School of Biological Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, UK
Correspondence
P. S. Poole
p.s.poole{at}reading.ac.uk
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ABSTRACT |
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Present address: Department of Microbiology, The Dental Institute, King's College London, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, UK.
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INTRODUCTION |
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In addition to the wild-type protein, there are many derivatives of GFP, which have increased levels of fluorescence emission, and shifted excitation or emission spectra (Cormack et al., 1996; Crameri et al., 1996
; Ellenberg et al., 1998
). GFPUV has mutations at F99S, M153T and V163A, produced by shuffle mutagenesis, which result in a 16-fold higher emission than wild-type GFP, but it retains the wild-type excitation spectrum (Crameri et al., 1996
). GFPUV appears to have a higher fluorescence emission because it is more soluble than wild-type GFP. Site-directed mutagenesis of wild-type gfp has been used to change F64L and S65T to produce a series of GFPmut derivatives that have a red-shifted excitation spectrum (excitation maximum 488 nm) and a 35-fold increase in fluorescence, giving them characteristics close to those of FITC, and therefore making them better suited to FAC sorters (Cormack et al., 1996
). Furthermore, the addition of a protease-targeting signal to GFPmut has led to the creation of a suite of GFPmut proteins with different stabilities (Anderson et al., 1998
). A further derivative, GFP+, has been produced that incorporates the chromophore change from GFPmut3.1 into the protein backbone of GFPUV, giving up to a 130-fold increase in fluorescence emission (Scholz et al., 2000
). This is due to the combination of the red-shifted chromophore of GFPmut3.1 with the greater solubility of GFPUV. GFP mutants with blue, cyan and yellowish-green emission spectra are now available, but none of these mutants has emission spectra at wavelengths longer than 529 nm, and as such are limited for dual-labelling experiments with GFP (Baird et al., 2000
). However, another fluorescent protein, DsRed, which is 28 kDa in size and originally isolated from corals of the genus Discoma (Baird et al., 2000
), shares certain structural and chromophore motifs with GFP, but has an emission maximum of 583 nm, and so can be used in conjunction with GFP. A disadvantage of wild-type DsRed is that it is tetrameric and is slow to mature compared to GFP. However, mutant derivatives, DsRedT.3 and DsRedT.4, have recently been isolated, which, while still yielding tetrameric proteins, mature much faster than the wild-type (Bevis & Glick, 2002
). In addition, a more rapidly maturing monomeric variant of DsRed has been developed, called monomeric red fluorescent protein (mRFP1) (Campbell et al., 2002
).
Due to the advantages of AFPs as reporter proteins, a large number of vectors incorporating them have been made (Allaway et al., 2001; Miller et al., 2000
; Stuurman et al., 2000
). However, we considered it would be of great use if a suite of these AFPs was available in the same polylinker background in two compatible vectors, enabling the easy switching of promoters between vectors. In many cases it is still desirable to use chromogenic reporter systems (GusA and LacZ), which have increased sensitivity relative to AFPs and have been the gold standard for decades. We therefore developed two families of stable vectors, containing a compatible polylinker upstream of various gfp derivatives, gusA, lacZ, dsRed derivatives and mRFP1, suitable for use in Gram-negative bacteria in the environment.
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METHODS |
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Measurement of reporter fusion activity.
GFP fluorescence was measured using a Tecan GENios fluorometer equipped with excitation filters of 390 nm (for GFPUV) and 485 nm (for GFPmut3.1 and all other GFP derivatives), and emission filter 510 nm. Strain 3841, containing various pOT2 derivatives with the dctA promoter cloned into them, was grown in AMS supplemented with 10 mM succinate or glucose. When the cells reached an OD595 of 0·40·6, the specific fluorescence was measured by dividing the fluorescence of the sample by the OD.
For measurement of -D-glucuronidase (GusA) activity on agar plates, AMA was supplemented with 5-bromo-4-chloro-3-indolyl
-D-glucuronic acid (X-Glc) to a final concentration of 40 µg ml1 and for LacZ, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-Gal) was added at a final concentration of 40 µg ml1. In liquid culture,
-glucuronidase activity was measured as previously described for
-galactosidase reactions (Lodwig et al., 2004
), except that p-nitrophenyl-
-D-glucuronide was substituted as the chromogenic substrate.
Microscopy.
Microscopy was performed with a Carl Zeiss Axioskop2.0 epifluorescence microscope with appropriate fluorescence sets. Images were captured using an Axiocam digital camera. For GFPmut3.1 and DsRed the FITC filter set (no. 10, 450470 nm excitation band pass), and the rhodamine filter set (no. 15, 450490 nm excitation band pass), respectively, were used.
Plant growth and inoculation.
Vetch (Vicia sativa) seeds were surface-sterilized in 95 % ethanol for 30 s and then immersed in a solution of 2 % sodium hypochlorite for 10 min. The seeds were washed extensively with sterile water and then allowed to germinate on Falcon tube slopes made from 0·75 % agarose containing nitrogen-free rooting solution (Poole et al., 1994a) for 3 days in the dark. The plants were then inoculated with 103105 c.f.u. bacteria. The tubes were then placed in a growth chamber (23 °C, 16 h/8 h light/dark period). Three to seven days post-inoculation, the plants were examined for the formation of infection threads.
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RESULTS AND DISCUSSION |
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It is often desirable to detect two different AFPs simultaneously. The only other AFP that can be detected at the same time as GFP is DsRed. However, the wild-type DsRed has the disadvantage that it is very slow to mature and is tetrameric. Faster-maturing variants of DsRed have been developed (Bevis & Glick, 2002) and a new monomeric red fluorescent protein, mRFP1, isolated (Campbell et al., 2002
). We therefore cloned DsRed2.0 and its fast-maturing derivatives (T.3 and T.4), as well as mRFP1, into pOT2, creating pRU1104, pRU1105, pRU1106 and pRU1144, respectively.
Construction of ultra-stable IncP-based vectors
The environmentally stable IncP plasmid, pTR102, was made by addition of the parABCDE genes (Weinstein et al., 1992), and this region was subsequently used to create a stable gusA reporter probe vector (pJP2) (Prell et al., 2002
). These vectors are very stable upon repeated subculturing and are completely retained in individual bacteroids in legume nodules, as revealed by histological staining (Prell et al., 2002
; Weinstein et al., 1992
). To construct a vector with tandem gusA and AFP, gfpmut3.1 and its associated multiple cloning site was cloned as a SacIHindIII fragment into pJP2, creating pRU1156. In order to check the expression of gfpmut3.1, the xylose promoter (xylA from pRU604) of R. leguminosarum was cloned as a PmeIHindIII fragment into pRU1156, creating pRU1157. The plasmid was conjugated into R. leguminosarum, which was grown on AMS medium with glucose or xylose as carbon source, and the specific fluorescence of GFPmut3.1 (764 versus 5890 fluorescence units, respectively) and GusA activity [719 versus 1895 nmol min1 (mg prot)1, respectively] was measured.
To enable monitoring on agar plates, gfpUV from pOT2 was cloned as a HindIIISacI fragment into pJP2, deleting the two C-terminal amino acids (YK) and forming pRU1064. The xylA promoter was cloned into pRU1064 and this was conjugated into R. leguminosarum. After growth on agar plates containing xylose, it was confirmed that expression was inducible and unaffected by the two-amino-acid deletion (data not shown).
To make a DsRed marked vector in the IncP background that is compatible with pOT2, mRFP1 (a monomeric and fast-maturing derivative of DsRed) was cloned into pJP2, creating pRU1161. As a test, the dpp promoter from strain 3841 was cloned into pRU1161 as a SpeIHindIII fragment to create pRU1164, resulting in strong expression (data not shown).
As a general observation, R. leguminosarum strains containing pOT-based plasmids gave higher fluorescence readings than pJP2-based plasmids. This is probably due to plasmid copy number, since RP4-based plasmids such as pJP2 have a modest copy number of around 25 in E. coli (Fang & Helinski, 1991) and yields of pOT plasmids isolated from E. coli are much higher than those of pJP2. However, this has not been confirmed by measurement of plasmid copy number in R. leguminosarum. For environmental work, the pJP2-based plasmids have the advantage of being ultra-stable, with no detectable curing even in single bacteroids stained for GusA activity (Prell et al., 2002
). R. leguminosarum carrying pOT plasmids retained the plasmid in 48 % of cells (78 colonies from 13 nodules) recovered from 4-week-old pea nodules. This result is similar to the value for vetch plants reported previously (Stuurman et al., 2000
) and indicates reasonable stability, but clearly pJP2-based vectors are superior for long-term environmental applications.
Monitoring gene expression in situ
AFPs are very useful to monitor gene expression of single cells in the environment. To infect plants, rhizobia must first attach to root hairs before growing down a plant-derived infection thread. This ultimately leads to bacteroid formation, where bacteria are engulfed by plant cortical cells. In order to test the expression of AFPs in infection threads, plasmids pRU1119 (GFPmut3.1) and pRU1127 (DsRedT.3), which are under the control of dctAp, were inoculated onto vetch seedlings. dctAp was chosen because its expression in bacteroids is essential for nitrogen fixation (Finan et al., 1981), but it is not known whether it is expressed in infection threads. It can be seen that dctAp : : DsRedT.3 was expressed throughout infection threads (Fig. 3
), and dctAp : : gfpmut3.1 gave a similar result (data not shown). However, dicarboxylates are not the only carbon sources available during nodule development, since dicarboxylate transport mutants develop into bacteroids (Finan et al., 1981
). Thus, while dicarboxylates are available from this early stage of contact between the bacteria and the plant, their absolute requirement for nitrogen fixation by mature bacteroids must be related to the final metabolic cycling with the plant (Lodwig et al., 2003
). Other compounds, such as sugars, polyols and amino acids, are likely to be available for growth of R. leguminosarum in the infection thread. The vector families developed here are powerful tools with which to investigate this problem.
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ACKNOWLEDGEMENTS |
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Received 29 June 2005;
accepted 11 July 2005.
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