Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa

Abstract

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.

Genomic Insights into Xylella fastidiosa Interactions with Plant and Insect Hosts

“…Transformation and recombination efficiencies increase with increments in the length of homologous flanking regions of inserts, up to 1 kb in size. In addition, efficiency reduces as the size of the non-homologous insert increases, with recombination not detected once the region was 6 kb in length (Kung et al 2013). Competency, as in other bacteria, appears to be mediated by a type IV pilus-like apparatus, with associated com genes that transport DNA fragments through the cell membrane.…”

“…Both the coffee and citrus isolates of subspecies pauca appear to have acquired alleles from subspecies multiplex (Nunney et al 2012). Likewise, subspecies multiplex has participated in recombination with subspecies fastidiosa (Nunney et al 2010;Kung et al 2013). This process of allele conversion has even gone as far as to produce new lineages with roughly equal contributions from subsp.…”

Explanatory note on DNA sequence similarity searches in the context of the assessment of horizontal gene transfer from plants to microorganisms

“…Recombination efficiencies also decrease with increasing size of the non-homologous insert in a double HR event. Under in vitro conditions the recombination efficiency drops about 1 log if the size of the non-homologous insert increases from 1 to 2 kb and no recombination is detected with a non-homologous insert of 6 kb length flanked by 1 kb homologous DNA on either side (Kung et al, 2013). While single HR may result in a gene addition or alteration of the DNA sequence of a gene (de Vries and Wackernagel, 2002), a double HR event can result in the replacement of the region between the two similar sequences by the inserted gene.…”

“…The efficiency of HR in bacteria depends on nucleotide sequence identity and length between the foreign and recipient DNA. Furthermore, in case of double HR (see below), the length of the non-homologous DNA to be transferred is of crucial importance for transfer rates (Kung et al, 2013). Two regions of sufficient sequence identity and length are necessary for a double HR event to take place, one at each side of the DNA sequence that would be transferred.…”

“…Homologous recombination becomes increasingly inefficient with decreasing length of sequences with high identity (de Vries and Wackernagel, 2002;Monier et al, 2007;EFSA, 2009;Overballe-Petersen et al, 2013) and reaches under in vitro conditions a plateau phase with about 1 kb of sequences with high identity at both stretches of DNA (Kung et al, 2013). Based on the above-mentioned literature data, a 200-bp stretch of identical sequence can be considered as a minimum length of continuous DNA sequence to take into account in the analysis of identifying GM plant DNA regions with increased recombination potential with microbial genomes.…”