Name: adherence of bacteria to plant surfaces measured in the laboratory
Uploaded: 2018-06-19T12:30:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

The author thanks Susan Whitfield for preparation of the figures and Camille Martin and Hillary Samagaio for assistance with some of the experiments.

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+Escherchia+coli.O157:H7+to+alfalfa,+human+epithelial+cells,+and+plastic+is+mediated+by+a+variety+of+surface+structures.">Differential binding of Escherchia coli.O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures.</a> Appl. Environ. Microbiol. 71, 8008-8015 (2005).</li><li>Matthysse, A. G., Deora, R., Mishra, M., Torres, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharides+cellulose,+poly-beta-1,6-n-acetyl-D-glucosamine,+and+colanic+acid+are+required+for+optimal+binding+of+Escherichia+coli+O157:H7+strains+to+alfalfa+sprouts+and+K-12+strains+to+plastic+but+not+for+binding+to+epithelial+cells.">Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells.</a> Appl. Environ. Microbiol. 74, 2384-2390 (2008).</li><li>Matthysse, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cellulose+overproduction+on+binding+and+biofilm+formation+on+roots+by+Agrobacterium+tumefaciens.">The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.</a> Mol. Plant Microbe Interact. 18, 1002-1010 (2005).</li><li>Matthysse, A. G., Kijne, J. W., Spaink, H. P., Kondorosi, A., Hooykaas, P. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Rhizobiaceae+to+plant+cells.">Attachment of Rhizobiaceae to plant cells.</a> The Rhizobiaceae. , 235-249 (1998).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+promotes+the+attachment+of+Agrobacterium+tumefaciens.strain+NT1+to+carrot+cells.">Conditioned medium promotes the attachment of Agrobacterium tumefaciens.strain NT1 to carrot cells.</a> Protoplasma. 183, 131-136 (1994).</li><li>Matthysse, A. G., McMahan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+Agrobacterium+tumefaciens.+attR+mutation+on+attachment+and+root+colonization+differs+between+legumes+and+other+dicots.">The effect of the Agrobacterium tumefaciens. attR mutation on attachment and root colonization differs between legumes and other dicots.</a> Appl. Environ. Microbiol. 67, 1070-1075 (2001).</li><li>Jeter, C., Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+binding+of+diarrheagenic+strains+of+E.+coli.to+plant+surfaces+and+the+role+of+curli+in+the+interaction+of+the+bacteria+with+alfalfa+sprouts.">Characterization of the binding of diarrheagenic strains of E. coli.to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts.</a> Mol. Plant Microbe Interact. 18, 1235-1242 (2005).</li><li>Matthysse, A. G., McMahan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+colonization+by+Agrobacterium+tumefaciens+is+reduced+in+cel,+attB,+attD,+and+attR+mutants.">Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants.</a> Appl. Environ. Microbiol. 64, 2341-2345 (1998).</li><li>Morton, E. 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V., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+seed+and+root+exudate+sugars:+composition,+utilization+by+Pseudomonas+biocontrol+strains+and+role+in+rhizosphere+colonization.">Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization.</a> Environ. 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S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20-26 (2005).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu. Rev. Microbiol. 54, 49-79 (2000).</li><li>Charkowski, A. O., Barak, J. D., Sarreal, C. Z., Mandrell, R. 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The author declares that she has no competing financial interests.

It is important to be aware of all of the surfaces to which bacteria can adhere during the experiment. Thus bacteria which are capable of binding to glass may be underestimated if viable cell counts are done using glass tubes and pipettes. If plants are grown in agar or soil and some of the agar or soil remains on the plants then the bacteria may bind to the adhering material rather than to the plants. On the other hand, washing the plant surface, particularly in the case of roots, may remove natural surface coatings such as mucous and thus alter the results of adherence tests.
It is important to be certain that the bacteria added to the incubation mixture remain alive during the experiment. Thus viable cell counts of free as well as attached bacteria should routinely be made. Some treatments or bacterial mutations may reduce bacterial growth rate or actually cause the death of a fraction of the bacterial population. Live and dead bacteria may not be distinguishable in the microscope unless special stains are used. There is a useful stain kit for live/dead bacteria which depends on the exclusion of dyes from the living bacteria. However, if a mixed population of bacterial species is present then viable cell counts of the species of interest is likely to be the easiest method to determine whether the incubation has resulted in bacterial death.
Medium composition will influence bacterial survival and growth. Root exudate and materials released from wound and cut sites will provide substrate to support modest bacterial growth. Phosphate, nitrogen, and iron tend to be limiting in these conditions. Divalent cations such as calcium and magnesium may influence adhesion. In some cases carbon source can influence adhesion. pH also matters. In general the pH of the rhizosphere is between 5.5 and 6.5.
It is necessary to be careful in using bacteria marked with antibiotic resistance. The antibiotics most often used are rifampicin and nalidixic acid. Resistance to these antibiotics is generally due to mutations in chromosomal genes (RNA polymerase and gyrase, respectively) and thus cannot easily be transferred to another strain during the incubation. This type of resistance also does not result in the degradation or modification of the antibiotic. Marking bacteria with a plasmid-borne gene marker is not recommended unless the plasmid cannot be transferred to any other bacteria. The antibiotic resistance must not be due to degradation or chemical modification of the antibiotic as antibiotic sensitive bacteria will then be able to grow on antibiotic plates if they are located close to the resistant bacteria.
The methods described in this paper are useful for small sample sizes and/or experiments where the samples need to be contained (for example, experiments involving human pathogens). For large sample sizes (above 100 g of material or more than 50 plants) other methods or drastic modification of these methods would be needed10,19,32,19,33,34,35,36. The presence of large numbers of microorganisms other than the species being studied can also pose significant problems. Possible solutions include the use of bacteria marked with a fluorescent protein or antibiotic resistance as described in steps 6.1.2 and 7.3. However, when the bacteria of interest are rare individuals in a large population of other microorganisms these markers may not be adequate to allow an unambiguous assessment of the numbers of the bacteria being studied.
All of the methods described here are laboratory based methods. Minor modifications would be needed for greenhouse studies. More major modifications are likely to be required for field studies where protozoa, insect and other animal predation and climate variation complicate the provision of defined conditions for the experiments. In the future these methods may be extended to include the interactions of two or more microorganisms on the plant surface.

The measurement of bacterial binding to plant surfaces has become important in three disparate situations. The first situation is the examination of the transmission of human pathogens on plant surfaces1,2,3. The goal here is to prevent bacterial binding or to remove or kill bound bacteria and thus to reduce the transmission of disease by plant material. The second situation is the examination of the binding of plant pathogens to plant surfaces4. Once again the goal here is to prevent binding or to remove or kill bound bacteria and thus to reduce disease. The third situation is the examination of the binding of symbiotic or plant-growth-promoting bacteria5,6. The goal here is to promote bacterial binding and thus to increase plant health and crop yields.
The techniques for measuring bacterial binding to plant surfaces described in this article are inexpensive and relatively easy to carry out. The only requirements are a microscope and materials generally found in a bacteriology laboratory. For some techniques a bath sonicator is useful. The techniques described are designed for binding experiments carried out using relatively small sample sizes. Measurements of binding are made in the laboratory, although it may be possible to modify some of these techniques for use in the greenhouse or in the field.
These techniques have been used to measure bacterial binding to roots, sprouts, cut leaves, cut fruits, and intact cherry tomatoes in the laboratory7,8,9,10,11,12,13,14,15. They have also been used to measure root colonization of plants growing in soil or sand in the laboratory16. The techniques have been used with many bacterial species including Agrobacterium tumefaciens, Sinorhizobium meliloti, Escherichia coli, Salmonella enterica, and Pseudomonas fluorescens. A useful description of methods for assessing the interaction of A. tumefaciens with surfaces can be found in Morton and Fuqua (2012)17. In all cases the sample sizes involved were small, generally less than 25 - 50 plants. The techniques described are suitable for use with human pathogens which need to be kept contained during the experiments.

<table border="0" cellpadding="0" cellspacing="0" style="width:731px;" width="731">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="56">
			<td height="56" style="height:56px;width:143px;">light microscope</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">seeds</td>
			<td style="width:164px;">any&#160;</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">make a note of the seed lot number and the cultivar</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">bleach</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">bath sonicator</td>
			<td style="width:164px;">any scientific supply company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Triton X-100</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">T9284</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">nutrient agar&#160;</td>
			<td style="width:164px;">Difco&#160;</td>
			<td style="width:125px;">001-01-8</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">soytone</td>
			<td style="width:164px;">Difco&#160;</td>
			<td style="width:125px;">24360</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Sand</td>
			<td style="width:164px;">sea sand Fisher Scientific</td>
			<td style="width:125px;">S25</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">sand</td>
			<td style="width:164px;">&#160;Sigma-Aldrich</td>
			<td style="width:125px;">S9887</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">conetainers</td>
			<td style="width:164px;">Stuewe &#38; Sons, Inc.</td>
			<td style="width:125px;">Ray-Leach cone-tainers</td>
			<td style="width:299px;">many different sizes are available to suit the type of plant you wish to grow</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">parafilm</td>
			<td style="width:164px;">any scientific supply company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">MS salts</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">M5524</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">parrafin</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">centrigfuge</td>
			<td style="width:164px;">any scientific company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">to pellet most bacteri only a small centrifuge with a max force of 10,000 XG is needed</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">vortex mixer</td>
			<td style="width:164px;">any scientific company</td>
			<td style="width:125px;"></td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">micrometer</td>
			<td style="width:164px;">ACCU-SCOPE Accessories</td>
			<td style="width:125px;">A3145</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Sedgwick-rafter counting cell</td>
			<td style="width:164px;">Hauser Scientific</td>
			<td style="width:125px;">HS3800</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">probe-clip press-seal incubatin chamber</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">Z359483</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">rifampicin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">R3501</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">naladixic acid</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">n8878</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Live/dead stain</td>
			<td style="width:164px;">In Vitrogen Molecular Bioprobes</td>
			<td style="width:125px;">L7007</td>
			<td style="width:299px;"></td>
		</tr>
	</tbody>
</table>

adherence of bacteria to plant surfaces measured in the laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

A. tumefaciens colonizes root surfaces. In order to determine whether bacterial production of cellulose plays a role in root colonization, the effects of bacterial mutations which prevent cellulose synthesis were examined16. The techniques described in steps 1.3 and 7.1 were used. Tomato seeds were surface sterilized and germinated in sterile water. When the roots were approximately 2 cm long they were dipped in a suspension of 105 bacteria per mL and planted in pasteurized soil in containers. The plants were grown for 14 days at 25 &#176;C on a 12 h light/12 h dark cycle. After the indicated times the plants were removed from the containers. The roots were washed and sonicated in a bath sonicator to remove bound bacteria. Bacterial numbers were determined using viable cell counts. Figure 2 shows the effect of two different cellulose-minus mutations on the ability of the bacteria to colonize tomato roots. Although the standard deviations of some measurements were as high as 0.9 log10 (a common problem with this type of measurement) the reduction in binding of the cellulose-minus mutants is clearly evident and we can conclude that bacterial production of cellulose aids the bacteria in the colonization of tomato roots.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56599/56599fig2.jpg" /> 
Figure 2: Root colonization by wild type and cellulose-minus mutants of&#160;A. tumefaciens.&#160;Log10&#160;total number of bacteria per cm root length recovered from tomato roots inoculated with wild type&#160;A. tumefaciens&#160;strain C58 and cellulose-minus mutants C58:1 and C58:A60. The numbers shown are the means from a minimum of four separate experiments. Bars indicate standard deviations of the means. Roots were inoculated by dipping them in a suspension of 105&#160;bacteria per mL for one min. The plants were grown in containers and the loosely adherent bacteria were removed by washing the roots in washing buffer in a glass vial. Tightly adherent bacteria were removed using a bath sonicator and the resulting suspension plated to determine viable cell counts16. This figure has been modified from Matthysse and McMahan.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The role of exopolysaccharides in the binding of E. coli and other bacteria to alfalfa sprouts was examined. Some outbreaks of diarrheal disease due to E. coli O157:H7 have been traced to contaminated alfalfa sprouts. Binding of the wild-type bacteria and mutants unable to make various exopolysaccharides was measured using the methods described in steps 1.1, 5.1, and 7.2. Alfalfa sprouts were surface sterilized and germinated for one day in sterile water at 25 &#176;C in the dark. Four sprouts with attached seed coats were placed in sterile plastic dishes containing 5 mL of water. Bacteria grown in Luria broth were added to a final concentration of approximately 5 x 103 per mL. The inoculated sprouts were incubated at 25 &#176;C in the dark for 3 days. The sprouts were washed twice in 5 mL sterile water in a vial by vigorous inversion and homogenized in washing buffer using a motor-driven Teflon glass homogenizer. Previous experiments using bacteria marked with a plasmid carrying the GFP gene showed no internalized bacteria although surface bacteria were easily observed. The results are shown in Table 1. Two strains of E. coli O157:H7 were examined. In both strains the production of poly-&#946;-1,6-glucuronic acid(PGA) appeared to make the largest contribution to the binding of pathogenic E. coli to plant surfaces. Colonic acid also played a significant role in binding. While the reduction in binding in cellulose-minus mutants was significant it was not as large as that for the other two polysaccharides.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="4">Effects of Mutations in Exopolysaccharide Production Genes on Binding of E. coli O157:H7 to sprouts and open seed coats</td>
		</tr>
		<tr>
			<td rowspan="2">Bacterial strain&#160;</td>
			<td rowspan="2">Mutation or genotype (relevant phenotype)</td>
			<td colspan="2">Log10 &#160;number of bacteria bound per sprout or seed coat&#160;</td>
		</tr>
		<tr>
			<td>Alfalfa sproutsb</td>
			<td>Open seed coats</td>
		</tr>
		<tr>
			<td>86-24</td>
			<td>None (wild type)</td>
			<td>4.7 &#177; 0.6</td>
			<td>5.6 &#177;&#160; 0.2</td>
		</tr>
		<tr>
			<td>8624N</td>
			<td>yhjN (cellulose-minus)</td>
			<td>2.9&#160; &#177;&#160; 0.7c</td>
			<td>3.5&#160; &#177;&#160; 0.6c</td>
		</tr>
		<tr>
			<td>8624C</td>
			<td>wcaD (colonic acid-minus)</td>
			<td>1.8&#160; &#177;&#160; 0.7c</td>
			<td>2.4&#160; &#177;&#160; 0.5c</td>
		</tr>
		<tr>
			<td>8624P</td>
			<td>pgaC (PGA-minus)</td>
			<td>&#60;1.0c</td>
			<td>1.0&#160; &#177;&#160; 1.0c</td>
		</tr>
		<tr>
			<td>DEC4A</td>
			<td>None (wild type)</td>
			<td>5.6&#160; &#177; 0.2</td>
			<td>6.1&#160;&#160; &#177;&#160; 0.3</td>
		</tr>
		<tr>
			<td>DEC4AN</td>
			<td>yhjN (cellulose-minus)</td>
			<td>4.8&#160;&#160; &#177; 0.8d</td>
			<td>4.1&#160;&#160; &#177; 0.8d</td>
		</tr>
		<tr>
			<td>DEC4AC</td>
			<td>wcaD (colonic acid-minus)</td>
			<td>3.9&#160;&#160; &#177; 0.5c</td>
			<td>4.8&#160;&#160; &#177; 0.8d</td>
		</tr>
		<tr>
			<td>DEC4AP</td>
			<td>pgaC (PGA-minus)</td>
			<td>&#60;1.0c</td>
			<td>1.2&#160;&#160; &#177; 0.7c</td>
		</tr>
		<tr>
			<td colspan="4">a mean &#177; standard deviation of a minimum of three measurements of the number (log10) of bacteria bound after 3 days.</td>
		</tr>
		<tr>
			<td colspan="4">b Sprouts were washed before measurement.</td>
		</tr>
		<tr>
			<td colspan="4">c Significantly different from the wild type: P &#60;0.01.</td>
		</tr>
		<tr>
			<td colspan="4">d&#160; significantly different from the wild type: P &#60;0.05.</td>
		</tr>
		<tr>
			<td colspan="4">This table has been modified from&#160; Matthysse, Deora, Mishra, and Torres10.&#160;&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Effects of mutations in exopolysaccharide production genes on binding of&#160;E. coli&#160;O157:H7 to sprouts. In order to determine the role of various exopolysaccharides and lipopolysaccharide in the binding of pathogenic&#160;E. coli&#160;O157:H7 strains to alfalfa sprouts, the binding of a set of mutants to the sprouts and open seed coats was examined using the methods described in step 6. The results showed that poly-&#223;-1, 6-N-acetyl-D-glucosamine (PGA) appears to be essential for binding to sprouts and that both cellulose and colanic acid are required for maximum binding of&#160;E. coli&#160;O157. This table has been modified from Matthysse, Deora, Mishra, and Torres10.
In order to determine if the production of PGA is sufficient to cause bacterial binding to plant surfaces, a plasmid (pMM11) carrying the cloned operon encoding the genes required for PGA production was introduced into two different bacteria which would not ordinarily be able to bind to tomato roots10. A. tumefaciens A1045 is a mutant of the wild type strain C58 which fails to make cyclic-&#946;-1,2 glucan and also fails to bind to plant surfaces29. Sinorhizobium meliloti 1021 which forms root nodules on alfalfa fails to bind to non-legumes including tomato12. The techniques described in steps 1.1, 5.1, 6.3, and 7.1 were used to determine if the ability to make PGA generally increased bacterial binding to root surfaces. Tomato seeds were surface sterilized and germinated in sterile water. Roots were cut into segments 1 cm in length and placed in sterile water and the bacteria were inoculated. As these two species of bacteria grow at different rates, binding was measured at different times to allow for roughly equal amounts of bacterial growth. The presence of the plasmid pMM11 caused a similar significant increase in the number of bound bacteria of both species (Table 2)10. A significant increase in binding was also seen in the light microscope but the binding was very different for the two species (Figure 3). A. tumefaciens A1045 bound as individual bacteria to the root surface. S. meliloti bound in large clusters in which only a few of the bacteria were directly attached to the root and the majority of the bacteria were attached to other bacteria. This example shows that simply analyzing the numbers of bacteria bound without including microscopic observations can give a misleading impression of the results of an experiment. Although both methods (viable cell counts and microscopic observations) show that pMM11 increased bacterial binding to tomato roots, the type of binding caused by the production of PGA was different for the two bacterial species10.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td colspan="3">The Effect of the Plasmid pMM11 on the Binding of Bacteria to Tomato Roots</td>
		</tr>
		<tr>
			<td>Bacterial strain</td>
			<td>Plasmid</td>
			<td>Number of bacteria bound per mm root length</td>
		</tr>
		<tr>
			<td>A. tumefaciens A1045a</td>
			<td>none</td>
			<td>0.25 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td></td>
			<td>pBBR1mcs (vector)</td>
			<td>0.25 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td></td>
			<td>pMM11 (PGA synthesis)</td>
			<td>10 x 103 &#177; 0.25 x 103</td>
		</tr>
		<tr>
			<td>S. meliloti 1021b</td>
			<td>none</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td></td>
			<td>pBBR1mcs (vector)</td>
			<td>none detected</td>
		</tr>
		<tr>
			<td></td>
			<td>pMM11 (PGA synthesis)</td>
			<td>50 x 103 &#177; 5 x 103</td>
		</tr>
		<tr>
			<td colspan="3">a Bacterial binding was measured after 2 hours</td>
		</tr>
		<tr>
			<td colspan="3">b Bacterial binding was measure after 18 hours&#160;</td>
		</tr>
		<tr>
			<td colspan="3">This table has been modified from&#160; Matthysse, Deora, Mishra, and Torres10.&#160;&#160;</td>
		</tr>
	</tbody>
</table>
Table 2: The effect of a plasmid carrying genes for the synthesis of PGA on binding of&#160;A. tumefaciens&#160;A1045 and&#160;S. meliloti&#160;1021 to tomato root segments. In order to examine the ability of poly-&#223;-1, 6-N-acetyl-D-glucosamine (PGA) to promote the binding of bacteria to plant roots, the effect of a plasmid conferring the ability to make PGA (pMM11) on the binding of two strains of plant-associated bacteria to tomato roots was examined. Neither strain of bacteria showed significant binding to tomato roots in the absence of the plasmid or in the presence of the plasmid without the genes encoding PGA synthesis (pBBR1mcs). The addition of the plasmid carrying PGA synthesis genes increased binding by both types of bacteria. Because&#160;A. tumefaciens&#160;grows faster than&#160;S. meliloti&#160;binding was measured after 2 h of incubation for&#160;A. tumefaciens&#160;and after 18 h for&#160;S. meliloti. The techniques used are those described in step 7. This table has been modified from Matthysse, Deora, Mishra, and Torres10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56599/56599fig3.jpg" /> 
Figure 3: The effect of the plasmid pMM11 carrying the PGA biosynthesis genes on the binding of&#160;A. tumefaciens&#160;A1045 and&#160;S. meliloti&#160;1021 to tomato root hairs.&#160;Binding to tomato root hairs of A)&#160;A. tumefaciens&#160;A1045, B)&#160;A. tumefaciens&#160;A1045 pMM11, C)&#160;S. meliloti&#160;1021, and D)&#160;S. meliloti&#160;1021 pMM11. Although the increase in binding of the two bacterial species is roughly similar the details of the binding as seen in the light microscope are quite different. The techniques used are those described in step 6. This figure has been modified from Matthysse, Deora, Mishra, and Torres10.&#160;<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is sometimes possible to use binding to non-biological surfaces to aid in distinguishing the contribution of the bacteria and of the plant in a specific interaction. The unipolar polysaccharide(UPP) of A. tumefaciens has been shown to be able to mediate bacterial binding to a variety of both biological and non-biological surfaces30. Calcium was observed to inhibit the binding of A. tumefaciens to plant surfaces mediated by the UPP28. In order to determine whether the inhibition by calcium ions of bacterial binding to plant surfaces is due to an effect on the bacteria or on the plant surface, the binding of the bacteria to nylon threads was examined. The techniques describes in step 8.2 were used. Tomato seeds were surface sterilized and germinated in water as described in step 1. The bacteria were grown in minimal medium with sucrose and added to tomato roots or nylon threads at a final concentration of approximately 105/mL as described in step 5.1. The effect of added CaCl2 on bacterial binding to tomato roots and nylon threads was examined in the microscope. Figure 4 shows a similar inhibition of binding by calcium using either surface suggesting that the effect of calcium is primarily on the bacteria10.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56599/56599fig4.jpg" /> 
Figure 4: The effect of calcium on the binding of A. tumefaciens to tomato root hairs and nylon threads. A. tumefaciens was incubated with tomato roots (A and B) or nylon threads (C and D) for 24 h in a 1:10 dilution of MS salts and a 1:20 dilution of AB minimal medium, 0.4 % sucrose (A and C) or in a 1:10 dilution of MS salts and 1:20 dilution of AB minimal medium, 0.4 % sucrose containing 60 mM CaCl2 (B and D)31. The added CaCl2 inhibited bacterial binding to both roots and nylon threads suggesting that the inhibition was primarily due to an effect on the bacteria rather than on the plant surface. <a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

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