Abstract
What mechanisms underlie the transitions responsible for the diverse shapes observed in the living world? While bacteria display a myriad of morphologies1, the mechanisms responsible for the evolution of bacterial cell shape are not understood. We investigated morphological diversity in a group of bacteria that synthesize an appendage-like extension of the cell envelope called the stalk2,3. The location and number of stalks varies among species, as exemplified by three distinct sub-cellular positions of stalks within a rod-shaped cell body: polar in the Caulobacter genus, and sub-polar or bi-lateral in the Asticcacaulis genus4. Here we show that a developmental regulator of Caulobacter crescentus, SpmX5, was co-opted in the Asticcacaulis genus to specify stalk synthesis at either the sub-polar or bi-lateral positions. We show that stepwise evolution of a specific region of SpmX led to the gain of a new function and localization of this protein, which drove the sequential transition in stalk positioning. Our results indicate that evolution of protein function, co-option, and modularity are key elements in the evolution of bacterial morphology. Therefore, similar evolutionary principles of morphological transitions apply to both single-celled prokaryotes and multicellular eukaryotes.
Stalks are a common feature in aquatic bacterial species living in oligotrophic environments3,6. When these species are subjected to nutrient limitation, stalks elongate to increase the effective length and surface area of the cells7, thereby increasing the rate of nutrient uptake2,8. The thin cylindrical stalk is composed of inner and outer membranes separated by peptidoglycan6, and compartmentalized by proteinaceous structures called “cross-bands”9,10 (Fig. 1a). In the Caulobacteraceae family, stalk synthesis occurs at a specific stage of a dimorphic life cycle in which a non-replicating motile swarmer cell differentiates into a sessile stalked cell11 (Fig. 1b). In C. crescentus, the stalk is positioned at a single cell pole; in Asticcacaulis excentricus, the stalk is synthesized at a sub-polar position off-center of a cell pole; and in Asticcacaulis biprosthecum, two stalks are positioned bi-laterally on the cell body4 (Fig. 1a).
The natural variation in stalk location provides an opportunity to study the mechanisms underlying the precise targeting of cell envelope growth zones to generate different morphologies. Stalks in C. crescentus are synthesized from their base12 by insertion of peptidoglycan within a small area of the cell body13,14. To test whether this mechanism is conserved in the Asticcacaulis genus, we used pulse-chase labeling with Texas Red Succinimidyl Ester (TRSE)15,16 to study cell envelope growth and a fluorescent D-amino acid (FDAA) to label regions of peptidoglycan synthesis13. The stalks of A. excentricus and A. biprosthecum are also synthesized by insertion of peptidoglycan at their base (Extended Data Fig. 1a and b), suggesting that all three species share the same stalk synthesis mechanism.
In light of the above results, we hypothesized that if a conserved stalk morphogen exists, it must localize to the base of stalks. Since many proteins localize at the pole in C. crescentus17, we took advantage of the non-polar localization of the stalks in the Asticcacaulis genus to identify stalk morphogen candidates. We constructed fluorescent protein fusions to orthologs of the pole-localized proteins from C. crescentus DivJ, PleC, PopZ, and SpmX and analyzed their localization in A. biprosthecum. Strikingly, only the regulatory histidine kinase DivJ18 (Extended Data Fig. 2a) and its localization and activation factor SpmX5 (Fig. 1c) localized at the base of the stalks in A. biprosthecum. During the cell cycle, A. biprosthecum DivJ-EGFP localized at the base of stalks only after cytokinesis, during swarmer to stalked cell differentiation (Extended Data Fig. 2b). In stark contrast, SpmX-EGFP localized to bilateral positions in the incipient swarmer half of the predivisional cell prior to cytokinesis and subsequent stalk synthesis (Extended Data figure 1c and e). Therefore SpmX localization precedes both DivJ localization and stalk synthesis, potentially marking the future site of stalk synthesis.
Interestingly, while the A. biprosthecum divJ− mutant still synthesized bi-lateral stalks (Extended Data Fig. 2a), the A. biprosthecum spmX− mutant was stalkless (Fig. 2a). Moreover, while newly synthesized peptidoglycan material co-localized with SpmX-GFP in wild-type cells, no bi-lateral foci of FDAA staining were observed in the absence of SpmX (Extended Data Fig. 1h and j), demonstrating that SpmX is required for stalk peptidoglycan synthesis in A. biprosthecum. Finally, stalk elongation only occurred when SpmX was expressed (Extended Data Fig. 1f), suggesting that SpmX is required both for the initiation and the elongation of stalk synthesis in A. biprosthecum. Similar results were obtained for A. excentricus (Fig. 1c, middle; Extended Data Fig. 1d, e, g, i, and k), suggesting that the role of SpmX is conserved in both Asticcacaulis species. Notably, SpmX is not required for stalk synthesis in C. crescentus5. Since the Caulobacter genus diverged earlier than the Asticcacaulis genus (Fig. 1d), we conclude that SpmX has been co-opted for stalk synthesis in the Asticcacaulis genus. However, despite its newly acquired role in stalk synthesis, the ancestral function of SpmX in DivJ localization has been retained in A. biprosthecum (Extended Data Fig. 2c).
To test the hypothesis that SpmX could play a pivotal role in the evolutionary transitions in stalk positioning, we carried out cross-complementation experiments by expressing heterologous SpmXs and SpmX fusions in wild-type or spmX mutant strains of the two Asticcacaulis species and quantitatively analyzed SpmX localization. (Fig. 2 and Extended Data Fig. 3-6). When we cross-complemented SpmX-EGFP in either the homologous or heterologous wild-type backgrounds, SpmX both localized and drove stalk synthesis at its host-specific location, suggesting that the endogenous SpmX may be able to recruit the heterologous SpmX (Extended Data Fig. 4b, c, h, and i). To test this possibility, we expressed heterologous SpmX in absence of the native spmX gene. Strikingly, when SpmX from the sub-polar stalked species A. excentricus (SpmXAE(S)-EGFP) was expressed in the bi-lateral stalked species A. biprosthecum spmX− mutant, it localized to and drove stalk synthesis at a sub-polar position (Fig. 2c; Extended Data Fig. 7). Therefore, A. excentricus SpmX can recruit the heterologous stalk synthesis machinery of A. biprosthecum to synthesize a stalk at an ectopic sub-polar position. In contrast, when SpmX from the bi-lateral stalked species A. biprosthecum (SpmXAB(L)-EGFP) was expressed in the sub-polar stalked species A. excentricus spmX− mutant, it localized mostly to poles where it induced stalk synthesis (Fig. 2d; Extended Data Fig. 7). These results indicate that while the sub-polar positional information exists in A. biprosthecum and can be recognized by SpmXAE(S), the specific bi-lateral positional information present in A. biprosthecum is absent or not recognizable in A. excentricus.
Remarkably, these observations also suggest that A. excentricus possesses the ability to synthesize polar stalks in the absence of its endogenous SpmX. Indeed, phosphate starvation, which stimulates stalk synthesis in wild-type strains of all three species (Extended Data Fig. 2f and g), rescued stalk synthesis in A. excentricus spmX− cells, but stalks were located at the pole (Extended Data Fig. 2e, g, and h). Using holdfast polysaccharide adhesin as a polar marker (Fig. 1c), we found that stalks from phosphate starved A. excentricus spmX− cells were tipped by a holdfast (Extended Data Fig. 2e), confirming that they were synthesized polarly.We infer that A. excentricus possesses an alternative polar stalk synthesis mechanism that is normally masked by the endogenous SpmX-driven sub-polar stalk synthesis mechanism (as detailed in Supplementary Information). In contrast, the A. biprosthecum spmX− mutant remained stalkless when starved for phosphate (Extended Data Fig. 2f), indicating that the spmX− independent pathway for stalk biosynthesis has been lost in A. biprosthecum, or is no longer regulated by phosphate starvation. We conclude that both Asticcacaulis species possess the ability to synthesize stalks at exogenous positions, which is masked by the effects of the endogenous SpmX in wild-type cells. We hypothesized that the exogenous positions of stalk synthesis are phylogenetically ancestral, and we next sought to infer the evolutionary trajectory of stalk positioning.
In order to improve the phylogenetic resolution of stalk positioning, we sequenced the genomes of several additional Asticcacaulis strains (Extended Data Fig. 5f and g) and inferred their phylogeny (see materials and methods; Fig. 1d). Based on parsimony, the emergence of the polar stalk morphology occurred before the divergence between the Caulobacteraceae and the Hyphomonadaceae family (Maricaulis maris and Oceanicaulis alexandrii) (Fig. 1d). No known Asticcacaulis isolates synthesize polar stalks, implying that the transition in stalk positioning from polar to sub-polar occurred very early. In addition, two sub-polar stalked strains, Asticcacaulis benevestitus and Asticcacaulis sp. AC466 (Fig. 1d, bracket), diverged from the same ancestor that led to the sub-clade containing A. biprosthecum, indicating that sub-polar stalk synthesis is ancestral to bi-lateral stalk synthesis. In conclusion, stalk positioning evolved from an ancestral single polar stalk to a single sub-polar stalk, and subsequently to bi-lateral stalks.
We next sought to understand how SpmX has evolved at the protein level by testing the requirement of its major domains for localization and stalk synthesis (Fig. 1e and Extended Data Fig. 8). We constructed a set of truncated alleles removing various domains of A. biprosthecum SpmX, which failed to localize or rescue the stalkless phenotype of the A. biprosthecum spmX− mutant (Extended Data Fig. 7d and e). The muramidase domain and the C-terminal region (intermediate region and transmembrane domains) of SpmX are indispensable for its localization and function. To determine what region of SpmX evolved to specify the location of stalk synthesis, we constructed chimeric SpmX proteins by mixing and matching the muramidase and the C-terminal regions of different SpmX proteins. In each case, the phenotype of the spmX− mutants expressing the various chimeras correlated with the source of their C-terminal region (Fig. 3, Extended Data Fig. 4, and Supplementary Information). We conclude that mutations in the SpmX C-terminal region are responsible for the evolution of SpmX’s ability to drive stalk synthesis from polar to sub-polar to bi-lateral positions.
Morphological transitions generate the diversity of biological forms. A few cases have been studied in eukaryotes, highlighting the importance of both changes in regulatory sequences and functional protein evolution19-23. Our study has begun to unravel the elusive mechanisms of morphological transitions in bacteria by showing that evolution of the SpmX morphogen underlies the evolutionary trajectory of stalk positioning in the Caulobacteraceae family. Polar stalk synthesis arose from non-stalked species before the divergence of the Caulobacteraceae and Hyphomonadaceae families, but C. crescentus SpmX is not required for stalk synthesis, likely representing the ancestral state. Through differential protein evolution, changes in the SpmX C-terminal region led to stalk synthesis and positioning functions in the Asticcacaulis clade. Interestingly, the ancient polar targeting mechanism is conserved in Asticcacaulis since SpmXCC(P) can localize to the pole in both A. excentricus and A. biprosthecum (Fig. 3b, Extended Data Fig. 4a, g and Fig. 9f). Inversely, both SpmXAE(S) and SpmXAB(L) can still localize to the polar target in the C. crescentus strains, suggesting the lack of recognizable alternative targets (Extended Data Fig. 9). During the transition from polar to sub-polar stalk positioning, the C-terminal region of SpmX evolved to position and coordinate the synthesis of stalks, coupled with its co-option at the sub-polar target in A. excentricus (Fig. 4). Further divergence of the C-terminal region of SpmX led to its ability to recognize new targets, combined with its co-option at the bi-lateral targets in A. biprosthecum (Fig. 4).
Our results highlight the modular nature of the positioning mechanism that directs the zonal peptidoglycan synthesis responsible for stalk synthesis. This modularity is evident in both Asticcacaulis species, since SpmX always localizes at the base of ectopically synthesized stalks in several genetically engineered strains (Fig. 2 and 3 and Extended Data Fig. 2d). In addition, the fact that changes in the abundance of SpmX alone can alter the number of stalks in A. excentricus (Extended Data Fig. 2d) suggests that simple changes in the regulation of SpmX expression could drive the evolution of a species with multiple sub-polar stalks. Conceptually, to position the stalk around the cell body, the cells only need to evolve to the ability to localize SpmX to a new sub-cellular position, where it recruits the stalk synthesis module. This morphogenetic modularity could be exploited in synthetic biology to generate the optimal cell shape for a given process.
Finally, this study has demonstrated that functional evolution of a regulatory protein into a morphogenetic module made the evolution of stalk positioning possible, which in turn generated distinct cellular morphologies. Therefore, protein evolution, co-option, and modularity can drive morphological transitions in both single-celled prokaryotes and multicellular eukaryotes, contributing to the diversity of Darwin’s “endless forms most beautiful”24 from the microscopic to the macroscopic world.
Methods Summary
Caulobacter crescentus, Asticcacaulis excentricus, and Asticcacaulis biprosthecum strains were used in this study. Strains were grown in liquid PYE medium at 30°C for C. crescentus and 26°C for the Asticcacaulis strains. A detailed list of strains and plasmids and their methods of construction is provided in the Supplementary Information and the Methods section. For the quantitative analysis of fluorescent protein fusion localization, cells were incubated for 18 hours in the presence of inducer, mounted on a 1% (w/v) agarose pad and imaged. Quantitative sub-cellular localization of fluorescent protein fusions was performed at sub-pixel resolution using a specifically developed plug-in for ImageJ25. For bioinformatics analysis, orthologs of SpmX were identified using the BLAST suite hosted by NCBI and Integrated Microbial Genomes (IMG). Phylogenetic trees were generated using the maximum likelihood method, and a concatenation of the products of six housekeeping genes was used to infer the phylogeny of species involved. All methods are detailed in the Methods section.
Supplementary Material
Acknowledgements
We thank members of the Brun lab and Clay Fuqua for critical comments on the manuscript. We thank David Kysela, Velocity Hughes, and Vidhya Silvanose for their help and efforts in environmental sampling and phylogenetic analysis, Luting Zhuo and Chunfeng Huang for their help in statistical analysis, Sidney Shaw for his advice on quantitative image analysis, Michael Lynch and Rudolf Raff for insightful discussions on evolution, and Matthew Hahn and the Center for Genomics and Bioinformatics at Indiana University for their help in sequencing and bioinformatics analysis. We thank the Indiana University Light Microscopy Imaging Center for their help with OMX super resolution microscopy, supported by National Institutes of Health Grant S10RR028697-01, and the Indiana Molecular biology Institute (IMBI) Electron Microscopy facility at Indiana University for their help with electron microscopy. We thank Martin Thanbichler, Jeanne Poindexter, Paul Caccamo and Patrick Viollier for providing us with Caulobacter strain and plasmids, Jeanne Poindexter, Judy Peterson, John Lindquist, and Alvaro Quinones for help in locating the strain collection of the late Jack Pate from which we obtained some of the A. excentricus and A. biprosthecum strains used in this study, and Mark Wortinger, Sally Green, Ellen Quardokus, and Jennifer (Wagner) Herman for early work with Asticcacaulis that helped set the stage for this study. This work was supported by National Institutes of Health Grant GM051986, National Science Foundation Grant MCB0731950, and by a grant from the Indiana University Metabolomics and Cytomics Initiative (METACyt) program, which was funded, in part, by a major endowment from the Lilly Foundation. P.J.B.B. was supported by National Institutes of Health National Research Service Award AI072992.
Footnotes
Author Contributions
C.J., P.J.B.B., and Y.V.B. designed the experiments. C.J. and A.D. performed the research. C.J., P.J.B.B., A.D., and Y.V.B. analyzed and interpreted the data. C.J. and Y.V.B. wrote the paper. C.J., P.J.B.B., A.D., and Y.V.B. edited the paper.
Accession numbers for new genomic data
Genomic data of strains sequenced in this study are deposited in Genebank under the accession numbers: AWGD00000000 (Asticcacaulis sp. AC460), AWGE00000000 (Asticcacaulis sp. AC466), AWGC00000000 (Asticcacaulis sp. AC402) , AWGF00000000 (Asticcacaulis sp. YBE204), and AWGB00000000 (Asticcacaulis benevestitus DSM16100).
The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.
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