Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA

Abstract

The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and single-molecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an α helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA–DNA aggregates are similar in morphology to protein–DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein–DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance.

Nucleoid-Enriched Proteomes in Developing Plastids and Chloroplasts from Maize Leaves: A New Conceptual Framework for Nucleoid Functions

“…The DnaJ domain protein pTAC5 (protein GRMZM2G031721_ P0) was abundant in nucleoids, but it did not pass our criteria to be a candidate nucleoid protein. Escherichia coli nucleoids contain a DnaJ-related protein, CbpA, with DNA-binding activity involved in DNA aggregation that protects DNA from degradation by nucleases (Cosgriff et al, 2010). Therefore, it seems possible that some of the nucleoid-localized DnaJ domain proteins interact directly with DNA.…”

Pervasive regulation of nucleoid structure and function by nucleoid-associated proteins

“…With circular plasmid DNA FIS binds at crossovers [36] stabilising a negatively supercoiled plectoneme with right-handed DNA coils (Figure 2). However at the tyrT UAS regulatory region three FIS dimers constrain a left-handed toroidal loop [31].…”

A Conserved Acidic Amino Acid Mediates the Interaction between Modulators and Co-Chaperones in Enterobacteria

“…9 This work revealed that the CbpA-CbpM system was amenable to analysis using the bacterial twohybrid (BACTH) assay system that detects proteinprotein interactions in vivo. 15 Note that this system relies on the fact that the E. coli cyaA− strain BTH101 is unable to produce cAMP and thus has a Lac− phenotype.…”

“…Our previous work also demonstrated that CbpA forms aggregates when bound to DNA that can be detected in vitro using gel shift assays. 9 Figure 1b-ii shows the outcome of such an experiment. Addition of CbpA to plasmid DNA results in the formation of aggregates that are trapped in the wells of the gel (compare lanes 1 and 2).…”

“…All assays were performed using cells grown in MacConkey broth as described previously. 9 The different CbpA derivatives tested are indicted below the chart. (a-ii) The graph shows results of BACTH assays performed to measure CbpA-CbpA interactions.…”