Use of green fluorescent protein as a marker for ecological studies of activated sludge communities

FEMS Microbiology Letters 149 (1997) 77^83

Use of green £uorescent protein as a marker for ecological studies of activated sludge communities Leo Eberl a *, Renate Schulze a , Aldo Ammendola a , Otto Geisenberger a , Robert Erhart a , Claus Sternberg b , SÖren Molin b , Rudolf Amann c ;

a b

M

Lehrstuhl fu ë r Mikrobiologie, Technische Universita ë t Mu ë nchen, Arcisstra e 16, D-80290 Munich, Germany

Department of Microbiology, The Technical University of Denmark, Building 301, DK-2800 Lyngby, Copenhagen, Denmark

c

M

Max-Planck-Institut fu ë r Marine Mikrobiologie, Celsiusstra e 1, D-28359 Bremen, Germany

Received 3 February 1997 ; accepted 6 February 1997

Abstract

The potential of the green fluorescent protein (GFP) as a marker gene for ecological investigations of an activated sludge community was assessed. By inserting the hybrid transposon mini-Tn5 gfp into the chromosome of Pseudomonas putida KT2442 a strongly fluorescent mutant was obtained. This strain was used for in vivo tracking of individual cells after introduction into a simple sludge microcosm. It is demonstrated that the observed reduction of introduced bacteria from sewage is mainly the result of predation by protozoa. The feasibility of combining detection of GFP fluorescence with whole cell hybridization employing fluorescently labeled, rRNA-targeted oligonucleotides in paraformaldehyde fixed samples is demonstrated. Keywords :

Green £uorescent protein; In situ hybridization; Protozoan grazing; Activated sludge

1. Introduction

In recent years the in situ hybridization with £uorescently labeled, rRNA-targeted nucleic acid probes has become a powerful tool for the analysis of various natural microbial communities [1]. For example, by employing this culture-independent rRNA approach the composition and structure of the bacterial consortia present in activated sludge could be studied in great detail. In contrast, only a minority of the bacteria present in activated sludge are culturable by standard methods. Moreover, the di¡erent * Corresponding author. Tel.: +49 (89) 2892 2656; fax: +49 (89) 2892 2360; e-mail: [email protected]

cultivation techniques tend to over- or underestimate certain groups of bacteria resulting in an incorrect picture of the actual population composition [2,3]. Recently, a novel reporter for gene expression studies, the green £uorescent protein (GFP) from the jelly¢sh Aequorea victoria, has become available [4]. Because the detection of GFP requires only irradiation with near-UV or blue light, it is not limited by the availability of substrates and thus provides an excellent marker system for studies of complex ecosystems [4^6]. Moreover, the GFP protein is extremely stable and persists not only through exposure to heat and extreme pH but also through treatment with formaldehyde allowing the detection of GFP £uorescence even in ¢xed samples [4].

0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 0 5 8 - X

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In this study we present a method for the detection of GFP £uorescence in paraformaldehyde-¢xed samples that are simultaneously used for in situ hybridization with £uorescently labeled probes. Furthermore, we ascertained the potential of GFP as a marker gene for on-line tracking of individual cells of a chromosomally tagged derivative Pseudomonas putida KT2442 in activated sludge. 2. Material and methods

2.1. Organisms and culture conditions Pseudomonas putida KT2442 is a rifampicin-resistant, TOL plasmid-cured derivative of P. putida mt-2 [7]. Strains were grown in Luria-Bertani broth (containing 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter) at 30³C. When appropriate, antibiotics were added at ¢nal concentrations of 50 Wg ml31 for rifampicin and for kanamycin. P. putida KT2442 was chromosomally marked with the green £uorescent protein by inserting the hybrid transposon mini-Tn5 gfp randomly into the chromosome of the strain using a three-factor mating procedure [8]. Since this transposon carries a promoterless gfp cassette, £uorescence intensity is a measure of the strength of adjacent promoters. Approximately 3000 colonies of random insertion mutants grown on selective medium were screened for strong green £uorescence upon irradiation of the plates at 366 nm with a hand-held long-wave UV source. One mutant, GREEN31, which gave rise to very bright £uorescence, was selected and used throughout this study. 2.2. Microcosm experiments

Grab samples of mixed liquor were collected from the aeration stage 1 of a local municipal sewage treatment plant (Muënchen-GroMlappen, about 2 million population equivalents) immediately before the beginning of an experiment. 150 ml portions of the sludge mixture were incubated in 500 ml Erlenmeyer £asks which were vigorously aerated by stirring at 20³C. Every 12 h the sludge was allowed to settle and the supernatant was exchanged with the cellfree supernatant of fresh sewage. The composition of the sludge community in this very simple micro-

cosm was found to be stable for at least 5 days as judged from a population analysis employing in situ hybridization with rRNA-targeted oligonucleotide probes speci¢c for the larger phylogenetic groups within the Bacteria [2,9]. 2.3. Fluorescent-oligonucleotide probes

The sequences and speci¢cities of the probes used in this study are listed in Table 1. Amino-linked oligonucleotides (MWG-Biotech, Ebersberg, Germany) were labeled with the activated £uorescent dyes carboxy£uorescein (Boehringer Mannheim, Germany), tetramethylrhodamine (Molecular Probes, Eugene, OR), Cy3, and Cy5 (Biological Detection Systems, Pittsburgh, PA) as described by Amann et al. [10]. 2.4. Cell ¢xation and in situ hybridization

Activated sludge samples were ¢xed by the addition of 3 volumes of 4% paraformaldehyde in 200 mM sodium phosphate bu¡er (pH 7.2) [10]. Samples were stored in a 1:1 mixture of PBS and 96% ethanol without any apparent loss of GFP £uorescence over at least 2 months. In situ hybridization was performed at 46³C in a bu¡er containing 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 0.01% sodium dodecyl sulfate (SDS), and 5 ng/Wl oligonucleotide probe for 90 min. For each probe, optimal hybridization stringency was established by the addition of formamide to following ¢nal concentrations: 35% for GAM42a, BET42a, and ACA; 25% for HGC; 20% for EUB338 and EUK. Following washing in a buffer containing 20 mM Tris-HCl (pH 7.4), 0.01% SDS, and NaCl (at concentrations to ensure su¤cient stringency for each probe: 225 mM for EUB338 and EUK, 160 mM for HGC, and 80 mM for GAM42a and BET42a) at 48³C for 15 min, the slides were rinsed with distilled water, air dried, and mounted in Citi£uor solution (Citi£uor Ltd., Canterbury, UK). 2.5. Microscopy

Routinely cells were viewed with a Zeiss Axioplan epi£uorescence microscope (Zeiss, Oberkochen, Germany) equipped with Zeiss ¢lter sets 09 for £uores-

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cein and 15 for tetramethylrhodamine. For Cy3 the Chroma HQ 41007 (Chroma Tech. Corp., Brattleboro, VT, USA) ¢lter set was used. Color micrographs were taken with Kodak Ektachrome 1600. Exposure times were 2^15 s for epi£uorescence and 0.01^0.03 s for phase contrast. Confocal laser microscopy was performed on a Carl Zeiss LSM 410 equipped with an argon ion and a helium-neon laser to supply excitation wavelengths of 488, 543 and 633 nm suitable for carboxy£uorescein and GFP, tetramethylrhodamine, and Cy5, respectively. Image analysis and processing was performed as described previously [11].

Fig. 1. Survival of

3. Results and discussion

3.1. Use of GFP for real-time monitoring of bacteria introduced into activated sludge The GFP-tagged strain

P. putida

GREEN31 was

inoculated into a simple activated sludge microcosm 6 1 and the survival of the strain at 5 10 cells ml

U

3

replicates.

green

£uorescent

Vorticella Aspidisca sp.

the genus

In

agreement

with previous studies which employed derivatives of the same

P. putida

within

various

protozoa,

this study (most dominating were attached ciliates of

tozoa

kanamycin.

cells

which were typical for the activated sludge used in

taining

and

GREEN31 in a sewage microcosm.

mycin. The graph represents typical data obtained from three

was followed by plating on selective medium conrifampicin

P. putida

CFU were determined on plates containing rifampicin and kana-

and among free-swimming proand

Bodo

sp.). Moreover, we

were able to study on-line the ingestion process of

strain [12,13] we observed that

GREEN31 cells by the di¡erent protozoa. Upon

cell numbers of GREEN31 decreased by 2^3 orders

prolonged incubation we frequently observed proto-

of magnitude during the ¢rst 3 days after addition to

zoa that contained many food vacuoles that were

activated sludge (Fig. 1).

entirely

By combined phase contrast and epi£uorescence microscopy

were

to

£uorescent

cells

(Fig.

that were highly £uorescent without containing any visible bacterial cells indicating cell lysis due to pro-

This allowed us to follow on-line the fate of this

gression of the digestion process. In agreement with

strain after introduction into the complex sludge

our plate count experiments, there were only very

community. Immediately after addition most cells

few £uorescent cells detectable 3 days after inocula-

were

sludge

tion. Most of the remaining cells were found to be

£ocks. However, already after 2 min we observed

incorporated into the sludge £ocks suggestive of a

swimming

visualize

green

individual

freely

able

with

GREEN31 cells due to their bright £uorescence.

found

we

¢lled

2a,b). Often, however, we observed food vacuoles

between

the

Table 1 Probe sequences and target sites of the oligonucleotide probes used for whole-cell hybridization Probe

Sequence

Target site

Ref.

EUB338

5P-GCTGCCTCCCGTAGGAGT-3P

16S (338^355)

[10]

EUK

5P-TAGAAAGGGCAGGGA-3P

16S (1379^1394)

[18]

BET42a

5P-GCCTTCCCACTTCGTTT-3P

23S (1027^1043)

[9]

GAM42a

5P-GCCTTCCCACATCGTTT-3P

23S (1027^1043)

[9]

ACA

5P-ATCCTCTCCCATACTCTA-3P

16S (652^669)

[3]

HGC

5P-TATAGTTACCACCGCCGT-3P

23S (1901^1918)

[19]

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Fig. 2. (a) Phase-contrast and (b) epi£uorescence micrograph of a ciliate (Aspidisca sp.) 6 h after addition of GREEN31 cells. Multiple food vacuoles containing several GFP-tagged cells were visualized with a Zeiss ¢lter set 09. (c and d) Simultaneous detection of bacterial cells (c) by in situ rRNA hybridization and (d) by GFP-mediated £uorescence signals. The paraformaldehyde-¢xed sludge sample containing GREEN31 cells was hybridized with the Cy3-labeled probe EUB338. (e) In situ hybridization of activated sludge containing GREEN31 cells with 23S rRNA-targeted oligonucleotide probes speci¢c for the beta (£uorescein-labeled) and gamma (tetramethylrhodamine-labeled) subclasses of

Proteobacteria.

In the double exposure epi£uorescence microphotograph (using Zeiss ¢lter sets 09 and 15)

GREEN31 cells appear yellow. (f) Detection of GREEN31 in activated sludge by confocal laser scanning microscopy. The sample was hybridized with the tetramethylrhodamine-labeled probes GAM42a and EUK, the £uorescein-labeled probe BET42a, and the Cy5-labeled probes ACA and HGC. The arti¢cial colors red, green, and blue were assigned to the data recorded in the tetramethylrhodamine, £uorescein, and Cy5 channels, respectively. Members of the genus

6

Acinetobacter

appear purple due to the simultaneous binding of ACA (blue)

and GAM42a (red). Note the protozoa (red) in the upper part of the picture.

protective role of the £ock from predatory protozoa,

ized by their green £uorescence indicating that GFP

which mainly feed on suspended bacteria [12,14].

persisted not only through cell ¢xation but also

Our observations strongly support previous stud-

through the hybridization procedure (Fig. 2d).

ies suggesting that predation by protozoa is a major

Recently, it has been demonstrated that by apply-

cause for the disappearance of introduced bacteria

ing three (or more) probes labeled with three di¡er-

from sewage [14,15]. Furthermore, it is demonstrated

ent £uorochromes up to seven di¡erent bacterial

that the use of GFP-tagged bacteria could be ex-

populations can be detected simultaneously [11]. To

tremely valuable for estimating instantaneous rates

investigate whether this approach could also be com-

of

Since

bined with the detection of GFP we hybridized a

GREEN31 cells are perfectly viable and the strain

sludge sample, which was taken 3 h after the addi-

is completely indistinguishable from the wild-type

tion of GREEN31 cells, with the tetramethylrhod-

with respect to growth and starvation survival this

amine-labeled probe GAM42a and the £uorescein-

approach may be superior to techniques employing

labeled probe BET42a. Using a £uorescein-speci¢c

monodispersed, £uorescently labeled bacteria [16] or

¢lter set (Zeiss ¢lter set 09) GFP-mediated green £u-

bacterial-sized £uorescent latex microspheres [17].

orescence of GREEN31 could not be distinguished

The possibility to monitor tagged cells on-line may

from cells detected by the £uorescein-labeled probe.

also allow light to be shed on some aspects of pro-

However, since

tozoan feeding physiology, e.g. measuring digestion

gamma subclass of

rates of bacteria or rates of vacuole formation.

also detected by the red-labeled probe GAM42a.

in

situ

protozoan

grazing

on

bacteria.

P. putida GREEN31 belongs to the Proteobacteria these cells were

As a consequence of the co-localization of green

3.2. Combining GFP detection with in situ rRNA hybridization

GFP £uorescence and red £uorescence conferred by probe GAM42a GREEN31 cells will appear yellow in a red-green double exposure microphotograph

A further advantage of GFP is the extreme stabil-

(Fig. 2e). Hence, this technique enabled us to simul-

ity of the protein and because its £uorescence per-

taneously target GREEN31 cells (yellow) and cells

sists even after treatment with formaldehyde, ¢xed

belonging to the beta (green) and gamma (red) sub-

preparations can also be examined [4]. This possibil-

classes of

ity prompted us to investigate whether GFP £uores-

ning image shown in Fig. 2f an example of the same

cence could be monitored in samples that are simul-

approach but in this case employing multiple probes

taneously used for in situ rRNA hybridization.

is shown. These results demonstrate that visualiza-

Proteobacteria.

In the confocal laser scan-

Samples taken 2 h after the addition of GREEN31

tion of community structures by in situ hybridization

were ¢xed with paraformaldehyde and hybridized

can be perfectly combined with the simultaneous de-

with the Cy3-labeled bacterial probe EUB338. As

tection of GFP £uorescence.

expected, the majority of the cells present in the

GFP has been demonstrated to be a very useful

sample could be detected by this probe (Fig. 2c).

marker for studying in vivo gene expression in com-

In addition, GREEN31 cells could be easily visual-

plex environments [5,6]. Work currently in progress

FEMSLE 7486 1-5-97

L. Eberl et al. / FEMS Microbiology Letters 149 (1997) 77^83

82

in our laboratories aims at the construction of bacterial reporter strains that respond to speci¢c environmental stimuli with the expression of GFP. Introduction of respective monitor strains into their natural environments should enable us to obtain information about the metabolic activities associated with their microhabitats. The combination of monitoring GFP expression (providing information on the ongoing activities) and in situ hybridization (providing

M.,

Tu,

Y.,

Euskirchen,

G.,

Ward,

W.W.

and

Prasher, D.C. (1994) Green £uorescent protein as a marker for gene expression. Science 263, 802^805. [5] Kremer, L., Baulard, A., Estaquier, J., Poulain-Godefroy, O. and Locht, C. (1995) Green £uorescent protein as a new expression marker in mycobacteria. Mol. Microbiol. 17, 913^ 922. [6] Valdivia,

R.H.,

Hromockyj,

A.E.,

Monack,

D.,

Ramak-

orescent protein (GFP) in the study of host-pathogen interac-

investigated

community

[4] Chal¢e,

should allow the mapping of the structure-activity the

the

and its application for in situ monitoring in

activated sludge. Appl. Environ. Microbiol. 60, 792^800.

rishnan and Falkow, S. (1996) Applications for the green £u-

of

on

Acinetobacter

structure)

pro¢le

information

rRNA-targeted oligonucleotide probe speci¢c for the genus

bacterial

consortium.

Such studies will improve our current understanding

tion. Gene 173, 47^52. [7] Bagdasarian, M., Lurz, B., Ruckert, B., Franklin, F.C.H., Bagdasarian, M.M., Frey, J. and Timmis, K.N. (1981) Specif-

of the distribution of metabolic functions within

ic-purpose plasmid cloning vectors. II Broad host range, high

multispecies communities and how these microhabi-

copy number, RSF1010-derived vectors for gene cloning in

Pseudomonas.

tats in turn a¡ect community architecture. Recently, GFP has been employed as a marker for monitoring the transfer of the TOL plasmid between bacterial cells [8]. Work is currently under way to

Gene 16, 237^247.

[8] Christensen, B.B., Sternberg, C. and Molin, S. (1996) Bacterial plasmid conjugation on semi-solid surfaces monitored with the green £uorescent protein (GFP) from

toria

Aequorea vic-

as a marker. Gene 173, 59^65.

adopt the combined detection of GFP with in situ

[9] Manz, W., Amann, R., Ludwig, W., Wagner, M. and Schlei-

hybridization £uorescence signals to assess the £ow

fer, K.-H. (1992) Phylogenetic oligonucleotide probes for the

of plasmids within complex communities and to in situ identify possible recipients of the respective plasmids.

major subclasses of proteobacteria : problems and solutions. Syst. Appl. Microbiol. 15, 593^600. [10] Amann, R.I., Krumholz, L. and Stahl, D.A. (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic,

and

environmental

studies

in

microbiology.

J. Bacteriol. 172, 762^770.

Acknowledgments

[11] Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H. (1996) In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178, 3496^

This work was supported by grants from the Bavarian Government (Bayerisches Staatsministerium fu ër

Landesentwicklung

und

Umweltfragen,

No.

6496-7/42-28154) and the Danish Center for Microbial Ecology. L.E. is supported by the Research Training Program of the EU (contract No. BIO4CT965025).

3500. [12] McClure, N.C., Weightman, A.J. and Fry, J.C. (1990) Survival of

Pseudomonas putida

UWC1 containing cloned catabolic

genes in a model activated-sludge unit. Appl. Environ. Microbiol. 55, 2627^2634. [13] Nu ë Mlein, K., Maris, D., Timmis, K. and Dwyer, D.F. (1992) Expression and transfer of engineered catabolic pathways harbored by

Pseudomonas

ssp. introduced into activated sludge.

Appl. Environ. Microbiol. 58, 3380^3386. [14] Mallory, L.M., Yuk, C.-S., Liang, L.-N. and Alexander, M.

References

(1983) Alternative prey : a mechanism for elimination of bacteria by protozoa. Appl. Environ. Microbiol. 46, 1073^1079. [15] Goldstein, R.M., Mallory, L.M. and Alexander, M. (1985)

[1] Amann, R.I., Ludwig, W. and Schleifer, K.-H. (1995) Phylogenetic identi¢cation and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143^169.

Reasons for possible failure of inoculation to enhance biodegradation. Appl. Environ. Microbiol. 50, 977^983. [16] Sherr, B.F., Sherr, E.B. and Fallon, R.D. (1987) Use of mono-

[2] Wagner, M., Amann, R., Lemmer, H. and Schleifer, K.-H.

dispersed, £uorescently labeled bacteria to estimate in situ

(1993) Probing activated sludge with oligonucleotides speci¢c

protozoan bacterivory. Appl. Environ. Microbiol. 53, 958^

for proteobacteria : inadequacy of culture-dependent methods for describing microbial community structure. Appl. Environ.

965. [17] Borsheim, K.Y. (1984) Clearance rates of bacteri-sized particles by freshwater ciliates, measured with mono-disperse £u-

Microbiol. 59, 1520^1525. [3] Wagner, M., Erhart, R., Manz, W., Amann, R., Lemmer, H., Wedi, D. and Schleifer, K.-H. (1994) Development of an

orescent latex beads. Oceologia 63, 286^288. [18] Hicks, R., Amann, R.I. and Stahl, D.A. (1992) Dual staining

FEMSLE 7486 1-5-97

L. Eberl et al. / FEMS Microbiology Letters 149 (1997) 77^83

83

of natural bacterioplankton with 4P,6-diamino-2-phenylindole

fer, K.-H. (1994) In situ probing of Gram-positive bacteria

and £uorescent nucleotide probes targeting kingdom-level 16S

with high DNA G+C content using 23S-targeted oligonucleo-

rRNA sequences. Appl. Environ. Microbiol. 58, 2158^2163.

tides. Microbiology 140, 2849^2858.

[19] Roller, C., Wagner, M., Amann, R., Ludwig, W. and Schlei-

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