MICROBIAL BIOTECHNOLOGY: SCOPE, TECHNIQUES CONTD
Over a quarter-century ago,luminescent bacteria were introduced as bio-sensors for the rapid assessment of toxic compounds in aquatic environments. The use of these organisms has now become “institutionalized” for
a wide range of toxicological assays. These assays are versatile because the
change in signal (bio luminescence) is linked directly to change in the global
metabolism of the cell independent of the cause.
The advent of genetic manipulation by recombinant DNA technology has
created a broad range of specific microbial bio-sensors. The great majority of
these are genetically engineered bacteria within which a promoter–operator
(the sensing element) responds to the stress condition (toxic organic or
inorganic compound, DNA damage, etc.) and changes the level of expression of a reporter gene that codes for a protein (the signal). The protein may be detected either directly (e.g., green fluorescent protein) or through its catalytic activity (e.g., formation of a fluorescent or chemiluminescent product).
VIBRIO FISCHERI CYTOTOXICITY TEST:
Toxicological assays that depend on the bioluminescence of V. fischeri NRRLB-11177 are used widely in detecting contaminants in aquatic environments, monitoring wastewater treatment, and generally in assessing the relative cytotoxicity of a wide range of compounds that are released into the environment as a direct or indirect consequence of human activity.
Vibrio fischeri NRRLB-11177 is a naturally bioluminescent marine bacterium. The
bioluminescence results from an oxidoreductase (reaction 1) and a luciferase (reaction 2) catalyzed reaction sequence:
and RCHO is palmitaldehyde.
Palmitaldehyde is regenerated by the following reaction:
The cytotoxicity assay is performed as follows:
1.Freeze-dried cells are reconstituted in buffer and incubated at the desired assay temperature.
2.Equal volumes of solution of analyte at different concentrations are added to equal
aliquots of bacterial suspension.
3.Luminescence of these solutions and of a control solution (lacking analyte) is measured.
4.The percent inhibition (I) is given by
where Ic is the bioluminescence of the control solution and Iathat of a solution containing analyte. The analyte concentration that gives 50% inhibition of bioluminescence
(designated EC50) provides a quantitative measure of toxicity under the conditions of
this assay. Because the intensity of bioluminescence is dependent on
the intracellular levels of ATP and NADPH, the assay effectively monitors
the metabolic status of the cell. Consequently, damage to the cytoplasmic
membrane,interference withtransport processes that bring metabolites into
the cell,interference with electron transport systems,and other perturbation
of the ion gradients across the cytoplasmic membrane all result in a decrease
in bioluminescence. This strong point of the assay is also a weakness. In
itself, the assay provides no information on the nature of the toxic effect or
the molecular target affected by the analyte. However, the assay has proved
to serve as a useful indicator of toxicity of a wide variety of compounds to
REPORTER GENE BIOASSAYS:
The above limitations are addressed by assays designed to detect specific
molecules. Following are three examples from among dozens of such assays.
Certain Staphylococcus aureus strains carry plasmid pI258, which contains the operon cadAcadC. This operon confers resistance to Cd ion and Zn ion and Cd ion also acts as an inducer. The bioluminescence of S. aureus, engineered to carry a construct in which the luciferase genes,luxAB of Vibrio harveyi, are placed under the control of the cadApromoter, allows detection of Cd ion over a concentration range of 1 to 100µM.
The Pseudomonas oleovorans pathway for octane sensing consists of a transcriptional activator, encoded
by alkS, which activates the alkB promoter in the presence of linear alkanes with chain lengths ranging from C6 to C12. This activator/promoter system can be utilized to express green fluorescent protein (GFP) in E. coli. Wild-type GFP is quickly degraded, so a particularly stable mutant of GFP was used in the
engineered octane-sensing E. coli strain. The bioluminescence of the octane-sensing E. coli showed a dose dependent response range from 0.01 to 0.1µM octane and allowed monitoring of mass transfer of octane through the gas phase or by diffusion from micro droplets through water.
Erwinia herbicola 299R is a colonizer of the plant leaf surface. This epiphytic bacterium was converted to a whole-cell sensor for local sugar availability. The bacterium was transformed with a plasmid, pPfruB-gfp[AAV], in which the promoter region of the operon responsible for fructose utilization in E. coli was fused to a variant of GFP that folds faster than wildtype GFP, gives a brighter fluorescence, and has a significantly reduced stability. These properties make the fluorescence of the engineered E. herbicola strain track closely the rate and level of the GFP gene expression. The engineered strain was sprayed on the surface of bean plant leaves and collected by rinsing sample leaves at intervals after one to 24 hours.The intensity of fluorescence emission from individual cells, measured by epifluorescence microscopy, provided information on the level of sugar on the leaves at the
various times. The results showed that the sugar level was relatively high
in the initial phases of the experiment and then declined as the bacteria
multiplied. Such single-cell sensors have enormous potential in the study
of interactions between microorganisms and their hosts, as well as those
CITED BY Kamal Singh Khadka
Msc Microbiology, TU.
Assistant Professor In Pokhara University, Pokhara Bigyan Thata Prabdihi Campus, PNC, NA, LA.
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