Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

Abstract

Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Alpha-Helical Protein KfrC Acts as a Switch between the Lateral and Vertical Modes of Dissemination of Broad-Host-Range RA3 Plasmid from IncU (IncP-6) Incompatibility Group

“…In the RA3 plasmid of the IncU group, KfrC could interact directly not only with KfrA but also with KorB ( Figure 8 A), one of the components of the active partition system [ 18 ]. It was shown that KfrA RA3 had the ability to form filaments [ 29 ] and interacted with both components of the partition apparatus, KorB and IncC. Hence, the accessory role of a scaffold built of Kfr complexes in the segregation of plasmid molecules to the progeny cells in a species-dependent manner was envisaged [ 18 ].…”

“…Within the stabilization module, upstream of the type Ia active partition system encoding KorB of the ParB family and IncC, the Walker-type ATPase of the ParA family, there are two structural genes for alpha-helical proteins KfrA and KfrC [ 22 , 28 ]. It has been shown recently that KfrA acts as a transcriptional autoregulator and is able to form long filamentous structures in the presence of plasmids carrying its cognate binding site [ 18 , 29 ]. Moreover, it forms a complex with KfrC and with both active partitioning proteins, KorB and IncC [ 18 ].…”

“…Moreover, it forms a complex with KfrC and with both active partitioning proteins, KorB and IncC [ 18 ]. The highly unstable test plasmid that was stabilized by the presence of the RA3 stability module displayed the increased segregation rate in some hosts when deprived of the kfrA operon [ 26 , 29 ] and/or kfrC [ 18 ]. To better understand the functions the Kfr proteins play in the RA3 plasmid biology, the present study focused on the detailed analysis of the KfrC properties and on a wide-range search for KfrC partners among the plasmid RA3- and host-encoded proteins.…”