Environmental Engineering Reference
In-Depth Information
6.4.1.6 Developing and testing molecular tracking recombinant
organisms
in situ
The success of any bioremediation process depends upon establishing favor-
able conditions for microorganisms to degrade the target contaminant. Fail-
ures in bioremediation more often result from a lack of knowledge about
ecological constraints than from information about the
genetic
capabilities of
the organism. Good data on population dynamics in bioremediation are
important for providing insights into better methods of inoculation, main-
tenance, and management of contaminated sites.
Although numerous molecular markers have been developed and used
for detection of microorganisms (Burlage et al., 1990; Recorbet et al., 1992;
Hwang and Farrand, 1997), including successful applications based on the
green flourescent protein (GFP) gene (Bloemberg et al., 1997), the addition
or alteration of genetic traits may have deleterious effects on the environ-
mental fitness of microorganisms (Lenski, 1991). Other molecular tracking
methods, such as amplified ribosomal DNA restriction analysis (ARDRA)
(Nüsslein and Tiedje, 1998), single-strand conformation polymorphism
(SSCP) (Lee et al., 1996; Schwieger and Tebbe, 1998), denaturing gradient gel
electrophoresis (DGGE) (Recorbet et al., 1992; Muyzer et al., 1993), and
terminal restriction fragment length polymorphism (T-RFLP) (Muyzer et al.,
1998), lack a quantitative component.
In situ
hybridization with fluorescently
labeled oligonucleotide probes (FISH) targeted to the rRNA sequences pro-
vides for quantitation and has proven useful in aqueous environments
(DeLong et al., 1989), but it is labor intensive, does not offer species-to-strain
level specificity, and proved difficult to use in soil.
Real-time PCR (RTm-PCR) was originally developed for measuring the
copy number of a targeted gene (Dölken et al., 1998; Heid et al., 1996; Higuchi
et al., 1993) and presented a promising tool for the detection and quantifi-
cation of microorganisms. In this method, a double-labeled probe is used to
measure the accumulation of fluorescence of a released reporter dye during
PCR. This fluorescence is then correlated to the amount of product formed
in real time during amplification when PCR is more quantitative (Higuchi
et al., 1993). The method has been successful in clinical studies with
Listeria
monocytogenes
(Bassler et al., 1995),
Yersinia pestis
(Higgins et al., 1998),
Myco-
bacterium tuberculosis
(Desjardin et al., 1998), and
Borrelia burgdorferi
(Pahl et
al., 1999), as well as for assessing ecology of denitrifying nitrite reductase
(Grüntzig et al., 2001). We applied this technique to study the population
dynamics in soil of a recombinant
Rhodococcus
RHA1(pRHD34::
fcb
) geneti-
cally engineered to grow on a chlorobiphenyl by quantifying both the 16S
rRNA gene and the introduced 4-chlorobenzoate
fcb
degradation operon
(Rodrigues et al., 2002).
Species-specific 16S rRNA regions were identified for
Rhodococcus
sp. strain
RHA1 using the phylogeny of these strain and close relatives. All candidate
primers included more than one species of the genus
Rhodococcus
because of
the inherent difficulty in designing a 16S rRNA-based strain-specific probe. The
