Microcosm Studies of GMMs

A long-term microcosm study to reproduce storage of commercial agricultural inoculants was undertaken to obtain information on the behavior of GMMs during a mandatory stage for any marketed PGPR (Plant Growth Promoting Rhizobacteria). The collaboration with Heligenetics S.p.A (Gaiba [Rovigo] Italy), a company manufacturing inoculants, allowed us to use state-of-the-art industrial technology and test

Fig.10.1. Map of the assembled gene cartridge used for the genetic modifications (redrawn with modifications from ref. 1).

Table 10.1. Expression level of the

introduced genes: fi-galactosidase activity and

resistance to mercury chloride

Strain

ß-gal activity (Miller U)

ß-gal activity (Miller U)

MIC (|g/ml) of

IPTG-induced

uninduced

HgCl2

1003 (wt)

38

40

< 1

1110

6.849

260

10

1111

12.982

15.165

10

1112

718

243

5

three different carrier formulations, namely; sterile liquid medium, y-irradiated sterile vermiculite, and non sterile peat. The latter was a suitable environment to assess possible gene transfer to indigenous peat microflora. The three GMMs, and the parent strain, were inoculated into the three different microcosms. Strain survival and marker stability were monitored by periodic sampling from stored packages for over 500 days. Results are shown in Figure 10.2. The liquid medium supported high viability during the first 100 days whereas vermiculite appeared more suitable for long term survival. The chromosomal integration was extremely stable and allowed a survival rate comparable to that of the parent. Strain 1110, carrying the regulated plasmid-borne cassette, demonstrated weaker persistence. By contrast, strain 1111 rapidly lost the plasmid carrying the unregulated construct, but plas-mid free segregates survived as well as the wild type. Marker loss was easily detected by plating bacteria on X-gal plates. A typical feature of the highly expressed lac genes in strain 1111 was the small size of its dark blue colonies compared to the larger and paler revertants. Horizontally-acquired marker genes were sought among peat indigenous bacteria but the background levels of lactose utilization and of HgCl2 resistance (respectively 20% and 0.07% of the total aerobic bacterial peat population) hindered such analysis. However, when 1650 HgR colonies were screened for lac phenotypes, 139 colonies showed both marker pheno-types and were tested by hybridization with probes from pDG3, (mercury resistance genes and the 59-mer synthetic promoter). Two colonies hybridized with the first probe and two different ones with the second one. When the isolates with homology to the synthetic promoter probe were checked with a lacZ probe no signal was detected. These fragmentary homologies may suggest that transformation by liberated DNA portions had occurred, but absolute proof was not obtained for gene transfer or exchange.

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