pMP2463原核荧光表达载体广宿主载体质粒图谱序列抗性

We developed two sets of broad-host-range vectors that

drive expression of the green fluorescent protein (GFP) or

color variants thereof (henceforth collectively called autofluorescent

proteins [AFPs]) from the lac promoter. These

two sets are based on different replicons that are maintained

in a stable fashion in Escherichia coli and rhizobia.

Using specific filter sets or a dedicated confocal laser scanning

microscope setup in which emitted light is split into

its color components through a prism, we were able to unambiguously

identify bacteria expressing enhanced cyan

fluorescent protein (ECFP) or enhanced yellow fluorescent

protein (EYFP) in mixtures of the two. Clearly, these vectors

will be valuable tools for competition, cohabitation,

and rescue studies and will also allow the visualization of

interactions between genetically marked bacteria in vivo.

Here, we used these vectors to visualize the interaction between

rhizobia and plants. Specifically, we found that

progeny from different rhizobia can be found in the same

nodule or even in the same infection thread. We also visualized

movements of bacteroids within plant nodule cells.

The following plasmids carrying genes expressing different autofluorescent proteins (AFPs) (pMP2463, green),

(pMP4516, cyan) and, (pMP4518, yellow) to mark Aur6, AMG443 and ISP42 strains;

(pMp4655, green), (pMP4641, cyan) and, (pMP4518, yellow).

Initially, the coding sequence for EGFP was transferred into

the broad-host-range vector pBBR1MCS-5 in frame with the

LacZ-a-peptide, which is expressed from the lac promoter. E.

coli harboring such a plasmid (two clones, pMP2444 and

pMP2463 were used) showed clear EGFP fluorescence when

viewed by fluorescence microscopy using the appropriate excitation

and emission filters. We noted that when Escherichia

coli was grown under high selective pressure (gentamicin at

40 μg/ml instead of 10 μg/ml) pMP2463 could be reproducibly

mutated into a form that produced much higher levels of

EGFP per cell than the unmutated form as detected by fluorescence

microscopy. Analysis of plasmid DNA isolated from

Corresponding author: Nico Stuurman; Telephone: +31 71 527 4909;

Fax: +31 71 527 4999; E-mail: stuurman@rulbim.leidenuniv.nl

/ Molecular P 1164 lant-Microbe Interactions

these cells suggested that this mutation strongly increased the

plasmid copy number (data not shown). The same effect on expression

was noted when one of the mutated isolates (pMP2464)

was transferred to various rhizobia species, suggesting that the

copy number effect is maintained across bacterial species.

To express other AFPs, the coding sequence for ECFP and

EYFP was inserted into pBBR1MCS-5, resulting in pMP4516

and pMP4518, respectively. E. coli transformed with each plasmid

indeed expressed the expected protein as determined by

fluorescence microscopy using the appropriate filter sets. When

crossed into various rhizobial species, cells became fluorescent

with the expected excitation and emission characteristics.

As a result, it was possible to mix two populations of rhizobia,

each expressing a different fluorescent protein, and to trace

the individual populations within the mixture. To specifically

detect bacteria expressing ECFP or EYFP, we used a confocal

laser scanning microscope that allows detection of emitted

light at freely selectable wavelengths. For ECFP, objects were

excited with light of 457 nm and emitted light was detected

around 480 nm, whereas for EYFP, excitation was at 488 nm

and detection around 540 nm. To avoid cross talk, the images

for each channel were acquired separately.

Insertion of the ECFP and EYFP coding sequence into

pMP2464 resulted in plasmids pMP4517 and pMP4519 (Fig.

1). Analysis of plasmid stability of these two plasmids in

various rhizobial species (also discussed below) showed, surprisingly,

that the high-copy, ECFP-containing derivative

pMP4517 was less stable in the absence of antibiotics in species

Mesorhizobium loti R7A and S. fredii USDA201. Although

this effect was not clearly noticeable with the EYFPcontaining

plasmid pMP4519, we decided not to use the highcopy-

number derivatives for infection studies.

A second set of AFP-expressing vectors was constructed

using plasmid pME6010, which is known to be very stable in

various soil bacteria (Heeb et al. 2000). The resulting plasmids,

pMP4657, pMP4656, and pMP4639, confer tetracycline

resistance and express the EGFP, ECFP and EYFP genes, respectively,

driven by the lac promoter.

The various constructs as well as the relative levels of AFP

expression in rhizobia transfected with these plasmids are

summarized in Figure 2. Results show that levels of fluorescence

correlate with the presumed copy number of the plasmid

replication origin.

Transformation of plasmid DNAto competent E. Coli cells

1. Thaw competent cells on ice. 20–200µL per tube

2. Add max. 1-3µL of plasmid

3. Mix very gently!

4. Incubate the tubes on ice for 30 min

5. Heat shock the cells for 45 sec to 2 min at 42°C

6. Place the tubes immediately on ice for at least 2      min

7. Add 800µL of SOC medium to each tube

8. Incubate for 1 hour at 37°C and shake vigorously

9. Spin down briefly and remove most supernatants

10. Resuspend cell pellet with the rest SOC medium in      the tube by pipetting

11. Plate out the suspension on a LB agar plate      containing the appropriate antibiotic. Incubate the plates overnight at      37°C