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

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 (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.

Transformation of Acinetobacter baylyi in non-sterile soil using recombinant plant nuclear DNA.

To provide estimates of horizontal gene transfer from transgenic crops to indigenous soil bacteria, transformation frequencies were obtained for naturally transformable Acinetobacter baylyi BD413 using a chromosomally integrated plant transgene. The transgene comprised sequences for two phenotypic markers: kanamycin resistance (npt II) and green fluorescent protein (gfp), expressed from their own bacterial promoters. Recipient bacteria carried a copy of these two genes, with deletions in their 3'-termini abolishing the marker activity, these genes were integrated into a 16S rRNA gene in the bacterial chromosomal genome or carried on a broad host range plasmid. Successful recombination between the plant transgene and the bacterial genome resulted in restoration of the markers, allowing detection through antibiotic selection and fluorescence. Transformation parameters of increasing complexity, without any enrichment steps, were used to approach the field conditions, while still obtaining measurable transformation frequencies. In pure culture filter experiments, transformation was detected using ground, chopped and whole leaves, as well as whole sterile seedlings, and ground roots. In sterile soil microcosms, transformation was detected using pure plant DNA (3.6 x 10(-8) transformants per recipient) and ground leaves (2.5 x 10(-11)). Transformation was also detected for the first time in non-sterile soil using pure plant DNA (5.5 x 10(-11)). Since the same constructs were used throughout, these data allow predictions of even more complex environmental systems where measurable frequencies are not easily obtainable.

Purification and characterisation of an antifreeze protein from Forsythia suspensa (L.).

Recrystallisation inhibition (RI) activity has been isolated from cold-acclimated Forsythia suspensa bark and leaves, which is stable when boiled, and not sensitive to reducing agents. The antifreeze activity has been purified to a single 20 kDa protein, using anion exchange, hydroxyapatite chromatography, and gel filtration. The protein is abundant in forsythia bark with 0.5microg pure protein obtained from 35 g bark. RI activity is seen with as little as 6 microg ml(-1) protein. Sequence homology was seen with dehydrins, and forsythia AFP contains the Y-segment, a conserved region found in many dehydrins.

Nitrification by plants that also fix nitrogen.

Nitrification is a key stage in the nitrogen cycle; it enables the transformation of nitrogen into an oxidized, inorganic state. The availability of nitrates produced by this process often limits primary productivity and is an important determinant in plant community ecology and biodiversity. Chemoautotrophic prokaryotes are recognized as the main facilitators of this process, although heterotrophic nitrification by fungi may be significant under certain conditions. However, there has been neither biochemical nor ecological evidence to support nitrification by photoautotrophic plants. Here we show how certain legumes that accumulate the toxin, 3-nitropropionic acid, generate oxidized inorganic nitrogen in their shoots, which is returned to the soil in their litter. In nitrogen-fixing populations this 'new' nitrate and nitrite can be derived from the assimilation of nitrogen gas. Normally, the transformation of elemental nitrogen from the atmosphere into a fixed oxidized form (as nitrate) is represented in the nitrogen cycle as a multiphasic process involving several different organisms. We show how this can occur in a single photoautotrophic organism, representing a previously undescribed feature of this biogeochemical cycle.