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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1998, p. 1902–1909 Copyright 1998, American Society for Microbiology Effect of Bacterial Distribution and Activity on Conjugal Gene Transfer on the Phylloplane of the Bush Bean BO NORMANDER,1 BJARKE B. CHRISTENSEN,2 SØREN MOLIN,2 AND NIELS KROER1* National Environmental Research Institute, Department of Marine Ecology and Microbiology, DK-4000 Roskilde,1 and Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby,2 Denmark Received 18 September 1997/Accepted 22 February 1998 Conjugal plasmid transfer was examined on the phylloplane of bean (Phaseolus vulgaris) and related to the
spatial distribution pattern and metabolic activity of the bacteria. The donor (Pseudomonas putida KT2442)
harbored a derivative of the TOL plasmid, which conferred kanamycin resistance and had the gfp gene inserted
downstream of a lac promoter. A chromosomal insertion of lacIq prevented expression of the gfp gene. The
recipient (P. putida KT2440) had a chromosomal tetracycline resistance marker. Thus, transconjugants could
be enumerated by plating and visualized in situ as green fluorescent cells. Sterile bean seedlings were
inoculated with donors and recipients at densities of approximately 105 cells per cm2. To manipulate the
density and metabolic activity (measured by incorporation of [3H]leucine) of the inoculated bacteria, plants
were grown at various relative humidities (RH). At 100% RH, the transconjugants reached a density of 3 3 103
CFU/cm2, corresponding to about one-third of the recipient population. At 25% RH, numbers of transconju-
gants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic
activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after
96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between
the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above
a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between
epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the
distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities
equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus,
aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.
Genetic exchange by conjugal plasmid transfer has been the presence of solid surfaces are believed to stimulate conju- observed in diverse aquatic (2, 3, 38, 43, 47) and terrestrial (28, gal transfer (15, 20, 35, 50).
32, 49, 51, 52) environments and has been suggested to be an An understanding of the factors that influence genetic ex- important mechanism in the adaptation of microbial commu- change by conjugation is pertinent for assessing the signifi- nities to changing environmental conditions (4, 31).
cance of conjugation in the evolution of microbial communities An important habitat in the terrestrial environment is the as well as for more pragmatic reasons, such as risk assessment phyllosphere. Gene transfer by conjugation between epiphytic of released genetically engineered bacteria. The aim of the bacteria is, however, poorly investigated. Lacy and Leary (30), present study was to investigate the significance of bacterial Knudsen et al. (25), and Bjo¨rklo¨f et al. (5) studied conjugation distribution and metabolic activity on conjugation on the phyl- on the phylloplane of bean. Transfer ratios up to 3 3 1021 loplane. To the best of our knowledge, this is the first report (number of transconjugants per recipients [T/R]) were ob- which relates conjugal transfer on the phylloplane to the bac- served at humidities close to 100% (30). In other studies, Lilley terial metabolic activity, and it is the first study in which the and Bailey (31) demonstrated transfer of natural mercury re- effect of cell distribution on transfer is directly assessed. To sistance plasmids from indigenous bacteria of the sugar beet accomplish these objectives, bean plants were grown at various phylloplane to an added pseudomonad.
relative humidities (RH) to simultaneously manipulate the The phylloplane can under many environmental conditions density and activity of the inoculated bacteria. In situ metabolic be considered a hostile habitat as the epiphytic bacteria are activity and distribution of transconjugant cells were deter- exposed to desiccation and solar UV radiation (8, 33, 45). On mined by incorporation of tritiated leucine (Leu) and by using the other hand, leaf exudates, such as carbohydrates, amino green fluorescent protein as plasmid reporter gene, respec- acids, and organic acids (37) may support bacterial densities of up to 5 3 107 CFU/g (fresh weight) under humid conditions (23). In addition, the structurally complex leaf surface, consist- ing of epidermal cells, interstitial spaces, trichomes, and sto- MATERIALS AND METHODS
mata (7, 22), may provide bacteria with survival habitats. Both Bacterial strains, plasmids, and growth media. Characteristics of the strains
availability of growth substrates, a high bacterial density, and and plasmids used are listed in Table 1. Pseudomonas putida KT2442::lacIq served as donor strain in biparental mating experiments. The strain harbored a derivative of the TOL plasmid which conferred kanamycin resistance and had the gfpmut3b reporter gene cloned downstream of the lac promoter, PA1/O4/O3 (con- * Corresponding author. Mailing address: Dept. of Marine Ecology structions are described below). As recipient strain, P. putida KT2440 with a and Microbiology, National Environmental Research Institute, Fred- chromosomal tetracycline resistance marker was used. Donors were grown in eriksborgvej 399, DK-4000 Roskilde, Denmark. Phone: 45 46 30 13 88.
Luria broth (LB) (36) supplemented with 50 mg of kanamycin per ml (KM50), Fax: 45 46 30 12 16. E-mail: [email protected].
while recipients were grown in LB with 15 mg of tetracycline per ml (TC15).
GENE TRANSFER ON THE PHYLLOPLANE OF THE BUSH BEAN TABLE 1. Bacterial strains and plasmids Strain or plasmid Source or reference P. putida KT2440 TOL plasmid-cured derivative of P. putida mt-2 P. putida KT2440-Tc KT2440 with mini-Tn5 insertion of Tcr P. putida KT2442 Rifr mutant of KT2440 P. putida KT2442::lacIq KT2442 with mini-Tn5 insertion of lacIq, Rifr P. fluorescens AS12 arg mutant of P. fluorescens R2f, Rifr E. coli MC1061 Self-transmissible 117-kb plasmid from P. putida mt-2 TOL with mini-Tn5 insertion of PA1/O4/O3::gfpmut3b, Kmr Self-transmissible 60-kb broad-host-range IncP plasmid, Kmr, Tcr, Ampr Transconjugants (P. putida KT2440/TOL) were enumerated on LB plates con- were kept in the dark for 4 to 5 days, after which they were transferred to the taining both KM50 and TC15. Plates were incubated at 30°C.
growth chamber. Prior to inoculation, all plants were incubated for 24 h at the In studies of transfer to indigenous epiphytic bacteria, the auxotrophic (arg RH to be used in the specific experiment.
mutant) Pseudomonas fluorescens AS12 containing plasmid RP4 was used. P. The growth chamber was equipped with two halogen-quartz-iodine-tungsten fluorescens AS12 was chosen as donor in these experiments because it, unlike for lamps (Osram Daylight HQI-T 250 W/D). Light intensities were 240 to 270 P. putida KT2442, is possible to counterselect this strain on transconjugant mmol/m2/s. RH was controlled by a vaporizer and measured by a Kane May 8004 selective plates (see below). RP4 was the plasmid of choice because it is a RH sensor and time logged by a Tinytalk datalogger (Orion Components [Chich- promiscuous plasmid that can be transferred to a large variety of bacterial species ester] Ltd., United Kingdom). Both sensors had an accuracy of 62% RH and an (26). The TOL plasmid, on the other hand, has a more narrow host range (40).
upper limit of 95% RH. An RH of approximately 100% was obtained by incu- Donors were enumerated on LB supplemented with KM50 and TC15, indige- bating plants in plastic containers (15 to 20 liters) covered with polyethylene film nous recipients on minimal medium (16) amended with 0.2% glucose, and and with water added to the bottom of the containers.
indigenous transconjugants on minimal medium amended with 0.2% glucose Inoculation of plants. Overnight cultures were washed twice in 10 mM phos-
with KM50 and TC15. To avoid overgrowth by fungi, media were supplemented phate buffer (pH 7.0) (7,740 g, 8 min in a Beckman JA20 rotor), starved for 24 h with 25 mg of natamycin (Delvocid; Gist-Brocades, Delft, Holland) per ml. Plates at room temperature and adjusted to approximately 108 cells/ml according to were incubated at 25°C.
predetermined optical density curves. The starvation period was used to reduce Construction of strains and plasmids. The lacIq gene (48) was inserted into
intracellular energy resources.
the chromosome of P. putida KT2442 by triparental mating (14) by using a Leaves of sterile 12- to 14-day-old plants were inoculated by carefully immers- modified pUT vector with resolvase sites flanking the npt gene (27). Subse- ing the green parts of the plants in a 1:1 mixture of the P. putida donor and quently, the npt gene was deleted by a second round of triparental mating and a recipient suspensions or in the P. fluorescens AS12/RP4 suspension for 10 to 15 s.
Kms transconjugant was picked.
Excess drops of liquid were removed by gentle shaking of the plants. Densities of To construct a PA1/O4/O3::gfpmut3b gene cassette, the gfpmut3b gene (12) was approximately 108 CFU per g (dry weight) or 105 CFU per cm2 were achieved amplified by PCR as a 0.7-kb SphI-HindIII fragment. The gfpmut3b gene is a (Fig. 1). In some instances seed inoculation was used. This was done by inocu- variant of the wild-type gfp gene in which two amino acids have been substituted.
lating the Hoagland solution of the sterile rock wool cubes (see above) with 107 These substitutions result in an enhanced fluorescent signal (12). To introduce a CFU/ml of the cells.
SphI restriction site in the start codon of gfpmut3b, the sequence was changed In the biparental mating experiments, the metabolic activity of the inocula was during PCR so that the gfpmut3b contained an Arg instead of a Ser residue at determined by incorporation of tritiated Leu (24, 28). One milliliter of a 0.01 mM position 2. The gfpmut3b fragment was cloned downstream from the promoter Leu solution containing 0.40 mCi of [4,5-3H]Leu (139 Ci/mmol; Amersham Life PA1/O4/O3 (34) in an optimal distance from the ribosome binding site of phage T5 Science) was added to 4-ml aliquots of the bacterial suspensions to give a final (RBSII) and upstream of a region with translational stop codons in all three Leu concentration of 2,000 nM. Killed controls were set up by addition of 500 ml reading frames, as well as two strong transcriptional terminators, T0 (from phage of 37% formaldehyde. The incorporation was terminated after 30 min by addi- lambda) and T1 (from the rrnB operon of Escherichia coli). The NotI fragment tion of 500 ml of 37% formaldehyde. Suspensions were filtered through 0.2-mm- from the resulting plasmid (pJBA27), containing RBSII, gfpmut3b, the transla- pore-size cellulose-nitrate filters (Sartorius GmbH, Go¨ttingen, Germany). Filters tional stop codons, and the transcriptional terminators, was inserted into the were rinsed with 5 ml of 10 mM phosphate buffer and counted on a Beckman NotI site of pUT-Km (13), resulting in a transposon delivery vector (pJBA28) LS1801 scintillation counter. The concentration of Leu required to reach the containing the PA1/O4/O3::gfpmut3b and npt gene cassette.
saturation level with respect to bacterial assimilation had been determined in a Insertion of the PA1/O4/O3::gfpmut3b cassette into the TOL plasmid was per- formed in two steps. First, pJBA28 was transferred to P. putida KT2440 by Sampling procedure. At each sampling time, both the metabolic activity and
triparental mating. Isolation on AB minimal plates (11), containing KM50 and 10 bacterial population size were determined. One leaf was excised from each of mM citrate, resulted in KT2440 derivatives carrying the PA1/O4/O3::gfpmut3b three replicate plants, and leaves were submerged individually in 5 ml of phos- cassette either on the chromosome or on the TOL plasmid. To isolate clones with phate buffer (10 mM, pH 7.0) containing 250 nM Leu and 1 mCi of [4,5-3H]Leu the cassette integrated on the plasmid, a second round of conjugation was (139 Ci/mmol; Amersham Life Science). Controls were set up by addition of 500 performed. All colonies from the selective plates (.1,000 per plate) were ml of 37% formaldehyde. Incorporation of Leu was stopped after 30 min by scraped off and suspended in 1 ml of 0.9% NaCl. Cells were then mixed with the transferring the leaves to new phosphate buffer without Leu. The bacteria were Kms P. putida KT2442::lacIq. Isolation on plates containing KM50 and 50 mg of then extracted by sonication for 7 min in a Branson 5210 ultrasonic bath followed rifampin per ml (RIF50) resulted in different Kmr derivatives carrying the mod- by 15 to 20 s of vortexing. Aliquots (4 ml) of the extracts were filtered through ified TOL plasmid. The clone chosen for the gene transfer experiments was able 0.2-mm-pore-size cellulose-nitrate filters. Filters were rinsed and counted as to grow on AB minimal plates supplemented with either 5 mM benzyl alcohol or described above.
5 mM benzoate (53); it showed green fluorescence upon addition of 1 mM IPTG Numbers of donors, recipients, and transconjugants were determined by seri- (isopropyl-b-D-thiogalactopyranoside) and illumination with blue light, and the ally diluting the remaining extract and plating on selective medium. To improve conjugation frequency of the gfp-tagged plasmid was similar to that of the the detection limit of transconjugants, aliquots of 400 ml were mixed with 1.6 ml wild-type TOL plasmid as tested on agar plates.
of 10 mM phosphate buffer and filtered through 0.2-mm-pore-size polycarbonate Sterilization and growth of plants. Seeds of bush bean (Phaseolus vulgaris cv.
membrane filters (Poretics Products, Livermore, Calif.). Filters were placed on Montana) were sterilized in a solution of 0.25% benzalkoniumchloride and 25% transconjugant selective media. Parallel to sampling, the significance of mating H2SO4 for 2 h followed by careful rinsing in sterile MilliQ-water. The sterilized on the transconjugant selective media was assessed. This was done by combining seeds were pregerminated on LB plates (to test for sterility) for 3 to 4 days in the extracts of leaves, inoculated with donors and recipients separately, and plating dark after which they were transferred aseptically to sterile rock wool cubes with on transconjugant selective media as described above. Plate mating constituted 5 ml of autoclaved Hoagland's plant nutrient solution (18) in 30-ml plastic pots.
less than 5% of the observed transconjugants. Reported numbers of transcon- The pots were incubated in a growth chamber at 26 to 28°C and a 23:1-h jugants (see Results) are corrected for plate mating.
light-dark cycle. The plants were watered with autoclaved Hoagland's plant Leaves were dried for 24 h at 110°C, and the dry weight was determined.
nutrient solution when needed. Untreated bean plants were grown in pots with Conversion of dry weight to surface area (both sides) was performed according soil from an uncultivated field at Risø, near Roskilde, Denmark. In this case, pots to the following equation: surface area (cm2) 5 0.747 3 dry weight (mg) (n 5 22; NORMANDER ET AL.
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FIG. 1. Survival of P. putida KT2442/TOL (donors, ‚) and P. putida KT2440-Tc (recipients, V) and appearance of KT2440-Tc/TOL (transconjugants, M) on bean leaves at 100% RH (A) and 90% 6 2% RH (B). Error bars are 6 SD of triplicate samples.
P , 0.0001). The equation was determined by measuring the dry weight of 1- by Data analysis. Plasmid transfer was calculated as T/R and T/D (number of
1-cm squares of the leaves.
transconjugants per number of donors) ratios and as the time- and density- Filter matings. Two different filter-mating experiments were performed. In
independent transfer coefficient, kt1 (44). kt1 was calculated for two data points one experiment, starved donor and recipient suspensions were filtered onto as (DT/Dt)/(D 3 R) under the assumption that D@T, R@T, and D and R were 0.2-mm-pore-size polycarbonate filters (Poretics Products) to a density of 107 constant (35).
CFU/cm2. A monolayer of cells was formed (verified by microscopy), which One-way analyses of variance on log10-transformed data and linear regression insured cell-to-cell contact. Filters were presoaked for 10 min in 10% (vol/vol) analysis were performed by using the SigmaStat software for Windows (Jandel Suprapur HCl (Merck, Darmstadt, Germany) and washed three times in 0.9% Corp., Erkrath, Germany).
NaCl (solid purity, 99.5%; Merck) in UV-treated MilliQ-water. The filters with the bacteria were floated on saline in acid-rinsed petri dishes and incubated in the dark at 26°C. In the other experiment, starved donors and recipients were filtered into the polycarbonate membranes to a density similar to that on the leaves, i.e., ca. 105 CFU/cm2. The filters were placed on agarose plates. In both Effect of RH on survival and conjugal transfer. Survival of
experiments, cell numbers and metabolic activity were determined at regular intervals as described above for the leaves. The concentration of dissolved or- the bacteria on the leaves depended on RH. At 100% RH, ganic carbon in the saline (,0.25 ppm) was measured on a Shimadzu TOC-5000 numbers of CFU of P. putida KT2442/TOL almost doubled during the 96-h incubation (Fig. 1A), whereas at 90% RH, Verification of transconjugants and identification of indigenous epiphytic
numbers declined by a factor of 200 within the first 24 h (Fig.
bacteria. Putative transconjugants were either tested for green fluorescence, to
show the presence of the TOL plasmid, or tested for their ability to act as donors 1B). At lower humidities (80, 55, and 25%), population den- of RP4 to E. coli MC1061 (Table 1).
sities of the donor were reduced further (Fig. 2). The P. putida Natural epiphytic isolates possessing different cell and/or colony morphology KT2440 recipient did not survive as well as the donor (P , were gram-identified by the KOH method (39). Subsequently, gram-negative 0.003) as numbers were reduced by a factor of 8 during the isolates were characterized by the API 20E and API 20NE test kits (Biomerieux SA, Marcy l'Etoile, France).
incubation at 100% RH (Fig. 1A). At 90% RH and lower In situ detection of bacteria on leaves. Epiphytic indigenous bacteria were
humidities, survival rates of donors and recipients were com- stained with 0.2 mm-pore-size-filtered (Nalgene sterilization filter; Nalge Com- parable (P . 0.17) (Fig. 1B and 2).
pany, Rochester, N.Y.) phenolic aniline blue (PAB) according to Jones et al.
RH also affected the appearance of transconjugants. At (21) and Hossell and Baker (19). Basically, a leaf was submerged in PAB for 1 to 2 min. A square of approximately 5 by 5 mm was excised and placed on a 100% RH, numbers of transconjugants reached a level of 3 3 microscope slide mounted in a drop of PAB. A Zeiss Axioplan microscope fitted 103 CFU/cm2 within 24 h, corresponding to about one-third of with a 12-V tungsten lamp was used for transmitted illumination. Digital images the recipient population (Fig. 1A). Relative to 100% RH, were recorded with a 12-bit cooled slow-scan charge-coupled device camera transconjugant densities were approximately 500, 1,200 and (KAF 1400 chip; Photometrics Ltd., Tucson, Ariz.).
The spatial distribution of transconjugant cells was determined by examining 8,000 times lower at 90, 80, and 55% RH, respectively (Fig. 2).
a 5- by 5-mm leaf square with a Zeiss Axioplan microscope equipped with an At 25% RH, no transconjugants were detected.
HBO-100 mercury lamp and Zeiss filter set 10 (BP 470- to 490-nm exitation filter, Incubation at low RH did not result in an irreversible decline 510-nm dichroic mirror, and BP 515- to 565-nm emission filter). Plan-Neofluar in the population size of the bacteria. In the experiment per- 403 and 633 oil immersion lenses and 203, 403, and 1003 dry lenses were formed at 55% RH, some plants were transferred to 100% RH Three-dimensional images were obtained by a Leica Lasertechnik TCS 4D after 96 h. After an additional 72 h at this humidity, densities confocal scanning laser microscope equipped with a 15-mW argon-krypton ion of donors, recipients, and transconjugants increased 70, 10, laser (excitation wavelength, 488 nm). To discriminate between the green fluo- and 100 times, respectively (data not shown).
rescence emitted by the cells and the red fluorescence emitted by the leaf, BP-510 and LP-515 emission filters (Leica) were used. Series of monochrome Maximal transfer ratios, calculated as T/D, ranged from 2-D sections along the optical axis were recorded and combined to create a 3-D 0.007 to 0.026 whereas ratios calculated as T/R ranged from image by use of the simulated fluorescent projection technique provided by the 0.01 to 0.343 (Table 2). Ratios did not appear to be directly Scanware 1.02 software (Leica). Stereo-pairs of 3-D images of the green fluo- related to RH. However, by exerting an effect on cell survival, rescent cells and the leaf surface were colored and combined within a red-green- blue display by using Adobe Photoshop for Windows 95 (Adobe Systems Inc., RH did have an effect on transfer, especially on T/R which was San Jose, Calif.).
fivefold lower at 90% than at 100% RH.
GENE TRANSFER ON THE PHYLLOPLANE OF THE BUSH BEAN The fluorescent signal was reduced if cells on microscope slides were exposed to desiccation. Similarly, the signal disap- peared after a few hours if the plants were incubated at 60% RH. The signal persisted the longest time in the substomatal cavities followed by the interstitial spaces. Hence, relative to the surface of the epidermal cells, these habitats most likely protected the bacteria against desiccation.
Examination of unsterile leaves grown at 100% RH revealed a distribution of the indigenous bacteria similar to that of the transconjugants (Fig. 3D and E). Microcolonies consisting of 100 to 1,000 cells were often found associated with the epider- mal interstices. Cells were observed in about 10% of the sto- mata. Plants incubated at 60% RH showed a similar distribu- tion of the bacteria; however, fewer cells were observed.
Densities of CFU were 6.1 6 5.0 3 102 CFU/cm2 and 1.8 6 1.1 3 105 CFU/cm2 (6 standard deviation [SD]) (n 5 6) at 60 and 100% RH, respectively.
In situ metabolic activity. Immediately after inoculation
onto the leaves, the per-cell metabolic activity increased four to eight times relative to the activity of the cells when in the inoculation buffer. For instance at 100% RH, the activity in- creased significantly (P , 0.0005), from 0.2 3 1022 to 1.6 3 1022 fmol of Leu/CFU/h (Fig. 4). Through the incubation, the FIG. 2. Density of donors (‚), recipients (V) and transconjugants (M) after activity decreased and approached the level of the inocula after 96 h of incubation as function of RH. No transconjugants were observed after 96 h of incubation at 25% RH. Error bars are 6 SD of triplicate samples.
96 h. At lower RHs, metabolic activities on the leaves de- creased to the level of the inocula after 4 h, following which activity could no longer be detected (Table 2). The metabolic In situ distribution of cells on leaves. Green fluorescent
activity was inversely correlated with cell density (r2 5 0.218; transconjugants were observable 5 to 6 h after inoculation of P , 0.0001) (Fig. 5).
the plants (100% RH). Thus, detection by microscopy was Effect of metabolic activity and density on conjugal transfer.
delayed about 4 h relative to detection by plating, due to an Metabolic activity and conjugal transfer on the leaves were not approximately 4-h processing time of the fluorophore (17).
correlated (r2 5 0.267; P . 0.05) (Fig. 6). Calculated kt1 values After 24 h of incubation, numerous green fluorescent cells ranged between 6.4 3 10211 and 1.4 3 1027 cm2/CFU/h and were found. Highest numbers were observed in the epidermal metabolic activities ranged between 0.0034 and 0.030 fmol of interstices (Fig. 3A and B), but transconjugants were also seen Leu/CFU/h (Fig. 6).
in 5 to 10% of the ca. 300 stomata investigated on five leaves.
Per-cell metabolic activities on filters placed on saline were From 1 up to more than 100 cells per stoma were observed about six times lower than activities measured on the leaves, (Fig. 3C). Occasionally, transconjugants were observed at the ranging between 2.6 3 1024 to 5.5 3 1024 fmol of Leu/CFU/h base of trichomes. Leaves inoculated directly with transconju- (Fig. 6). Although cell densities on the filters were 100 times gants showed an identical distribution. The distribution of the higher than on the leaves, no transconjugants were observed by cells did not depend upon the inoculation procedure, i.e., plating or microscopical examination for fluorescent cells.
transconjugants were distributed as described above, when Transfer ratios were not correlated to cell density (P . 0.11) sterile seeds were inoculated with either transconjugants or a on leaves with densities around 106 CFU/cm2 (not shown).
1:1 mixture of donors and recipients.
However, maximal numbers of transconjugants and maximal transfer ratios were about 100 and 35 times lower, respectively, at densities between 103 and 104 CFU/cm2 (Table 2).
Transfer to indigenous epiphytic bacteria. The highest num-
TABLE 2. Metabolic activity and maximal transfer ratios bers of indigenous bacteria that had received the RP4 plasmid were attained after 6 h of incubation, after which the popula- tion size remained stable at 1.5 3 103 CFU/cm2 (Fig. 7). Under Metabolic activityb (fmol of Leu 3 103/CFU/h) the conditions employed here, more than 95% of the cultur- able indigenous bacteria were prototrophic and thus were po- tential recipients of RP4. The T/R ratio, however, was 23 times 8.66 3 BDc lower than the maximal ratio for the biparental mating with the TOL plasmid (Table 2). RP4 was transferred to six different indigenous Pseudomonas spp., to Stenotrophomonas malto- philia, and to four unidentified gram-negative isolates.
Low inoculum density (100% RH; 103–104 CFU/cm2) Nonsterile leaves (100% RH) Filter on agarose (100% RH) Filter on saline (100% RH; This is the first study on effects of bacterial distribution and metabolic activity on conjugal gene transfer on the phyllo- plane. The experiments demonstrated that the phylloplane of a Inoculum density was 105 CFU/cm2 unless otherwise specified.
bean is a habitat conducive to conjugal transfer. Transfer pri- Maximal (0 h) and minimal (96 h) activities.
c BD, below detection.
marily took place in the interstitial spaces and stomata (Fig. 3), d ND, not determined.
and numbers of transconjugants were positively related to RH

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FIG. 3. Confocal scanning laser microscopy photographs showing green fluorescent transconjugant cells in the interstices of epidermal cells (A), in the interstices of vein cells (B), and inside a stoma (C). Photographs (D) and (E) show charge-coupled device-recorded images of indigenous bacteria in the interstices of epidermal cells and in the interstices of vein cells, respectively. Bars represent 10 mm.
and inoculum concentration (Fig. 2 and Table 2). The meta- Compared to results of the rhizosphere, however, the maximal bolic activity of the bacteria inoculated onto the leaf surface T/R (0.02) was high. For instance, Smit et al. (46) and Rich- was stimulated, possibly due to leaf exudates (Fig. 4). No aume et al. (41) reported T/R values of RP4 in the range of correlation, however, between conjugal transfer on the leaves 1026 to 1024 between added pseudomonad donors and indig- and metabolic activity was observed (Fig. 6).
enous soil or wheat rhizosphere bacteria.
The observed T/Rs of up to 0.34 in the biparental mating In order to estimate the in situ activity of the donors and experiments (Table 2) are similar to results of earlier studies of recipients it was necessary to use sterilized plants. Although the phylloplane (5, 30). A literature comparison of transfer to this gnotobiotic model system does not completely reproduce indigenous bacteria is not feasible, however, as only one study the complexity of the natural situation, it allowed us to specif- has been published and no transfer ratios were reported (5).
ically address the importance of metabolic activity by eliminat- Relative to the biparental mating experiment at 100% RH, ing the large numbers of uncontrolled parameters of a more maximal transfer ratios to indigenous bacteria were 23 times complex system.
lower (Table 2). Although RP4 is transmissible to a wide range Possibly as the result of growing the plants aseptically, the of gram-negative and a few gram-positive bacteria (26), trans- metabolic activity of the bacteria increased upon inoculation fer to all epiphytic bacteria would not be expected. Further- onto the leaves (Fig. 4). Most likely, accumulated exudates more, only 95% of the indigenous bacteria were prototrophic initially stimulated the bacterial activity. During incubation, and would be scored as transconjugant on the selective media.
however, the surplus exudates were used up and the bacterial GENE TRANSFER ON THE PHYLLOPLANE OF THE BUSH BEAN FIG. 4. Metabolic activity of the donor prior to inoculation (‚), metabolic FIG. 6. kt1 as a function of metabolic activity (100% RH). Open symbols (E) activity of the recipient prior to inoculation (V), and the mean activity of donors represent individual leaves, while filled symbols (F) represent individual filters and recipients after inoculation onto leaves at 100% RH (M). Error bars are 6 floating on saline (see text for details).
SD of triplicate samples.
ulated conjugal transfer, and Bjo¨rklo¨f et al. (5) proposed that activity approached the level of the starved inocula (Fig. 4). A availability of nutrients could be responsible for the high trans- negative correlation between density and metabolic activity fer ratios on the phylloplane.
was observed at 100% RH (Fig. 5). The relatively low numbers No relationship between metabolic activity and transfer on of CFU at RHs below 100% should, according to Fig. 5, result the phylloplane was found in the present study (Fig. 6); i.e., in an elevated activity of the surviving cells. This, however, was metabolic activity was not rate limiting. However, a minimum not the case (Table 2). Possibly, the Leu uptake was impeded level of activity appeared to be necessary for transfer to occur.
by the lower water potential.
This was demonstrated by the filter mating experiment in The physiological state of the bacteria has been suggested to which the bacteria were kept at low activity on saline (,0.9 3 be important for conjugal transfer due to the energy required 1023 fmol of Leu/CFU/h). In this case, no transfer was ob- for synthesis of a pilus and replication of the plasmid DNA (35, served. Since a monolayer of cells was present on the filters, 42). For instance, the k the required cell-to-cell contact was achieved. Our data suggest t1 for transfer of RP4 from E. coli to Rhodobacter capsulatus in batch cultures was found to be pro- that the threshold level of metabolic activity must have been portional to substrate concentration (35). van Elsas et al. (51) somewhere between 0.9 and 3 3 1023 fmol of Leu/CFU/h (Fig.
suggested that root exudates in the rhizosphere of wheat stim- FIG. 7. Survival of P. fluorescens AS12/RP4 (‚), indigenous recipients (V), FIG. 5. Relationship between bacterial density and metabolic activity at and appearance of transconjugants (M) on bean leaves at 100% RH. Error bars 100% RH. Data points represent single leaves.
are 6 SD of triplicate samples.
NORMANDER ET AL.
APPL. ENVIRON. MICROBIOL.
The hypothesis that metabolic activity is not limiting for Metabolic activity, on the other hand, was not rate limiting for conjugation in planta is supported by recent evidence by Kroer conjugal transfer in this habitat. Most likely, very little energy et al. (28), who reported a lack of causal relationship between is required for completion of transfer, and the leaf exudates transfer and Leu uptake in the rhizosphere of water grass insured that the activity of the bacteria was well above the (Echinochlora crusgalli). In their study, measured metabolic threshold value.
activities were in the interval of 8 3 1023 to 16 3 1023 fmol of Leu/CFU/h and, hence, above the estimated threshold activity level observed for the phylloplane in this study.
It may be argued that accumulated exudates on sterile leaves This research was partly financed by grants from the Danish Envi- supported bacterial activity at levels that were not limiting for ronmental Protection Agency and The Plasmid Foundation.
We thank Tamar Barkay for critically reviewing the manuscript.
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