Two pKM101‐encoded proteins, the pilus‐tip protein TraC and Pep, assemble on the Escherichia coli cell surface as adhesins required for efficient conjugative DNA transfer

Abstract

Summary Mobile genetic elements (MGEs) encode type IV secretion systems (T4SSs) known as conjugation machines for their transmission between bacterial cells. Conjugation machines are composed of an envelope-spanning translocation channel, and those functioning in Gram-negative species additionally elaborate an extracellular pilus to initiate donor-recipient cell contacts. We report that pKM101, a self-transmissible MGE functioning in the Enterobacteriaceae, has evolved a second target cell attachment mechanism. Two pKM101-encoded proteins, the pilus-tip adhesin TraC and a protein termed Pep, are exported to the cell surface where they interact and also form higher-order complexes appearing as distinct foci or patches around the cell envelope. Surface-displayed TraC and Pep are required for efficient conjugative transfer, ‘extracellular complementation’ potentially involving intercellular protein transfer, and activation of a Pseudomonas aeruginosa type VI secretion system. Both proteins are also required for bacteriophage PRD1 infection. TraC and Pep are exported across the outer membrane by a mechanism potentially involving the β-barrel assembly machinery. The pKM101 T4SS thus deploys alternative routing pathways for delivery of TraC to the pilus tip or both TraC and Pep to the cell surface. We propose that T4SS-encoded, pilus-independent attachment mechanisms maximize the probability of MGE propagation and might be widespread among this translocation superfamily.

Conjugative Transfer of IncP-9 Catabolic Plasmids Requires a Previously Uncharacterized Gene, mpfK , Whose Homologs Are Conserved in Various MPF T -Type Plasmids

“…Deletion of the pep gene from pKM101 led to a 10 2 -fold decrease in conjugative transfer frequency, and similar decreases were observed for the four point mutants of pep, each of which possessed the substitution at one of the four conserved Cys residues (27). Our detailed analysis of various mpfK mutants of NAH7 in this study exhibited the following properties: 10 2 -to 10 3 -fold reduced transferability of the four Cys-to-Ala single-substitution mutants and two double-substitution mutants (MpfK C39A-C88A and MpfK C44A-C54A ) and no transferability of the four other double-substitution mutants or a quadruple-substitution mutant (Fig.…”

“…S2) encode the MpfK homologs with high (76% to 100%) similarities to the MpfK protein. Although some non-IncP-9 conjugative plasmids encode the proteins with this superfamily domain, the experimentally analyzed proteins with the TrbM superfamily domain are limited to the pep product (44% amino-acid sequence similarity with MpfK) from an IncN plasmid pKM101 and the orf104 product (44% similarity) from another IncN plasmid, pCU101; both of these proteins are important for the efficient conjugative transfer (25)(26)(27).…”

“…2b). Gonzales-Rivera et al (27) recently constructed the four Pep derivatives, each of which possessed the substitution mutation at one of the conserved Cys residues. Their subsequent biochemical analysis showed that such Cys residues play an important role in the stabilization or function of Pep protein at the cell surface, although the cysteine pair(s) involved in the disulfide bond (DSB) formation remained unclear.…”

“…The pKM101-encoded Pep protein has been reported to function as an adhesin for stable contact between donor and recipient cells during the conjugation process (27). However, MpfK and the orf104 product from pCU1 were located at periplasmic space (Fig.…”

Role of Recipient Susceptibility Factors During Contact-Dependent Interbacterial Competition

“…Because T6SS firing is also induced by membrane-disrupting compounds such as polymyxin B, the authors concluded that the T6SS counterattack results from Mpf-mediated membrane disruption. Recent studies further showed that the production of two adhesins (TraC and Pep), or the formation of a T4SS channel, but not assembly of conjugative pilus, is capable of activating a T6SS counterattack (Gordon et al, 2017;González-Rivera et al, 2019). Therefore, T6SS firing could be a defensive weapon in response to various assaults challenging membrane integrity ( Figure 5).…”