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

Published online by Cambridge University Press:  12 September 2007

Deborah J. Simpson ,
Lisa F. Dawson ,
John C. Fry ,
Hilary J. Rogers  and
Martin J. Day
Deborah J. Simpson
Affiliation:
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK
Lisa F. Dawson
Affiliation:
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK
John C. Fry
Affiliation:
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK
Hilary J. Rogers
Affiliation:
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK
Martin J. Day
Affiliation:
Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, Cardiff, CF10 3TL, UK

Abstract

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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 Acinetobacterbaylyi 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 (nptII-gfp; 2400 bp) between similar homologous regions. To investigate the effect of smaller regions of flanking homology (100 – 2000 bp), a partial nptII-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 BD413natural transformationpredicting transfer frequencygene transfer
Type
Research Article
Information
Environmental Biosafety Research , Volume 6 , Issue 1-2: Thematic Issue on Horizontal Gene Transfer , January 2007 , pp. 55 - 69
Copyright
© ISBR, EDP Sciences, 2007

References

Arber, W (2000) Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS Microbiol. Rev. 24: 1–7 CrossRef
Asai T, Condon CN, Voulgaris J, Zaporojets D, Shen B, Al-Omar M, Squires C, Squires CI (1999) Construction and initial characterization of Escherichia coli strains with few or no intact chromosomal rRNA operons. J. Bacteriol. 181: 3803–3809
Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19: 327–336
Bertolla F, Van Gijsegem F, Nesme X, Simonet P (1997) Conditions for natural transformation of Ralstonia solanacearum. Appl. Environ. Microbiol. 63: 4965–4968
Dasgupta C, Radding CM (1982) Polar branch migration promoted by RecA-protein - effect of mismatched base-pairs. Proc. Natl. Acad. Sci. USA 79: 762–766
Davidoff-Abelson R, Dubnau D (1973) Conditions affecting the isolation from transformed cells of Bacillus subtilis of high molecular weight single-stranded deoxyribonucleic acid of donor origin. J. Bacteriol. 116: 146–53
de Vries J, Wackernagel WM (2002) Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc. Natl. Acad. Sci. USA 99: 2094–2099
Elhai J (1993) Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol. Lett. 114: 179–184
Fry JC (1993) Biological Data Analysis: A Practical Approach, IRL Press, Oxford.
Gebhard F, Smalla K (1998) Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl. Environ. Microbiol. 64: 1550–1554
Gerischer U, Ornston LN (2001) Dependence of linkage of alleles on their physical distance in natural transformation of Acinetobacter sp. strain ADP1. Arch. Microbiol. 176: 465–469
Gralton EM, Campbell AL, Neidle EL (1997) Directed introduction of DNA cleavage sites to produce a high-resolution genetic and physical map of the Acinetobacter sp. strain ADP1 (BD413UE) chromosome. Microbiology 143: 1345–1357
Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91: 12501–12504
Heinemann JA, Traavik T (2004) Problems in monitoring horizontal gene transfer in field trials of transgenic plants. Nat. Biotechnol. 22: 1105–1109
Juni E (1972) Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J. Bacteriol. 112: 917–931
Juni E, Janik A (1969) Transformation of Acinetobacter calcoaceticus (Bacterium anitratum). J. Bacteriol. 98: 281–288
Khasanov FK, Zhvingila DJ, Zailhudlin AA, Prozorov AA, Bashkirov VI (1992) Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology. Mol. Gen. Genet. 234: 494–497
Koonin EV, Makarova KS, Aravind L (2001) Horizontal gene transfer in prokaryotes: quantification and classification. Ann. Rev. Microbiol. 55: 709–742
Li X, Zhang G, Ngo N, Zhao X, Kain SR (1997) Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J. Biol. Chem. 272: 28545–28549
Lorenz MG, Wackernagel WM (1994) Bacterial gene-transfer by natural genetic-transformation in the environment. Microbiol. Rev. 58: 563–602
Majewski J, Zawadzki P, Pickerell P, Cohan FM, Dowson CG (2000) Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation. J. Bacteriol. 182: 1016–1023
Nielsen KM, Townsend JP (2004) Monitoring and modelling horizontal gene transfer. Nat. Biotechnol. 22: 1110–1114
Nielsen KM, van Weerelt MDM, Berg TN, Bones AM, Hagler AN, Van Elsas JD (1997) Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms. Appl. Environ. Microbiol. 63: 1945–1963
Palmen R, Hellingwerf KJ (1997) Uptake and processing of DNA by Acinetobacter calcoaceticus – a review. Gene 192: 179–190
Palmen R, Vosman B, Buijsman P, Breek CKD, Hellingwerf KJ (1993) Physiological characterisation of natural transformation in Acinetobacter calcoaceticus. J. Gen. Microbiol. 139: 295–305
Pettersen A-K, Bøhn T, Primicero R, Shorten P, Soboleva T, Nielsen KM (2005) Modelling suggests frequency estimates are not informative for predicting the long-term effects of horizontal gene transfer in bacteria. Environ. Biosafety Res. 4: 223–233
Pifer ML, Smith HO (1985) Processing of donor DNA during Haemophilus influenzae transformation: analysis using a model plasmid system. Proc. Natl. Acad. Sci. USA 82: 3731–3735
Rochelle PA, Day MJ, Fry JC (1988) Occurrence, transfer and mobilisation in epilithic strains of Acinetobacter of mercury-resistance plasmids capable of transformation. J. Gen. Microbiol. 134: 2933–2941
Shen P, Huang HV (1986) Homologous recombination in Escherichia coli: Dependence on substrate length and homology. Genetics 112: 441–457
Stahl FW, Crasemann JM, Stahl MM (1975) Rec-mediated recombinational hot spot activity in bacteriophage lambda. III. Chi mutations are site-mutations stimulating rec-mediated recombination. J. Mol. Biol. 94: 203–212
Strätz M, Mau M, Timmis KN (1996) System to study horizontal gene exchange among microorganisms without cultivation of recipients. Mol. Microbiol. 22: 207–215
Thomas CM, Nielsen KM (2005) Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat. Microbiol. Rev. 3: 711–721
Timms-Wilson TM, Bailey MJ (2001) Reliable use of green fluorescent protein in fluorescent pseudomonads. J. Microbiol. Meth. 46: 77–80
Vaneechoutte M, Young DM, Ornston LN, De Baere T, Tanny AN, van Der Reijden EC, Tjernberg I, Dijkshoorn L (2006) Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi. Appl. Environ. Microbiol. 72: 932–936
Watt VM, Ingles JC, Urdea MS, Rutter WJ (1985) Homology requirements for recombination in Escherichia coli. Proc. Natl. Acad. Sci. USA 82: 4768–4772
West SHE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148: 81–86
Williams HG, Day MJ, Fry JC, Stewart GJ (1996) Natural transformation in river epilithon. Appl. Environ. Microbiol. 62: 2994–2998