Common Requirement for the Relaxosome of Plasmid R1 in Multiple Activities of the Conjugative Type IV Secretion System

Abstract

b Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N 1-992 ]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.

Comparative Genomics of the Conjugation Region of F-like Plasmids: Five Shades of F

“…Expression of F conjugative functions is controlled by three transcriptional regulators: TraM, TraJ, and TraY (Frost and Koraimann, 2010; Arutyunov and Frost, 2013). From these three proteins, TraM and TraY play an additional role in relaxosome assembly (Wong et al, 2012; Lang et al, 2014). TraJ is the key activator of the Py promoter, responsible for the transcription of tra genes (Finnegan and Willetts, 1973; Frost and Koraimann, 2010).…”

Type IV Secretion in Gram-Negative and Gram-Positive Bacteria

“…Pioneering work on the nature and recruitment of T4SS substrates, with focus on conjugative plasmids from Grambacteria, has been performed by the groups of Llosa and Zechner (Fernandéz-Gonzaléz et al, 2011;Zechner et al, 2012;Lang et al, 2014;Gruber et al, 2016). All conjugative T4SSs encode relaxases, which initiate substrate processing by a nucleophilic attack of the active site tyrosyl-hydroxyl group of the enzyme on the scissile phosphate group within oriT, releasing the bridging oxygen and forming a long-lived ssDNAprotein conjugate.…”

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion

“…It is possible that there are additional unidentified proteins required for DNA secretion in gonococci beyond those that have been identified in the GGI. It also may be that the DNA secretion substrate is a limiting factor for type IV secretion (Lang et al, 2014). A single oriT sequence is located on the chromosome in the yaf-ltgX intergenic region (Salgado-Pabón et al, 2007), and since gonococci are naturally diploid (Tobiason and Seifert, 2006), exactly two DNA substrates would be expected to be produced per cell.…”