Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer

Abstract

Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes.IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo. For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

“…Disruption of the arcB iTIS by synonymous mutations at the start codon and the 331 upstream SD-like sequence did not affect the synthesis of the full-length ArcB but 332 abrogated the production of ArcB-C ( Figure 3E). These results demonstrate that 333 the iTIS within the arcB gene may indeed be utilized in the cell for synthesis of a 334 stand-alone ArcB-C protein, detached from the membrane-bound ArcB ( Another remarkable example of a 3'-proximal iTIS that likely directs 355 production of a functional protein is found in homologous rpnA, rpnB, rpnC, rpnD 356 and rpnE genes found in the E. coli genome that encode nucleases involved in 357 DNA recombination (Kingston et al, 2017). All the rpn genes in the E. coli show 358…”

Comparative Genomic Analysis of a Novel Strain of Taiwan Hot-Spring Cyanobacterium Thermosynechococcus sp. CL-1

“…One distinct feature in the CL-1 genome is the presence of 14 copies of RPN family genes that encode recombinationpromoting nuclease/putative transposases (Kingston et al, 2017). Most are located in two tandem gene arrays (nine and four copies, respectively) (FFX45_10335-10375 and FFX45_07895-07910) except for one gene (FFX45_07205).…”

Peer Review #1 of "Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12 (v0.1)"

“…Interestingly, in a recent article, these authors describe results suggesting that more than one mechanism could contribute to these RecA-independent recombination phenomena (Kingston et al, 2017). In this work we present PCR-based evidence of RecA-independent recombination in two genetic models while in vivo evidence of this phenomenon was attained in only one of them.…”