BUSCADOR RECOLECTA

(a) Reconstruction of deuterostome ALGs R and B1 based on pairwise comparisons. Using the same logic as described for S6 Fig, sea star POC12 and POC18 appear to correspond to sea urchin SPU3 and SPU17, respectively (b), supporting the conclusion that their LCA possessed these 2 chromosomes (echinoderm ALGs R and B1). Comparison between hemichordate PFL and echinoderm species revealed that POC12/SPU3 and POC18/SPU17 correspond to a single hemichordate chromosome PFL9 (c and d). This observation suggests a fusion event occurred in the ambulacraria ancestor leading to PFL9 or a split event leading to POC12/SPU3 and POC18/SPU17. Using amphioxus BFL as an outgroup, the analysis showed that POC18/SPU17 corresponds to BFL10 (e and f), while amphioxus orthologs of POC12/SPU3 genes spread in the genome and no single BFL chromosome could be assigned to POC12/SPU3. Another outgroup scallop PYE was then used, revealing that POC12/SPU3 and POC18/SPU17 respectively correspond to PYE13 and PYE12 (h and i). Based on these comparisons, 3 major inferences can be made: (1) both deuterostome and ambulacraria ancestors possessed the 2 distinct chromosomes (deuterostome/ambulacraria ALGs R and B1); (2) at least in the LCA of hemichordates PFL and SCA, ALGs R and B1 were fused, leading to PFL9/SCA5; (3) in amphioxus, orthologous genes of deuterostome ALG R were dispersed to other chromosomes. Notably, in addition to POC12/SPU3, PYE13 also corresponds to POC22/SPU15, explaining the comparability between PYE13 and the hemichordate PFL1 and amphioxus BFL4 (j and k) and suggesting a fusion event led to PYE13. Consistent with this idea, the hemichordate PFL9 (fused from ALGs R and B1) corresponds to BFL10 (ALG B1) (g). It has been proposed that all chromosomes of vertebrates correspond to amphioxus chromosomes [19,20], suggesting that one ancestral chromosome (ALG R) spread to other chromosomes in the LCA of chordates. The scallop chromosome name was labeled and sorted according to chromosome size. Here, PYE12 is chromosome number 13 and PYE13 is chromosome number 12 in the previous study [25]. The data underlying this figure can be found in S1 Data., Peer reviewed

(a) Reconstruction of deuterostome ALGs J2, C1, A2, A1, I, and O1 based on pairwise comparisons among amphioxus BFL, hemichordate PFL, sea urchin SPU, sea star POC, and scallop PYE. First, the comparison of POC with SPU showed that POC16, POC8, POC21, POC1, POC11, and POC22 have one-to-one correspondence with SPU10, SPU13, SPU2, SPU5, SPU11, and SPU15, respectively (b), suggesting that these 6 chromosomes were already present in their LCA (echinoderm ALGs J2, C1, A2, A1, I, and O1). These 6 chromosomes also have one-to-one correspondence with hemichordate PFL5, PFL21, PFL3, PFL14, PFL20, and PFL1 (c and d), indicating that the existence of these 6 chromosomes could be traced further back to the ambulacrarian LCA (ambulacraria ALGs J2, C1, A2, A1, I, and O1). Comparisons with the amphioxus BFL genome showed that both POC16/SPU10/PFL5 and POC8/SPU13/PFL21 correspond to a single amphioxus chromosome BFL2 (e–g). Similarly, POC21/SPU2/PFL3 and POC1/SPU5/PFL14 correspond to amphioxus BFL1; POC11/SPU11/PFL20 and POC22/SPU15/PFL1 correspond to amphioxus BFL4. To infer the deuterostome ancestral condition, scallop PYE was used as an outgroup. This analysis showed that the 6 ambulacraria chromosomes correspond to 6 distinct PYE chromosomes (PYE4, PYE9, PYE16, PYE5, PYE11, and PYE13, see h–j), supporting the conclusion that these 6 chromosomes are ancient and were present in the deuterostome LCA (deuterostome ALGs J2, C1, A2, A1, I, and O1). Accordingly, the 3 amphioxus chromosomes (BFL 2, BFL1, and BFL4) correspond to the aforementioned 6 PYE chromosomes (k). Therefore, the amphioxus BFL2, BFL1, and BFL4 were formed from respective fusion events between deuterostome ALGs J2 and C1, ALGs A2 and A1, and ALGs I and O1. These 3 fusion events are likely amphioxus-specific because the 6 deuterostome ALGs correspond to 6 vertebrate ALGs [19,20], which support the notion that these 6 chromosomes remained intact in the LCA of chordates (chordate ALGs J2, C1, A2, A1, I, and O1). The data underlying this figure can be found in S1 Data., Peer reviewed

(a) A Hi-C contact map of P. flava genome assembly based on the HiC-pro pipeline [91]. Note that the 3′ end of PFL3-1 interacts with the 5′ end of PFL3-2, and the 5′ end of PFL3-1 interacts with the 3′ end of PFL3-3 (green arrows). The boxed area is magnified to show the chromosomal interactions around the PFL chromosome 23. (b) The 3′ end (right side) of PFL23-1 interacts with the 3′ end of PFL23-2 (blue arrow), suggesting that the 2 scaffolds are closely linked at their 3′ ends. These 2 scaffolds also highly interact with several smaller scaffolds (blue arrowheads). Similarly, the 3′ end of PFL3-4 interacts with the 5′ end of PFL3-3 (green arrow). Based on the contact information, PFL3-1 to PFL3-4 were assembled in the order of PFL3-4, PFL3-3, PFL3-1, and PFL3-2. PFL3-2 and PFL3-3 also interact with several smaller scaffolds (green arrowheads). P. flava chromosome #3 (PFL3) was thus assembled by joining PFL3-1 to PFL3-4; PFL23 was assembled by joining PFL23-1 and PFL23-2. The data underlying this figure can be found in S5 Data., Peer reviewed

Cartoon representation of the dimers observed in the crystal structures of Rap3T, Rap105, RapJ (PDB 4GYO), RapH (PDB 3Q15), RapI (PDB 4I1A), and RapF (PDB 4I9E). All proteins show a similar dimerization mode interacting mainly with the TPR domains (orange-yellow) mainly through the C-terminal helix (in tones of red) with the 3HB domains (blue tones) facing outward from the dimer., pbio.3002744.s003.tiff, Peer reviewed

(a) RMSD calculation from the superimposition of Cα atoms of Rap3T structures in complex with peptides SRGHTS, RGHTS, RRGHTA, and RRGHTAS. RMSD were calculated from individual monomers (lower) and dimers (upper). Number of atoms used in RMSD calculation is showed between parenthesis. (b) Superposition of 4 Rap3T-Peptide complexes with different pheromone variants in monomeric state RRGHTAS (blue), RRGHTA (pink), RGHTS (light green), and SRGHTS (orange). (c) Superposition of dimers for the 4 Rap3T complexes with peptides RRGHTAS (blue), RRGHTA (pink), RGHTS (light green), and SRGHTS (orange)., pbio.3002744.s004.tiff, Peer reviewed

pbio.3002744.s005.tiff, Peer reviewed

Peer reviewed

Peer reviewed

Disc diffusion assay for testing fosfomycin resistance of WT AB5075, ΔcavA deleted mutant, vfr::Tn transposon mutant and their complemented derivative strains (ΔcavA+vfr and vfr::Tn+vfr respectively), as well as ΔcavA mutant overexpressing vfr (ΔcavA+vfr). CAMH agar inoculated with each corresponding strain and having a fosfomycin (50 μg) disc were assessed after 24 h at 37°C. Representative images (A) and the measured zone of inhibition in millimetres (B) are shown. Data represents the averages ± SD of biological triplicates. ns p>0.05, *p<0.05, ***p<0.001, ****p<0.0001 –One-Way ANOVA with Dunnet post-hoc test (B). All controls used are presented in S10B Fig, Peer reviewed

Cyclic di-GMP levels measured using CensYBL-Ab biosensor in ΔcavA mutant with low cAMP and ΔcavA+cavA complemented strain with high cAMP levels (A) as well as vfr::Tn mutant (B) compared to WT AB5075. Deletion of cavA reduced c-di-GMP levels by 66% (A) while disruption of vfr decreased them by 49% (B). Cultures were diluted in LB broth supplemented with apramycin (100 μg/ml) and biosensor expression was induced with anhydrotetracycline (50 ng/ml). Harvested cells were resuspended in sterile PBS and YFP and mCherry fluorescence signals were measured. The average normalised fluorescence (YFP/mCherry) ± SD from three independent biological repeats is presented. Data was analysed using One-Way ANOVA with Tukey post-hoc test (A) and Unpaired t-test (B) (* p<0.05, ** p<0.01). Strains harbouring empty miniTn7 were used as controls (S9B Fig)., Peer reviewed