Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 8, Num. 1, 1998

Australasian Biotechnology,
Volume 8 Number 1, January/February 1998, pp. 43-46

Molecular Markers in Environmental Biotechnology

Joanna M. Shepherd and Pak-Lam Yu
Biotechnology Group, Department of Process and Environmental Technology, Massey University, Private Bag 11-222, Palmerston North, New Zealand email: P.Yu@massey.ac.nz

Code Number:AU98011
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In the past decade, advances in molecular biology have greatly expanded the `tools' available for monitoring the activities of microorganisms in the degradation of environmental pollutants. These molecular markers (reporter genes) produce signals which are easily detectable by change in colour or giving off light at a certain wave length. Bioluminescent and fluorescent markers are a class of molecular markers that can be used to monitor cell viability and concentration without the enrichment and disruption of the cell growth in complex environment such as the soil. Molecular markers have wide applications in the field of environmental biotechnology.

Introduction

A molecular marker (reporter gene) is a unique gene that enables detection of a particular bacterial strain in the environment as distinct from the indigenous populations. The ability to track bacteria introduced into the environment is essential for assessing their persistence and dispersal. This is important for risk assessment of genetically engineered microorganisms (GEMs) that have been constructed for environmental applications such as the bioremediation of toxic chemicals, biological pest control, and plant growth promotion. Molecular markers are particularly useful in assessing the potential impact that introduced strains or GEMs may have on the environment through their effects on established populations or by possible exchange of genetic material. A key advantage of molecular markers as tools in microbial ecology is that they enable closely related strains of bacteria to be readily distinguished, and provide a rapid means of identifying the strain of interest. The extent to which these advantages are realised depends largely on the properties of the reporter gene used.

Selection of Useful Molecular Markers

There are a number of criteria that a molecular marker must meet in order to be of use in environmental biotechnology and selection of a strain that is not adversely affected by the marker is necessary. It is important that the characteristics conferred by a molecular marker are not exhibited by the indigenous population in the system being studied, this is a factor that greatly limits the range of potential markers. For a molecular marker to be extensively applicable it must be simple to detect, highly sensitive and cost effective. The product of the marker should exhibit minimal interference with the environmental system and with physiological processes within the cell; the growth, activity and viability of the marked strain should be similar to that of the wild type strain. Expression of the marker gene should not place a metabolic load on the host bacteria; markers have been designed with inducible promoters to reduce metabolic load (Winstanley et al., 1991). The marker gene must be stable within the host organism in the absence of selective pressure, favouring chromosomal rather than plasmid markers, and there must be no transfer of the marker gene to other organisms.

Any gene that is unique to the system being assessed could be used provided that a sensitive, rapid method of detection is available. Some molecular markers that are currently being used for environmental monitoring of bacteria are presented in Table 1. Antibiotic resistance genes are not considered here because of the wide spread resistance to antibiotics already observed in the environment and the general wish not to magnify this situation, and because of the effect that antibiotics may have on the microbial ecology of the environment being studied. Most molecular marker systems require cultivation techniques to be employed before enumeration of the marked organisms is possible, the most commonly used molecular marker systems in environmental monitoring of bacteria include b-galactosidase, b-glucuronidase, bioluminescence, and green fluorescent protein. All these marker systems provide the ability to determine viable and total cell concentrations. An important advance provided by bioluminescent (lux/luc) and fluorescent (GFP) marker systems is the ability to measure marked cell activity without the requirement for extraction of cells and the need to culture organisms. Real time, in situ metabolic activity can be measured using luminescence from lux-marked organisms and fluorescence from GFP marked organisms providing for the first time the ability to quantify and localise the activity of a specific organism in the presence of indigenous microbial communities. These markers therefore have significant advantages over other markers for environmental biotechnology studies.

Table 1. Some molecular markers used for environmental monitoring of bacteria

Molecular marker		Source	Mode of action/detection	Comments	Reference
b-Galactosidase Lactose permease lacZY		Escherichia coli	Cleavage of X-gal produces a blue green colour	High levels of endogenous enzymes in plants and bacteria requires procedures that eliminate background activity	(Drahos et al., 1986)
b-Glucuronidase gusA		Escherichia coli	Hydrolyses a number of glucuronide substrates giving rise to coloured or fluorescent products	No background activity in plants or in many bacteria of economic and agricultural importance	(Wilson et al., 1995)
b-Glucosidase ceIB		Pyrococcus furiosus	Cleavage of X-gal produces a blue green colour	Half life of 85 hours at 100°C	(Sessitsch et al., 1996)
Bioluminescence lux (luciferase)	Vibrio fischeri Vibrio harveyi		Luciferase activity leads to light production	Requires a long-chain aldehyde,O2 and a source of reducing equivalents	(Silcock et al., 1992)
Bioluminescence luc (luciferase)	Photinus pyralis (firefly)		Oxidative decarboxylation of luciferin results in light production	ATP-dependent	(Palomares et al., 1989)
Green Fluorescent Protein GFP	Aequorea victoria (Pacific jellyfish)		Fluoresces green when excited by blue light	Operates independently of cofactors	(Gage et al., 1996)
Catechol 2,3- dioxygenase xyIE	Pseudomonas putida TOL plasmid pWWO		Converts colourless catechol to the bright yellow product2-hydroxymuconic semialdehyde by a meta-cleavage reaction	2-Hydroxymuconic semialdehyde is water soluble Common to many aromatic compound-degrading bacteria found in soil	(Winstanley et al.,1991)
2,4-Dichloro phenoxyacetate monooxygenase tdfA	Pseudomonas sp.		Converts phenoxyacetate to phenol which can be assayed by GC or by production of a red dye on reaction with 4-aminoantipyrine	The assay is more complex than the other marker gene assays	(King et al., 1991)

Detection of Bioluminescent/Fluorescent Marked Organisms

Light output (luminescence, fluorescence) can be detected and quantified in a number of ways. Colonies on agar plates can be detected by eye in the darkroom or by using photographic and X-ray film, and Charge Couple Device (CCD) imaging. Populations in the soil can be detected by
luminometry, and single cells can be detected in soil using CCD-enhanced microscopy. Development of CCD technology has greatly enhanced the power of light-based bacterial detection in environmental samples. These techniques enable assessment of viable and non-culturable cells without the need to extract the cells and additionally provide information on their metabolic activity. Luminometry is a rapid and sensitive technique that correlates closely with other measures of activity and is selective in assessing the activity of the marked population only, against a background of indigenous organisms. CCD imaging is a powerful technique that provides information on the spatial distribution of the individual cells and of colonies (Meikle et al., 1995).

Bioluminescent Markers

The eucaryotic, luc , and procaryotic, lux, genes for luciferase are both used as marker genes to express bioluminescence in host microorganisms. Luciferase activity leads to light production which is an easily detectable reaction product. The eucaryotic and the procaryotic luciferase enzymes carry out different reactions that produce light. The luciferase from the North American firefly Photinus pyralis catalyses the ATP-dependent oxidative decarboxylation of luciferin resulting in the production of light in the most efficient bioluminescent reaction known. The assay for the enzyme is extremely sensitive and essentially instantaneous (Palomares et al., 1989). The procaryotic lucifease genes have been cloned from the marine bacterium Vibrio fischeri. There are seven genes involved located on two operons. The structural genes, luxA and luxB encode luciferase, and luxC, luxD, and luxE code for the synthesis and recycling of the aldehyde substrate, while luxI and luxR are involved in regulation of luciferase production. Light production catalysed by luciferase requires a long-chain aldehyde and a source of reducing equivalents, usually reduced flavin mononuleotide (FMNH2). Most of the marker gene constructs contain only the luxAB genes in order to minimise the metabolic load, this means that n-decyl aldehyde must be supplied to the marked strains as an exogenous substrate in order for light to be produced (Hastings and Nealson, 1977). Variation in luminescence per cell may reflect the physiological state of the organism or the copy number of the marker.

A number of environmental studies have successfully made use of bioluminescent marker genes. Single bioluminescent Pseudomonas syringae cells marked with the luxAB genes from Vibrio harveyi were able to be detected in soil slurries using charge couple device-enhanced microscopy, and additionally the extent of competition from indigenous soil bacteria could be monitored. This technique is considered to offer great potential for tracking and determining the spatial distribution of genetically marked organisms in the environment (Silcock et al., 1992). Pseudomonas sp. tagged with the luxAB genes demonstrated in the rhizosphere that bioluminescence is at least 1,000-fold more sensitive than b-galactosidase-based systems (de Weger et al., 1991). Rhizobium meliloti has been stably tagged with the firefly luciferase gene, luc, using a mini-Tn5 delivery vector. Studies revealed a good correlation between cell biomass and bioluminescence (Cebolla et al., 1993).

Green Fluorescent Protein

Green fluorescent protein is a spontaneously fluorescent protein isolated from coelenterates. The green fluorescent protein from the jellyfish Aequorea victoria fluoresces green (508 nm) when excited by long wave ultraviolet light (395 nm) (Chalfie et al., 1994). This protein autocatalytically forms a chromophore of three amino acids within its primary structure and in contrast to other bioluminescent molecules GFP operates independently of cofactors (Reid and Flynn, 1997). It appears that the folding of the chromophore requires oxygen as active GFP is not produced in anaerobic conditions, and GFP does becomes active on exposure to oxygen. GFP possesses a number of significant advantages over other molecular markers, such as real-time in vivo visualization, which is independent of exogenous co-factors, small molecular size (2.69kDa) and monomeric nature. GFP is stable after exposure to heat, extreme pH and chemical treatments (Bokman and Ward, 1981).

GFP has been used successfully for environmental monitoring of marked bacteria. GFP marked bacteria have been used to visualise the early events of symbiosis between Rhizobium meliloti and alfalfa. Growing cells could be seen in the rhizosphere, the root tip, and inside infection threads. Cells that were not active did not produced observable fluorescence (Gage et al., 1996). Another study quantitatively measuring GFP expression in E. coli found that after GFP levels peak there is a decrease in GFP concentration while the fluorescent remains high (Albano et al., 1996). The survival of bacteria introduced to activated sludge has been studied used GFP marked organisms, the numbers of marked organisms were observed to decrease in the sludge due to predation by protozoa. The fluorescence from the marked bacteria allowed them to be visualised inside the protozoa (Eberl et al., 1997). GFP has also been used successfully to monitor the survival and tracking of genetically engineered bacteria in aquatic environments (Burlage et al., 1996), however the researchers note that further study is required to determine if GFP is produced continually or if initial GFP stocks in the cells persist (Leff and Leff, 1996). Studies of GFP-tagged Pseudomonas fluorescens suggest that the GFP persists in the cell for long periods of time, probably until cell lysis and that fluorescence which is observed to increase during starvation is probably due to cell shrinkage causing concentration of the GFP (Tombolini et al., 1997).

Summary

GFP and luminsecence (lux/luc) marker genes have great advantages over other molecular marking systems employed for environmental monitoring of bacteria. They allow rapid monitoring of the marked strains without the need for culturing. The sensitive of detection of light output using CCD imaging technology, luminometry and microscopy techniques make these markers a powerful tool which is now being applied to ecological studies of a range of organisms. The information being obtained is of enormous value for risk assessment studies for environmental release of GEMs, in determining activity and potential activity of culturable and nonculturable cells. Of equal importance is the ability of these techniques to increase our inderstanding of the environmental factors controlling microbial growth, activity, survival and interactions in natural environments.

References

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