Analysis of Bacterial ParABS System

Our experts have a keen interest in the study of liquid-liquid and liquid-solid phase transitions in bacterial cells. We have cutting-edge super-resolution imaging or infinite diffraction microscopy apparently combined with single molecule trafficking methods, and computer analysis platforms to analyze key microbial biomolecular condensates undergoing LLPS, as well as the formation and organization of biomolecular condensates within the intracellular space. Here, CD BioSciences is committed to analyzing intrinsically disordered bacterial ParABS system.

Introduction of Bacterial ParABS System

The ParABS system is responsible for the segregation and faithful inheritance of most chromosomes and plasmids in bacteria. The system is composed of three components: (1) a mitotic sequence (parS); (2) a dimeric DNA-binding protein (ParB) that binds parS; and (3) a Walker A ATPase (ParA). ParB is abundant and is able to bind high-affinity parS sites to form tight nucleoprotein complexes. Multiple interactions are involved in the formation of ParB-parS partition complexes, including specific interactions of ParB with parS, disordered and low-complexity region-mediated weak ParB-ParB dimer interactions, and non-specific interactions with surrounding DNA, among others. Many pieces of evidence backing the "Nucleation and Caging" model point to ParB-parS partition complexes as condensates.

Fig. 1. CTP-controlled liquid-liquid phase separation of ParB. (Babl L, et al., 2022)

Customized Services

Despite the fundamental role of the ParABS system in prokaryotic cell biology, the underlying molecular nature of the interactions between ParB-parS partition complexes remains unclear. We offer several models of partition complex assembly to analyze the liquid-liquid phase separation (LLPS) of the ParABS system partition complexes.

• The "Diffusion" model.
• The "propagation, bridging, and circulation" model.
• The "Nucleation and caging" model.
• The "clamping and sliding" model.

ParABS system partitioning complexes have liquid-like properties and meet multiple marker criteria for LLPS. CD BioSciences provides professional services to analyze the structural dynamics and biochemical functions of intrinsically disordered ParABS systems in bacteria. Our services include：

• Characterization of ParABS LLPS in Vitro
  Our laboratory has successfully established a rational model system from Corynebacterium glutamicum to analyze ParAB-driven phase separation in vitro, including:

• We provide single particle reconstruction by single-molecule localization microscopy (SMLM) to characterize the LLPS of ParB-parS partition complexes.
• We can analyze the dynamic exchange of ParB between the two partition complexes by a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence lifetime imaging microscopy measurements.
• We provide size-exclusion chromatography-coupled multi-angle light scattering (SEC-Mals) to analyze changes in ParB molecular weight when changing ionic strength.

• Characterization of ParABS LLPS in Vivo
  We provide single-molecule tracking and super-resolution microscopy to characterize parS and ParB binding in vivo to form nanometer-sized membrane-free spherical condensates. Our technical team assembles plasmids with three parS sites uniformly distributed around the sequence and uses centrifugation-based assays and turbidity measurements to analyze the potential impact of these plasmids on the ParB condensation phase boundary. The centrifugation-based assay provides accurate concentration data for the dilute phase after LLPS, and turbidity provides an estimate of the total amount of phase-separated material present in the sample.

CD BioSciences offers a simple modular biomolecular platform to characterize the LLPS of intrinsically disordered ParABS system. Novel methods based on our high-throughput sequencing, single-molecule imaging, single-molecule biophysics, and traditional biochemistry and genetics will continue to provide data on the self-assembly of the ParB-parS system. If you have any special requirements for our services, please feel free to contact us.