although active, is no 448 longer used to lyse bacterial cells. Instead, the active domain plays a role in localizing the SpmX 449 protein for its function in developmental regulation and morphogenesis and has become part of a 450 core gene in Caulobacterales. The co-option of phage genes for housekeeping function is likely a 451 common event in nature, but identifying such genes may require a careful search. We suggest 452 that the characteristics of SpmX may recommend future strategies for their detection: searching 453 for phage gene homologues that have long histories of vertical inheritance and signs of bacterial 454 innovation.

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Acknowledgements 457 We thank members of the Brun, Vernet, and Dessen laboratories for support, advice and 458 encouragement. Many thanks to members of the Brun Laboratory, Ernesto Vargas, Farrah 459 Bashey-Visser, Jay Lennon, Daniel Schwartz, and Breah LaSarre for critical reading and editing 460 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  BLAST hits from non-SpmX muramidases (n = 60), and (iv) SpmX muramidases (n = 66), and 490 organized in a cladogram to resemble the sequence cluster tree diagram in Figure S2. Amino 491 acids are color-coded according to chemical properties, with uncharged polar residues in green, 492 neutral residues in purple, basic residues in blue, acidic residues in red, and hydrophobic residues 493 in black. The height of each letter is proportional to the relative frequency of a given identity and (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    This study 7380 pET22b-spmX-Mur-Cc-E19A (Amp) "E11A" This study . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This tool uses reverse position-specific BLAST, a method that compares query sequences to 635 databases of position-specific score matrices and obtains E-values, such as in PSI-BLAST 636 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. and EcoRI to insert P22Lyso. In all pET28a plasmids, the constructs were cloned in frame with 665 the N-terminal His-tag and a stop codon to eliminate the C-terminal His-tag. For pET22b, the 666 vector was linearized with EcoRI and the C-terminal His-tag was preserved. Point mutants in 667 expression vectors were obtained by using standard quick-change procedures and primers with 3' 668 single stranded overhangs for increased efficiency. 669 Integrating plasmids for allelic exchange (pNPTS138): For allelic exchange, the desired 670 mutation was engineered into pNPTS138, bracketed by 1 kb up-and downstream of the 671 corresponding genetic region. Integrants were isolated by antibiotic selection and secondary 672 recombination events were selected by sucrose counter-selection using standard procedures. The 673 resulting clones were confirmed by PCR and sequencing isolated genomic DNA. 674 For genomic deletions of spmX in Asticcacaulis, pNPTS138 was linearized with EcoRI 675 and codons on either end of the gene were retained to avoid introducing frame-shifts in the 676 surrounding area. Therefore the final gene deletion in A. excentricus lacks residues 5-808 and in 677 A. biprosthecum lacks residues 5-815. For SpmX∆mur truncations, resides 2-150 were removed 678 in all three species. In all cases pNPTS138 was linearized with EcoRV, except for SpmXAb-679 E19A, where pNPTS138 was linearized with EcoRI. Point mutations E19A and N91R were 680 integrated into the ∆spmX background for ease of clone isolation and included full-length SpmX 681 flanked by 1 kb genetic context. pNPTS138 containing mutated SpmX were constructed using 682 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; Gibson cloning with fragments on either side of the intended mutation and overlapping primers 683 containing the mutation amplified from genomic DNA. In all cases pNPTS138 was linearized 684 with EcoRV, except for SpmXAb-E19A, where pNPTS138 was linearized with EcoRI. 685 Integrating plasmids for insertional eGFP fusions (pGFPC): The last 600 bp of spmX 686 from C. crescentus was amplified from genomic DNA and cloned into pGFPC-2 using Gibson 687 cloning.

688
Integrating plasmids for replacement at the ∆spmX locus (pMCS-2). These constructs 689 were designed to allow insertion of various SpmX mutants fused to C-terminal sfGFP into the 690 ∆spmX locus in C. crescentus. For SpmX-sfGFP and SpmX-E19A-sfGFP, fragments containing 691 1 kb of genomic DNA upstream of spmX and spmX or spmX-E19A were amplified from existing 692 pNPTS138 constructs and fused to a fragment containing monomeric sfGFP amplified from and sfGFP with the same linker. P22Lyso and P22Lyso-E11A were amplified from pET28a 698 plasmids containing these genes. Chimeras with SpmX muramidase from Brevundimonas 699 subvibrioides (residues 1-140) and Parvularcula bermudensis (residues 1-168) were similarly 700 made with the muramidase fragments amplified from genomic DNA and synthetic gene strands 701 (Eurofins), respectively. Cleavage reactions were passed through Ni-NTA resin to remove free His-SUMO tag and His-722 Ulp1, and untagged protein was collected in the flow through. Flow-through fractions were 723 concentrated with Amicon Ultra Centrifugal filter units with a molecular weight cutoff of 10 kDa 724 (Millipore) and were injected onto an ENrich TM SEC650 10x300 gel-filtration column (Biorad). 725 SpmX-Mur-Ae was eluted with buffer D (25 mM Tris-HCl (pH 8.0), 150 mM NaCl) and again 726 concentrated with Amicon Ultra Centrifugal filter units. Protein concentration was measured 727 using absorbance at 280 nm. 728 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

729
Protein crystallization and structure determination. High-throughput crystallization trials 730 were performed with a Cartesian PixSys 4200 crystallization robot (Genomic Solutions, U.K.). 731 Hanging drops containing 100 nl of protein (25 or 12.5 mg/ml) and 100 nl of reservoir solution 732 were set up in 96-well Crystal Quick plates (Greiner) and incubated at 20°C. Initial crystal hits 733 were refined manually by setting up hanging drops containing 1 µl of protein (25 or 12.5 mg/ml) 734 and 1 µl of reservoir solution in 24-well plates (Molecular Dimensions) incubated at 20°C. 735 Large needle-shaped crystals (dimensions of about 40 x 40 x 400 µm) were finally obtained for 736 SpmX-Mur-Ae in 0. deposited at the Protein Data Bank (PDB, http://www.rcsb.org) and were assigned PDB entry 755 code 6H9D. The data collection and refinement statistics are summarized in Table S2).

757
Protein production for in vitro assays. Fresh BL21(DE3) competent cells (Novagen) were 758 transformed with pET28a constructs containing various muramidase genes with N-terminal His-759 tags (Table S3) and grown overnight in LB with 1% glucose and 50 µg/mL kanamycin. 760 Overnight cultures were diluted 100-fold in LB medium with 1% glucose and 50 µg/mL 761 kanamycin. Typically 500 mL cultures of cells were grown for 1.5-2 hours to an OD600 of 0.6-762 0.7 and shifted to 20°C. When the OD600 reached 0.8−0.9, the cells were induced with 0.5 mM 763 IPTG. After growing for 4 h at 20 °C, cells were harvested and resuspended in 30 mL lysis 764 buffer (25 mM HEPES pH 7.5, 100 mM NaCl, 20 mM imiadazole, 5 mM BME) with a EDTA-765 free Protease Inhibitor Mini Tablet (Pierce) and phenylmethanesulfonyl fluoride (PMSF, 1 mM). 766 The 30 mL cell mixture was lysed on ice using a sonicating horn and spun down at 10,000g for 767 20-30 min. The clarified lysate was loaded onto a 5 ml HiTrap Chelating HP cartridge (GE 768 Healthcare) charged with Ni 2+ and pre-equilibrated with lysis buffer. After loading, the column 769 was washed with lysis buffer followed by an elution via a 0-100% linear gradient of buffer B (25 770 mM HEPES pH 7.5, 100 mM NaCl, 500 mM imidazole, 2 mM BME). Muramidase-containing 771 fractions were pooled based on SDS-PAGE analysis and concentrated to 2.5 mL. Imidizole was 772 removed by passing the concentrated fraction over a PD10 desalting column (GE Healthcare) 773 equilibrated with 25 mM HEPES pH 7.5, 100 mM NaCl, 2 mM BME. 774 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

775
Sacculi preparation and RBB labeling. Sacculi were prepared from all species in the same 776 manner. For a typical 2L prep, cells were grown to an OD of 0.5-0.7 in their respective medium 777 (see bacterial strains and growth conditions) and harvested by centrifugation at 6,000g for 20 778 minutes. C. crescentus cells usually required multiple centrifugation steps to collect all the cells. 779 Cells were resuspended in 25 mL water (or PBS for E. coli) and added drop-wise into 50 mL of 780 boiling 7.5% SDS under stirring. The mixture was boiled for 30 minutes and then allowed to 781 cool to room temperature. Sacculi were then pelleted by ultracentrifugation at 100,000g for 30 782 minutes at room temperature. The resulting pellets were resuspended in 100 mL pure water, and 783 washed repeatedly until SDS was no longer detected in the supernatant. The pellet was 784 confirmed to be clear of SDS by mixing 0.2 mL of the supernatant with 1 uL 0.5% methylene 785 blue, 0.1 mL 0.7M NaPO 4 pH 7.2, and 0.6 mL chloroform and checking to make sure that, after 786 vortexing and allowing to settle, the solution had an upper blue phase and a lower clear phase 787 [82]. At this point, the pellets were resuspended in 10 mL PBS with 20 mM MgSO 4 , 250 U/uL 788 Pierce Universal Nuclease (Thermo Fisher Scientific), and 10 mg/mL amylase (Sigma). The 789 mixture was incubated at 37°C for 1-4 hours. Afterwards, 10 mg/mL trypsin and 10 mM CaCl 2 790 was added and the mixture incubated overnight at 37°C. 800 uL of 15% SDS were then added to 791 the mixture and it was brought to a boil for about 10 minutes and allowed to cool to room 792 temperature. The sample was then pelleted (100,000g, 30 min, room temperature) and 793 resuspended in 4-5 wash steps until SDS was no longer detected. The final pellet was then 794 resuspended in 2 mL water and added to 0.8 mL 0.2M remazol brilliant blue (Sigma), 0.4 mL 795 5M NaOH, and additional water to 8 mL. The mixture was incubated, shaking, overnight at 796 37°C. After neutralizing the solution with 0.4 mL 5M HCl. The mixture was then pelleted 797 (21,000g, 20 minutes, room temperature), and resuspended in water until the supernatant became 798 clear.

799
To calibrate the concentration of RBB-labeled sacculi for dye-release assays and 800 peptidoglycan-binding assays, activity curves with Hen Egg White Lysozyme (Sigma) were 801 produced using different dilutions of the RBB-labeled sacculi. The RBB-labeled sacculi were 802 used at the dilution that resulted in an A595 of 0.5 when 5 µL of the RBB-labeled sacculi were 803 incubated with 4 uM HEWL. 804 805 Peptioglycan-binding assays. 5µL of calibrated RBB-labeled sacculi were incubated with 1 µM 806 of purified protein (protein constructs used are shown in Fig. S4 normalized against absorbances measured for reactions with HEWL that were run in tandem for 819 every measurement to correct for differences in different sacculi preparations. 820 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. visualized on 1% agar pads made with PBS using the methods described in microscopy. 866 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  sequences of the muramidase domains from genes listed in Table S1, a subset of which were 883 used for the gene tree in Fig. 1. with SpmX-Mur-Cc-E11A, the catalytic mutant. Due to poor expression and instability, SpmX-907 Mur-E11A was not concentrated to higher than 12 µM and its activity is compared to WT SpmX 908 at 12 µm. Error bars are ± standard deviation from two replicates. (B) RBB assays with SpmX-909 Mur-Cc-L20D, the mutant that restores the ancestral D20. showed signs of spheroplast formation and cell lysis but the other tested constructs did not. 912 . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Compare to growth curves and microscopy shown in Fig. 4CD. (D Normalization is to Hen Egg White Lysozyme (HEWL) assays (see methods). 919 920 Figure S5.  . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018;   SpmXMur-Ae . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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The copyright holder for this preprint . http://dx.doi.org/10.1101/418723 doi: bioRxiv preprint first posted online Sep. 16, 2018; CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

A B
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