A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments

Gene, 173 (1996) 53 58 © 1996 Elsevier Science B.V. All rights reserved. 0378-1119/96/$15.00

53

GENE 09376

A transposon for green fluorescent protein transcriptional fusions: application for bacterial transport experiments * (Tn5GFP1; C/Co; soil column)

Robert S. Burlage, Zamin K. Yang and Tonia Mehlhorn Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6036, USA Received by D.C. Youvan: 16 May 1995; Revised/Accepted: 18 August/23 August 1995; Received at publishers: 21 September 1995

SUMMARY

The movement of bacteria through groundwater is a poorly understood process. Factors such as soil porosity and mineralogy, heterogeneity of soil particle size, and response of the bacteria to their environment contribute to the pattern of bacterial flow. The identification of transported bacteria is often a limiting factor in both laboratory and field transport experiments. Two bacterial strains were modified for use in bacterial transport experiments: a strain of Escherichia coli harboring the p G F P plasmid and a strain of Pseudomonas putida modified with a Tn5 derivative, Tn5GFP1. The T n 5 G F P 1 transposon incorporates the gene (gfp) encoding green fluorescent protein (GFP) and can be used to mutagenize G r a m - bacteria. Fluorescent colonies were suspended in phosphate-buffered saline (PBS) at a concentration of a p p r o x . 109 bacteria/ml. A 10-cm glass column packed with quartz sand (diameter range 177-250 lam) was equilibrated with PBS prior to the forced flow introduction of the bacteria. Collected fractions were analyzed and the bacteria quantitated using a fluorescence spectrometer. Results demonstrate that the bacteria can be accurately tracked using their fluorescence, and that the intensity of the signal can be used to determine a C/Co ratio for the transported bacteria. The data show a rapid breakthrough of the bacteria followed by a characteristic curve pattern. A lower limit of detection of 105 cells was estimated based on these experiments. The Tn5GFP1 transposon should become a valuable tool for labeling bacteria.

INTRODUCTION

A thorough understanding of bacterial transport through porous material is vital to the success of environmental biotechnology. For example, it is a key process in delivery of microbes to contaminated sites for bioreCorrespondence to: Dr. R.S. Burlage, Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6036, USA. Tel. (1-615) 574-7321; Fax (1-615) 576-8543; e-mail: [email protected] * Presented at the Palo Alto Institute of Molecular Medicine Symposium on 'Fluorescent Proteins and Applications', Hyatt Rickeys, Palo Alto, CA, USA, 6 7 March 1995. Abbreviations: A, absorbance (1 cm); ATCC, American Type Culture Collection; Ap, ampicillin; bp, base pair(s); C, designates the fluorescent signal from each sample; Co, designates the fluorescence signal of the SSD1 0378-1119(95)00672-9

mediation of chemicals. However, relatively little is known about the factors (biological, geochemical and hydrologic) controlling the mobility of bacteria within subsurface systems. Heterogeneities in hydrological and geochemical properties can significantly retard or enhance transport relative to that in homogeneous media. input bacterial culture; cfu, colony-forming unit(s); Cm, chloramphenicol; GF, green fluorescence; GFP, GF protein; gfp, gene encoding GFP; Gm, gentamicin; IPTG, isopropyl-[~-o-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); mob, cis-acting mobilization sequence; nt, nucleotide(s); PBS, phosphate-buffered saline (137 mM NaC1/2.7 mM KC1/4.3 mM NazHPO4.7H20/1.4 mM KHzPO4); PCR, polymerase chain reaction; R, resistant/resistance; Tc, tetracycline; Tn, transposon; tnp, transposase-encoding gene; UV, ultraviolet; [], denotes plasmid-carrier state.

54 The significance and predictive importance of microbial properties that influence bacterial mobility (e.g., the size, surface charge and hydrophobicity, and motility of the microorganisms, as well as relevant physiological functions, such as p r o d u c t i o n of extracellular biopolymers) require intense investigation ( G a n n o n et al., 1991). Laboratory-scale c o l u m n studies can provide useful information ( H a r v e y et al., 1993). A m e t h o d to quickly and specifically identify the transported bacteria can facilitate the study of transport (Keswick et al., 1982). Selective agars can be used to determine colony-forming units (cfu) that are transported t h r o u g h a column of material (Wollum and Cassel, 1978). We have used molecular techniques, such as the use of D N A probes and PCR, to identify several strains without requiring a selectable phenotype (Burlage et al., 1995). Both conventional and molecular techniques have substantial drawbacks. Conventional plating on selective agar requires a unique selectable marker on the transported bacterial strain, such as antibiotic resistance. However, antibiotic resistance m a y be c o m m o n in indigenous bacteria. Molecular techniques have the advantages of precision and great sensitivity, although they require more sample preparation and manipulation. In a screening experiment for bacterial m o v e m e n t in a variety of soil types and conditions, the sample preparation time m a y hinder rapid analysis. We utilized two bacterial strains that contain the gfp gene, which allows their detection by their G F signal. G F P forms a cyclic peptide t h r o u g h an autocatalytic reaction; this protein is highly fluorescent and does not require any other substrate, additional gene or co-factors. G F P fluorescence is stable after exposure to heat, extreme p H and chemical treatments ( B o k m a n and Ward, 1981; Ward and Bokman, 1982).The m o v e m e n t of G F P - l a b e l e d strains t h r o u g h a relatively h o m o g e n e o u s m e d i u m are reported here. Samples can be analyzed rapidly using a fluorescence spectrometer, and the data are amenable to C / C o analysis. These studies are a prelude to more difficult experiments involving heterogeneous soil systems, as well as measurements of bacterial transport on a microscopic scale.

EXPERIMENTAL AND DISCUSSION

(a) Transport of the E. coil strain The p G F P plasmid (Clontech, Palo Alto, CA, USA) was introduced into Escherichia coli strain N M 5 2 2 by transformation ( S a m b r o o k et al., 1989) and selection for Ap R (50 ~tg Ap/ml) on LB agar plates. After induction with I P T G , this strain fluoresces brightly when exposed to U V light. Fig. la shows the results of a transport experiment using a suspension of E. coli[pGFP]. As expected for transport t h r o u g h a well-defined, h o m o g e n e o u s medium, the bacterial suspension moves t h r o u g h the medium with little tailing at the leading edge. W h e n the bacteria are tracked using G F , b r e a k t h r o u g h of the bacteria (detection of G F above b a c k g r o u n d ) is observed in fraction 18 and increases quickly. The G F signal also attenuates rapidly, and a tailing of the signal is observed for the remainder of the experiment. These results correlate exactly with the results from cfu determination (Fig. lb). The lower limit of detection in this experiment is approx. 105 bacteria. Below this n u m b e r the b a c k g r o u n d G F obscures the detection of the bacteria. The use of more sensitive equipment will improve our description of the bacterial b r e a k t h r o u g h curve. The ratio of bacteria in each sample to the total input bacteria (C/Co) is presented in Fig. lc. For transport experiments it is not essential to obtain absolute numbers of bacteria; it is m o r e useful to obtain the C/Co value. Greater than 95% of the input bacteria were detected using this technique. (b) Construction of transposon Tn5GFP1 A transposon vector for the formation of transcriptional fusions using gfp as a bioreporter gene was constructed (Fig. 2). A Tn5 derivative was utilized in order to create transposon mutants of G r a m - bacteria. The resulting T n 5 G F P 1 can be introduced into G r a m bacteria either by conjugation or electroporation. The latter m e t h o d was used to mutagenize Pseudomonas putida mt-2 (ATCC No. 33015), using a BTX 600 (BTX, San Diego,

Fig. 1. Transport of the E. coli strain as observed in a sand column experiment. Cell suspensions were created by scrapping cells from agar plates and resuspending them in 1 ml PBS with vortexing. Cells were used immediately after resuspension. A glass column with TeflonT M fittings was used for all transport experiments. This column was filled with a slurry of 8 g of clean quartz sand (diameter range 177 250 pm) in PBS. Length of the sand column was approx. 10 cm. A LKB peristaltic pump continuously delivered a reservoir of PBS at a rate of 1.2 ml/min to the column. Fractions (0.5 ml) were collected at the bottom of the column in plastic UV-transparent cuvettes. The volume was increased to 2 ml for each sample using PBS, and the fluorescence measured on a Perkin-Elmer fluorescence spectrometer model LS50. Dilutions were made as necessary for samples that were beyond the range of the instrument. (a) The fluorescent signal for each sample is plotted. Each sample designates successive 0.5 ml fractions. PBS was added to each sample for a total volume of 2 ml. (b) The cfu were counted on LB agar plates after incubation for 24 h at 37C. Serial dilutions were prepared in PBS prior to spread plating. (c) C/Co values were calculated by comparison to the fluorescent signal from the input bacterial sample. All values are standardized for dilution factors. The C/Co value reports the fraction of the input bacteria found in that sample.

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Fig. 2. Construction of Tn5GFP1 in plasmid pESD15. The Tn5-B21 transposon on plasmid pSUP102 (Simon et al., 1989) is the source of the transposable element. Two aliquots of pSUP102 were cut with restriction enzymes. One was digested with BamHI, the other was digested with BamHI + EcoRI. The BamHI digestion yielded a fragment of approx. 7 kb containing Cm R, mob and Gm*. The BamHl+EcoRI digestion yielded a fragment of approx. 5.5 kb containing tnp and Tc R. These fragments were isolated from an agarose gel using Spin Bind DNA Extraction Units ( F M C Bioproducts, Rockland, ME, USA). The gfp gene was obtained by cutting the p G F P plasmid (Clontech) with BamHl+EcoRI, yielding a 750-bp fragment that contains gfp. These three fragments were ligated together and transformed into E. coli NM522 (Stratagene, La Jolla, CA, USA) to form plasmid pESD15, containing the Tn5GFP1 transposon. Structure of the plasmid was confirmed by restriction mapping. E, EcoRI site; B, BamHI site. Dotted lines show fragments that were isolated for the construction of pESD15. Filled triangles show location of inverted repeat sequences.

CA, USA) electroporation system and selecting on LB plates containing 50 lag Tc/ml (Sambrook et al., 1989). Transformants were screened by resuspending colonies in PBS at an A60o n m between 0.1 and 0.3 and measuring fluorescence using a Perkin Elmer Luminescence Spectrometer LS50 (excitation, 395 nm; emission, 509 nm). Fluorescence was also observed using an epifluorescence microscope (Nikon Fluephot). A strain (designated 2FL) that demonstrated constitutive fluorescent activity was selected and used for the transport experiments.

(c) Transport of the P. putida strain The results of the transport experiment using P. putida 2FL are shown in Fig. 3a. Fraction 12 is the first point showing a detectable signal in this experiment. This represents the leading edge of the transported bacteria, and is equivalent to approx. 1 x 105 bacteria in the 0.5-ml fraction. This number represents the lower limit of detection for the strain. It is possible that this number could

be an order of magnitude lower, since microscopic analysis of the strain demonstrates a broad variability in G F signal of the bacteria (data not shown). There is a substantial fraction of the total bacteria that emit little or no GF. As for the E. coli strain, the transported bacteria move through the sand matrix in a tight pattern. Tailing of the signal is evident after > 90% of the bacteria have exited the sand column. Fig. 3b demonstrates that cfu counts correlate well with the G F data. Fig. 3c describes the C/Co values for this experiment. In this relatively simple experiment, almost complete recovery of the input bacteria ( > 9 5 % ) is observed through analysis of the samples. Only a small percentage was undetected either through method error or through bacterial retention in the column.

(d) Conclusions (1) Bacterial strains carrying the gfp gene are easily detectable in bacterial transport experiments, allowing rapid identification of bacterial breakthrough. (2) The Tn5GFP1 transposon effectively introduces the gfp gene into P. putida. (3) Results obtained with this method are as accurate as standard plate counts. (4) The limit of detection using this method is approx. 105 bacteria. (5) C/Co values are easily calculated from these data.

ACKNOWLEDGEMENTS

We thank L. Liang and J. McCarthy for their excellent technical advice. Research supported by the Subsurface Science Program of the Office of Health and Environmental Research, US Department of Energy, and by Defense Programs/US Department of Energy under a Cooperative Research and Development Agreement (CRADA) No. D O E 92-0077 between Lockheed Marlin Energy Research Corp. and the National Center for Manufacturing Sciences. Oak Ridge National Laboratory is managed for the US Department of Energy under contract DE-AC05-96OR22464 with Lockheed Marlin Energy Research Corp. Publication No. 4558, Environmental Sciences Division, ORNL.

Fig. 3. Transport of the P. putida strain as observed in a sand-column experiment. Approx. 5 x 109 bacteria were loaded onto a quartz sand column, which was constructed as described in the legend to Fig. 1. Panels a, b and c represent experiment described in the legend to Fig. 1, using P. putida rather than E. coll.

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58 REFERENCES Bokman, S.H. and Ward, W.W.: Renaturation of Aequeora greenfluorescent protein. Biochem. Biophys. Res. Commun. 101 (1981) 1372-1380. Burlage, R.S., Palumbo, A.V. and McCarthy, J.: Method efficiency and signal quantitation of bacteria for a groundwater transport experiment. In: Hinchee, R.E. (Ed.): Proceedings of the Third International In Situ and On Site Bioreclamation Symposium, San Diego, CA. Lewis Publishers, Boca Raton, EL, 1995, in press. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C.: Green fluorescent protein as a marker for gene expression. Science 263 (1994) 802 805. Gannon, J.T., Manilal, V.B. arid Alexander, M.: Relationship between cell surface properties and transport of bacteria through soil. Appl. Environ. Microbiol. 57 (1991) 190-193. Harvey, R.W., Kinner, N.E., MacDonald, D., Metge, D.W. and Bunn, A.: Role of physical heterogeneity in the interpretation of small-

scale laboratory and field observations of bacteria, microbial-sized microsphere, and bromide transport through aquifer sediments. Water Resources Res. 29 (1993) 2713-2721. Keswick, B.H., Wang, D. and Gerba, C.P.: The use of microorganisms as ground-water tracers: a review. Groundwater 20 (1982) 142-149. Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Simon, R., Quandt, J. and Klipp, W.: New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80 (1989) 161 169. Ward, W.W. and Bokman, S.H.: Reversible renaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21 (1982) 4535 4550. Wollum, A.G. and Cassel, D.K.: Transport of microorganisms in sand columns. Soil Sci. Soc. Am. J. 42 (1978) 72 76.