Fig. 4: Distribution and evolution of key Caulobacter crescentus morphogenic genes.

a Schematic illustration of the cell curvature-generating cytoskeletal protein crescentin (CreS) and S-layer cell envelope protein RsaA in C. crescentus. OM outer membrane, PG peptidoglycan, IM inner membrane. b Presence (triangles) of crescentin (creS; K18642; blue), S-layer (rsaA; K12544; orange), and S-layer secretion machinery (rsaDEF; K12533–K12535; black) across Caulobacterales genomes. The phylogeny shows the tree from Fig. 1a. Phen., Phenylobacterium; Acau., Acaudatibacter; Caul., Caulobacter; CAIM., “CAIMFV01”; Brev., Brevundimonas. See Supplementary Data 4 and 5g, h for the full dataset. c ML tree of crescentin (CreS) homologs inferred using IQ-TREE¹⁰⁹ with the Q.pfam + C60 + R9 model of evolution and 1000 ultrafast bootstraps (356 sequences with 902 amino acid alignment positions), and rooted using the major Beta-/Gammaproteobacteria clade. ufBS is indicated with circles and specified with numbers for key branches. Greek letters indicate Pseudomonadota (Proteobacteria) classes, and uppercase letters indicate alphaproteobacterial orders (see Supplementary Fig. S12a). The numbers of sequences per collapsed clade are shown in parentheses. Scale bars here and in (d) indicate the number of substitutions per site in the phylogenies. The full tree is presented in Supplementary Fig. S12a. d ML tree inferred separately for the subset of putative crescentin homologs that form a supported clade (100% ufBS) labeled “Crescentin-like proteins” in the tree shown in (c), using IQ-TREE¹⁰⁹ with the JTT + C60 + R5 model of evolution and 1000 ultrafast bootstraps (135 sequences with 407 amino acid alignment positions). ufBS is indicated with numbers for key branches. Schematic cell silhouettes are drawn roughly in scale (thick scale bar: ~1 µm) based on microscopy images presented in Supplementary Fig. S4 for Brevundimonas goettingensis, C. crescentus, and Chelatococcus reniformis, and from published material for remaining species (Methylocystis parva, Methylocystis sp. Rockwell, and Roseiarcus fermentans; references are listed in Supplementary Table S1). gen. genus, fam. family, ord. order. The full tree is presented in Supplementary Fig. S13. e Representative micrographs (from two independent experiments each) of C. crescentus NA1000 and Ch. reniformis B2974^T from cultures in mid-exponential phase, grown in PYE at 30 °C and R2A at 28 °C, respectively. Scale bars: 5 µm. f Complementation of C. crescentus NA1000 ΔcreS with native creS or the crescentin homolog from Ch. reniformis B2974^T (creS_{Ch.reniformis}) expressed from the xylose-inducible P_xyl promoter on the pBXMCS-4 vector. Representative micrographs (from one experiment) are shown for mid-exponential cultures before (0 h) and after the addition of 0.3% xylose. Scale bars: 5 µm. EV empty vector. See Supplementary Fig. S14 for corresponding experiments done in the wild-type NA1000 background.