Dataset Information

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

ABSTRACT: In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used conjugal DNA transfer, combining a classical conjugation donor, HfrH, with modern genome engineering methods and whole genome sequencing analysis to enable interrogation of genetic dependence of integration mechanisms and characterization of recombination products. As in classical experiments, genomic DNA transfer begins at a unique position in the donor, entering the recipient via conjugation; antibiotic resistance markers are then used to select recombinant progeny. Different configurations of this system were used to compare known mechanisms for stable DNA incorporation, including homologous recombination, F'-plasmid formation, and genome duplication. A genome island of interest known as the immigration control region was specifically replaced in a minority of recombinants, at a frequency of 3 X 10(-12) CFU/recipient per hour.

SUBMITTER: Kingston AW

PROVIDER: S-EPMC4498929 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

Kingston Anthony W AW Roussel-Rossin Chloé C Dupont Claire C Raleigh Elisabeth A EA

PloS one 20150710 7

In bacteria, mechanisms that incorporate DNA into a genome without strand-transfer proteins such as RecA play a major role in generating novelty by horizontal gene transfer. We describe a new illegitimate recombination event in Escherichia coli K-12: RecA-independent homologous replacements, with very large (megabase-length) donor patches replacing recipient DNA. A previously uncharacterized gene (yjiP) increases the frequency of RecA-independent replacement recombination. To show this, we used ...[more]

PMID: 26162088

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Project description: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.

Project description:BACKGROUND: Bacterial motility is a crucial factor in the colonization of natural environments. Escherichia coli has two flagella-driven motility types: swimming and swarming. Swimming motility consists of individual cell movement in liquid medium or soft semisolid agar, whereas swarming is a coordinated cellular behaviour leading to a collective movement on semisolid surfaces. It is known that swimming motility can be influenced by several types of environmental stress. In nature, environmentally induced DNA damage (e.g. UV irradiation) is one of the most common types of stress. One of the key proteins involved in the response to DNA damage is RecA, a multifunctional protein required for maintaining genome integrity and the generation of genetic variation. RESULTS: The ability of E. coli cells to develop swarming migration on semisolid surfaces was suppressed in the absence of RecA. However, swimming motility was not affected. The swarming defect of a DeltarecA strain was fully complemented by a plasmid-borne recA gene. Although the DeltarecA cells grown on semisolid surfaces exhibited flagellar production, they also presented impaired individual movement as well as a fully inactive collective swarming migration. Both the comparative analysis of gene expression profiles in wild-type and DeltarecA cells grown on a semisolid surface and the motility of lexA1 [Ind-] mutant cells demonstrated that the RecA effect on swarming does not require induction of the SOS response. By using a RecA-GFP fusion protein we were able to segregate the effect of RecA on swarming from its other functions. This protein fusion failed to regulate the induction of the SOS response, the recombinational DNA repair of UV-treated cells and the genetic recombination, however, it was efficient in rescuing the swarming motility defect of the DeltarecA mutant. The RecA-GFP protein retains a residual ssDNA-dependent ATPase activity but does not perform DNA strand exchange. CONCLUSION: The experimental evidence presented in this work supports a novel role for RecA: the promotion of swarming motility. The defective swarming migration of DeltarecA cells does not appear to be associated with defective flagellar production; rather, it seems to be associated with an abnormal flagellar propulsion function. Our results strongly suggest that the RecA effect on swarming motility does not require an extensive canonical RecA nucleofilament formation. RecA is the first reported cellular factor specifically affecting swarming but not swimming motility in E. coli. The integration of two apparently disconnected biologically important processes, such as the maintenance of genome integrity and motility in a unique protein, may have important evolutive consequences.

Dataset Information

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

Publications

Novel recA-Independent Horizontal Gene Transfer in Escherichia coli K-12.

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