Independent research project conducted under Dr. Ann Matthysse and faculty advisor Eric Scott in BIOL 421L course - Microbiology Lab With Research

1. Analysis of cellulose synthesis mutations in Agrobacterium tumefaciens by transposon
mutagenesis
Taylor Beard, Thalita Cortes, Justin Fung, Maryanna Parker, Eric Scott*
Microbiology Lab Research 421L, Biology Department, University of North Carolina,
Chapel Hill, NC
*faculty advisor
Abstract: 195 words
Text: 1486 words

2. Abstract:
The production of cellulose fibrils is involved in the attachment of Agrobacterium
tumefaciens C58 to a wounded plant host. Previous studies have shown that bacteria
synthesize cellulose fibrils, not plant cells, since they continued to be synthesized after A.
tumefaciens attached to dead plant cells. In this study, we examine cellulose under-
producing mutants of A. tumefaciens that were constructed using transposon mutagenesis.
Tn5 transposon was introduced into the A. tumefaciens and incubated on Luria agar
containing tetracycline. Mutational analysis of the resulting colonies was conducted by
cellufluor staining and examining for fluorescence under UV light. Extra dark mutants
failed to produce cellulose and indicated a successful insertion of the Tn5 transposon into
operon gene, celABC or celDE, which would have altered the synthesis of cellulose
synthase. Extra bright mutants are caused by an overproduction of cellulose, which can
be a result of the transposon insertion in a repressor regulating cellulose synthesis.
Incubation of 404 suspected A. tumefaciens mutants for 5 days resulted in 54 extra dark
colonies with a successful mutation frequency of 13%. Therefore, the dark colonies
produced less cellulose, and further research of these isolated colonies can determine how
cellulose synthesis is regulated.
Introduction:
Cellulose (b 1,4-glucan), synthesized by plants and some bacteria, is one of the
most abundant polysaccharides on Earth.1
It represents an essential component of plant
cell walls, but is also found in bacteria. Bacterial cellulose produced by some species of
the genus Agrobacterium differs from plant cellulose. Agrobacterium tumefaciens is a
rod-shaped, Gram-negative plant pathogen that is dependent on attaching to its host
through anchoring factors in order to induce crown gall tumors.2
Virulent strains of A.

3. tumefaciens form cellulose fibrils, which serve to anchor the bacteria to each other and to
stabilize the interaction with the plant cell wall.2
Mutants that under-produce cellulose
bind less tightly and fail to form biofilms, while mutants that overproduce cellulose result
in an increase in biofilm production.3
A. tumefaciens serves as the model organism for this study of cellulose synthesis.
A. tumefaciens strain C58 has a unique genome consisting of a circular chromosome, and
two plasmids.4
Cellulose synthesis by A. tumefaciens C58 is encoded on two closely
linked operons, celABC and celDE, located on the linear chromosome.5
Two genes, celA
and celB, are responsible for producing the enzyme cellulose synthase.6
The celA-celB
complex produces cellulose fibrils by catalyzing a single polymerization step, utilizing
UDP-glucose as the substrate.7
Given the importance of cellulose fibril for attachment, we
constructed and examined mutants of A. tumefaciens with altered ability to synthesize
cellulose fibrils. This study describes the construction and properties of the mutants.
Materials and Methods:
The first step in this experiment was to prepare the plasmid DNA. About 2 mL of
a fresh overnight culture of E.coli containing the pRL27 plasmid grown with tetracycline
in a T broth was added to a sterile 2 mL Eppendorf tube. The tube was spun in a
centrifuge for 1 min. The supernatant was poured off, while the remaining pellet was
suspended in in 100 μL of solution P1 (100 μg/mL RNase A, 50 mM Tris-CL pH 8.0, 10
mM EDTA) via vortexing. An additional 100 μL of solution P2 (800 μL water, 100μL 2
M NaOH, 100 μL 10% SDS) was added and mixed via inversion. The solution was then
incubated at room temperature for 5 minutes.

4. Following incubation, 100μL of solution P3 (3 M K acetate adjusted to pH 5.0
with acetic acid) was mixed via inversion. The Eppendorf was then centrifuged for 10
minutes. Afterwards, the aqueous phase was transferred to a new tube and 210 μL of
isopropanol was added to precipitate DNA. The tube was centrifuged another 10
minutes.
The supernatant was carefully separated with a micropipette and discarded. Then 1
mL of 70% ethanol was added to the remaining pellet in the tube and inverted. This
solution was centrifuged for 10 minutes. The supernatant was once again removed, and
the pellet was dried in a speed vac for 40 minutes. Once thoroughly dried, 35 μL of
sterile water was added to the dried pellet and mixed with the pipette tip until the pellet
was in solution.
The prepared plasmid was then introduced into a culture of A. tumefaciens C58,
which was grown overnight in YEP and diluted, and then grown again in YEP until it
reached mid-log phase. 2 mL of cells were collected and centrifuged in the microfuge for
1 min. The supernatant was poured off, and the pellet was resuspended in 0.1 mL of ice-
cold sterile 20 mM CaCl2. The cells were kept on ice as 10 μL if the prepared plasmid
DNA was added. The solution was frozen in liquid nitrogen, then thawed via incubation
in a 37°C water bath for 5 minutes. Finally, 1 mL of YEP medium was added to the tube,
and the solution was incubated overnight on a roller drum at room temperature.
After 1 day, 0.1 mL of the cells and a 0.1 mL of a 10-1
dilution of the cells (0.1 mL
cells, 0.9 mL 0.9% NaCl) were each plated on a nutrient agar plate containing
tetracycline, allowing only cells which had taken up the tetracycline resistant plasmid to
grow. These plates were then allowed to grow for 5 days at room temperature, 25°C.

5. The plates for screening cellulose under-producing mutants were prepared. The
exact proportion of each chemical used is given in Table 1. The substances were added
and sterilized with an autoclave. Before pouring into petri dishes, 0.5 mL of tetracycline
was added to the beaker. The plates were then poured and allowed to solidify.
After the bacterial colonies grew for 5 days (incubated at 25°C), 404 colonies were
randomly picked and placed onto 8 cellufluor and tetracycline containing screening
plates. The presence of the antibiotic tetracycline served to kill any A.tumefaciens cells
that did not take up the plasmid (only the plasmid contained genes conferring tetracycline
resistance). The bacteria grew for 4 days (25°C), and then were screened using a UV
light. Under ultraviolet light, cellufluor causes cellulose producing colonies to glow in
the light, meaning that the brighter a colony, the more cellulose produced. Colonies that
did not glow as brightly as others were deemed to be cellulose under-producing mutants
and were marked.
All the under producing mutants were tallied, and some were randomly selected
and streaked on another screening plate. Each plate was allowed to grow for an
additional 4 days, and then an isolated colony was re-streaked on a screening plate. One
of these double isolated mutant colonies was then used to create a stock solution for
future study.
Results:
Of the 404 colonies selected with tetracycline, 54 were cellulose under-producing
mutants. Of the eight experimental plates, the mean frequency of cellulose under-
producing mutants, as indicated by extra dark colonies under UV light, was 0.13 ± 0.12.

6. The large standard deviation suggests that a larger sample size would have been useful.
No cellulose overproducing mutants were found in our total sample.
Calculations:
Discussion:
Transposons were successfully inserted into A. tumefaciens C58 via E. coli pRL27 Tn5
plasmid. Although only 13% of bacteria were shown to be mutated (table 2), the
transposon inserted in many more genes that were not screened for in this experiment.
Extra bright colonies on celluflour and tetracycline plates indicate cellulose
overproduction, caused by mutation to a repressor for cellulose synthesis. No extra
bright colonies were found in this study. This indicates that, in the 54 A. tumefaciens
mutated in cellulose production, the Tn5 transposon did not insert into the repressor gene
for cellulose synthesis. This could mean that there are fewer genes responsible for the
inhibition and control of cellulose synthesis than genes that confer cellulose production.
By having fewer genes regulating cellulose production (like a repressor), there is less of a
chance of the transposon inserting in these sequences and disrupting their function, which
would lead to fewer extra bright colonies than extra dark.

7. Extra dark colonies indicate under-production of cellulose, caused by a mutation
in one of the cellulose synthesis genes. This was the only type of mutant found, making
up 13% of the mutations among all mutants selected with tetracycline. Cellulose under-
production results in inability to form biofilms, thus reducing virulence of the bacteria A.
tumefaciens.2
The transposon may have inserted into either operon gene, celABC or
celDE, or into the genes for cellulose synthase enzyme, celA or celB. If there are more
genes responsible for cellulose synthesis than there are for repression, a greater mutation
rate for under-producing mutants is expected. Further studies are necessary to determine
which gene was interrupted in order to cause the mutated phenotypes, as the data from
this experiment is insufficient. This can be done by DNA sequencing and comparison of
the mutated genome with that of normal A. tumefaciens.
Errors:
There are multiple points where error could have been introduced into the data. Figure
1 shows a photograph of two plates under the UV light. Extra dark cellulose mutants
were selected by hand from these fluorescent colonies, and, as the image demonstrates,
the extra dark mutants were not necessarily extremely different from the normally
producing colonies. This added potential room for error, because subjectivity could be
introduced into the selection process. Also, brightness of a colony under UV light is
dependent on amount of produced cellulose. While darker colonies can signify cellulose
under-producing mutants, it could also just mean that a colony is growing slower than
surrounding colonies. This could easily be the case for a colony found at the edge of a
plate, where nutrients could be scarcer than in the middle of the plate, leading to slower

8. growth. The colony could be darker than surrounding ones, but because of competition
for nutrients as opposed to being an actual cellulose producing mutant.
Also, this experiment was conducted as a group, with pooled data and results; this
means that error could be introduced by different individuals using different techniques
for various processes, like the generation of the competent transposon or picking colonies
onto selection plates. This could lead to difference in results that are due to protocol
differences instead of actual experimental differences.

9. Figure 1: Photograph of two selection plates under UV light.

10. Table 1. List of substances and exact amounts used to create screening plates.
Ingredient Amount
Water 500 mL
Tryptone 10.053 g
Yeast Extract 5.078 g
NaCl 5.059 g
3M NaOH 0.5 mL
Bacto-agar 7.016 g
Cellufluor 0.014 g
Table 2. Frequency of cellulose under-producing mutants.
Plate 1&
2
3 4 5 6 7 8 TOTA
L
Mutant
colonies
(per
plate)
12 2 3 4 8 21 4 54
Total
colonies
(per
plate)
100 54 44 44 54 54 54 404 Mea
n
Std.
deviatio
n
Frequenc
y of
Mutants
0.1
2
0.03
7
0.06
8
0.09
1
0.1
5
0.3
9
0.07
4
0.13 0.12

11. References:
1. Delmer DP, Amor Y (1995) Cellulose biosynthesis. Plant Cell 7:987–1000
2. Matthysse AG, Holmes KV, Gurlitz RH. 1981. Elaboration of cellulose fibrils by
Agrobacterium tumefaciens during attachment to carrot cells. J. Bacteriol. 145:583–
595.
3. Matthysse AG, Marry M, Krall L, Kaye M, Ramey BE, Fuqua C, White AR. 2005.
The effect of cellulose overproduction on binding and biofilm formation on roots by
Agrobacterium tumefaciens. Mol. Plant Microbe Interact. 18:1002–1010.
http://www.ncbi.nlm.nih.gov/pubmed/16167770
4. Matthysse AG. 1983. Role of bacterial cellulose fibrils in Agrobacterium tumefaciens
infection. J. Bacteriol. 154:906 –915.
5. Wood DW, et al. 2001. The genome of the natural genetic engineer Agrobacterium
tumefaciens C58. Science 294:2317–2323.
6. Matthysse AG, Thomas DL, White AR. 1995. Mechanism of cellulose synthesis in
Agrobacterium tumefaciens. J. Bacteriol. 177:1076 –1081.
7. Saxena IM, Kudlicka K, Okuda K, Brown RM, Jr. 1994. Characterization of genes in
the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications
for cellulose crystallization. J. Bacteriol. 176:5735– 5752.