A. Cresswell et al., THE CONSTRUCTION, DETECTION AND USE OF BIOLUMINESCENT RHIZOBIUM-LEGUMINOSARUM BIOVAR TRIFOLII STRAINS, Journal of Applied Bacteriology, 77(6), 1994, pp. 656-665
The gene encoding the firefly luciferase enzyme (luc) was introduced t
o Rhizobium leguminosarum biovar trifolii strains with a view to using
the resulting bioluminescent strains to study the survival of genetic
ally-engineered rhizobia in soil microcosms. The genetically-engineere
d micro-organisms (GEMs) behaved similarly to their parent strains wit
h respect to growth rate in laboratory media and in their symbiotic pe
rformance with their host plants. No gene transfer could be detected i
n laboratory mating experiments. When inoculated onto a non-sterile so
il the population of the GEM declined sharply from an initial cell den
sity of 2 x 10(7) g(-1) soil to approach a stable cell density of appr
oximately 3 x 10(2) g(-1) after 150 d. Direct photography of biolumine
scent rhizobia enabled the detection of colonies as small as 0.1 mm in
diameter without the need for transferring colonies onto filter paper
. When a Rhizobium strain carrying the luc marker on a plasmid was use
d as inoculant it was possible to visualize differences in colonizatio
n of the rhizosphere of white clover and ryegrass by contact print and
colour transparency films. The photographic detection methods describ
ed here demonstrate the possibilities of using bioluminescent rhizobia
for assessing their survival in soil, and for looking at rhizosphere
populations which may be an important site for potential gene transfer
.