Influence of flanking homology and insert size on the transformation frequency of Acinetobacter baylyi BD413

Environ. Biosafety Res. 6 (2007) 55–69 c ISBR, EDP Sciences, 2007  DOI: 10.1051/ebr:2007027 Available online at: www.ebr-journal.org Thematic Issue on Horizontal Gene Transfer Influence of flanking homology and insert size on the transformation frequency of Acinetobacter baylyi BD413 Deborah J. SIMPSON, Lisa F. DAWSON, John C. FRY* , Hilary J. ROGERS and Martin J. DAY Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK RecA-mediated recombination requires regions of homology between donor and recipient DNA for successful integration. This paper investigates the effect of the relationship between the length of gene-sized inserts (434, 733, 2228 and 2400 bp) and flanking sequence homology (100 – ca. 11 000 bp) on transformation frequency in Acinetobacter baylyi strain BD413. Both insert size and size of the homologous region were varied, which improves on previous studies that kept insert size constant and varied only the homologous flank size. Transfer frequency of a non-homologous single small gene for gentamicin resistance (aac (3)I; 773 bp) was increased 18-fold when flanking homology was changed from about 2000 bp to 8000 bp, but was reduced 234-fold when two genes were inserted (npt II-gfp ; 2400 bp) between similar homologous regions. To investigate the effect of smaller regions of flanking homology (100 – 2000 bp), a partial npt II-gfp deletion (434 bp) was restored. This confirmed that a minimum of 500 bp on each flank was required for transformation to be affected by flanking homology. The data obtained allowed development of a multiple regression equation to predict transformation frequency from homology, insert size and total fragment size for gene insertions. We also show that the ratio of flanking homology to insert size and not the total size of donor DNA is the most important variable determining transformation frequency. The equation developed was consistent with results previously reported by others, and so will be useful when using A. baylyi as a model for gene transfer by transformation in the laboratory, environment and for biosafety. Keywords: Acinetobacter baylyi BD413 / natural transformation / predicting transfer frequency / gene transfer INTRODUCTION Natural transformation is the uptake and subsequent integration of exogenous DNA by competent bacteria (Lorenz and Wackernagel, 1994; Thomas and Nielsen, 2005). Recombination is mediated by RecA, and requires sequence homology between incoming donor DNA and sites in the bacterial genome (Dasgupta and Radding, 1982). Integration is essential for the expression of DNA in transformed cells. The amount of homology needed for recombination in well studied bacteria such as Escherichia coli (Watt et al., 1985), Ralstonia solanacearum (Bertolla et al., 1997) and Bacillus subtilis (Khasanov et al., 1992) is about 20–70 bp, with increasing transformation frequency as homology increases. Bacteria in the genus Acinetobacter are common in many natural environments and Acinetobacter baylyi shows particularly high natural transformation frequencies (ca. 10−3 transformants per cell; Juni, 1972; Lorenz * Corresponding author: fry@cardiff.ac.uk and Wackernagel, 1994; Vaneechoutte et al., 2006). Many studies using this organism have shown that sequence homology of regions flanking the DNA to be transferred affect transformation frequency. For example, recombination was detected with 183 bp homology (de Vries and Wackernagel, 2002) but there are no published data for smaller regions. Gerischer and Ornston (2001) showed that transfer frequencies of two point mutations fell as distance increased from 2 base pairs to 10.5 kb. Transformation is more efficient with homology on both sides of a sequence, but integration is also possible with onesided homology, but at reduced frequency (de Vries and Wackernagel, 2002). Sequence identity also affects transformation efficiency: for example, a decrease from 100% to 90% sequence similarity resulted in a 40-fold decrease (Majewski et al., 2000; Shen and Huang, 1986). In addition to a requirement for sufficient homology, Palmen and Hellingwerf (1997) also highlight the importance of intra-cellular nucleases affecting transformation. They have shown that for A. baylyi approximately 500 bp of the incoming DNA is degraded during transfer of a point Article published by EDP Sciences and available at http://www.ebr-journal.org or http://dx.doi.org/10.1051/ebr:2007027 D.J. Simpson et al. mutation. Thus there is a body of literature indicating the importance of the length of flanking homology in relation to transformation frequency, but no systematic study of this effect in Acinetobacter in relation to insert size. Arber (2000) states that a major force in generating diversity is DNA acquisition. Thus the ability to exchange genes horizontally, even at low frequencies, binds all organisms together evolutionarily. Plant DNA is expected to be present in large amounts and ubiquitously in the environment, and Koonin et al. (2001) have proposed that the uptake of this DNA by bacteria would be a significant evolutionary force. Since A. baylyi is naturally competent and transformation proficient, it is a good model organism for studying gene transfer between plants and bacteria in nature. The size of the insert relative to the amount of homology required for the integration of nonhomologous DNA is of relevance both to this evolutionary process and also to the debate on genetically modified plants, as it is a likely limiting factor for horizontal gene flow of whole plant genes or transgenes into environmental populations of bacteria. The aim of this work was to determine the amount of flanking homology required for efficient integration of whole genes into A. baylyi, and to determine the relationship between the insert size, the amount of flanking sequence homology and transformation frequency. RESULTS AND DISCUSSION Constructs used in experiments Two types of genetic construct have been used in these experiments as donor DNA. The first type of construct used partial A. baylyi 16S rRNA gene flanks to act as homologous regions around firstly a gentamicin resistance gene (Aac(3)I) and secondly two adjacent genes, encoding green fluorescent protein (gfp) and kanamycin resistance (nptII). The gentamicin construct (called BC3) acted as donor to provide a 773 bp insert for transfer and the nptII-gfp donor insert (called PC1) was either 2228 bp or 2400 bp depending on the promoter used (SP6 and psbA respectively). In both cases the 16S rRNA gene homology allowed transfer into one or more of the seven 16S rRNA genes in the wild type A. baylyi recipient genome (Gralton et al., 1997). Using 16S rRNA genes in this way as sites for transformation is neither lethal (Strätz et al., 1996) nor does it reduce growth rate (Asai et al., 1999). In experiments using lysates of A. baylyi carrying these constructs as donor DNA, the genes were inserted into the genome of th (...truncated)