INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Many reporter genes have been described for use in estimating rates of transcription of target genes in prokaryotic systems. Some of the more common reporter genes include lacZ, luxAB, and gusA. Others, such as inaZ or gfp, are not as widely used or have only recently been described (for a general review, see reference 22). Generally, reporter genes are used in transcriptional fusions to measure the transcriptional activity of a particular promoter or gene under various environmental or physiological conditions or, when fused to a constitutively expressed promoter, can be used as marker genes in in situ studies. For example, the gene (gfp) encoding the green fluorescent protein (GFP) (6) has been used primarily as a marker gene in which a transcriptional fusion to a strong constitutive promoter is introduced into a bacterial or eukaryotic cell, conferring a fluorescent phenotype that can be used as a tag in localization studies in situ. GFP has also been used to provide a qualitative measure of gene expression (6, 9, 15, 39). Unlike other reporter genes, such as lacZ or gusA, gfp has not been widely used to quantify promoter strength.

As an increasing number of reports describing reporter gene systems are published, limitations have been discovered. Not all reporter genes can be used under all circumstances. For example, LuxAB-dependent bioluminescence and GFP fluorescence require the presence of molecular oxygen (5, 12). Therefore, studies using either of these two reporter genes under anaerobic conditions would not be possible. LuxAB bioluminescence also requires the flavin mononucleotide FMNH_2. Thus, estimates of the transcriptional activity of luxAB fusions are strongly influenced by levels of metabolic activity; cell activity must be high enough that the concentrations of FMNH₂ are not limiting. For this reason, a common use of lux gene fusions is an indirect assessment of the metabolic state of cells constitutively expressing the lux operon (10, 27). Additionally, the usefulness of lacZ fusions in studies involving eukaryotic cells is limited by the high background level caused by the presence of both bacterial and eukaryotic -galactosidases (22), as well as pigments or particulate material which interferes with enzymatic assays. For use in environmental studies in situ, where the population size of an environmental microorganism will often be very low compared to that in laboratory studies, the efficiency of the reporter gene is also an important issue. If a large number of cells is required to yield a detectable enzymatic activity, as in the case of lacZ-encoded -galactosidase, it may not be possible to use such reporter genes for environmental studies.

Reporter genes also differ in their utility in detecting transcriptional activity at the population or individual-cell level. lacZ and inaZ transcriptional fusions are generally used to determine gene expression and gene regulation in a population of cells, since the activity of individual cells is not easily measured. Ice nucleation events, in particular, cannot be attributable to individual cells in a population. Recently, several studies using gfp fusions have illustrated the capacity to detect the transcriptional activity of single cells harboring such gene fusions (2, 38). Microscopes equipped with charge-coupled devices can be used to quantify the fluorescence of individual cells. Likewise, cells differing in fluorescence can be differentiated using a fluorescence-activated cell sorter (2, 38).

Carbon compounds have been shown to be the most limiting resource on plants (40). Several common sugars, including sucrose, are among the most abundant carbon sources on leaves (28). While sugars are depleted on plants during colonization by bacteria, some sugars apparently remain after colonization ceases (28). Thus, the interactions of microbes with plants with regard to nutrition are apparently complex, and new tools are needed to better understand such interactions.

In this report, we describe the construction of three sucrose-regulated transcriptional fusions, p61RYTIR, p61RYlac, and p61RYice, which contain the gfp, lacZ, and inaZ reporter genes, respectively, fused to a sucrose-responsive promoter. These fusions have been mobilized into Erwinia herbicola strain 299R, a common epiphytic bacterium capable of exploiting plant surfaces and of producing the plant hormone indole-3-acetic acid (4), to create a set of whole-cell biosensors that can be used to estimate the concentrations of sucrose in the cell environment. These biosensors have also been used to compare, for the first time, the relative efficiencies of these reporter genes. The transcriptional activity exhibited by a population of cells will also be correlated with measurements of fluorescence in cells harboring GFP fusions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Construction of the sucrose promoter and control plasmids p61RYTIR, p61RYlac, p61RYice, and pKT-bla. The scrY promoter from pMU3001 (7) was amplified using oligonucleotides SCRPCR4 (5'-GGGAATTCTCAACCGTCAGTTTTAATCC-3') and SCRPCR5 (5'-GGGGATCCTGCTGTTGGCATCCCAAAAG-3'). PCR was carried out for 30 cycles of 1 min at 95°C, 2 min at 56°C, and 3 min at 72°C. The PCR product was then digested with EcoRI and BamHI and cloned into the multiple cloning site of pVSP61 (24) to create the plasmid p61Y. The sucrose repressor gene, scrR, was first cloned as a BamHI-SphI fragment from pRSL26-1, a scrA:: derivative of pJOE637 (34), into pGFP (Clontech, Palo Alto, Calif.) to create the plasmid pSCRR. A 1.9-kb BamHI-HindIII fragment from this plasmid was then cloned into p61Y to create the plasmid p61RY, which has a unique BamHI site between P_scrY and scrR. In order to construct the sucrose promoter-reporter gene fusion plasmids, the inaZ and lacZYA genes had to be subcloned to generate BamHI sites on both ends of the reporter genes. A 3.7-kb EcoRI fragment from pTn3-Spice (20) was cloned into pUC1813 (18) to create pUC1813ice (D. Roberts, unpublished data). To construct pUC1813lac, the 5-kb fragment containing lacZYA was isolated from BamHI- and StuI-restricted pRS551 (37). This fragment was filled in with DNA polymerase (Klenow fragment) and cloned into SmaI-digested pUC1813. BamHI fragments from pGreenTIR (29), pUC1813ice, and pUC1813lac were cloned into the unique BamHI site of p61RY to create the plasmids p61RYTIR, p61RYice, and p61RYlac, respectively.

Media, chemicals, and growth conditions. E. herbicola cells were routinely grown at 24°C. The responsiveness of the various E. herbicola whole-cell biosensors to different concentrations of sucrose and fructose was determined by growing cells for 12 to 16 h in M9 minimal medium supplemented with 0.2% Casamino Acids (MCA) and containing various concentrations of the sugar under investigation. The cells were then washed and resuspended in fresh MCA containing the same test concentration of sugar and grown for 2.5 h. The cells were then harvested, and the reporter gene activity was measured as described below. When appropriate, rifampin and kanamycin were added to the medium at 100 and 50 µg/ml, respectively. Restriction and DNA-modifying enzymes were obtained from New England Biolabs (Beverly, Mass.) or Stratagene, Inc. (La Jolla, Calif.). All chemicals were purchased from Sigma Chemicals (St. Louis, Mo.) or Fisher Scientific (Pittsburgh, Pa.).

Enzyme assays. -Galactosidase and ice nucleation assays were performed as described previously (21, 37). Fluorescence was measured on a Perkin-Elmer LS50B luminescence spectrometer at an excitation wavelength of 490 nm, an emission wavelength of 510 nm, and excitation and emission slit widths of 8 nm; intensity readings are represented by arbitrary units and were normalized to a cell density of 10⁹ cells/ml. The host strain, 299R, demonstrates minimal but measurable fluorescence at the above wavelengths. Therefore, in order to correct for the background fluorescence of 299R, eight independent measurements of the fluorescence of 299R cultures in MCA were taken. The mean fluorescence of these cultures was then subtracted from the values for 299R(p61RYTIR) at each of the sucrose concentrations. The -galactosidase and ice nucleation activities of 299R were negligible and were not corrected for in the calculations of reporter gene activities.

Plant inoculation and bacterial cell recovery. Cells from stationary-phase cultures of strain 299R(p61RYice) or strain 299R(p61RYTIR) grown in MCA containing 0.2% glucose were washed twice in potassium phosphate buffer (10 mM, pH 7.0) (PPB) before being resuspended in distilled water at 1 × 10⁵ or 2 × 10⁷ cells ml¹, respectively. The cell suspension was immediately applied with an atomizer to bean plants (Phaseolus vulgaris cv. Bush Blue Lake 274) (three replicate pots of eight plants per pot) at the first trifoliate leaf stage. The plants were then bagged and incubated at 24°C. Two primary leaves were sampled at random from each pot 48 h after inoculation for examination of 299R(p61RYTIR) cells directly on leaves by confocal laser scanning microscopy, or for recovery of cells to be examined by epifluorescence microscopy following fluorescence in situ hybridization (FISH). For assessment of bacterial populations and P_scrY-inaZ expression on plants, 15 leaves were sampled at regular time intervals. Each sampled leaf was placed in 20 ml of sterile PPB. Cells were removed from leaves by sonication for 7 min followed by vigorous agitation. Bacterial populations were estimated by plating appropriate dilutions on Luria-Bertani agar supplemented with kanamycin. Also, leaf washings were assayed for ice nucleation activity as described above. Prior to FISH and microscopy, cells removed from leaves were concentrated by filtration of the cell suspension through a 0.4-µm-pore-size polycarbonate filter (Millipore, Bedford, Mass.).

Microscopy and image analysis. Bacterial cells recovered from plants were fixed promptly and prepared for FISH with the rhodamine-labeled probe Eh-299R, which is specific to E. herbicola 299R, as described by Brandl et al. (M. T. Brandl, B. Quiñones, and S. E. Lindow, submitted for publication). For cytological analyses of cells grown in vitro, we used five 100-µl aliquots of the same cultures for which fluorescence was measured with a fluorimeter. The culture aliquots were centrifuged, and the cells were washed and resuspended in PPB to a final concentration of approximately 5 × 10⁷ cells ml¹. Ten microliters of this suspension was spotted on Polysine microscope slides (Erie Scientific Company, Portsmouth, N.H.), left to dry at room temperature for 10 min, mounted in glycerol-PBS (pH 8.0; 50:50 [vol/vol]), and examined immediately. When indicated, cultured cells were fixed with 4% paraformaldehyde prior to cytological analysis following a protocol described by Amann (1). Cells recovered from cultures or from plants were visualized on an Axiophot microscope fitted with a Plan-NEOFLUAR 100×/1.30 oil objective (Carl Zeiss, Oberkochen, Germany) using phase-contrast and epifluorescence illumination. GFP and rhodamine fluorescences were detected with an Endow GFP filter set and a rhodamine filter set (Chroma Technology Corp., Brattleboro, Vt.), respectively. The microscope was equipped with a Princeton ST138 cooled charge-coupled device camera (Princeton Instruments, Inc., Trenton, N.J.). Acquisition of digitized 12-bit images were performed with the MacIntosh version 3.1 of the IPLab Spectrum software package (Scanalytics, Inc., Vienna, Va.). Two fields of at least 100 individual cells were captured per sample at an exposure of 5 s, which was within the linear range of detection of the camera for all tested samples. The fluorescent grey scale intensity of each pixel within each cell was quantified with the MacIntosh version 3.2 of IPLab Spectrum by overlay of the binary mask from the phase-contrast image on the corresponding fluorescence image. Mean pixel intensity per cell was computed from the sum of the intensities of all of the individual pixels averaged over the total number of pixels forming the cell profile.

Statistical methods. All statistical calculations were performed with the program Statistica (version 5.1) (StatSoft, Tulsa, Okla.). Cumulative normal probability values (normal score), analysis of variance, and the probability values for the Kolmogorov-Smirnov D statistic were calculated with the SAS program (version 6.03; SAS Institute Inc., Cary, N.C.).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Construction and specificity of the PscrY transcriptional fusions. In order to compare the lacZ, gfp, and inaZ reporter genes, a set of transcriptional fusions, isogenic apart from the reporter gene, was constructed using the scrY promoter (P_scrY) from the Salmonella enterica serovar Typhimurium plasmid pUR400 (7) (Fig. 1). The three promoter plasmids, p61RYTIR, p61RYice, and p61RYlac, contain the genes, devoid of their native promoters, encoding the GFP (gfp) from Aequorea victoria (6), the ice nucleation protein (inaZ) from Pseudomonas syringae pv. syringae (11), and -galactosidase from E. coli, respectively. The sucrose repressor gene, scrR, is also present on each plasmid and located immediately downstream of the reporter gene. Transcription from P_scrY has been shown to be regulated by the ScrR repressor and induced during cell growth on sucrose, fructose, and fructose-containing oligosaccharides (7). To determine if transcription from P_scrY in our constructs is induced exclusively by sucrose and fructose, E. herbicola strain 299R(p61RYice) was grown in MCA with various sugars at a concentration of 0.2%. The ice nucleation activity of 299R(p61RYice) after growth on each of the substrates was measured (data not shown). As expected, both fructose and sucrose induced transcription from P_scrY to a high level (10^2.13 and 10^1.45 ice nuclei/cell, respectively). Since ice nucleation activity increases approximately with the square of the increase in the cellular abundance of InaZ (20), the ca. 6,000-fold increase in ice nucleation activity in MCA-sucrose compared to MCA alone (10^5.24 ice nuclei/cell) corresponds to an approximately 80-fold increase in transcriptional activity from P_scrY. Sorbose induced the highest level of ice nucleation activity (10^0.5 ice nuclei/cell), which was about 9-fold higher than that observed for sucrose and about 43-fold higher than for fructose. Sorbose and fructose are both ketohexoses and are diastereomers of each other. Therefore, other diastereomers of fructose (e.g., psicose and tagatose) might be presumed to induce P_scrY, but this possibility was not tested.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   General organization of the sucrose promoter-reporter gene fusion plasmids. The common elements of each plasmid are (clockwise) a sucrose-responsive promoter (PscrY) (7), the sucrose repressor gene (scrR) (7), and the following components derived from the parental vector, pVSP61 (24): the P15a origin of replication from pACYC184, a kanamycin resistance gene (nptII), and a broad-host-range origin of replication (VS1 replicon). Promoterless reporter gene fragments (gfp, inaZ, and lacZYA) containing BamHI sites at both ends were cloned into the unique BamHI site between PscrY and scrR to create the plasmids p61RYTIR, p61RYice, and p61RYlac, respectively. The lac promoter present on pVSP61 is located immediately downstream of the HindIII site in a transcriptional orientation opposite to that of scrR. Restriction enzymes: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII. *, EcoRI site present in p61RYTIR only; **, EcoRI site present in both p61RYTIR and p61RYice

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Dose response of the sucrose biosensor strain 299R(p61RYice) when grown in MCA amended with various concentrations of sucrose or fructose. Each data point is the mean of the activities of at least three replicate cultures.

Characterization and comparison of the induction response for the three reporter gene fusions. Once the specificity of the gene fusions for various sugars had been demonstrated, we wished to use these constructs to compare the relative sensitivities of the gfp, inaZ, and lacZ reporter genes to sucrose as an inducer when used in a whole-cell biosensor. Plasmids harboring these gene fusions were first mobilized into strain 299R, and the resulting transconjugants were grown in MCA in which sucrose concentrations were varied by about 10,000-fold (1.45 µM to 29 mM). The three whole-cell biosensors exhibited a qualitatively similar pattern of reporter gene activity as a function of sucrose concentration in growth media (Fig. 3). For example, maximum activity was observed at about 10³ M or higher sucrose in all cases (Fig. 3). Likewise, activity increased rapidly with sucrose concentrations over the range of about 10⁵ to 10³ M. All three strains showed similar maximum proportional levels of induction at high sucrose concentrations compared to sucrose-free media: 183-fold in 299R(p61TYTIR) (Fig. 3A), 103-fold in 299R(p61RYlac) (Fig. 3B), and 122-fold (corresponding to a 15,000-fold difference in ice nucleation activity) in 299R(p61RYice) (Fig. 3C). However, there were quantitative differences between the three strains in their responses to low sucrose concentrations. Little reporter gene activity was observed in all three strains at the lowest concentration of sucrose tested (1.45 µM). However, whereas GFP fluorescence in 299R(p61RYTIR) and -galactosidase activity in 299R(p61RYlac) were induced less than 2-fold at 14.5 µM sucrose, inaZ expression in 299R(p61RYice) was induced 6.5-fold (35-fold higher ice nucleation activity) at the same sucrose concentration. In fact, while no induction of GFP fluorescence or -galactosidase activity was seen in MCA containing 2.9 µM sucrose, significantly higher ice nucleation activity was observed at that sucrose concentration. In MCA containing 29 µM sucrose, GFP fluorescence and -galactosidase activity in both 299R(p61RYTIR) and 299R(p61RYlac), respectively, were induced 11- to 13-fold but inaZ expression in 299R(p61RYice) was induced 35-fold. It is likely that with all three reporter genes tested in this study the level of transcriptional activity mediated by low sucrose concentrations was underestimated since the host strain, E. herbicola 299R, is capable of rapid catabolism of sucrose. Thus, the cells probably depleted the concentration of this inducer, at least partially, at low initial sucrose concentrations, during the 3-h period of exposure of the whole-cell biosensor to sucrose. Therefore, some cells produced during the later part of this exposure period may have experienced lower sucrose concentrations than that estimated from initial sugar concentrations. Furthermore, mutants of E. herbicola that could take up but not catabolize sucrose might be preferable hosts for P_scrY reporter gene fusions in studies in which sucrose biosensors are to be used to estimate only initial concentrations of this sugar in a habitat rather than the process of sugar consumption, since they would not be capable of altering the abundance of the compound to which they were responsive.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Dose response of the sucrose biosensor strains 299R(p61RYTIR) (A), 299R(p61RYlac) (B), and 299R(p61RYice) (C) when grown in MCA amended with various concentrations of sucrose. Each data point is the mean of the activities of four replicate cultures.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Comparison of the sensitivities of the three sucrose biosensor strains. The minimum detectable cell population is defined as the number of cells necessary to achieve the limit of detectability for each enzyme assay: 1 Miller unit, 5 fluorescence units, and 10 ice nuclei for LacZ, GFP, and InaZ, respectively. Each data point was calculated using the reporter gene activity values shown in Fig. 3.

Analysis of P_scrY-gfp expression in individual cells. Analysis of digital images of the fluorescence of individual cells of 299R(p61RYTIR) obtained by epifluorescence microscopy enabled the quantitative assessment of the mean pixel intensity of each cell within a population of bacterial cells. The parental strain 299R exhibited negligible fluorescence at the excitation and emission wavelengths used in this study, when grown in MCA containing glucose. The mean fluorescence intensity of 299R(p61RYTIR) cells grown in MCA containing 0 to 14.5 µM sucrose was not different from that of cells that did not harbor the P_scrY-gfp fusion (Fig. 5). However, the mean fluorescence intensity per cell increased significantly at sucrose concentrations of 29 µM and higher (Fig. 5 and 6). The frequency of cells showing fluorescence intensities higher than background levels (mean pixel intensity >125 units) increased with increasing sucrose concentrations, and average fluorescence intensities of 146, 223, 781, and 984 pixel intensity units for a large collection of cells cultured in 29 µM, 58 µM, 580 µM, and 29 mM sucrose were observed (Fig. 5 and 6). Thus, quantitative digital image analysis revealed a response of the biosensor to various concentrations of sucrose at the population level that was similar to that obtained for fluorescence of aliquots of the same culture measured with a fluorimeter (Fig. 3A).

View larger version (36K): [in this window] [in a new window]   FIG. 5.   Histograms of the distribution of the mean pixel intensity per cell of E. herbicola 299R(p61RYTIR) cells containing a PscrY-gfp transcriptional fusion and grown in MCA supplemented with sucrose at 0 µM (A), 14.5 µM (B), 29 µM (C), 58 µM (D), 580 µM (E), and 29 mM (F).

View larger version (28K): [in this window] [in a new window]   FIG. 6.   Cumulative probability plot of the log-transformed estimate of mean fluorescence intensity of individual cells of E. herbicola 299R(p61RYTIR) cells cultured in MCA containing various sucrose concentrations.

View larger version (33K): [in this window] [in a new window]   FIG. 7.   Histograms of the distribution of the mean pixel intensity per cell of E. herbicola 299R(pKT-bla) cells containing a Pbla-gfp transcriptional fusion and grown in MCA supplemented with 58 µM (A) and 29 mM (B) sucrose. The Kolmogorov-Smirnov statistics for these two distributions are D = 0.064 and P = 0.43 (A) and D = 0.035 and P = 0.52 (B).

Quantification of P_scrY-gfp expression in situ on plants. We compared the utility of the gfp- and inaZ-based sucrose biosensors in estimating sucrose availability on bean leaf surfaces, a common bacterial habitat. We were particularly interested in knowing if the estimates of sugar variability on leaves measured at the single-cell level with the gfp-based biosensor construct in E. herbicola provided results consistent with that of the population-level estimates of the inaZ-based biosensor and whether they both were sufficiently efficient to detect the low concentrations of sugar expected to be on moist leaf surfaces (28). Qualitative estimates of the distribution of GFP fluorescence among cells of E. herbicola 299R(p61RYTIR) was obtained by direct visualization of leaf surfaces 48 h after inoculation by confocal laser scanning microscopy (CLSM). The large majority of cells of the biosensor strain on leaves were dim and could be visualized only in long-duration exposures. A few cells in most fields of view, however, exhibited bright GFP fluorescence; these cells often occurred as dispersed individual cells that were markedly brighter than surrounding cells, although occasionally several bright cells were found in close proximity (Fig. 8). The bright cells appear larger than the dimmer cells in an image due to an optical artifact associated with their brightness and hence their "overexposure" in images captured to visualize many cells in a field. We did not pursue spatial scale studies to determine if such sites of apparent localized sugar concentration were associated with particular features of the leaf, but we can conclude that such localizations were uncommon on leaves.

View larger version (215K): [in this window] [in a new window]   FIG. 8.   CLSM image of cells of E. herbicola 299R(p61RYTIR) expressing high levels of PscrY-gfp on a stomate and at the junction of plant cells of a colonized bean leaf 2 days after inoculation. The image is an overlay of two projected z series captured with channel 1 (em = BP 525/50 to detect GFP fluorescence) and channel 2 (em = LP 590 to detect the autofluorescence of leaf tissue) of a Leica TCS4D confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) equipped with argon and krypton lasers. Bar, 5 µm.

View larger version (22K): [in this window] [in a new window]   FIG. 9.   Histogram of the distribution of GFP fluorescence among individual cells of E. herbicola 299R(p61RYTIR) recovered from bean leaves 48 h after inoculation.

4.5

2.5

View larger version (18K): [in this window] [in a new window]   FIG. 10.   Time course expression of PscrY-inaZ in the bean phyllosphere. The population (closed circles) and ice nucleation activity (open squares) of E. herbicola 299R(p61RYice) were determined over time after inoculation onto bean plants. The bars represent the standard errors of the mean log-transformed bacterial population size per gram of leaf tissue and of the mean number of log-transformed ice nuclei per cell.