American Journal of Biochemistry and Biotechnology 1 (3): 121-124, 2005

ISSN 1553-3468
2005, Science Publications

Role of Plasmid in Production of Acetobacter Xylinum Biofilms

Abbas Rezaee, Sanaz Solimani and Mehdi Forozandemogadam

Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Abstract: Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected
to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a
complex system of plasmid DNA molecules. A 44 kilobases (kb) plasmid was isolated in wild type of
A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of
cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there
is considerably difference in cellulose production. In order to study the relationship between plasmid and
the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for
preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common
molecular techniques, such as digestion and transformation, with high efficiency.

Key words: Acetobacter xylinum, plasmid, biofilm, cellulose, curing

INTRODUCTION were recognized for the biofilms[3,5]. Its successful

application by dermatologists and plastic surgeons
Cellulose is the most abundant biopolymer on included human second and third degree skin burns,
earth, recognized as the major component of plant skin grafts, face peelin, infectious enhanced propertions
biomass, but also a representative of microbial of fibroblasts, collagen, blood vessels and granulation
extracellular polymers[1]. Bacterial Cellulose (BC) tissue as a result of the cellulosic pellicle use were seen
belongs to specific products of primary metabolism and in the healing wound. Wider application of this
is mainly a protective coating, whereas plant cellulose polysaccharide is obviously dependent on the scale of
plays a structural role[2]. Cellulose is synthesized by production and its cost. Therefore, basic studies run
bacteria belonging to the genera Acetobacter, together with intensive research on strain improvement
Rhizobium, Agrobacterium and Sarcina. Its most and production process development. In the research
efficient producers are Gram-negative, acetic acid was studied, about role of plasmid on production of
bacteria Acetobacter xylinum (reclassified as cellulose by elimination of plasmid DNA ,curing,.
Gluconacetobacter xylinus), which have been applied Curing is an important step in identifying the
as model microorganisms for basic and applied studies phenotypic traits encoded by a given plasmid. Plasmid
on cellulose[3]. Acetobacter xylinum produce an curing studies have been focused on Escherichia coli,
extracellular gel- like material or pellicle, which Salmonella typhimurium, Staphylococcus aureus, to a
comprises of a random assembly of cellulose ribbons lesser extent, strains of degradative Pseudomonads. A
composed of a number of microfibrils. Extracellularly multitude of different chemicals including inhibitors of
synthesized microbial cellulose differs from plant DNA replication, intercalating drugs, bacterial surface
cellulose with respect to its high crystallinity and purity agents and Sodium Dodecyl Sulphate (SDS) at elevated
(free of legnin and other biogenic products), high temperature has been used as curing agents. However,
water-absorption capacity and mechanical strength in apart from its spontaneous loss, very little is known
the wet state[4]. Several application have been proposed about how to cure the plasmids of A. xylinum. The
for this cellulosic layer. Because of the unique plasmids may prove biotechnologically important as it
properties, resulting from the ultrafine reticulated has been implicated in the production of microbial
structure, BC has found a multitude of applications in cellulose[6]. In this study, Acridin orange properties
paper, textile and food industries and as a biomaterial in were exploited for curing of the A. xylinum plasmid.
cosmetics and medicine[5]. One of applications of
bacterial cellulose is use as burn infection and heal MATERIALS AND METHODS
lesions. Modern medical biotechnology has accepted
artificial skins as a valid prospect. Several properties Bacterium: A freeze-dried culture of A. xylinum was
advantageous for its use as a temporary skin substitute obtained from the Persian Culture Type Collection of

Corresponding Author: Abbas Rezaee, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Fax: +98 21 8013030
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Microorganisms. Following recultured in Schramm freshly prepared lysis buffer (0.2 N NaOH containing
Hestrin (SH) medium , the culture was found to be pure 3% SDS) was added and after the contents were rapidly
and biochemical and morphological tests revealed that and thoroughly mixed without vortexing, the Eppendorf
the organism conforms to the generic description listed tube was allowed to stand on ice for 5 min. An
for A. xylinum in Bergeys Manual of aliquot (150 L) of ice-cold neutralizing solution
Systematic Bacteriology[7]. (acidic potassium acetate constituted by mixing
11.5 mL of glacial acetic acid, 28.5 mL of distilled
Culture conditions: The bacteria was inoculated with water and 60 mL of 5 M potassium acetate) was added
108 to 109 cell per mL in SH medium at 28C for a week and the contents were gently mixed by inversion to
under static conditions to produce cellulose pellicles. neutralize the pH in the cell lysate which was noted to
For plasmid isolation, Acetobacter xylinum was grown be viscous. The SDS and associated proteins as well as
in SH agar. cell debris were then removed by centrifugation at
12000 rpm for 5 min at 4C. The supernatant
Determination of wet and dry weights of containing the plasmid DNA and possibly some RNA
pellicles:The pellicle was lifted from the plate with a was transferred into a fresh Eppendorf tube containing
bent glass rod and allowed to drip for 30 min. The 20 L of DNase-free RNase (10 mgmL-1) to ensure the
pellicle was then placed in a pre weighed plastic removal of any traces of RNA. After the addition of
weighing plate. The weighing plate and pellicle were DNase-free RNase and incubation for 20 min in 37C,
then placed in a drying oven for 6 h at 80C. After an equal volume of phenol/chloroform/isoamyl alcohol
being dried and removed from the oven, the weighing (DNA grade) was added to the supernatant. The
boat and pellicle were weighed immediately. Pellicles contents of the Eppendorf tube were mixed gently but
were also washed with running tap water prior to the thoroughly and then centrifuged at 12000 rpm for 5 min
wet and dry weight determinations. There were no at 4 C in a microfuge. With the aid of a pipette, the
percentage differences between the wet and dry weight supernatant (upper layer) was transferred to a fresh
comparisons of pellicles that had not been washed Eppendorf tube and great care was taken to avoid the
before being allowed to drip for 30 min[8]. transfer of the white precipitate present at the interface,
if the upper layer was not clear owing to the presence of
Rapid screening for plasmids: A small number of large quantities of polysaccharides, this step was
cells were picked by touching a colony with a repeated. Two volumes of ice-cold ethanol were then
toothpick. They were then inoculated into a microfuge added to the supernatant and after thorough mixing and
tube containing 300 L of SH broth. After overnight incubation at 70 C or room temperature for 60 min,
growth, the cells were pelleted in a microfuge the plasmid DNA was precipitated by centrifugation at
(10,000 rpm, 2 min). The cells were then suspended by maximum speed in a microfuge at room temperature for
vortexing in 20 L of gel-loading mix (0.25% 10 min, the supernatant was discarded and the
bromophenol blue and 30% glycerol). Then 40 L Eppendorf tube was then allowed to stand inverted on a
each of chloroform and phenol (saturated with 1.0 M paper towel to drain away any excess ethanol. The
Tris-HCl, pH 8.0) was added. The mixture was plasmid DNA pellet was then washed twice with 1 mL
vortexed at full speed for 1 min followed by of ethanol (70%) at 4 C and after discarding the
centrifugation for 10 min at 12,000 rpm. Then 10 L of supernatant and removing large droplets of ethanol with
the aqueous fraction was subjected to electrophoresis the aid of sterile cotton buds or fine tissues, any traces
on 0.7% agarose minigel (5.26.0 cm) with TAE buffer of ethanol were further removed. Electrophoresis of the
(40 mM Tris-acetate, pH 8.0 containing 2 mM plasmid was carried out overnight on 0.7% agarose gels
Na2-EDTA) at 100 volts for 30 min. The gel was at 3.0 Vcm-1 in TAE buffer. Restriction fragments,
stained with ethidium bromide (0.5 gmL-1) and however, were electrophoresed using TBE buffer at
the DNA bands were visualized under a 6.0 Vcm-1 for 4 h as previously described[8,10,11].
UV transilluminator.
Curing of Acetobacter xylinum: Acetobacter xylinum
Plasmid isolation: Plasmid isolation was performed by cured by growth at 28C in SH medium containg a
a modified method of Sambrook[9]. The protocol is as subinhibitory concentration of Acridin orange (5 to
follows: Because Acetobacter xylinum produces 600 g). Cultures containing the highest concentration
cellulose in broth culture, SH medium was used. The of Acridin orange which growth was clearly visible
bacterium were grown in SH medium supplemented were diluted and spread into SH plates. Resulting
with chloramphenicol, bismuth nitrate and sodium clonies were tested for chloramphenicol sensitivity and
salicylate . The bacteria were suspended by mixing with electrophoresis. A cell lysate of A .xylinum and its
a vortex mixer in suspension buffer, TE buffer (10 mM cured derivative were cultivate as described in culture
Tris, 1 mM EDTA), pH 8.0. An aliquot (200 L) of conditions of materials and methods and the presence or
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absence of plasmid DNA was tested using agarose gel

digested with
electrophoresis. A purified sample of A. xylinum

Plasmid
Marker

BamHI
plasmid as well as lambda digested with HindIII)
were also included.

Restriction analysis of isolated plasmid DNA:

Plasmids isolated from A. xylinum were digested with
two restriction enzymes (BamHI and HindIII) at the
same time. Aliquots of A. xylinum plasmid DNA,
prepared as described above, were digested overnight at
37C with ten units of various restriction enzymes and
the fragments thus generated were fractionated by
electrophoresis on 0.7% agarose gel as described above.
The bands, which were visualized under ultraviolet
light, were compare with standard marker.

RESULTS AND DISCUSSION

A. xylinum is an acetic acid bacterium which has

the ability to produce cellulose extracellurarly. To Fig. 1: Agarose gel electrophoresis of A. xylinum
improve the cellulose-producing ability of A. xylinum, plasmid digested with BamHI
recombinant DNA techniques seems to be proper
method. The genus A. xylinum contains several species mucoid strains of A.xylinum. The method for plasmid
which are being extensively studied in connection with isolation was not suitable for each bacteria because of
acetic acid production and cellulose formation[12]. There the inhibitory effects of bismuth. Thus, removal of
have been several reports of spontaneous mutation in contaminating bacterial surface structures enabled the
A. xylinum strains affecting both morphological and rapid isolation and characterization of plasmids from
physiological properties, including loss of the ability to
mucoid isolates, without the use of organic solvents,
produce cellulose[13,14]. Bacteria of this genus are not
CsCl gradients, or expensive, disposable columns[16]. In
genitically well characterized and genetic investigations
this study, a simple and rapid method for screening
of the phenotypic instabilies have not been reported. To
plasmids of A.xylinum has been developed. It is
improve the cellulose producing ability of A. xylinum,
especially useful when sample sizes are large. This
we studied role of the plasmid in production of
method can be achieved in one step simply by
cellulose. Most strains of A. xylinum examined contain
a rather complex system of plasmids[15]. Alterations in phenol/chloroform treatment of the cells pelleted from a
the plasmid profile were found in both cellulose volume as small as 300 L. The experiment from
negative mutants and cellulose producing revertants of growing cells to the completion of plasmid extraction
A. xylinum. Cells of A. xylinum produce large amounts can be carried out using the same microfuge tube. The
of exopolysaccharide, which interferes with the whole procedure takes about 40 min from harvesting
extraction of plasmid DNA[16,17]. To repress capsular the bacterial cultures to visualization of the DNA bands
polysaccharide production, bacteria were cultured in in agarose gel. Using this method, detected plasmids in
SH medium containing bismuth nitrate and sodium A. xylinum have detected. In conclusion, this new
salicylate. After treatment, bacterial cells were more method for the isolation of plasmid DNA from
readily lysed in alkaline detergents. The resulting lysis-resistant A. xylinum has major advantages over the
plasmid preparations contained virtually no capsular classic method that requires phenol-extraction and
polysaccharide and relatively small quantities of cesium chloride gradient centrifugation. This new
lipopolysaccharide and protein, yet they produced method does not employ highly toxic reagents, is
yields of nucleic acids similar to those of conventional considerably cheaper and is easy to perform. This
plasmid preparations. Conventional preparations from method provides high yields of pure DNA, which can
encapsulated organisms were largely insoluble and be used for restriction analysis, transformation and
appeared as smears following agarose gel other molecular techniques. This plasmid isolation
electrophoresis, with indefinite plasmid banding. technique is also applicable to other Gram-negative
Plasmids prepared by the new method were highly bacteria and will improve the efficient engineering
soluble in conventional buffers and exhibited high- of biotechnologically and therapeutically useful
resolution plasmid banding patterns in agarose gels. A. xylinum recombinants for cellulose production. To
The method proved effective with encapsulated or construct a physical map, isolate plasmid was cut with
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different restriction endonucleases and subjected to 7. Hestrin, S. and M. Schramm, 1954. Synthesis of
agarose gel electrophoresis. The results showed that it Cellulose by Acetobacter xylinum. Biochem. J.,
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