Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination

Abstract

The active uptake of extracellular DNA and its genomic integration is termed natural transformation and constitutes a major horizontal gene-transfer mechanism in prokaryotes. Chromosomal DNA transferred within a species can be integrated effectively by homologous recombination, whereas foreign DNA with low or no sequence homology would rely on illegitimate recombination events, which are rare. By using the nptII(+) gene (kanamycin resistance) as selectable marker, we found that the integration of foreign DNA into the genome of the Gram-negative Acinetobacter sp. BD413 during transformation indeed was at least 10(9)-fold lower than that of homologous DNA. However, integration of foreign DNA increased at least 10(5)-fold when it was linked on one side to a piece of DNA homologous to the recipient genome. Analysis of foreign DNA integration sites revealed short stretches of sequence identity (3-8 bp) between donor and recipient DNA, indicating illegitimate recombination events. These findings suggest that homologous DNA served as a recombinational anchor facilitating illegitimate recombination acting on the same molecule. Homologous stretches down to 183 nucleotides served as anchors. Transformation with heteroduplex DNA having different nucleotide sequence tags in the strands indicated that strands entered the cytoplasm 3' to 5' and that strands with either polarity were integrated by homologous recombination. The process led to the genomic integration of thousands of foreign nucleotides and often was accompanied by deletion of a roughly corresponding length of recipient DNA. Homology-facilitated illegitimate recombination would explain the introgression of DNA in prokaryotic genomes without the help of mobile genetic elements.

Figure 1

The plasmids used for the analysis of recombination after natural transformation. The nptII is represented by arrows, shaded areas indicate regions of homology, and dashed lines indicate nonhomologous recipient DNA. (A) The donor DNA pBlue-Km1 carries the nptII⁺ gene on a 1.87-kb HindIII-BamHI fragment from Tn5. Restriction sites used for the linearization of the donor DNA are indicated. (B) The DNA corresponding to the HindIII-BamHI fragment of the donor DNA contains in pMR7 a 10-bp deletion and in pMR30 a substitution of the 51 C-terminal nucleotides of nptII plus the following 716 bp by eukaryotic tg4 terminator DNA. The vector plasmid of pMR7 and pMR30 was pKT210.

Figure 2

Locations of illegitimate recombination sites of 20 transformants obtained with one-side homologous donor DNA. For each recombinant the fusion between donor and recipient DNA is shown by a line. The positions of forward and reverse primers complementary to the donor and recipient DNA used for the localizations are indicated by arrows.

Figure 3

The nucleotide sequence of illegitimate recombination sites in 20 transformants. Nucleotides identical in donor (D) and recipient (R) sequences are indicated by shading. The sequences present in the transformants are underlined. The core regions of identity (boxed) contain the presumptive fusion sites, and their nucleotide number is indicated in brackets.

Figure 4

Dependence of the transformation frequency with one-side homologous donor DNA on the length of the homologous region. The arrows represent the nptII gene and its deletion derivatives in the donor DNA, and the white arrowhead symbolizes the selective marker region. Shading indicates homology between donor and recipient (pMR30) DNA. (A) Deletion derivatives of pBlue-Km1 obtained by removal of nucleotides (dotted lines) between the HindIII site and the other indicated restriction sites. (B) Frequency of transformation (number of transformants per donor molecule) with pBlue-Km1 and the deletion derivatives. Data are given with SD (n = 3).

Figure 5

Structures of HD molecules HD1, HD2, and HD3 and frequencies of genomic integration of the strands. (A) Nucleotide sequences including the stop codon of nptII (bold face) and the tag site of the donor plasmids. Restriction recognition sites are boxed in the wild-type (WT) and mutant (mut.) version of the plasmid. (B) Frequencies of genomic integration of strands with the selective marker region (black bar) on the 3′ or 5′ side of the homology (waved line). Restriction sites used for the formation of the transforming fragment are indicated. The arrowheads symbolize the 3′ ends of the marker-containing DNA fragment that would be released by a double strand cut in either the left or right flanking region of the marker. The sizes of the flanking regions are given. Mismatches are indicated by a tine in the mutant strand. The source (ds, double-stranded plasmid DNA; ss, single-stranded phage DNA), genotype (WT and mut.), and integration frequency are given for each strand. The numbers of transformants analyzed are indicated.

Figure 6

Models for genomic integration of a heterologous 3′ (A A′) and 5′ end (B) of one-side homologous DNA. Homology between the donor (thin line) and recipient (bold lines) DNA is indicated by shading. In the donor DNA the homologous (waved) and selective marker (black bar) regions are indicated. The dotted arrows indicate newly synthesized DNA. For details see Discussion.