Investigation of the ParB_Bb features near the CTP binding pocket and their impact on <i>parS</i>_<i>Bb</i> recognition.

Related to Fig 4. (A) parSBb sequence matches with the consensus. Top: parS sequence from B. bacteriovorus. Bottom: a sequence logo generated by WebLogo 3.0 [1], using parS sequence alignments from [5] as input. (B) ParBBb is a CTPase. Continuous monitoring of the released inorganic phosphate (Pi) by recording the absorbance at 360 nm overtime at 25°C. The NTP hydrolysis of ParBBb was also monitored in the presence of ATP, GTP, or UTP, with a 22 bp parSBb DNA duplex. (C) ParBBb does not cluster without parS or on non-cognate parS. Left: representative phase contrast and fluorescence images of MG1655 E. coli strain constitutively producing ParBBb-msfGFP from a plasmid carrying no parS sequence (GL1661) or the C. crescentus parS (parSCc; GL2024). Right: histograms representing the percentage of cells with zero, one, or two ParBBb-msfGFP foci in the same strains. The scale bar is 2 μm; schematics illustrate parBBb-msfgfp expression plasmids. (D) ParB from C. crescentus (ParBCc) is more promiscuous to parS binding than ParBBb. BLI analysis of the interaction between 1 μM ParBCc and a 40 bp cognate parSCc (grey) or a non-cognate parSBb (blue). ParBCc binds both parS sequences. (E) ParBCc can bind to its cognate parSCc and a non-cognate parSBb in E. coli. Left: representative phase contrast and fluorescence images of MG1655 E. coli strain constitutively producing ParBCc-msfGFP from a plasmid carrying parSCc (left, GL2025) or parSBb (right, GL2026); fluorescent foci are observed in both cases. The scale bar is 2 μm; schematics illustrate the parBBb-msfgfp expression plasmid. (PDF)