Part:BBa_K4968009

CsgC-CsgD

CsgC (BBa_K1583001) and CsgD (BBa_K805015) are pivotal regulators in the Csg protein family, essential for curli fiber assembly in bacterial biofilms. CsgD controls expression, while CsgC aids proper extracellular assembly. Since the genome of the JF1 strain lacks a complete curli operon, CsgC-CsgD is required as an important part of the plasmid to assist the transcriptional expression and self-assembly of the CsgA-AG4 fusion protein. We intend to use Congo Red Dye to stain the amyloid (mainly CsgA) to prove that CsgC-CsgD successfully helps fusion protein expression and secretion.

Figure 1 | Congo red staining of polycarbonate membrane with CsgA+Ag4 fusion protein. This picture shows the CsgA-AG4 fusion protein on a polycarbonate membrane (EMD) after simple isolation, purification, and filtration. Here the curli fiber membrane has undergone Congo red staining (amyloid can be stained by Congo red). This could prove that the function of CsgC, and CsgD seem well.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage & biology

The bacterial Csg protein family is characterized by its involvement in the formation of curli fibers, which are essential components of the extracellular matrix in various bacteria, including adhesive curli fibers in Escherichia coli (Jain and Chapman, 2019). These fibers, also known as Tafi (thin aggregative fimbriae), are integral to biofilm formation and serve as mediators of bacterial adhesion to surfaces and host cells (Gibson et al., 2007). The process of curli fiber assembly involves multiple components, and two crucial regulators in this pathway are CsgC and CsgD.

CsgC and CsgD each have distinct roles in the regulation of curli fiber assembly and biofilm formation. According to Evans et al. (2015), CsgC is involved in preventing the premature polymerization of curli subunits, ensuring that the assembly occurs outside the bacterial cell where it's intended. This chaperone-like protein binds to curli subunits and prevents their aggregation within the cell, enabling controlled and organized assembly once they are transported outside (Taylor et al., 2016).

On the other hand, CsgD functions as a master regulator that orchestrates the expression of the genes necessary for curli fiber assembly (Sokaribo et al., 2020). Its presence is vital for initiating the transcription of csgBAC/ymdA operon, which encodes for the components required for curli biogenesis (Brombacher et al., 2006). CsgD responds to various environmental cues and can adjust csgBA transcription in response to changes in cell density. This adaptability allows bacteria to optimize curli production depending on the availability of resources and the need for biofilm formation.

In our specific case, the JF1 strain lacks the entire curli operon due to genetic modifications. This presents an opportunity to investigate the roles of CsgC and CsgD in curli fiber assembly and biofilm formation. By introducing plasmids containing CsgC and CsgD genes into the JF1 strain, we aim to restore and study the curli fiber formation process. Successful expression and secretion of proteins by JF1 cells would serve as evidence of the efficacy of these genetic components in facilitating the assembly of curli fibers.

Figure 2 | Congo red staining of polycarbonate membrane with CsgA+Ag4 fusion protein. This picture shows the CsgA-AG4 fusion protein on a polycarbonate membrane (EMD) after simple isolation, purification, and filtration. Here the curli fiber membrane has undergone Congo red staining (amyloid can be stained by Congo red). This could prove that the function of CsgC, and CsgD seem well.

Figure 3 | The function of CsgC and CsgD in MC4100 strains. CsgC is involved in preventing the premature polymerization of curli subunits, ensuring that assembly occurs outside the bacterial cell where it's intended. This chaperone-like protein binds to curli subunits and prevents their aggregation inside the cell. CsgD is used as a transcription factor to ensure the expression of the fusion protein CsgA-AG4.

Figure 4 | The function of CsgC and CsgD in BL21(DE3) strains. CsgC is involved in preventing the premature polymerization of curli subunits, ensuring that assembly occurs outside the bacterial cell where it's intended. This chaperone-like protein binds to curli subunits and prevents their aggregation inside the cell. CsgD is used as a transcription factor to ensure the expression of the fusion protein CsgA-AG4.

In a broader context, understanding the roles of CsgC and CsgD could have implications for biofilm-related research, bacterial adhesion mechanisms, and the development of strategies to control biofilm formation in both clinical and industrial settings. These proteins' regulatory functions make them potential targets for interventions aimed at modulating biofilm formation in bacteria, which could have applications in various fields, including medicine and biotechnology.

Source

The sequence of CsgC and CsgD are from NCBI.

References

Brombacher, E. et al. (2006) ‘Gene expression regulation by the Curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion’ J Bacteriol. 188(6):2027-37. Available at: https://doi:10.1128/JB.188.6.2027-2037.2006.

Evans, L.M. et al. (2015) ‘The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation’ Mol Cell, 57(3): 445–455. Available at: https://doi.org/10.1016/j.molcel.2014.12.025

Gibson, D.L. et al. (2007) ‘AgfC and AgfE facilitate extracellular thin aggregative fimbriae synthesis in Salmonella enteritidis’ Microbiology 153(Pt 4):1131-1140. Available at: https://doi:10.1099/mic.0.2006/000935-0. PMID: 17379722.

Jain, N. and Chapman, M.R. (2019) ‘Bacterial functional amyloids: Order from disorder’ Biochim Biophys Acta Proteins Proteom. 1867(10):954-960. Available at: https://doi:10.1016/j.bbapap.2019.05.010.

Taylor, J.D. et al. (2016) ‘Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones’ Scientific Reports, bind 6, 24656. Available at: https://doi.org/10.1038/srep24656