Isolation and Analysis of Donor Chromosomal Genes Whose Deficiency Is Responsible for Accelerating Bacterial and Trans-Kingdom Conjugations by IncP1 T4SS Machinery

Abstract

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans-kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (ΔfrmR, ΔsufA, and ΔiscA) were isolated, which showed an increase by one order of magnitude in both E. coli–E. coli and E. coli–yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (ΔfrmRΔsufA and ΔiscAΔfrmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea.

Genomic insights into the adaptation of Acinetobacter johnsonii RB2-047 to the heavy metal-contaminated subsurface mine environment

“…However, this gene was not found in the RB2-047 genome. Moreover, a metal/formaldehyde-sensitive repressor was found to regulate the IncP1-type plasmid conjugation (Zoolkefli et al 2021 ).…”

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

“…qRT-PCR was performed using six biological and three technical replicates, on a ViiA7 system of QuantStudio Real-Time PCR System (Applied Biosystems, USA) using the SYBR™ Select Master Mix (Applied Biosystems, Cat #: 472908, USA) under the following conditions: 50°C for 2 minutes, 95°C for 2 minutes followed by 40 cycles of: 95°C for 1 second, 60°C for 30 seconds. Expression levels were normalized against two reference genes ( rrsA and cysG ) as previously described [ 29 ]. Primer sequences used for the qRT-PCR expression analyses are listed in Supplemental Table S2 .…”

“…While conjugation typically occurs between bacterial species, cross-kingdom conjugation from bacteria to yeast and algae has been demonstrated [24][25][26][27]. Despite recently optimized protocols [28,29], conjugation to yeast still suffers from relatively low conjugation frequency compared to prokaryotic recipients.…”