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MethodsMicrobiological

Journal of Microbiological Methods 35 (1999) 187–199

Review

Applications of the green fluorescent protein as a molecular markerin environmental microorganisms

a b b b b bD. Errampalli , K. Leung , M.B. Cassidy , M. Kostrzynska , M. Blears , H. Lee ,b ,*J.T. Trevors

aAgriculture and Agri-Food Canada, P.O. Box 1210, Charlottetown, PEI, Canada C1A 7M8bDepartment of Environmental Biology, University of Guelph, Guelph, ON, Canada N1G 2W1

Received 23 November 1998; accepted 18 January 1999

Abstract

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmentalmicrobiology research. The GFP and its variants are reviewed and applications in plant–microbe interactions, biofilms,biodegradation, bacterial–protozoan interactions, gene transfer, and biosensors are discussed. Methods for detectingGFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new researchthat will increase our understanding of microorganisms in the environment. 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Bacteria; Biotechnology; Environmental; Green fluorescent protein; Marker; Microorganisms; Soil

1. Introduction natural ecosystems and possible effects that mayresult from dissemination of novel genetic infor-

Genetically engineered microorganisms (GEMs) mation to indigenous microbial populations is essen-are being developed and assessed for their beneficial tial to understand the fate of GEMs in the openuses in agricultural pest control and bioremediation environment.of toxic chemicals in the environment. Bioremedia- Detection methods are required to monitor surviv-tion of soils and sediments contaminated with chemi- al of GEMs in a background of large numbers ofcal pollutants by naturally occurring microorganisms heterogenous microbial populations. In the past,and GEMs may provide a cost effective alternative to researchers depended on conventional microbiologi-physical and chemical remediation procedures. Cur- cal methods, such as activity measurements and therent interest in the possible release of GEMs into the isolation and cultivation of pure cultures on selectiveopen environment has raised concerns over issues of media. However, many microorganisms present inhuman and environmental health. Knowledge about soil and water are difficult to cultivate and onlythe survival and expression of traits in GEMs in about 0.1–10% of the microorganisms present in the

soil are culturable (Brock, 1987). Recently, molecu-lar detection methods including gene probing, DNA*Corresponding author. Tel.: 1 1-519-824-4120 (extn. 3367); fax:hybridization, polymerase chain reaction (PCR) and1 1-519-837-0442.

E-mail address: [email protected]. (J.T. Trevors) reporter genes have been used in addition to tradi-

0167-7012/99/$ – see front matter 1999 Elsevier Science B.V. All rights reserved.PI I : S0167-7012( 99 )00024-X

188 D. Errampalli et al. / Journal of Microbiological Methods 35 (1999) 187 –199

tional detection methods. These molecular detection victoria has attracted considerable attention as amethods assist in the identification of genetically marker / reporter system. The objective of this manu-marked microorganisms in environmental samples. script is to review applications of the GFP as a

Molecular markers, genes conferring specific molecular marker in environmental microbiologyphenotypes, are used to detect and enumerate micro- research.organisms after their introduction into the environ-ment (Masson et al., 1993). Monitoring GEMs inspecific and diverse environments requires uniqueand easily identifiable markers. The marker should 2. Green fluorescent protein (GFP)facilitate detection of the introduced microorganismsfrom diverse indigenous microbial populations. The green fluorescent protein (GFP) is a 27 kDaMoreover, genes such as the gfp introduced into the polypeptide, which converts the blue chemilumines-

21bacterial chromosome are more stable than plasmid- cence of the Ca -sensitive photoprotein, aequorin,borne genes and minimize the potential transfer of into green light (Chalfie, 1994; Cody et al., 1993).marker DNA to indigenous environmental micro- Inouye and Tsuji (1994a) showed the active chromo-organisms. phore is a tripeptide and is dependent on the

Considerable research has been conducted on the presence of oxygen to maturate. Wild-type GFPuse of molecular markers for detection of GEMs in absorbs blue light at 395 nm and emits green light atthe environment and the literature has been reviewed 510 nm in bioluminescent organisms and whenup to 1994 (Akkermans et al., 1994; Greer et al., purified in solution (Ward et al., 1980).1993; Prosser, 1994). Akkermans et al. (1994) In 1994, a GFP-based reporter system was de-discussed the importance of marker genes and DNA veloped in which visible fluorescence was created byprobes when studying the ecology of GEMs and molecular biological techniques. Prashar et al.wild-type microorganisms that are non-culturable. (1992) cloned gfp cDNA from Aequorea victoriaMolecular biology techniques, including reporter and Chalfie et al. (1994) showed the expression ofgenes and DNA probes used in the isolation and the cloned gfp gene produces green fluorescence inmonitoring of pollutant degrading bacteria, have various organisms such as Drosophila and Es-been reviewed by Greer et al. (1993). Prosser (1994) cherichia coli. The GFP can be used as a reporter forpresented a comprehensive review of molecular the visualization of gene expression and proteinmarker systems including antibiotic resistance, subcellular localization (Chalfie et al., 1994). InouyelacZY (b-galactosidase), xyIE (catechol 2,3-dioxy- and Tsuji (1994b) showed that the GFP can begenase), tfdA (2,4-dichlorophenoxyacetate), and lux introduced into cells and intact organelles within(luciferase) used for detection of GEMs in the cells.environment. In 1994, a novel marker system, the GFP is an attractive marker system to monitorgreen fluorescent protein (GFP) became available bacterial cells in the environment (Fig. 1a). Detection(Chalfie, 1994). Chalfie (1994) has also reviewed of GFP requires irradiation by blue light or near-properties of the native GFP, and the expression of ultraviolet (UV) light and does not require anyGFP in heterologous systems. Research on the exogenous substrate, complex medium, or expensivebiochemical characterization of GFPs has been car- equipment. These, and other characteristics (Tables 1ried out for over 35 years. For more information, the and 2), make the GFP more attractive than otherreader is directed to a comprehensive review of the genetic markers such as the luxAB luminescencebiochemistry of GFP published by Tsien (1998) and marker, which requires aldehyde substrates to bea recent text on bioluminescence and chemilumines- visualized (Chalfie, 1994; Kremer et al., 1995;cence (Hastings et al., 1997). Jansson and Prosser Valdivia et al., 1996). The GFP is stable in the(1997) have reviewed bacterial monitoring methods presence of many denaturants and proteases, andwith an emphasis on the quantitation of specific persists at high temperatures (658C), pH values (6–organisms and their activity. In the past 4–5 years, 12) (Ward et al., 1980) and can withstand parafor-the GFP of the bioluminescent jellyfish, Aequorea maldehyde treatment, thereby allowing detection of

D. Errampalli et al. / Journal of Microbiological Methods 35 (1999) 187 –199 189

Fig. 1. (a) Two large green fluorescent colonies of GFP-marked Pseudomonas fluorescens R2fGI and indigenous soil microbial coloniesfrom an agricultural soil plated on 1/10 tryptic soy broth (TSB) agar. These colonies were viewed and photographed under a Leicafluorescent stereo dissecting microscope using a GFP-plus filter set (excitation filter 480 nm; beam-splitting mirror, 505 nm; barrier filter,510 nm). (b) Six macro-colonies (2–3 mm in diameter) of GFP-marked Pseudomonas fluorescens R2fGI cells grown on TSB agarillustrating the intensity of red-shift mutant gfp. Colonies were viewed and photographed as in (a). (c) Protozoan ciliate (Tetrahymena sp.)after ingestion of gfp-tagged Moraxelia sp. G21 cells. Note the vacuoles and GFP fluorescence inside the protozoan after cell walls havebeen digested (arrow). The GFP is stable and capable of fluorescence after the Moraxella cells have been ingested and lysed (average size ofthis protozoan is 40 pm diameter). This photograph was taken using a Nikon Labophot epifluorescent microscope equipped with a GFP filterset (Chroma Technology Corn., Brattleboro, VT: excitation wavelength, 420–470 nm; emission wavelength 490–530 nm). (d) GFP-markedPseudomonas fluorescens R2fGI micro-colonies (not individual cells) on and around surface of tomato root. Tomato plant was grown inagricultural soil inoculated with R2fG1 cells for 4 days. Root segment was embedded in sterile 1 /10 TSB agar and incubated for 24 h at308C. Root and GFP-marked cells were viewed and photographed as in (a) (Magnification factor, 50).

green fluorescence in fixed samples (Eberl et al., 10 000 GFP molecules in the cytoplasm of a tissue1997). culture cell were necessary to accurately detect

Early studies showed expression from a single microscopically. However, a mutant strain of GFPcopy of wild-type gfp in the chromosome of a marked Psedomonas putida cells resulted from abacterium was not strong enough to be detected by mutated GFP protein which was more fluorescent,fluorescent microscopy (Errampalli et al., 1997). more soluble and more evenly distributed throughoutPatterson et al. (1997) suggested that approximately the cytoplasm than the wild-type, and allowed de-


Table 1Comparison of the major bacterial genetic markers

Genetic marker Substrate requirement Principle of detection Major quantification methods

gfp No Fluorescence (1) Epifluorescent microscopy(2) Fluorimetry(3) Flow cytometry(4) Plate counting

luxAB/ luc n-decanal / luciferin Luminescence (1) Luminometry(2) CCD digitized camera(3) Flow cytometry(4) Plate counting

lac ZY x-gal Colour change Plate counting(colourless → blue)

xyl E Catechol Colour change Plate counting(colourless → yellow)

Heavy metal Heavy metals Heavy metal resistance Plate countingresistance genesAntibiotic Antibiotics Antibiotic resistance Plate countingresistance genes

Genetic marker Single-cell detection Real-time in situ detection References

gfp Yes (high resolution) Yes Chalfie et al. (1994)lux AB/ luc Yes (low resolution) Yes Prosser (1994)lac ZY No No Drahos et al. (1986)xyl E No No Winstanley et al. (1989)tfd A No No King et al. (1991)Heavy metal No No de Lorenzo (1994)resistance genesAntibiotic No No Hagedorn (1994)resistance genes

tection of single cells in situ (Eberl et al., 1997). tant that produces blue fluorescence with excitationRecently, high-fluorescent-intensity red-shifted gfp and emission maxima at around 385 and 445 nm,variants have been developed with excitation wave- respectively (Heim et al., 1994; Stauber et al., 1998;lengths shifted to 481–501 nm (Cormack et al., Yang et al., 1998), and a yellow–green fluorescent1996). At these wavelengths, the fluorescence signals mutant with excitation and emission wavelengths at

öf the red-shifted GFP mutants are 20–35 times 513 and 527 nm, respectively (Ormo et al., 1996;stronger than the wild-type GFP (Cormack et al., Clontech, Palo Alto, CA, USA, Cat. No. 6004-1).1996) (Fig. 1b) and the effect of photobleaching at Using specific optical filters, three different formsthese longer wavelengths is lower than at 395 nm have been detected simultaneously in samples of(Cubitt et al., 1995). Single cell detection of bacteria living bacteria (Heim and Tsien, 1996). Patterson etlabelled chromosomally with one copy of the red- al. (1997), in comparing properties of five GFPshifted gfp is possible using epifluorescence micro- variants relevant to quantitative imaging in live cells,scopy or flow cytometry (Delagrave et al., 1995; suggested that no single variant was ideal for allHeim et al., 1994; Cormack et al., 1996). applications, but each one offers advantages and

A series of GFP mutants with various excitation disadvantages for imaging living cells.and emission wavelengths have been developed Although plasmid-based gfp vectors have been(Table 3). This development allows monitoring of used in eukaryotic systems, and some gfp-broad-multiple species of bacteria simultaneously in a host-range plasmids have been successfully used tocomplex microbial community. They include a mu- label certain species of bacteria, there are two


Table 2Advantages and disadvantages of GFP marker in environmental applications

AdvantagesEase of detectionNo exogenous substrate neededNo processing of cells requiredAble to monitor single cellsNo fixing or staining of samples /cells necessary; but detection of fluorescence still possible in

formaldehyde fixed cellsNon-destructive; detection without disruption of microbial communityPossible to monitor on-line or in real timeExtremely stable—heat (658C); pH (6–12); resistant to denaturants and proteasesGFP expressed in cytoplasm; should have minimal effect on cell–surface dynamicsContinually synthesized; minimizes fluorescence signal dilution during bacterial replicationAllows analysis of living cells; repeated readings under various conditions for the same cell is possibleNo GFP background in indigenous bacterial populationsDual detection possible with different coloured markers

DisadvantagesVariability of GFP expression in different species unknownPlasmids may be unstable—use chromosomal insertionInfluence of environmental conditions on GFP expression is unknownInterference by other fluorescent particles or bacteriaExtended lifetime of fluorescence once cell had died or lysedGFP may not work under anaerobic conditions

Table 3Major variants of GFP

GFP variant Major Major Referenceexcitation emissionwavelength wavelength(nm) (nm)

Wild type 395 509 Chalfie et al. (1994)Red-shifted variants 471–490 502–511 Heim et al. (1994);

Cormack et al. (1996);Heim et al. (1995);Stauber et al. (1998);Yang et al. (1998)

Ÿellow–green variant 513 527 Ormo et al. (1996)OGFP 385 510 Crameri et al. (1996)(enhanced fluorescence)GFP variants 488 507 Andersen et al. (1998)with short half-lives

limitations to applying these vectors for bacterial of the genetic marker to other microorganisms (Fig.strains used in environmental studies. First, due to 2). The second limitation is a low sensitivity forconcerns about plasmid stability under natural en- single-cell detection of cells containing a single copyvironmental conditions, bacterial strains used in of the gfp marker. To circumvent these limitations, aecological studies should be chromosomally marked Tn10- (Stretton et al., 1998) and several Tn5-basedwith a single copy of the gfp gene to maximize (Burlage et al., 1996; Eberl et al., 1997; Matthysse etgenetic stability as well as reduce the risk of transfer al., 1996; Suarez et al., 1997; Tombolini et al., 1997)


Fig. 2. Construction of pUTGFPI and pUTGFP2. The PUTmini-Tn5 was digested with Notl and the Notl fragment containing luxAB wasreplaced with: (A) PCR fragment ( gfp 1 lac promoter), amplified with primers GFP3 (3) and GFP5 (5) and digested with NotI to yieldpUTGFPI and (B) PCR fragment ( gfp), amplified with primers GFP4 (4) and GFP5 (5) and digested with Notl to yield pUTGFP2. Ap, blagene which confers resistance to many b-lactam antibiotics; ori, R6K origin of replication; lac, lac promoter; mob RP4, the RP4 onT region;N, NotI; O and I, Tn5 end regions; TC, tetracycline (reprinted from Errampalli et al., 1998).

transposon suicide delivery vectors have been de- 3. Environmental applications of greenveloped. They have been used to mark various fluorescent proteinGram-negative bacteria including, Agrobacteriumtumefaciens, Alcaligenes eutrophus, Moraxella sp., Since 1996, gfp-based marker systems have beenPseudoalteromonas sp., Pseudomonas fluorescens, developed for use in environmental applicationsPseudomonas putida, Psych robacter sp., and Vibrio (Table 4). For example, the gfp has been used tosp. To ensure single cell detection of the labelled study dynamics and/or distribution of gfp-labelledbacteria, red-shifted gfp mutants with enhanced bacteria in soils, water systems, rhizospheres, ac-green fluorescence intensity and a strong bacterial tivated-sludges, biofilms, and root nodules. In addi-promoter are normally used in these constructs. To tion, the gfp has been used to study gene transferour knowledge, there have not been any studies on between bacterial populations in biofilms and onGram-positive bacteria chromosomally engineered phylloplanes. The gfp system has also been used towith a gfp genetic marker. study gene expression in bacteria. Since gfp-labelled


Table 4Bacterial strains marked with GFP for environmental detection and monitoring

Bacterial strain Insertion site Environment Reference

Agrobacterium tumefaciens C58 Chromosome Rhizosphere Matthysse et al. (1996)Alcaligenes eutrophus Chromosome Buffer Suarez et al. (1997)Escherichia coli Plasmid Activated sludge Olofsson et al. (1998)Moraxella sp. G21 Chromosome Soil Tresse et al. (1998)Pseudoalteromonas sp. S91 chromosome Marine Stretton et al. (1998)Pseudomonas aeruginosa PAQi Plasmid Biofilm Bloemberg et al. (1997)P. fluorescens A5O6 Chromosome Rhizosphere Tombolini et al. (1997)P. fluorescens R2fG1 Chromosome Rhizosphere Our laboratoryP. fluorescens WC5365 Plasmid Rhizosphere Bloemberg et al. (1997)P. putida KT2442 Chromosome Activated sludge Eberl et al. (1997)P. putida KT2442T0L Plasmid Phylloplane Normander et al. (1998)P. putida JB167, JB279, JB340, JB391, JB396 Chromosome Buffer Andersen et al. (1998)

¨P. putida RiTOL Plasmid Biofilm Moller et al. (1998)P. putida UWCI Chromosome Buffer Matthysse et al. (1996)Pseudomonas sp. Chromosome Buffer Burlage et al. (1996)Pseudomonas sp. B13 Chromosome Buffer Suarez et al. (1997)Pseudomonas UGI4 Chromosome Soil Errampalli et al. (1998)Phychrobacter SW5H Chromosome Marine Stretton et al. (1998)Rhizobium meliloti Plasmid Root hairs and nodules Gage et al. (1996)Serratia marcescens Plasmid Activated sludge Olofsson et al. (1998)Vibrio sp. S141 Chromosome Marine Stretton et al. (1998)

bacteria can be visualized by fluorescent microscopy, marked chromosomally with a red-shift gfp variantsingle cells of the labelled microorganisms can be (Tombolini et al., 1997). Because of the improveddetected in various samples, providing information fluorescent intensity of the red-shift gfp and a strongabout spatial location, survival or specific gene constitutive promoter (PpsbA from Amaranthus hy-expression. bridus), individual cells were visualized on the root

surface of Lotus japonicus plants using epifluores-cent confocal laser scanning microscopy.

4. Plant–microbe interactions

Gage et al. (1996) labelled Rhizobium meliloti 5. BiofilmsMB5OI with the gfp marker via the broad-host-rangeplasmid, pTB93F. The heterologous gfp was ex- The gfp system is ideal for studying multiple-pressed constitutively in this microorganism. Visuali- species bacterial communities in biofilms and adhe-

¨zation of the R. meliloti cells during infection of the sion of bacteria to flocs in activated sludge. Moller etroot and subsequent nodulation was observed. The al. (1998) demonstrated that gfp-labelled Pseudo-gfp system has also been used to visualize bacterial monas putida RI tended to colonize the outermostdistributions on root surfaces (Fig. 1d). Bloemberg et layer of a mixed-species biofilm while anal. (1997) constructed a gfp plasmid (pGBS) which Acinetobacter sp. strain C6 primarily attached to thewas maintained in Pseudornonas fluorescens substratum of the biofilm. Skillman et al. (1998)WC5365 cells for 7 days without antibiotic selective visualized bacterial distribution in dual-speciespressure. The gfp was constitutively expressed in the biofilms using a gfp-labelled Enterobacter. ag-cells without nutrients being supplied. Under labora- glomerans strain and an unlabelled Kiebsiella pneu-tory conditions, the gfp-labelled WCS36S cells were moniae strain. Sixteen-hour biofilms were stainedobserved to form microcolonies at the borders of using propidium iodide (P1) (did not mask the GFP),adjacent epithelial cells on the root surface of tomato which enabled the reseachers to visualize the spatialseedlings. Pseudomonas fluorescens A506 has been distribution of the gfp-labelled cells in green or


yellow, and the P1 stained cells as red using a tial location and degradative capability are all im-violet-blue excitation filter (395–446 nm) on a portant factors that provide valuable information tomicroscope. The two bacteria were often closely advance our understanding of the processes occur-associated in microcolonies, and the reseachers con- ring. The use of the GFP as a marker has allowedcluded that surface-associated macromolecules form researchers to detect the added cells, and determinethe basis of these interactions. They suggested that more precise data about cell counts, survival andthis convenient technique enabled them to observe spatial location than has previously been possible. Inthese unique interactions between the bacteria and to our laboratory, the GFP marker was employed todetermine the true properties of the resultant biofilm. study the survival of a p-nitrophenol (PNP)-degrad-

A gfp-labelled Pseudomonas putida has been used ing Moraxella strain in soil. The chromosomallyto study bacterial survival in activated sludge (Eberl gfp-labelled Moraxella sp. strain G21 had beenet al., 1997). Free swimming cells were observed constructed using a mini-Tn5-gfp suicidal plasmidbetween sludge flocs immediately after their intro- (see Fig. 2) (Tresse et al., 1998). The labelledduction and after 2 mm, protozoan grazing on the bacteria degraded up to 1440 pM PNP, both in brothlabelled cells was observed. After 3 days incubation, and soil, to the same extent as the parent strain.few fluorescent cells were detectable, but most of Survival studies indicated that individual greenthese cells were incorporated into the sludge flocs fluorescent colonies of G21 were detected up to 2suggesting a protective role of the floc from preda- weeks after inoculation in soil samples. It istory protozoa. The association between the GFP9 noteworthy that although only 0.1% of the originalmarked cells and flocs was visualized using epi- inoculum was detected by plating, 58% of thefluorescent microscopy. Using gfp-labelled E. coli radiolabelled PNP was mineralized. This suggestsand Serratia marcescens strains, Olofsson et al. that not all cells were culturable, or that there may(1998) determined that cell surface hydrophobicity have been an indigenous population of PNP-degrad-was an important factor in bacterial adhesion to ing microorganisms present in the soil. Errampalli etflocs. Epifluorescent microscopy enabled the visuali- al. (1998) detected GFP-marked Pseudomonas sp.zation and enumeration of free GFP marked cells UGI4Gr up to 13 months after inoculation intowhich could be distinguished in the activated sludge. creosote contaminated soil. By inserting the mini-Confocal laser scanning microscopy (CLSM) of the Tn5-gfp into the chromosome of a phenanthrene-gfp-labelled bacteria enabled a 3-dimensional profile degrading bacterium, a green fluorescent mutant,to be produced which showed that the two bacteria Pseudomonas sp. UGI4Gr, was obtained. The gfphad very different attachment patterns in the flocs, was stable in UGi4Gr and did not affect miner-

14and that cells can penetrate the flocs through chan- alization of C-labelled phenanthrene which verifiednels and pores. The authors suggested the combina- the gfp insertion had not interfered with phenan-tion of epifluorescent and confocal laser scanning threne-degradation genes. Colony forming units weremicroscopy to visualize GFP-marked cells offers used to enumerate GFP-marked cells from soilgreat potential for further investigations. Their ap- samples and UG14Gr colonies on solid agar platesproach resulted in a better understanding of bacterial were detected using a hand-held UV lamp. Detectionadhesion mechanisms and may assist in improving of GFP-marked cells for more than a year afterthe effectiveness of wastewater treatment. inoculation into soil microcosms demonstrated the

GFP marker can be useful for long-term bacterialsurvival in soil biodegradation or studies.

6. Biodegradation

In many biodegradation studies in the laboratory, 7. Bacterial transport through environmentalbacterial cells which are metabolically capable of matricesdegrading or mineralizing a pollutant are added tocontaminated environmental samples to determine Understanding bacterial transport through environ-biodegradation of target compound(s) in the environmental matrices, such as soil is critical to the successment. Cell counts, survival, metabolic activity, spa- of delivering microorganisms to contaminated sites


for bioremediation of pollutants. However, little is as green fluorescent cells using an epifluorescentknown about the factors controlling mobility of microscope and enumerated by plating. The authorsmicroorganisms in the subsurface. Burlage et al. observed a high frequency of conjugal transfer of the(1996) were able to track gfp-labelled Pseudomonas marker between donor and recipient cells (at 100%putida cells through a soil microcosm using a relative humidity, density of the transconjugants was

3 2fluorescence spectrophotometer. Although the lower 3 3 10 CFU/cm , corresponding to about one-thirdlimit of detection of the cells taken directly from soil of the recipient population) in specific microhabitats

5samples was 10 cells /0.5 ml fraction, the research- on the phylloplane of bush bean plants. The fre-ers suggested that the detection limit could be quency of horizontal plasmid transfer was about 30improved with more sophisticated equipment. They times higher on the phylloplane than on a polycarbo-also concluded that the GFP-tagged strain was easily nate filter which indicated the phylloplane had adetectable and the results obtained with this method stimulatory effect on gene transfer between bacteriawere as accurate as plate counts. aggregated into microhabitats on the leaf surface.

They suggested that this was the first study in whichthe effect of cell distribution on gene transfer was

8. Bacterial–protozoan interactions directly assessed. The GFP allowed them to visualizetransconjugant cell distributions in situ, which had

Ingestion of bacteria by protozoa is a major not previously been possible. Christensen et al.process controlling survival of bacteria in the en- (1996) investigated details on the distribution ofvironment. Eberl et al. (1997) observed green cells active in conjugation. Their approach was tofluorescent cells within various ciliate protozoa such monitor the flux of the gfp-tagged TOL plasmidas Vorticella and free-swimming protozoa Aspidisca transfer between two Pseudomonas strains estab-sp. and Bodo sp. within 2 min of addition of GFP- lished in a biofilm community. Although they ob-marked Pseudomonas putida Green3I cells to acti- served plasmid transfer, the fraction of GFP 1 cellsvated sludge. They suggested that the use of GFP- was small, and the signal was faint. However, theytagged bacteria may be valuable for following bac- suggested that the GFP system offers great potentialterial fate on-line, and estimating instantaneous rates for gene transfer studies due to the simple detectionof in situ protozoan grazing on bacteria. Similarly, and high sensitivity of the GFP signal.Olofsson et al. (1998) observed ingested GFP-mark-ed bacterial cells within predators during their studyof flocs in activated sludge. In our laboratory, we 10. Biosensorsvisualized ingestion of GFP-marked Moraxella sp.G21 cells by a ciliate strain (Tetrahymena sp.) in an By fusing reporter genes to specific catabolic geneaqueous microcosm using epifluorescence micro- promoters, biosensors can be designed to switch onscopy (Fig. 1c). in the presence of a particular pollutant. This ap-

proach has been successfully employed with lux-based (luminescent) systems. For example, Burlage

9. Gene transfer et al. (1990) developed a lux-modified bacterialbiosensor to identify the presence of naphthalene,

The GFP system is also useful for studying and Selifonova et al. (1993) constructed biolumines-horizontal gene transfer between microorganisms in cent biosensors to detect bioavailable Hg (II) in thethe environment. For example, Normander et al. environment. To our knowledge, there have been no(1998) used Pseudomonas putida donor cells with a published studies on the use of gfp-tagged cells asderivative of the TOL plasmid conferring kanamycin biosensors of environmental pollutants. Since thereresistance and had the gfp inserted downstream of a are many advantages to using the GFP for en-

qlac promoter. A chromosomal insertion of lacI vironmental studies (Table 2), our laboratory isprevented expression of the gfp gene. The P. putida currently developing a GFP-based biosensor forrecipient had a chromosomal tetracycline resistance environmental pollutant detection. Bioluminescent-marker. Transconjugants could be visualized in situ based biosensors have also been used to measure


acute toxicity as they respond to the presence of of the population was active by using a CCD cameratoxic compounds with a decline in light production, and a luminometer (unpublished results or personalreporting the effect of the toxicant on bacterial communication 1998). This approach, which allowsmetabolic activity. Since the GFP is not dependent monitoring of bacterial growth, persistence andon metabolic activity for its fluorescence, it may not activity by luminometry, flow cytometry, and CCD-be a useful system for toxicity assessment. enhanced epifluorescence microscopy, may be ex-

tremely useful for many environmental applications.

11. GFP applications with other technologies12. Detection methods for GFP

Although the GFP has proven to be a useful toolon its own, combining the use of GFP technology GFP-marked bacteria can be detected by severalwith other luminescent markers or probes is also methods. Direct microscopy of gfp-marked bacteriapromising to provide answers to challenging ques- is simple using an epifluorescent microscope with ations in environmental research (Eberl et al., 1997; GFP filter kit, and bacterial counts are rapid andErrampalli et al., 1998). Eberl et al. (1997) studied precise (Leff and Leff, 1996). In our laboratory,community structures with GFP and tetra- using cells marked with the red-shifted mutant, and amethylrhodamine-labelled probe, GAM42a and filter kit (e.g., Chroma Technology Corp., Brattle-simultaneously detected GFP-marked P. putida boro, VT: excitation wavelength, 420–470 nm; emis-Green3l and cells belonging to beta and gamma sion wavelength 490–530 nm) individual cells aresubclasses of Proteobacteria. Community structures easy to see and count, fluorescing bright green on awere visualized by in situ rRNA hybridization and black background. A model 120es 1.2 million pixelsimultaneous detection of GFP fluorescence. Christ- digital camera system (Pixera Corp., 140 Knowlesensen et al. (1996) used gfp labelled donor cells, and Drive, Los Gatos, CA 95030, USA) interfaced with aluxAB recipient cells to visually distinguish the two PC is routinely used for imaging individual cells andstrains in their study on distribution of cells active in colonies. Bloemberg et al. (1997) observed one or a

3conjugation in the transfer of the TOL plasmid. In few GFP-marked cells in a background of . 10our laboratory, GFP-marked Pseudomonas sp. unlabelled cells on a wet mount slide. An epifluores-UG14Gr was co-inoculated with luxAB-marked cent microscope equipped with a standard fluoresceinPseudomonas aeruginosa UG2Lr cells in creosote isothiocyanate (FITO) filter set is also effective forcontaminated soil (Errampalli et al., 1998). The two the detection of gfp red-shifted mutants which havebacteria were detected simultaneously and unam- excitation and emission maxima at 488 and 510 nm,biguously in soil samples for 390 days after inocula- respectively (Cormack et al., 1996). Interfacing antion. These studies demonstrate the potential of epifluorescent microscope with a charged-coupledtracking several introduced microorganisms at the device (CCD) camera and image analysis softwaresame time in bacterial survival and bioremediation can enhance and allow quantification of the fluores-studies. cence of individual gfp-tagged bacteria. Since the

Since the GFP is so stable and persists after cell wild-type gfp only has a minor excitation peak atdeath, researchers in the lab of Dr. J. Jansson (Dept. 475 nm, visualization of bacteria labelled with theOf Biochemistry, Stockholm University, Stockholm, wild-type gfp through this filter set will not beSweden) are combining genetic inserts of both the optimal. Although a DAPI (496-diamidino-2-gfp and Iuc (luciferase) or lux genes to chromo- phenylindole) filter set with excitation and barriersomally tag pollutant-degrading bacteria to compare filters of 330–380 and 435 nm, respectively, arefluorescent counts with metabolic viability. By tag- suitable for wild-type gfp detection, the fluorescentging with both the gfp and a metabolic-based marker protein is subject to a high level of photobleachingbehind a strong promoter (luc or lux), they de- under short wavelength excitation.termined total counts using fluorescent microscopy Green fluorescence can be observed in coloniesor flow cytometry, and determined what percentage cultured on agar media under a hand-held long-wave


(365 nm) UV lamp. This is a simple approach with activated sludge (Eberl et al., 1997; Olofsson et al.,minimal capital cost to enumerate culturable gfp- 1998), on rhizospheres (Bloemberg et al., 1997;labelled microorganisms from environmental sam- Tombolini et al., 1997), and phylloplane (Normanderples. For example, in our laboratory, survival of a et al., 1998). The major disadvantage is the high costp-nitrophenol (PNP)-degrading gfp-labelled Morax- of the CLS microscope.ella sp. G21 was monitored in PNP-spiked soil for Flow cytometry (including fluorescence activated14 days (Tresse et al., 1998), and Errampalli et al. cell sorting) is another method to quantify gfp-(1998) monitored survival of phenanthrene-degrad- labelled bacteria. This approach allows the detectioning gfp-labelled Pseudomonas sp. UGI4Gr in and quantitation of individual fluorescent cells withincreosote-contaminated soil. In both studies, soil a population since the flow cytometer measures andsamples were diluted and plated onto tryptic soy agar analyzes the optical properties of hundreds of singleand incubated. Plates were examined visually under cells per second passing through a focused lasera UV lamp (365 nm) to detect and enumerate GFP- beam. It can also quantify fluorescence intensity ofpositive colonies. Although useful for general de- various groups of gfp-labelled microorganisms.tection and plate counting, this method is not suitable Tombolini et al. (1997) showed that a pseudomonadfor studying cells or colonies over time due to the bacterium chromosomally labelled with one copy ofpotential for DNA damage. gfp had a lower fluorescence intensity than an E. coli

Another method is the fluorometric detection of strain containing multiple copies of a gfp-plasmid.gfp-labelled cells in solution. This method can be The authors also observed an increase in the fluores-useful for screening, or confirmation of cell growth cence signal per cell during starvation. They sug-or survival in environmental samples since it is less gested that this was due to an increase in gfpsensitive than other methods. For example, Burlage concentration in the cells as a result of cell size

3et al. (1996) reported a detection limit of 10 CFU/ reduction during starvation.ml Pseudomonas putida GFP-marked cells from soil Tombolini and Jansson (1998) provide furthersamples suspended in phosphate-buffered saline and information on these methods to monitor GFP-tagged

3in our laboratory a detection limit of 10 CFU/ml cells, including procedures describing protocols forPseudomonas fluorescens GFP-marked cells from direct visualization and single-cell detection bypure culture was determined (unpublished results). In epifluorescent microscopy arid flow cytometry.our laboratory, we have investigated use of fluoro- The GFP gene sequence is known, and the geneticmetric detection for most-probable-number (MPN) constructs are well-defined in most cases. Therefore,enumeration of bacteria using both a standard spec- the detection of gfp-tagged cells is also possible bytrofluorophotometer for 1–3 ml samples in cuvettes traditional molecular methods such as gene probing,and a fluorescent microplate reader for 96-well DNA hybridization, and PCR.microtitre plates. Depending on the application,researchers have choices in the experimental ap-proach they wish to take. 13. Summary

Confocal laser scanning microscopy (CLSM) is apowerful technique to visualize gfp-labelled micro- The information presented in this review dem-organisms with high resolution, and with 3D image onstrates the usefulness of the novel GFP marker inanalysis, enables the visualization of three dimen- environmental microbiology research. Although onlysional distribution of labelled bacteria in complex available as a marker for a few years, GFP hascommunities. The CLS microscope commonly uses become another convenient and effective tool in thean argon laser (488 nm wavelength) which provided toolkit of microbiologists studying environmentaloptimal excitation and resulting visualization of red- microorganisms in complex biological systems. Theshifted-gfp mutants. Confocal laser scanning micro- GFP system has numerous advantages over existingscopy has been used to demonstrate spatial arrange- marker systems and is particularly useful in visualiz-ment of labelled bacteria in mixed-species biofilms ing spatial distribution and relationships in situ with

¨(Christensen et al., 1996; Moller et al., 1998), existing technology, such as the use of epifluorescent


Crameri, A., Whitehorn, E.A., Tate, E., Stemmer, W.P.C., 1996.or confocal laser scanning microscopy. The versatili-Improved green fluorescent protein by molecular evolutionty and effectiveness of the GFP in combination withusing DNA shuffling. Nat. Biotechnol. 14, 315–319.

existing microbiological techniques has been demon- Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A.,strated. GFP has been, and will continue to be, useful Tsien, R.Y., 1995. Understanding, improving and using green

fluorescent proteins. TIBS 20, 448–455.in studying various applications of environmentalDelagrave, S., Hawtin, R.E., Silva, C.M., Yang, M.M., Youvan,microorganisms from biofilms to bioremediation.

D.C., 1995. Red-shifted excitation mutants of the green fluores-cent protein. Bio /Technology 13, 151–154.

de Lorenso, V., 1994. Designing microbial systems for geneexpression in the field. Trends Biotechnol. 12, 365–371.Acknowledgements

Drahos, D., Hemming, B.C., McPherson, S., 1986. Trackingrecombinant organisms in the environment: b-galactosidase as

This research was supported by an NSERC a selectable non-antibiotic marker for flourescent pseu-domonads. Bio /Technology 4, 439–444.(Canada) Strategic Grant to H. Lee and J.T. Trevors.

Eberl, L., Schulze, R., Ammendola, A., Geisenberger, O., Earhart,Research by J.T. Trevors is also supported by anR., Steinberg, C., Molin, S., Amann, R., 1997. Use of green

NSERC Operating Grant. fluorescent protein as a marker for ecological studies ofactivated sludge communities. FEMS Microbiol. Lett. 149,77–83.

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