System to study horizontal gene exchange among microorganisms without cultivation of recipients

Abstract

Ribosomal RNA genes are characterized by highly conserved sequences and are present in multiple copies in most prokaryotic chromosomes. In principle, therefore, they might serve as sites for homologous recombination between unrelated microorganisms. Plasmids containing 23S ribosomal gene sequences, from different bacteria, which had been interrupted by insertion of a kanamycin-resistance gene, were used to transform Acinetobacter sp. DSM587 (former name: Acinetobacter calcoaceticus BD413-ivl10). In all cases, homologies between the 23S rRNA genes of phylogenetically distant bacteria and Acinetobacter sp. DSM587 were sufficient for replacement recombination events. The integration events, resulting in inactivation of any one of the seven rrn operons of Acinetobacter sp. DSM587, had no observable influence on cell growth. These results suggest the possibility of rRNA genes serving as natural vehicles for horizontal gene transfer. They also provide the basis of a novel strategy to analyse gene transfer without selection or cultivation of recipient cells. Because of the highly conserved structure of bacterial rrn operons, recombination events subsequent to gene transfer can be readily identified by polymerase chain reaction amplification of the recombinant sequence using a universal forward primer for the 16S rRNA gene and a reverse primer specific for the integrated marker gene.

Natural Transformation of Acinetobacter sp. Strain BD413 with Cell Lysates of Acinetobacter sp., Pseudomonas fluorescens , and Burkholderia cepacia in Soil Microcosms

“…in soil and may, in addition, protect DNA from rapid inactivation. Furthermore, if DNA homology is present (21,29,32,47,53), gene transfer by natural transformation might provide populations of Acinetobacter cells with a mechanism for generating genetic variability (e.g., mosaic genes) by enabling them to take up chromosomal DNA released from various bacterial donors in their surroundings.…”

Implications of alternative classifications and horizontal gene transfer for bacterial taxonomy.

“…An integrase gene system with the specific function of facilitating gene exchange has been reported to be widespread in bacteria (Hall & Collis, 1995 ;Mazel et al, 1998), together with evidence for the presence of introduced functional genes. Perhaps most striking, the transfer of parts of 16S rRNA sequences has been suggested between genomic species in Aeromonas (Sneath, 1993) ; reports that the highly conserved state of 16S rRNA makes it a prime candidate as a vector for gene transfer (Stra$ tz et al, 1996) and evidence for its recombination Yap et al, 1999) are especially thought-provoking. The exchange of house-keeping genes or of extended chromosomal sequences containing large numbers of genes that are central to bacterial metabolic activity has not been documented extensively, although there are tantalizing indications of this possibility (Syvanen, 1994).…”

Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil

“…A first hypothesis deals with the presence of the molecular machinery permitting bacteria to develop a competence state. Tests should be conducted on bacteria belonging to the 99% nonculturable fraction of the soil microflora (28). Another hypothesis to explain the transformants we obtained with A. tumefaciens and P. fluorescens deals with a mechanism that would not be related to the well-defined natural transformation.…”