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Sodium dodecyl sulfate-agarose gel electrophoresis for the detection and

isolation of amyloid curli fibers

Article  in  Analytical Biochemistry · October 2010

DOI: 10.1016/j.ab.2010.09.038 · Source: PubMed

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SDS-Agarose Gel Electrophoresis for the Detection and Isolation of Amyloid Curli

Fibers

Chris Sitaras, Mahsa Naghavi, and Muriel B. Herrington*

Biology Department, Concordia University, Montreal, QC H4B 1R6 Canada

Running title: SDS-Agarose Gel Electrophoresis of Curli

*
Corresponding author

Biology Department,

Concordia University,

7141 Sherbrooke St. W.

Montreal, Quebec,

H4B 1G2

Canada

email: muriel@alcor.concordia.ca

Phone:
 Most strains of Escherichia coli and Salmonella enteritdis contain the genes

required to make fimbriae called curli, thin aggregative fibers or SEF17. However, curli

are only expressed by some strains and only under certain environmental conditions.

They are important for attachment to surfaces and have been implicated in infection [1-

5]. The fibers are composed of two similar proteins, CsgA and CsgB with CsgA making

up most of the fiber and CsgB serving as a nucleator protein for the formation of fibers

[6,7]. The mature fibers have many properties in common with amyloid fibrils from

eukaryotes [8].

A variety of methods can be used to detect whether cells are making curli fibers.

The simplest assay is to grow cells on Congo Red plates. Strains that make curli form red

colonies whereas those that do not remain white or pink [3]. However, in our experience,

the differentiation between the two types of cells varies from batch to batch of plates.

Curli mediate binding to some proteins such as fibronectin, and this binding can be used

to determine if cells are curliated or not [1]. Curli, either on cells or in extracts, can be

visualized by electron microscopy [9,10]. The fibers cannot be dissociated into their

constituent monomers by SDS treatment. However, formic acid treated crude extracts or

purified samples can be fractionated by SDS-PAGE [9]. This permits detection of the

CsgA protein by Coomassie blue staining or Western blotting with specific antibody.

Yeast prions are amyloid proteins that are also difficult to disrupt into monomers

and analyzed by SDS-PAGE. Kryndushkin et al. [11] used SDS-agarose gels to analyze

the PSI(+) protein. We asked if this would provide a rapid method for the analysis of

curli fibres.
Materials and Methods

Bacterial strains and Media.

Escherichia coli K-12 strains DH5α, MC4100, MG1655, MHR204, MH828, and Ymel

were from our stocks. LB [12] and YESCA [13] media were supplemented with 50

μg/ml thymidine. When required, LB-Thy was solidified with 15 g/l agar and YESCA-

Thy with 20 g/l. 1X Min A [12] was used to dilute cells. Congo Red plates were

prepared by adding 10 mg/l Congo red and 20 mg/l Coomassie Brilliant Blue G to

YESCA-Thy agar [13]. TCR plates contained 10 g/l tryptone, 15 g/l agar and 100 mg/l

Congo red [4].

Preparation of Cell Extracts

Overnight cultures were prepared by inoculating LB-Thy with single colonies and

incubating with shaking at 37 °C for 16 -24 hours. The cultures were diluted 100 fold

with 1X Min A and 100 μl were spread on YESCA-Thy plates. The plates were

incubated at 30°C for up to three days.

The cells were scraped off the agar surface using a rubber policeman and were

suspended in 3 ml (for one plate) 10 mM tris, pH6.8. The OD600 was measured and the

cell suspensions were adjusted to a standard OD600 (usually 20 but occasionally less).

Equal volumes of cells and extraction buffer (125 mM Tris-glycine, pH 1.8, 4% sodium

dodecyl sulfate, 20% glycerol) were combined and heated at 100˚C for 10 min. The
resultant crude extract was mixed well, and centrifuged at 16,000 g for 10 minutes. The

pellet was washed once with distilled water and was suspended in water. This procedure

is a modification of the quick purification by Collinson et al [14].

Protein was assayed by the bichinconcinic acid (BCA) assay following the

instructions provided by the manufacture (Pierce (Thermo Scientific)). Bovine serum

albumin (BSA) was used as the standard. Before assaying protein content,

macromolecules in the cell extracts were precipitated with TCA to remove glycine [15].

Washed extracts and cell samples were harvested by centrifugation at 16,000 g. Samples

were then solubilized in 5% SDS, 0.1 N NaOH for assay.

Gel Electrophoresis

SDS-agarose gels were prepared as described [11, 16]. We used a homemade gel

box with a tray that was 7 cm wide and 10 cm long and held 25 ml of SDS-agarose.

Samples were normally spun down at 16,000 g for 10 min, suspended in sample buffer

(20 mM tris-acetate, 0.5 mM EDTA, 5% v/v glycerol, 250 μg/ml bromophenol blue) and

boiled for 10 min before loading them into the wells of the gel. Gels were normally run

at 72 volts for 45 min.

For quantitation, samples were suspended in 100 μl sample buffer, boiled for 10

min and then mixed with 100 μl of hot 2 X concentrated SDS-agarose. The mixtures

were transferred to molds and allowed to solidify. The molds were 2 ml polypropylene

collection tubes with the bottom removed and the top sealed with Parafilm. When
solidified, the pucks were removed from the molds, were placed in the middle of the

electrophoresis tray and were covered with hot SDS-agarose. Wells were formed at the

top of the gel. These gels were run for 10 min, stopped and BSA standards were loaded

in the wells. Once the standards were loaded the gel was electrophoresed for another 35

min.

Gels were washed for 30 min in distilled water, stained with either EZ-Blue

(Sigma) or Easy Blue (Bioshop Canada Inc.) for 1-16 hours and were destained with

water. We agitated gels gently to avoid displacing material from the wells.

SDS-PAGE was carried out on 12% acrylamide gels and stained as described

[17].

Gels were imaged on an Odyssey Imager (Li-Cor Biosciences) [18]. Image

analysis was done with ImageJ [19].

Results and Discussion

Strain MC4100 is a well characterized curli proficient strain [3, 20]. Its

derivative, strain MHR204 has an insertion in the csgA gene [13]. Since this gene codes

for curlin, the major component of curli fibres, strain MHR204 is not able to make curli.

When we ran extracts on SDS-agarose gels and stained the gels, we observed stained

material both in the wells and in the gel (Fig. 1A). There was consistently more staining

material in wells of the MC4100 extracts than in the wells of MHR204 extracts (fig. 1A

and B) suggesting that curli are unable to enter the gel. In contrast, the yeast PSI(+)
protein could be dissociated into smaller aggregates that entered the gel and their

migration could be compared to known large proteins [11, 16].

Since we often observed material in the wells of MHR204 extracts we tested a

variety of extraction procedures to see if we could reduce this insoluble material. None

of the tested procedures was an improvement over the method used here.

Curli fibres are very robust and can only be dissociated into monomers by treating

with formic acid. We compared extracts with and without formic acid treatment (Fig.

1C). With the MC4100 extract, the formic acid treatment reduced the amount of material

that stayed in the well. The MHR204 extract had very little material in either well. This

suggested that a major portion of the material in the wells of extracts of MC4100 was

curli and the remainder was either undissociated curli or other insoluble material that

stains with coomassie blue.

The stained material in the gel was likely SDS-soluble proteins that were not

removed during the extraction and washing. The material in the wells took up the stain

rapidly, whereas the material in the gel required a longer exposure to the staining

solution. We often included a sample of molecular weight markers or BSA to check the

efficiency of staining (fig. 1 A and D). SDS-agarose gels can resolve mixtures of very

large proteins [11, 16] but they do not separate mixtures of smaller proteins although

individual proteins migrate according to size (fig. 1 D).

 SDS-PAGE was used to evaluate the extraction process (Fig. 2). Cells boiled in

sample buffer had bands spread over a wide range of sizes. In contrast, the extract

prepared by boiling at low pH had fewer bands particularly above approximately 60 KD,

and had a prominent band at the junction of the stacking gel and resolving gel. This

suggests that the conditions result in protein aggregation. When the boiled extract is

centrifuged the supernatant contains the same proteins in similar amounts. In contrast,

the washed sample contains small amounts of relatively few proteins.

Highly purified curli are prepared by a series of steps culminating in a preparative

SDS-PAGE gel. The curli do not enter the gel and so can be isolated from the wells [9].

We isolated curli in this way, and also isolated them from the well of an SDS-agarose gel.

Both preparations were made with strain MC4100. After formic acid treatment samples

were electrophoresed on SDS-PAGE gels (Fig. 3). Both had a band migrating at 17 KD,

the expected migration of curlin. There were small amounts of staining material that

runs below the presumed curlin band present in both samples. This supports our

conclusion that a significant proportion of the material that remains in the well of an

SDS-agarose gel is curli.

We prepared washed extracts from several strains and subjected these extracts to

SDS-Agarose gel electrophoresis. We also streaked the cells on TCR plates. On these

plates curli proficient strains are red and non-proficient strains are white. There was a

good correlation between samples that had stained material in the wells of the gel, and

were red on the TCR plate (Fig. 4). Strains MC4100 [20] and the commonly used wild
type K-12 strain MG1655 [21] were curli proficient. Strains MH828 [22] and DH5α

[23], like most K-12 strains, were not able to make curli.

The strain Y-Mel is reportedly curli proficient [10] but we always observed white

growth both on TCR plates and on reliable Congo Red plates. When we first observed

this approximately 15 years ago, we obtained a second sample of Y-Mel from the Coli

Genetic Stock Center, and it also was white. Curli expression requires active RpoS and

many strains of E. coli K-12 do not make RpoS because of a nonsense mutation in rpoS.

It was assumed that Y-Mel was curli proficient because it is a nonsense suppressor [10].

We confirmed that our isolate was still able to suppress nonsense mutations (T. MacRae

and M. B. Herrington, unpublished), suggesting that the isolate used by Olsen et al. [10]

was proficient for other reasons.

Although a rough estimate of the relative amount of curli can be obtained from

measuring the intensity of stain in the wells the potential for loss during washing and

staining the gel, and the packing of the protein at the bottom of the well contributes to the

inaccuracy of such measurements. We tested whether imbedding the sample in agarose

prior to electrophoresis would facilitate quantitation (fig. 5A ). The intensity of stain

both in the pucks and in the soluble protein (blobs) was proportional to the amount of

sample loaded into the agarose puck (fig. 5B). The intensity of the BSA standards, which

were loaded into wells, was proportional to the amount of protein except for the highest

concentration. In repeated gels, the BSA standards were highly variable, possibly due to

losses from the wells during loading, or an uneven distribution of protein through the
thickness of the gel. Loading the standards into agarose pucks would probably improve

the proportionality and the reproducibility.

In conclusion, we have demonstrated that SDS-agarose gel electrophoresis can be

used to detect, isolate and quantify curli fibers. Given that the equipment for these gels is

readily available in most laboratories, the preparation is less time consuming that for

SDS-PAGE, and the reagents used are not toxic this method will be useful for further

studies on curli.

Acknowledgements

This research was supported by a Discovery Grant (6727) awarded to M.B.H. by

the Natural Sciences and Engineering Council of Canada.

References

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Fig. 1. SDS-Agarose Gels of washed extracts and individual proteins. The specified

volumes of washed extracts were centrifuged and the pellets were suspended in sample

buffer and boiled prior to electrophoresis. (A) Extracts of strains MC4100 (Lanes 1-3)

and MHR204 (Lanes 4-6) were subjected to electrophoresis. Lanes 1 and 4: 20 μl, 2 and

5: 40 μl, 3 and 6: 60 μl (B) Wells of an SDS-agarose gel loaded with 100 μl of washed

extract and electrophoresed. Lane 1 MC4100 extract, Lane 2 MHR204 extract. (C)

Pellets from 100 μl of washed extracts were treated with formic acid and dried in a speed

vac before electrophoresis. An equivalent amount of untreated sample was also analysed.

Only the wells of the gel are shown. Lane 1: MC4100, untreated; Lane 2: MC4100,

treated; Lane 3: MHR204, untreated; Lane 4: MHR204, treated. (D) Migration of

different proteins in an SDS-agarose gel. Proteins (30 μg each) had migrated

approximately 2-3 cm into the gel. Lane 1: lysozyme; Lane 2: ribonuclease A; Lane 3:

BSA; Lane 4: molecular weight markers (contrast in this region of the image was

enhanced because the markers were relatively faint).

Fig. 2. SDS-PAGE of MC4100 cells and extracts. Lane 1: Whole cells (15 μg protein)

were boiled in sample buffer; Lane 2: Crude extract (15 μg protein); Lane 3:

Supernatant (15 μg protein)after centrifugation of the crude extract; Lane 4: Washed

extract (45 μg protein). Parallel samples of MHR204 gave similar results except that the

intensity of bands in the washed extract was greater, suggesting more SDS soluble

protein in the sample. Sizes of the molecular weight markers are in KD.

Fig. 3. SDS-PAGE of material recovered from the wells SDS-polyacrylamide and SDS-

agarose gels. Lane 1: Extracts of MC4100 were purified in a multistep process and

samples were recovered from the wells of a preparative SDS-polyacrylamide gel. The
samples were treated with formic acid prior to SDS-PAGE [9] Lane 2: Material was

recovered from the well of an SDS-agarose gel after a washed extract of MC4100 was

electrophoresed. The sample was treated with formic acid before SDS-PAGE.

Molecular weight markers are indicated in KD.

Fig. 4. SDS-Agarose electrophoresis and Congo Red Binding of different strains. The

upper panel shows the wells of an SDS-agarose gel of 100 μl of washed extracts of

different strains. The lower panel show streaks of the different strains on a TCR plate.

Lane 1: MC4100; Lane 2: MHR204; Lane 3: Y-Mel; Lane 4: MG1655; Lane 5: MH828:

Lane 6: DH5α

Fig. 5. SDS-Agarose electrophoresis of immobilized washed extract from MC4100.

Different amounts of washed extract was suspended in SDS-agarose pucks that were then

imbedded approximately 5 cm from the top of an SDS-agarose gel. Samples of BSA

were placed in the wells of the gel. A. The region of the gel with the BSA samples. This

was from the gel shown in B, with the contrast increased. B. The SDS-agarose gel. The

pucks are indicated by p, and the soluble protein that comes out of the pucks is indicated

by b (blobs). C. A comparison of the intensity of staining of different elements of the

gel. The integrated density is the average pixel intensity times the area of the selection (a

standard selection rectangle was used for each measurement). The relative amount of

sample was based on the maximum amount (15 μg BSA) or volume (100 μl washed

extract containing 62 μg) used.

 Fig. 2

fig 1. Fig 3.
 Fig. 4

Fig 5

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