A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales

ABSTRACT The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria. Using Rhodobacter sphaeroides, we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides’ divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria. Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.


FIG S1
Predicted lipoprotein signal peptide of DalA.SignalP 6.0 was used to predict the presence of lipoprotein signal peptide and the cleavage site of protein DalA.

FIGFIG S3
FIG S2DalA-mCherry binds PG. (A) Average growth curves of strains dalA-mCherry (dashed orange line), dalA-ng (green line) and WT (purple line), obtained from 4 independent cultures grown at 30°C.(B) Western blot analysis of in vitro binding of DalA to PG. DalA-mCherry was incubated with or without PG on ice 30 min then centrifuged.The supernatant fraction was recovered (SN) after a first centrifugation and the PG pellet washed 3 times.The first wash fraction (W) and the pellet (P) containing the PG were recovered and separated by electrophoresis.

FIG S4
FIG S4 Pearson correlation of fluorescence signals.Pearson correlation coefficients were calculated to quantify the colocalization of different fluorescence signals using MicrobeJ.In dalA-ng cells, coefficients were calculated between signals DalA-NG and HADA and as a control between DalA-NG and FM4-64.Coefficients of DalA-NG and Pal-mCherry were calculated in cells containing both proteins.Finally, a strain containing dalA-ng and ftsZ-mCherry was used to test the correlation between these two proteins.The values range from -1 (anti-correlation) to 1 (perfect correlation, 0 being no correlation) and are represented as violin plots.Black dots represent the mean of each biological replicate, and the red dot is the global mean with the corresponding value as text aside.P values were computed from mean values of biological replicates by unpaired two-tailed t-tests with the control DalA-NG/FM4-64 as reference group (***= p ≤ 0.001).
FIG S6 ∆pal impacts DalA levels and localization.(A) Box plot quantification of levels of DalA-NG obtained from western blots with cells dalA-ng, ∆pal, and ∆pal+pIND5 dalA-ng (downstream of its native promoter).Black dots represent intensity value from each replicate and red dot is the average.(B) Fluorescence microscopy image of ∆pal cells containing pIND5 dalA-ng.Scale bar represents 2 µm.
FIG S7 ftsZ-mCherry Z-rings localization and dalA-mC.(A) The relative position of membrane constrictions in cells was determined in ftsZ-mCherry and WT strains.For WT cells, FM4-64 was used to ensure the presence of membrane constrictions.The distribution of the relative distance of constrictions from midcell is displayed as violin plots, 0 being at the midcell and 0.5 at the pole.(B) Micrographs showing DalA-mCherry fluorescence signal in strain dalA-mCherry (right), scale bars represent 2 µm.Corresponding demograph (left) of DalA-mCherry fluorescence profiles measured along long axis, cells were sorted longitudinally by length and laterally by pole intensity.

FIG
FIG S9 MreB and FtsZ localization in R. sphaeroides.(Left) Micrographs showing brightfield channel and mCherry-MreB fluorescence of cells containing a mCherry-mreB fusion.(Right) Demograph of the mCherry-MreB fluorescence in cells containing a mCherry-mreB fusion and FtsZ-YFP fluorescence in cells containing pIND5 ftsZ-YFP.For cells containing pIND5 ftsZ-YFP, 10µM IPTG was added to cultures to increase expression of the ftsZ-YFP fusion.Cell profiles were sorted by length from top to bottom and from left to right to display the most intense pole on right.Scale bar represents 2 µm.

FIG
FIG S11Phylogenetic tree of DalA homologues.Homologues amino acid sequences were obtained by Blastp against a database of 450 unique Alphaproteobacteria with sequenced genomes.Protein sequences were aligned using MUSCLE and tree was inferred by using the Maximum Likelihood method and JTT matrix-based model.Analyses were conducted in MEGA X software(1)

Table S1 :
Strains, plasmids and primers used