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Microbiology

Biogenesis of Bacterial Cellulose has been revealed. Since bacterially synthesized cellulose is
chemically identical to that of higher plants and other organ-
Robert E. Cannon, Ph.D. and Steven M. Anderson, isms, this may suggest a probable common evolutionary origin
Ph.D. among cellulose producers. By studying the synthetic process
using microbes, which are easily maintained and manipulated,
it may be easier to uncover the details of genetics and bio-
ABSTRACT chemistry of cellulose biosynthesis.
Could the cellulose produced by bacteria like A. xylinum be
Cellulose is the most abundant biological polymer on Earth. put to productive use commercially? Will it replace or su-
It is found in wood and cotton, and forms the basic structural percede the cellulose that we now get from trees for paper or
foundation of the cell wall of almost all eukaryotic plants. cotton plants for fiber? Probably not, but bacterial cellulose
Bacteria are known to secrete cellulose as part of their metab- does have some advantages over that produced by other or-
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olism of glucose and other sugars. The focus of this review is ganisms. One of the advantages of Acetobucfer cellulose is
upon bacterial cellulose synthesis. We emphasize recent lit- that it does not need to be delignified before processing as is
erature directed primarily upon Acefubucferxylinum, which the case with woody plant cellulose that is processed into paper.
has been most widely studied. Our review covers the following A . Xylinm probably has the greatest potential for commer-
topics relating to cellulose synthesis: genetics, biochemistry, cialization since other cellulose-synthesizing microbes do not
ultrastructure, growth conditions, and ecological considera- product sufficient quantities of the cellulose polymer for in-
tions as they relate to the diversity of microbes capable of dustrial purposes. Acetobucfercellulose holds water well when
synthesizing this abundant, unique polymer - cellulose. left undried and has strong tear resistance. It can assume or be
woven into various shapes and it retains its shape well. One
1. INTRODUCTION current example of an application for bacterial cellulose comes
from the S O W Corporation, which uses cellulose produced
Cellulose is the most abundant natural polymer on Earth. It by A . xylinum to make sensitive diaphragms for stereo head-
phones. Microbial cellulose is also available as a food product
For personal use only.

forms the cell wall of eukaryotic plants and algae, and is also
found to be the major constituent of the cell wall of fungi. called Nata in the Philippines.* Weyerhauser and Cetus Cor-
Cellulose is also synthesized by bacteria, especially by the poration have recently developed a strain of Acerubucter that
Gram-negative bacterium Acetobarer xylinum, which is the is capable of large-scale cellulose production under fermen-
primary focus of this review. This microorganism has been the tative conditions with agitati~n.~ The fibers produced termed
most widely studied of the few bacteria that produce cellulose, Cellulon, have a number of potential commercial applica-
although a number of other bacterial cellulose producers are tions, such as binders for ceramic powders and minerals, thick-
discussed briefly. The following topics are covered: survey of eners for paint, ink, adhesives, and even foods. Another possible
cellulose-synthesizing bacteria, growth and cultivation require- use for Cellulon is as a paper coating because of its high purity.
ments, ultrastructure of bacterial cellulose synthesis, biochem- Also recently reported was the use of Acefubucrercellulose as
istry of cellulose synthesis, genetics of biosynthesis, and ecology a temporary skin substitute to protect burned tissue as normal
of cellulose producers. skin regenerate^.^ This is the first example of specific medical
Cellulose which is synthesized by bacteria as a secondary applications of bacterially produced cellulose. The cellulosic
metabolite is a polymer of glucose. The molecules of glucose material, identified by the trademark BioFill, has been used
are linked together as I+4p-glucan chains. Cellulose synthe- successfully for treatment of severe bums, skin grafting, and
sized by A. xylinum is polymerized into long microfibrils out- chronic skin ulcers. Advantages of this treatment were pain
side of the cell wall and is of the same chemical constituency relief, good adhesion, an effective barrier to infection, fast
as the cellulose made by higher plants. This similarity makes healing, good fluid (water and electrolyte) retention, low cost,
A. xylinum an ideal model system to study the cellulose bio- and short treatment time as normal, healthy skin grows to
genesis process. The synthetic pathways of many glucose poly- replace the artificial cellulosic skin substitute. A potential dif-
*
mers are fairly well understood and the synthase enzymes ficulty to human use of cellulose may be the need to remove
involved have been well characterized (e.g., chitin synthetase).
The biosynthetic pathway for bacterially produced cellulose is
still not completely understood, thus providing a fertile area
for research. The biosynthetic process is multi-step involving
both the production of cellulose and its crystallization into
extracellular fibrils. Our understanding of these processes is
increasing rapidly as more information concerning the enzyme,
cellulose synthase, and its organization in the cell membrane

1991
435
Critical Reviews In

endotoxic components that may contaminate bacterial cellu- producing Acetobacters is production of a floating cellulosic
losic products. pellicle when the organisms are cultured statically. The pellicle
Our knowledge of the process of cellulose synthesis carried forms from the organization of cellulose fibrils synthesized and
out by bacteria exemplified by A . xylinum has increased greatly secreted by the bacterium. Many strains oxidize ethanol to
over the past decade. This basic research has formed the foun- acetic acid. They are ubiquitous and commonly found on de-
dation for future attempts at potential commercialization of caying fruits and vegetables, vinegar, fermented beverages,
bacterially produced cellulose, which are currently in their sugar cane juice, garden soil, etc.
formative stages. Other bacteria that produce cellulose in lesser quantity than
A . xylinum include Sarcina ventriculi, which is the sole Gram-
II. SURVEY OF CELLULOSE-PRODUCING positive, cellulose producer.6 Also, a number of bacterial spe-
BACTERIA cies have been found to produce exocellular cellulose fibrils
when isolated from activated sewage sludge. The cellulose
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Many Gram-negative bacteria secrete extracellular polysac- fibrils were believed to account for floc formation. The specific
charide material, but only a few have been shown to produce genera isolated included Pseudomonas, Achrombacrer, Al-
cellulose (Table 1). A . xylinum, the most studied of bacterial caligenes, Aerobacter, Rhizobium, Agrobacterium, and Azo-
cellulose producers, is a Gram-negative, aerobic, rod-shaped tobacter. Cellulose fibril formation apparently plays an important
organism. It is classified in Bergeys Manual of Systematic part in the infection of plants by Agrobacterium tumefaciens
Bacteriology (Volume 1, 1984) in Section 4 -Gram Negative by aiding in attachment of bacteria to plant tissue eventually
Aerobic Rods and Cocci5 It is a member of the family Ace- leading to gall formation.8 A more detailed discussion of the
tobacteraceae. Species within the genus, Acerobacrer include possible role(s) of cellulose for the bacteria that produce it is
A . aceti, A . liquifaciens, A . pasteurianis, and A . hansenii. A . presented in the ecology section of the review.
xylinum is treated in scientific literature as a species, but for
the purpose of strict classification, it has been considered a
subspecies of A . aceti. For the purpose of this review, A . 111. BACTERIAL GROWTH
xylinum will be used throughout to indicate the primary, cel- CHARACTERISTICS, REQUIREMENTS, AND
For personal use only.

lulose producing bacterium. Members of the genus Acetobacter IDENTIFICATION OF CELLULOSE


are chemoorganotrophscommonly producing pale colonies when
grown on solid media. A prominent characteristic of cellulose- The undefined medium that is characteristically used to cul-

Table l
Survey of Bacterial Cellulose Producers

Organism (genera) Cellulose produced Cellulose assay Biological Role

Acetobacter Extracellular pellicle Cellulose To hold in aerobic environment


Cellulose ribbons synthase To colonize natural substrates
X-ray diffraction
Alkali insolubility
Fluorescent
brightener
Degradation by
cellulase
Agrobacterium Short extracellular fibrils X-ray diffraction Attach to plant tissue
Alkali insolubility
Fluorescent
brightener
Degradation by
cellulase
Rhizobium Short extracellular fibrils Same criteria as Attach to host plants
Agrobacterium
Pseudomonas No distinct fibrils Alkali insolubility Flocculation in wastewater
Degradation by
cellulase
Sarcina Amorphous celluloseb Alkali insolubility Unknown

a Cellulose I - highly crystalline, metastable microfibrils, native cell~lose.~


Cellulose 11 - crystalline allomorph of Cellulose I with antiparallel glucan chains, rare in nature.

436 Volume 17, Issue 6


Microbiology

ture Acetobacter qlinum to maximize both growth and cel- the microfibrillar product is cellulose rather than other non-
lulose production was developed by Hestrh and Schramm (Table cellulosic polysaccharides.l3 Fibrils of cellulose are extremely
2)." Other carbon sources have been shown to be suitable for strong, and insolubility in alkali (2% NaOH) has therefore been
growth, but they have not been as useful for production of used as a criterion for presence of cellulose. However, other
cellulose. A . xylinum grows well over a temperature range of glucose-based polymers (e.g., p-I ,2-D-glUCan,p-l,3-D-ghl-
25 to 30"C, but its temperature optimum is 30C. The pH of can) are also insoluble in base and therefore one should be
the undefined medium is 6. Historically, to maximize cellulose careful in using this as the sole criterion for demonstration of
production, A. xylinum has been cultured statically allowing cellulose production. Also, the crystalline structure of the pol-
for the formation of a pellicle composed of cellulose holding ymer is resistant to treatment by a combination of acetic and
within it bacterial cells floating on the surface of the medium. nitric acid. Cellulose gives a characteristic diffraction pattern
If cultures are aerated by shaking, bacteria grow faster, but upon X-ray crystallography, which is the ultimate method dem-
produce less cellulose. Rotary shaking results in production of onstrating its presence. Several inhibitors have been identified
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round balls of cellulose rather than the flat, sheet-like pellicle that interfere with cellulose synthesis.l4 These include cou-
of static cultivation. Doubling time for A. xylinurn held in static marin and dichlorobenzonitrile, which apparently disrupt gly-
culture is between 8 and 10 h, while in aerated culture (by cosylation in the synthesis process. In addition, substances
shaking), the organism doubles every 4 to 6 h. When A. xy- called fluorescent brighteners, e.g., Calcofluor White ST or
h u m is cultured on solid medium, colonies that are formed Tinopal LPW, have been used to confim cellulose production.
by cellulose-producing strains have a dry, wrinkled appear- When these stilbene derivatives react with the glucan chains
ance. This contrasts to celluloseless strains (cellulose-negative of cellulose by hydrogen bonding, they prevent normal micro-
mutants), which appear as smooth, spreading colonies that are fibril formation and assembly of cellulosic ribbons by A. q-
usually two or three times the size of cellulose-synthesizing l i n ~ r nDespite
.~ the disruption of normal ribbon formation, the
strains after a comparable period of growth. binding of these substances causes the bands of cellulose to
fluoresce brightly when viewed under ultraviolet light. It is
Table 2 possible to incorporate the fluorescent brighteners into the solid
Undefined and Synthetic Media for medium. Colonies of A . xylinum and other cellulose-synthes-
For personal use only.

Cultivation of Acetobacter xylinum izing bacteria, which are actively synthesizing cellulose flu-
oresce, while cellulose-negative strains do not. Unfortunately,
Undefined" Synthetic'* these brighteners bind to other exopolysaccharides, so addi-
tional tests must be done to c o n f m that cellulose has indeed
Glucose 2.0% Glucose 1.0%
been synthesized by the bacteria.
Yeastextract 0.5% NH.,Cl 0.1%
Peptone 0.5% Citric acid 0.115%
Sodium 0.27% N%HPO, 0.33% IV. ULTRASTRUCTURE AND MODEL FOR
phosphate
Citric acid 0.115% KCI 0.01%
CELLULOSE SYNTHESIS
MgSO, * 7H,O 0.025%
Nicotinamide 7.5 mg/I The ultrastructure of the cellulose synthesis apparatus is best
understood in A. xylinurn. Transmission electron microscopy
To be able to manipulate growth and know exact chemical and freeze-etching have revealed the presence of a row of pores
constituents, we have developed a synthetic minimal medium on the longitudinal axis of cells. These particles or pores may
for A. xylinurn that permits good growth (Table 2, Figure 1) contain or be composed of the last enzyme(s) involved in cel-
and maintains cellulose production.'2 The synthetic minimal lulose synthesis, and glucan chains that eventually form into
medium is useful for genetic manipulation of A. qlinum be- the composite ribbon of cellulose may be secreted through these
cause it can be used to isolate auxotmphic mutants. A culture pores.9 Figure 2 is a depiction of this hypothetical model. Each
grown in minimal medium with 1% glucose added produced cell that is actively synthesizing cellulose produces a cellulosic
about half as much cellulose as one in the undefined medium, ribbon ranging in width from 40 to 60 nm, which appears to
at a slightly slower growth rate. If any of the ingredients of be parallel to the longitudinal axis of the bacterial cell. Al-
the medium were omitted, no growth occurred. The synthetic though A. xylinum is nonmotile, it can be pushed by the for-
medium was used successfully to isolate a number of amino mation of the ribbon of cellulose at a rate of 2 pdmin. The
acid- and nucleotide-requiring auxotrophs after mutagenesis of ribbon of cellulose is composed of microfibrils, which are the
A . xylinum 'by N-methyl-N'-nitro-N-nitrosoguanidine. form in which cellulose is secreted through extrusion sites in
To insure that the biosynthetic product made by A. xylinurn the outer membrane of the bacterium. These microfibrils appear
is cellulose, a number of different techniques are employed. to be about 1.5 nm wide and aggregate into 3 to 4 nm micro-
The techniques vary in complexity and accuracy, and it may fibrils via crystallization of adjacent glucan chains. Finally,
be necessary to rely on a number of methods to be sure that these microfibrils band together to form the larger cellulosic

1991 437
Critical Reviews In

I e
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c.l - M a 8 Yedlum (Undellned)


to I --+ - Ylnlmel 111)Olucosa
w - YInImal 211) Olusoae
+

A--+ - Ylnlnel 0.6% OIUCOS.


.=-a - Ylnlmal 0.2611) O I U C O ~ ~

FIGURE 1. Growth curves for A. xylinum in undefined and minimal media at four glucose
concentrations (2, 1, 0.5, and 0.25%).Cells incubated statically at 2 6 T , but shaken briefly
at each time point to release cells consistently from the pellicle.*
For personal use only.

about these processes comes from studies employing fluores-


cent brighteners and cellulosic derivatives that alter ribbon
and Bundle Initiation
formation. Fluorescent brighteners such as Calcofluor bind to
LPS envelop. extruded glucan chains preventing microfibrils from banding
together into complete ribbons. Carboxymethylcellulose and
other cellulose derivatives affect ribbon assembly at a different
level of organization. I6They commonly stop fasciation of bun-
dles of microfibrils so that individual microfibrils remain sep-
flGURE 2. Possible model of cellulose assembly. Glucan chain aggregation
arated, never forming into highly organized fibrils. More
requires multienzyme complexes followed by crystallization of microfibrils recently, Kai and Kitamura and Haigler and Chanzy* have
through extrusion pores. Ribbons of cellulose form at the cell ~urface.~ used techniques of X-ray and electron diffraction to study the
effects of fluorescent brighteners upon cellulose structure in
ribbon. Figure 3 shows the presence of the extrusion pores on A. xylinum. Kai and Kitamura reported that brighteners do not
the side of an A. xylinum cell, a typical ribbon secreted by a effect the extrusion of monomolecular layers of cellulose I from
cell, and a more highly magnified view of individual ribbon^.^ cells. At high concentrations of brightener, a diffraction pattern
Bureau and Brown have demonstrated that cellulose synthesis resembling neither cellulose I nor II appeared. As brightener
occurs in the cytoplasmic membrane of A. xylinum rather than concentration was lowered, the cellulose I pattern returned.
in the outer membrane as had been previously thought. Final Haigler and Chanzy, using different washing methods than Kai
crystallization of microfibrils may still occur in or near the and Kitamura to prepare cellulose samples prior to diffraction
outer membrane and be dependent upon the ordered nature of techniques, did not observe altered cellulose. They speculate
the extrusion pores that are seen in freeze fracture preparations that the harsh treatment with alkali and alcohol used by Kai
of cells. and Kitamura might have introduced some form of crystallin-
The enzymes involved in the terminal stages of cellulose ity. Haigler and Chanzys results also showed that cellulose I
fibril biogenesis are cell-associated, probably within either in- could be reformed from amorphous cellulosic material that is
ner or outer membranes of the Gram-negative bacterium (see produced after dye treatment. In the final analysis, when po-
biochemistry section). It is not completely understood how the lymerization and crystallization processes are disrupted, nor-
individual glucan molecules polymerize and assemble into the mal microfibrillar ribbons of cellulose cannot form.
microfibrillar bundles of cellulose. Much of what is known Strong magnetic field strength has also been shown to alter

438 Volume 17, Issue 6


Microbiology
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For personal use only.

FIGURE 3. (Top) Freeze-etching of cells of A. xylinwr that are synthesizing cellulose. Particles in
the iipopolysaccharide layer are thought to be groups of multienzyme complexes involved in polymer-
ization of cellulose. (Middle) Typical twisting ribbon of cellulose seen by negative staining. (Bottom)
Higher magnification of cellulose ribbons negatively stained showing striation. Four separate ribbons
cross with larger striations corresponding to microfibrillar bundles of c e l l ~ l o s e . ~

ribbon assembly in A. xylinum, although specific mechanisms V. BIOCHEMISTRY OF CELLULOSE


are unknown at present.19 SYNTHESIS
The overall model to account for cellulose ribbon assembly
has been described as "hierarchical" by a number of inves-
tigators. First, small glucan chains aggregate by a self-assem- Even though cellulose is a relatively simple polymer, we
bly mechanism into the 3 to 4 nm microfibrils. This event is still do not completely understand the biochemistry of its syn-
followed by banding of the microfibrils into bundles, which thesis. Difficulties in both development of an in v i m assay
then form into the complete ribbon. The high degree of or- for cellulose synthesis and purification of the enzymes involved
ganization of the extrusion pores on the surface of the outer in this process (e.g., cellulose synthase) have hindered bio-
membrane facilitates the coordination of the assembly process. chemical analyses. These problems are being overcome through
An even higher degree of organization of cellulose fibrils can use of combined genetic and biochemical techniques with the
be observed when whole, washed pellicles are viewed under organism A. xylinum, and a clearer picture of the biochemical
scanning electron microscopy. Structures resembling tunnels, steps in cellulose synthesis is beginning to emerge.
which would indicate a considerable degree of regularity in In addition to glucose, other substrates such as hexoses,
the organization of microfibrils, appear." These structures pro- hexonates, pyruvate, glycerol, and dihydroxyacetone can be
vide an even higher level of fibril orientation than ribbon for- used in the biosynthesis of cellulose.z'-23Many of these sub-
mation described above and argue that synthesis of cellulose strates are metabolically associated with either the pentose or
by A. xylinum is not occumng randomly. The diameter of citric acid cycles, indicating that cellulose synthesis may be
tunnels is about 7 pm, with sufficient room for bacteria to tied to oxidative carbohydrate metabolism." However, since
move through them. cellulose formation in Acetobucter can occur independently of

1991 439
Critical Reviews In

h
protein synthesis,25it does not appear to be an indispensable
'OO
feature of carbohydrate metabolism.
Knowledge regarding the proposed biochemical pathway
leading from exogenous glucose to cellulose has come from Cellulose
two sources: (1) studies on the enzymes involved in glucose- . Glucose
carbohydrate metabolism, and (2) labeling studies with isotopic
glucose used to delineate precursor-product relationships. A \ ,
group of enzymes involved in carbohydrate metabolism have
been found in crude and partially purified extracts of A. xy-
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Within this group of enzymes, it has been pro-


linum.14*24*26*27
posed that glucokinase, phosphoglucomutase, and UDF'G-
pyrophosphorylase are most likely to be involved in the path-

-
way leading to cellulose synthesis (Figure 4).
Water soluble
*A-A A
Glucokinase Phosphoglucomutase
Glucose- - - - - - - > Glucose-6-phosphate-- -- -- - -- - - - - - >
Alkali rolubk

Chloroform soluble
--
For personal use only.

-
4 - A

FIGURE 4. The proposed biochemical pathway for cellulose synthesis in


Acetobacter xylinum.

The phosphorylation of hexose sugars provides a common


intermediate for both cellulose synthesis and oxidation of car-
bohydrate via the pentose and citric acid cycles. Phosphoryl-
ation of exogenous sugars is carried out by glucokinase and
fructokinase in A. xylinum.26Glucokinase activity is always
present in cells regardless of which exogenous sugar is pro-
vided, whereas fructokinase activity, like that of other sugar-
metabolizing enzyme systems, appears to be subject to glucose
repression. The flux of phosphorylated sugars through the two
pathways described above will in all likelihood determine the
level of cellulose produced. In the cellulose pathway, phos-
phorylated glucose in the form of glucose-6-phosphate (G6P)
is enzymatically converted to glucose-1-phosphate (G1P) by
phosphoglucomutase.24 UDPG-pyrophophorylase is then re-
1 5 . - 15 30 45 60
sponsible for the synthesis of UDP-glucose from GlP and
UTP.= Even though all four nucleoside sugars may act as Time (minutes)
precursors for cellulose synthesis, there is no evidence for any
other nucleoside diphosphoglucose pyrophosphorylase in A. FIGURE 5. The time course for a pulse-labeling experiment during which
xylin~m.'~ This observation is also consistent with UDP-glu- cellulose is synthesized from "C-glucose by wild-type Acetobocter xylinum
cells. Incorporation of the label into both cellular fractions (e.g., chloro-
cose acting as primary precursor for cellulose synthesis. This
form-,water-, and alkali-soluble fractions) as well as specific cellular com-
proposed pathway has been substantiated through the use of ponents (e.g., UDP-glucose) is demonstrated."
isotopic-labeling studies in which the flow of carbon from I4C-
glucose to celrulose has been examined.28After pulse-labeling
A. xylinum cells, the incorporation of label into glucose-phos- Labeled G6P, GlP, and UDP-glucose appear shortly after cells
phate and UDP-glucose was determined. The results of this are exposed to L4C-glucoseand their subsequent decrease is
study are presented in Figure 5 and support the roles of G6P, linked to an increase in the incorporation of I4C into cellulose,
G1P, and UDP-glucose as precursors for cellulose synthesis. e.g., an alkali insoluble cell fraction.

440 Volume 17,Issue 6


Microbiology

UDP-glucose is the substrate for cellulose synthase (E.C. 0


2.4.1.12, UDP-Glucose: 1,4-~-~-glucan4~-D-,olucosyltrans- \ /O'
ferase) that is believed to be the major enzyme involved in the
last step(s) of cellulose biosynthesis. The development of both /p\
an in vitro assay and a purification protocol for cellulose syn-
thase has provided for the initial characterizationof this enzyme.
Hesmn and Schramm" and G l a ~ e ?initially~ described the
in vitro synthesis of an alkali insoluble polymer from UDP-
glucose by a particulate fraction from A . xylinum. Unfortu-
nately, the rate of synthesis was <O. 1% of the in vivo rate and
therefore did not provide an adequate system to study either
the structure of the cellulose produced or the mechanism of
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synthesis. Aloni et al.30*31were able to achieve a 400-fold


increase in the in vitro rate of synthesis after stabilization of
the particulate/membranous fraction with either PEG 4000 or
calcium ions and upon activation of the system with GTP.
They also showed that is was necessary to add back a regulatory
protein from the soluble fraction to obtain maximal activity.
Recently, Lin and have been able to achieve partial 0/p\ 0-
purification (>90% homogeneity) of cellulose synthase from
detergent solubilized membranes. A 96-fold purification was FIGURE 6. The structure of the diguanylic acid activator of cellulose
obtained after two rounds of a cellulose entrapment purification synthase.y
protocol. This involves adding substrate and enzyme activators
to a detergent-solubilized membrane preparation and trapping phosphodiesterases, which are involved in the degradation of
the enzyme by centrifugation of the synthesized cellulose prod- the activator. One phosphodiesterase is inhibited by Cat+ and
For personal use only.

uct. Their preparations contain two major polypeptide products hydrolyzes the opening of the cyclic nucleotide and the second,
of 83 and 93 kDa. The 83 kDa polypeptide has been demon- which is not affected by Ca2+, converts the dinucleotide into
strated to be a glycoprotein by lectin affiiity chromatography, 5' GMF'. According to this model, the rate of polymerization
Schiff periodate, and fluoroscein isothiocyanate-Concanavalin of UDP-glucose into cellulose is dependent on the intracellular
A staining. Column chromatography results were consistent levels of diguanylic activator and the level of Ca2+. The in-
with cellulose synthase being a multimeric enzyme. Lin and teractions of these components is summarized in the model
Brown suggest that the 93 kDa polypeptidechain may represent presented in Figure 7. Amikan and Ben~iman'~ have recently
a second subunit of the multimeric complex. More recently, shown the possible involvement of the same activator in the
Lin et al.33have demonstrated by photoaffinity labeling with modification/regulation of cellulose synthesis in Agrobacter-
an analog of UDP-glucose that the 83 kDa subunit is the cat- ium tumefaciens.This observation suggests that both organisms
alytic subunit for cellulose synthase. In a membranous or de- may utilize a similar regulatory mechanism, even though the
tergent-solubilized extract this analog, [(P-32P)-5N3-UDP- final cellulose product produced (bundles and simple flocs in
glucose], would photoincorporate into both 57 and 83 kDa Agrobacterium vs. complex ribbons produced by Acetobacter)
polypeptides. Further analysis using competition assays with and the rate of cellulose synthesis are markedly different.
UDP-glucose have corroborated that the active site for the
enzyme is contained in the 83 kDa polypeptide. They suggest VI. GENETICS OF CELLULOSE SYNTHESIS
that the 57 kDa polypeptide is phosphoglucomutase,which has
been labeled due to contamination of the photoprobe prepa- Studies of the genes and gene products involved in cellulose
ration with trace levels of 32P-Glucose-1-Phosphate. synthesis have been canied out in both Acetobacter xylinum
As mentioned earlier, cellulose synthase activity in in vitro and Agrobacterium tumefaciens. The development of these
preparations could be enhanced by the addition of guanosine model systems should provide not only an understanding of
nucleotides or analogs of guanosine. The work of Ross et al." the genetics of cellulose biosynthesis in bacterial systems, but
has further demonstrated that the specific cellulose synthase also an insight into this process in higher plants.
activator is-bis - (3' * 5 ' ) - cyclic diguanylic acid depicted The genetic analysis of cellulose biosynthesis in Acetobacter
in Figure 6 . They suggested that cellulose synthase activity is qdinum has included the isolation of mutants that affect cel-
controlled by a multicomponent regulatory system that includes lulose production, characterization of indigenous plasmid spe-
(1) diguanylic cyclase, the enzyme involved in the synthesis cies, and the cloning of genes involved in this-process. A
of the cyclic diguanylic activator and (2) the activities of two number of researchers, beginning with Schramm and H e ~ t r i n , ~ ~

1991 441
Critical Reviews In

Cellulose fibril
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IMg+i
UDP-glucose

FIGURE 7. A proposed model for the regulation of cellulose synthesis by the cyclic diguanylic acid activator of cellulose synthase. Levels
of activator are determined by the synthetic activity of diguanylate cyclase, the degradative action of phosphodiesterase A and B, and the
inhibitory effect of Ca2+on phosphodiesterase activity."

have described the isolation of cellulose-negative (cel -) mu- Table 3


tants. Both spontaneous and mutagen-induced variants have Characteristics of Acetobacter xylinum Strains
For personal use only.

been isolated. Many reports have indicated that apparent spon-


taneous celluloseless mutants arise at a high rate when wild-
type cells are grown in aerated liquid The frequency I Characteristic

Colony morphology
Wild type

Small, rough
Cel-negative

Large, mucoid
Overproducer

Small, rough
with which these mutants occur also increases with the age of
Fluorescence with Bright Dull Bright
the culture. The spontaneous mutants have a mucoid appear- Tinopal
ance on solid agar like true cel- mutants, but the majority Pellicle production Pellicle No pellicle Thick pellicle
revert back to wild type when grown statically in broth culture. Relative cellulose 1 .o 0 . m . 1' 5.0-6.0
The most likely explanation for such behavior is that these are production (wet
weight)
not true genetic mutants, but instead represent alterations in
the phenotypic expression of genetic sequences involved in a
The trace amounts of cellulose produced in these pellicledeficient cultures
cellulose synthesis. These phenotypic mutants may be due to are thought to be cellulose II.
changes in gene expression, e.g., the repression of cellulose
synthesis genes during growth under aerated conditions, alter- will determine if these genetic changes represent mutations in
ations in the cell walkell membrane components induced by the structural or regulatory genes involved in cellulose bio-
the culture conditions, or other nongenetic events. A variety synthesis or pleiotropic mutations, e.g., those affecting the
of mutagens have been used in an effort to induce mutations lipopolysaccharide composition of the cell membrane.
in Acetobacter xylinuh. Nitrosoguanidine, ethyl methane sul- The existence of indigenous plasmids in A. xylinum was first
fonate, and nitrous acid are very effective mutagens for A . described by Valla et al?' They examined the plasmid content
xylinum, whereas hydroxylamine and ultraviolet light are rel- of a wild-type strain (ATCC 10245) and 13 cel- mutants de-
atively ineffective. 12.37*39 Cellulose-negative mutants can be rived from the wild type and showed that A. xylinum has a
identified by altered colony morphology (mucoid vs. rough), complex pattern of plasmids ranging in size from 16 to 300
fluorescence on agar plates containing tinopal or calcofluor, kb. A significant number of the cel- mutants (8/13) had iden-
and/or inability to produce a cellulose pellicle in liquid culture. tifiable changes in plasmid content. These changes included
In addition to cel- mutants, cellulose overproducer mutants both the loss of specific plasmids as well as apparent rear-
have also been isolated. These strains produce cellulose with rangements of plasmid sequences. A further analysis of cel-
the same characteristics as wild type, but at five to six times and transposon (Tn l)-induced mutants confirmed the complex
the wild-type level resulting in a thickened pellicle (Table 3). nature of the Acetobacter plasmid ~ o n t e n t . ~This
' series of
There has been little characterization of the overproducer strains mutants showed changes in plasmid content, changes in plas-
to date. Further study of these mutants as well as cel- mutants mid copy number, and often rearrangements between plasmid

442 Volume 17, Issue 6


Microbiology

and chromosomal sequences. Valla et al. were unsuccessful in different plasmid species. Because some wild-type, cellulose-
attempts to cure A . xylinum of any of these indigenous plasmid producing strains contained no plasmid sequences, they also
species. Their results were consistent with both plasmid genes concluded that plasmid genes probably have little involvement
being involved in cellulose synthesis and the existence of es- in cellulose synthesis.
sential genes on Acerobucfer plasmids. Acefobucter can act as both a recipient and donor for the
We have recently analyzed the plasmid content of another conjugative transfer of broad host range plasmids from incom-
wild-type strain of A . xylinum (ATCC 23769).42In contrast to patibility groups P and Q.4345 While plasmid sequences can
A . xylinum ATCC 10245, this wild-type strain has only a sin- be transferred between Escherichiu coli and A. xylinum or
gle, low copy number plasmid of approximately 58 kb. Com- between two A . xylinurn strains, there is no evidence of the
parison of the plasmid content of this strain with a series of mobilization of chromosomal genes during conjugation. The
nitrosoguanidine-induced,cellulose-negative mutants showed conjugative transfer of plasmids has been useful in the intro-
no gross differences in plasmid content (Figure 8). Restriction duction of transposons such as Tn 1 and Tn 5,~ which can
Critical Reviews in Microbiology Downloaded from informahealthcare.com by East Carolina University on 09/05/13

digestion analysis with a variety of enzymes revealed only a then be used to tag and isolate specific genes. In addition,
few minor differences in these plasmid species. We were un- conjugation has provided a means to introduce cloned DNA
able to cure this plasmid with a variety of known plasmid curing sequences into Acerobucferand, by complementation of known
agents, e.g., ethidium bromide, naladixic acid, mitomycin C, mutants, has led to the identification of the UDPG-pyrophos-
and sodium dodecyl sulfate. Our results suggest cellulose syn- phorylase gene.* Transformation methodologies described for
thesis is not necessarily linked to the presence or absence of other Gram-negative organisms have not proved successful for
plasmids in strain ATCC 23769. A . ~ylinum.~.~However, a transformationprocedure and clon-
Saxena and have also examined the plasmid content ing vectors for the related species A . uceri have been de-
of a variety ofA. xylinum wild-type strains. The various strains scribed,49 and the possibility exists that a variation on this
had remarkably different plasmid content, with 0 to 10 plasmids method may be successful with A . xylinum. As an alternative
found per strain, and sizes ranging from 2 to 230 kb. Southern to the above methodologies, we have recently demonstrated
blot analysis revealed little sequence homology between these that high voltage electroporationS0can be used to transform A.
For personal use only.

WT cel-4 cel-10 cel-13 cel-15 cel-20

plasmid

chr

FIGURE 8. The plasmid content of an Acerobacter qlinum wild-type strain (ATCC 23769) and cellulose-
negative (ce1-4, cel-10, cel-13, cel-15, and cel-20) rn~tants.~

1991 443
Critical Reviews In

xylinum with broad host range plasmids (pUCD2 and pRK host cell. Much of what is known concerning cellulose syn-
248). Transformation efficiencies of approximately lo7 colo- thesis in this organism centers around the role of cellulose in
nies per microgram of plasmid DNA can be achieved with this
electroporation. Therefore, as genetic exchange mechanisms A variety of mutants have been isolated that affect the ability
are further developed and improved, our understanding of the of Agrobacrerium to attach to plant cells. Included in this group
genetics of A. xylinum should increase. afe mutants deficient in cellulose produ~tion~.~as well as those
Recently, two genes encoding enzymes involved in cellulose still capable of cellulose ~ynthesis.~ Further analysis of these
biosynthesis, UDPG-pyrophosphorylaa and the catalytic mutants has led to the proposal that certain low molecular
subunit of cellulose synthase, have been cloned. The UDPG- weight glucans are important in the initial attachment of bac-
pyrophosphorylase gene was isolated from a partial library of terium to plant cell, whereas the synthesis of cellulose fibrils
Acerobacrer DNA by its ability to complement both cel- mu- promotes both the anchorage of the bacterium to the plant cell
tants of A. xylinum and a galU mutant of E. coli. Cellulose- surface and the formation of bacterial aggregates. Genetic anal-
Critical Reviews in Microbiology Downloaded from informahealthcare.com by East Carolina University on 09/05/13

negative mutants that received recombinant plasmid pVK ysis has indicated that many genedgene products are involved
lOO(240) were able to synthesize cellulose at the wild-type rate in cellulose synthe~is~..~ and that the majority of genes map
and had near wild-type levels of UDFG-pyrophosphorylase. to chromosomal sites. In fact, many genes are clustered, sug-
Assays for diguanylate cyclase, phosphodiesterase A, phos- gesting that a specific region of the Agrobacrerium chromo-
phoglucomutase, UDPG-pyrophosphorylase, and cellulose some is concerned with the interaction of the bacterium with
synthase showed that the three cel- mutants that could be the plant host cell. What remains to be determined is if the
complemented with pVK lOO(240) were deficient in only clustering of genes is necessary for the coordinate expression
UDPG-pyrophosphorylase activity. The 2.8 kb Hind III frag- of these sequences and if other cellulose-synthesizing organ-
ment cloned in pVK lOO(240) also complemented an E. coli isms have a similar genetic organization.
galU mutant containing a structural gene mutation in UDPG- In fact, recent work by Wong et al. suggests that at least
pyrophosphorylase. Valla et al.& have suggested celA, as the some of the genes involved in cellulose synthesis in Acero-
designation for this gene. bacter xylinum are also organized in an operon (bcs, bacterial
Saxena et aLS1used a different strategy to isolate the gene
For personal use only.

cellulose synthesis). These genetic sequences were localized


for the catalytic subunit of cellulose synthase. They used a to a segment of chromosomal DNA that could complement
synthetic oligonucleotide probe homologous to the amino ter- . cellulose-negative strains deficient in cellulose synthase. The
minus of this 83 kDa polypeptide to ident@ and clone a 9.5 operon contains at le& four protein-coding regions (bcsA,
kB Hind III fragment of Acerobacter DNA. Sequence analysis bcsB, bcsC, and bcsD), with the catalytic subunit of cellulose
has shown that this DNA fragment contains an open reading synthase encoded by the bcsB region. The functions of the
frame sufficient to code for a 80 kDa polypeptide. In addition, other genetic sequences are not known, but their expression
the amino terminal protein sequence deduced from the nucleic (along with bcsB) appears to be necessary for maximal cellulose
acid sequence provides a match to the 83 kDa subunit, indi- synthesis in vivo.
cating that in all likelihood it is the catalytic subunit for cel-
lulose synthase.
The genetic analysis of Acerobacter has provided a number VII. ECOLOGICAL ASPECTS OF
of interesting observations including (1) the existence of a CELLULOSE PRODUCING BACTERIA
complex pattern of plasmids; (2) the utility of chemical and
transposon mutagenesis to induce cel- mutants; (3) the ap- Because A. xylinum was observed to produce a pellicle dur-
parent genetic instability in this organism, e.g., the high rate ing Static culture, and since it was considered to be a strict
of induction and reversion of spontaneous mutants, chromo- aerobe, investigators proposed that the role of the pellicle was
somal and plasmid reakangements; and (4) the use of recom- to hold the organisms in an aerobic environment to facilitate
binant DNA technology to isolate genes involved in cellulose their ability to obtain ~ x y g e n . ~ ~
Could
. ~ there be other eco-
synthesis. The ability to clone the genes involved in cellulose logical roles for cellulose secreted by A. xylinum?
biosynthesis will not only provide a better understanding of Undried cellulosic pellicles retain water well. Thus, one
this process but also lead to potential commercialization of hypothetical role for cellulose could be moisture retention in
microbial cellulose production. the microenvironment to allow for bacterial growth and prevent
Because Agrobacterium tumefaciens can cause disease in a dessication. Also, our experiments cultivating A . xylinum in
wide variety of plants, it has been the object of study and candle jars, 5% carbon dioxide gassed enclosures, and Biobags
genetic analysis for quite some time.52Much attention has been show that A. xyxylinum can grow microaerophilically while con-
paid to the genetic interactions between bacterium and plant tinuing to synthesize cellulose,39 so the argument that the pel-
cell in the development of crown gall tumors. One aspect of licle is vital for aerobic growth is no longer as compelling.
this interaction is the specific attachment of bacterium to plant Laboratory experiments also indicated that pellicles could pro-

444 Volume 17, Issue 6


Microbiology

tect bacteria from the killing effects of ultraviolet light. This are four genera of Gram-negative bacteria that synthesize cel-
protective effect could be of considerable importance to the lulose. Sarcina is the sole genus of Gram-positive bacteria to
organism as it grows on decaying fruits just as it could be do so. The quality or kind of cellulose synthesized varies among
holding water for the biochemical degradation of substrates for the various microbes from the long microfibrils that form into
nutrition. Since A. xylinum is found primarily on decaying pellicles for Acetobacter to the short cellulosic fibrils of Agro-
fruits, could it be possible that cellulose pellicle synthesis plays bacterium and Rhizobium, which permit bacterial attachment
a role in successful colonization of a particular habitat? To to plant tissue. Pseudomonas and Sarcina secrete cellulosethat,
answer this question, a series of experiments were performed while crystalline in form, is more amorphous in structure.
in which pieces of apple were inoculated with various strains Cellulose producers are found among both prokaryotes and
of cellulose-producingand celluloselessA . xylinum, and assays eukaryotes; thus one assumes a possible evolutionary relation-
were done to determine density of various organisms growing ship in the biosynthetic processes of bacterial and higher or-
on the apples. Celluloseless and uninoculated controls tended ganisms. Brownlo has speculated that Sarcina was probably
the first cellulose producer. With the evolution of aerobiosis,
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to become dry over the course of the experiment. Molds, fungi,


and other bacteria tended to predominate on the pieces of apples came the other bacterial cellulose producers that are Gram-
that had been inoculated with the cellulose-negative strain. negative. The genetic information for cellulose synthesis by
When pellicles from a wild-type and a cellulose-over-producing eukaryotes probably came via an endosymbiotic mechanism
strain completely covered the pieces of apple, A. xylinum pre- from prokaryotes. When nucleic acid andlor protein sequence
dominated over other microbes growing on the fruit. These information becomes available for cellulose synthases of bac-
results indicate that the ability to synthesize a cellulosic pellicle teria and higher organisms, the relationships among these var-
may allow A. xylinum to colonize food sources more effectively ious cellulose synthesizers will become clearer.
than strains of A. xylinum that do not make cellulose. This Cellulose fibrils of Acetobacter xylinum are the most highly
colonization ability may be important in the microenvironment organized of those produced by all currently identified bacteria.
as A . xylinum seeks nutrients in competition with other de- The fibrils are synthesized at or near the cytoplasmic membrane
composing organisms such as other bacteria and fungi. probably from UDP-glucose via cellulose synthase that requires
Cellulose fibrils have also been implicated in the infection a cyclic diguanylic acid activator. The molecules of glucose
For personal use only.

process of plant tissue by Agrobacterium tumefaciens, which that comprise the fibrils are polymerized as they pass through
is another example of the need of a microorganism to use an the various membranes of the Gram-negative cell, possibly
exopolymer to hold to a particular substrate.8 Cellulose fibrils with final extrusion through pores in the outer membrane. The
anchor bacteria to plant surfaces. Cellulose-negative mutants fibrils have the capacity to form into a highly ordered and
of Agrobacterium produced by transposon mutagenesis secrete complex structure that under scanning electron microscopy re-
cellulose fibrils so no bacterial aggregates form on plants even sembles tunnels. The function of this complex extracellular
though the bacteria are still virulent. The ability of cellulose- matrix may be as simple as to hold the bacteria in the aerobic
negative mutants to form tumors at the site of inoculation was environment or it may serve a diversity of functions such as
decreased if the site was rinsed with water. The absence of protection from ultraviolet light, retention of moisture, and
cellulose fibrils apparently reduced the ability of the bacteria colonization of substrates.
to anchor and eventually infect plant tissue. This past decade has seen an explosion of information about
Cellulose fibril formation to enhance flocculation in acti- the biology of bacterially synthesized cellulose, but there is
vated sludge permits the aggregated growth of a variety of still much that we do not know. In the future, we should know
Gram-negative bacteria. The ability to form a floc could allow more about evolutionary relationships between prokaryotic and
for enhanced microbial growth by giving cells a surface upon eukaryotic cellulose biogenesis. Further ecological analyses are
which to cling while they are participating in the process of needed to understand the role(s) of cellulose for the bacteria
wastewater decomposition. in their natural habitats. A better understanding of the actual
To be successful in the environment, many microorganisms synthesis process as it occurs in the cell membrane may even-
have evolved efficient mechanisms to colonize surfaces whether tually permit the large scale in vitro synthesis of cellulose and
it be the bottom of a ship, the surface of teeth, or decaying provide potentially an important alternative source of this nat-
fruit. Colonization typically has involved production of exo- ural polymer. Detailed genetic analyses will increase our
polysaccharides that aid in attachment, act to prevent drying, knowledge of the basic mechanisms of cellulose synthesis and
pH changes, and effects of toxic molecules upon the colonizer. may eventually lead to many commercial applications of bac-
Cellulose is a highly organized exopolysaccharide, which may terial cellulose.
Play a role-similar to exopolymers secreted by other bacteria.
ACKNOWLEGMENTS
VIII. SUMMARY AND CONCLUSIONS
The authors wish to acknowledge the editorial assistance of
Acetobacter, Agrobacterium, Pseudomonas, and Rhizobium Barbara Randolph-Anderson and Janne Cannon and the sec-
1991 445
Critical Reviews In

retarial help of Pat McCarron. They also appreciate the will- methylcellulose and other cellulose derivatives, J. Cell B i d , 94, 64,
ingness of authors to provide permission for use of some of 1982.
the figures. Some of the authors research was supported by 17. Kai, A. and Kitamura, H.,The structure of cellulose produced by
Acetobucrer xylinum in the presence of a fluorescent brightener. The
the Research Council of the University of North Carolina at
influence of concentrationof a brightener in the medium on the structure
Greensboro. of cellulose, Bull. Chem. SOC.J . . 58, 2860, 1985.
18. Haigler, C. H. and Chanzy, H., Electron diffraction analysis of the
altered cellulose synthesized by Acetobucter xylinwn in the presence
of fluorescent brightening agents and direct dyes, 1. Ultrustruct. Mol.
S t r u t . Res., 98, 299, 1988.
19. Gretz, M. R., Folsome, D. B., and Brown, R. M., Jr., Cellulose
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446 Volume 17, Issue 6


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1991 447