Kam Tin Leung

Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds

Increasing levels of environmental pollution demand specific and sensitive methods for detection of genotoxic agents in water, food products and environmental samples. Tests for genotoxicity assessment are often based on biosensor strains that respond to DNA damage induced by chemicals. In the present study, fluorescent reporter Escherichia coli strains have been developed, which contain a plasmid-borne transcriptional fusion between the DNA-damage inducible recA promoter and the green fluorescent protein gene (gfp) or a gene encoding a red-shifted, higher intensity GFP variant (mutant 3). GFP-based biosensors allowed the detection of a dose-dependent response to genotoxic agents such as mitomycin C (MMC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and nalidixic acid (NA). A reporter strain carrying recA-gfp mutant 3 fusion gave more dramatic and sensitive response than a strain containing the wild-type gfp. These results indicate that recA-gfp mutant 3-based biosensor is potentially useful for detection of genotoxins.

Using a green fluorescent protein gene-labeled p-nitrophenol-degrading Moraxella strain to examine the protective effect of alginate encapsulation against protozoan grazing

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.

Quantitative detection of pentachlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number/polymerase chain reaction protocol

Abstract Traditionally, biodegradation of pentachlorophenol (PCP) in soil is studied by disappearance of the parent compound and its intermediates. Because of the lack of methods to enumerate PCP-degrading bacteria in environmental samples, little is known about the dynamics of these organisms in soil. In this study, the efficiency of a modified most-probable-number/polymerase chain reaction (MPN/PCR) protocol was compared to the traditional MPN/[14C]PCP mineralization assay to quantify the density of PCP-degrading Sphingomonas sp. UG30 cells inoculated in an agricultural soil. A 753-bp tetrachlorohydroquinone reductive dehalogenase gene (pcpC) fragment of UG30 was targeted for the MPN/PCR amplification. The MPN/PCR protocol had a detection limit of 3 CFU/g dry soil. A good correlation was established between the MPN/PCR estimations and initial inoculum densities ranging from 30 to 2×109 CFU/g of soil. However, the MPN/[14C]PCP mineralization protocol underestimated the inoculum density by 70 to 740-fold. Survival of UG30 in soil was monitored by the MPN/PCR assay. Cell density of the UG30 inoculum decreased from 1.8×108 to 1.9×105 cells/g of soil in the first 20 days of incubation and stabilized at 1.9×104 cells/g of soil after 50 days. When the soil was autoclaved prior to inoculation, UG30 cell density remained at 6.7×107 cells/g of soil after 50 days of incubation.

Extraction and amplification of DNA from the rhizosphere and rhizoplane of plants

Direct extraction of nucleic acids (DNA and RNA) from water, sediment, bulk and rhizosphere soil, and plant surfaces can be used to overcome limitations once caused by the necessity to culture microbial cells to allow subsequent detection of specific gene(s) and/or particular microbial species. DNA and RNA are always present in viable bacteria, even though the cells may not be culturable on a variety of microbiological media. For example, a specific DNA sequence can be detected using DNA hybridization techniques, which are among the most accurate and sensitive detection methods (Ford and Olsen 1988; Trevors and van Elsas 1989; Dijkmans et al. 1993; Jackman et al. 1992), especially when extracted DNA is amplified by the polymerase chain reaction (PCR) prior to hybridization analysis (Smalla et al. 1993). Therefore, DNA extraction followed by hybridization and/or PCR analysis will be valuable in studying the molecular ecology of rhizosphere and rhizoplane bacteria. For additional information on PCR instrumentation and use of PCR protocols in genetics, the reader is referred to Innis et al. (1990).

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.

Simultaneous detection of gfp-marked Moraxella sp. G21r and lux-marked Ralstonia eutrophas H850Lr using most-probable-number method.

The green fluorescent protein encoded by gfp gene and the luminescent protein encoded by luxAB genes were used as markers to detect p-nitrophenol (PNP)-degrading Moraxella sp. G21r and polychlorinated biphenyl (PCB)-degrading Ralstonia eutrophas H850Lr cells, respectively, in mixed liquid cultures and in soil samples using a most-probable-number (MPN) assay. Population estimates for both gfp-marked G21r and lux-marked H850Lr by using MPN assays were similar to viable colony counts. The MPN assay with microtiter plates permitted the simultaneous detection of fluorescent and luminescent bacteria in soil samples faster than conventional plate counting.