Abstract
KfrC proteins are encoded by the conjugative broad-host-range plasmids that also encode alpha-helical filament-forming KfrA proteins as exemplified by the RA3 plasmid from the IncU incompatibility group. The RA3 variants impaired in kfrA, kfrC, or both affected the host’s growth and demonstrated the altered stability in a species-specific manner. In a search for partners of the alpha-helical KfrC protein, the host’s membrane proteins and four RA3-encoded proteins were found, including the filamentous KfrA protein, segrosome protein KorB, and the T4SS proteins, the coupling protein VirD4 and ATPase VirB4. The C-terminal, 112-residue dimerization domain of KfrC was involved in the interactions with KorB, the master player of the active partition, and VirD4, a key component of the conjugative transfer process. In Pseudomonas putida, but not in Escherichia coli, the lack of KfrC decreased the stability but improved the transfer ability. We showed that KfrC and KfrA were involved in the plasmid maintenance and conjugative transfer and that KfrC may play a species-dependent role of a switch between vertical and horizontal modes of RA3 spreading.
Highlights
The existence of a complex filamentous network called the cytoskeleton that spatially organizes the content of a cell has long been regarded as typical for eukaryotic cells
A wide genomic approach was undertaken to search for putative partner proteins encoded in the E. coli and A. veronii genomes since Aeromonas spp are the most widely spread RA3 hosts in the aquatic environments [35]
Double transformants of E. coli BTH101 with compatible plasmids encoding CyaA fragment T18 or T25 fused to the analyzed proteins from N- or C-terminus were tested on indiplasmids encoding CyaA fragment T18 or T25 fused to the analyzed proteins from N- or C-terminus were tested on indicator cator MacConkey plates with maltose as a carbon source and by β-galactosidase assays in the liquid cultures
Summary
The existence of a complex filamentous network called the cytoskeleton that spatially organizes the content of a cell has long been regarded as typical for eukaryotic cells. The conjugative plasmids of the IncP, IncU, IncW, and PromA groups that are able to replicate, be stably maintained, and efficiently disseminate in a broad range of hosts encode the alpha-helical, coiled-coil-containing, DNA-binding proteins, designated KfrAs [13,14,15,16,17,18,19,20]. 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] It forms a complex with KfrC and with both active partitioning proteins, KorB and IncC [18].
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