Critical Reviews in Microbiology

ISSN: 1040-841X (Print) 1549-7828 (Online) Journal homepage: http://www.tandfonline.com/loi/imby20

Biogenesis of Bacterial Cellulose Robert E Cannon & Steven M. Anderson To cite this article: Robert E Cannon & Steven M. Anderson (1991) Biogenesis of Bacterial Cellulose, Critical Reviews in Microbiology, 17:6, 435-447, DOI: 10.3109/10408419109115207 To link to this article: http://dx.doi.org/10.3109/10408419109115207

Published online: 25 Sep 2008.

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Microbiology

Biogenesis of Bacterial Cellulose Robert E. Cannon, Ph.D. and Steven M. Anderson, Ph.D.

ABSTRACT

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Cellulose is the most abundant biological polymer on Earth. It is found in wood and cotton, and forms the basic structural foundation of the cell wall of almost all eukaryotic plants. Bacteria are known to secrete cellulose as part of their metabolism of glucose and other sugars. The focus of this review is upon bacterial cellulose synthesis. We emphasize recent literature directed primarily upon Acefubucferxylinum, which has been most widely studied. Our review covers the following topics relating to cellulose synthesis: genetics, biochemistry, ultrastructure, growth conditions, and ecological considerations as they relate to the diversity of microbes capable of synthesizing this abundant, unique polymer - cellulose.

1. INTRODUCTION

*

Cellulose is the most abundant natural polymer on Earth. It forms the cell wall of eukaryotic plants and algae, and is also found to be the major constituent of the cell wall of fungi. Cellulose is also synthesized by bacteria, especially by the Gram-negative bacterium Acetobarer xylinum, which is the primary focus of this review. This microorganism has been the most widely studied of the few bacteria that produce cellulose, although a number of other bacterial cellulose producers are discussed briefly. The following topics are covered: survey of cellulose-synthesizing bacteria, growth and cultivation requirements, ultrastructure of bacterial cellulose synthesis, biochemistry of cellulose synthesis, genetics of biosynthesis, and ecology of cellulose producers. Cellulose which is synthesized by bacteria as a secondary metabolite is a polymer of glucose. The molecules of glucose are linked together as I+4p-glucan chains. Cellulose synthesized by A. xylinum is polymerized into long microfibrils outside of the cell wall and is of the same chemical constituency as the cellulose made by higher plants. This similarity makes A. xylinum an ideal model system to study the cellulose biogenesis process. The synthetic pathways of many glucose polymers are fairly well understood and the synthase enzymes 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

has been revealed. Since bacterially synthesized cellulose is chemically identical to that of higher plants and other organisms, this may suggest a probable common evolutionary origin 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 biochemistry of cellulose biosynthesis. Could the cellulose produced by bacteria like A. xylinum be put to productive use commercially? Will it replace or supercede the cellulose that we now get from trees for paper or cotton plants for fiber? Probably not, but bacterial cellulose does have some advantages over that produced by other organisms. One of the advantages of Acetobucfer cellulose is that it does not need to be delignified before processing as is the case with woody plant cellulose that is processed into paper. A . Xylinm probably has the greatest potential for commercialization since other cellulose-synthesizing microbes do not product sufficient quantities of the cellulose polymer for industrial purposes. Acetobucfercellulose holds water well when left undried and has strong tear resistance. It can assume or be woven into various shapes and it retains its shape well. One current example of an application for bacterial cellulose comes from the S O W Corporation, which uses cellulose produced by A . xylinum to make sensitive diaphragms for stereo headphones. Microbial cellulose is also available as a food product called Nata in the Philippines.* Weyerhauser and Cetus Corporation have recently developed a strain of Acerubucter that is capable of large-scale cellulose production under fermentative conditions with agitati~n.~ The fibers produced termed “Cellulon”, have a number of potential commercial applications, such as binders for ceramic powders and minerals, thickeners for paint, ink, adhesives, and even foods. Another possible use for Cellulon is as a paper coating because of its high purity. Also recently reported was the use of Acefubucrercellulose as a temporary skin substitute to protect burned tissue as normal skin regenerate^.^ This is the first example of specific medical applications of bacterially produced cellulose. The cellulosic material, identified by the trademark “BioFill”, has been used successfully for treatment of severe bums, skin grafting, and chronic skin ulcers. Advantages of this treatment were pain relief, good adhesion, an effective barrier to infection, fast healing, good fluid (water and electrolyte) retention, low cost, and short treatment time as normal, healthy skin grows to replace the artificial cellulosic skin substitute. A potential difficulty to human use of cellulose may be the need to remove

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Critical Reviews In endotoxic components that may contaminate bacterial cellulosic products. Our knowledge of the process of cellulose synthesis carried out by bacteria exemplified by A . xylinum has increased greatly over the past decade. This basic research has formed the foundation for future attempts at potential commercialization of bacterially produced cellulose, which are currently in their formative stages.

II. SURVEY OF CELLULOSE-PRODUCING

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BACTERIA Many Gram-negative bacteria secrete extracellular polysaccharide material, but only a few have been shown to produce cellulose (Table 1). A . xylinum, the most studied of bacterial cellulose producers, is a Gram-negative, aerobic, rod-shaped organism. It is classified in Bergey’s Manual of Systematic Bacteriology (Volume 1, 1984) in Section 4 -Gram Negative Aerobic Rods and Cocci5 It is a member of the family Acetobacteraceae. Species within the genus, Acerobacrer include A . aceti, A . liquifaciens, A . pasteurianis, and A . hansenii. A . 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 . xylinum will be used throughout to indicate the primary, cellulose producing bacterium. Members of the genus Acetobacter are chemoorganotrophscommonly producing pale colonies when grown on solid media. A prominent characteristic of cellulose-

producing Acetobacters is production of a floating cellulosic pellicle when the organisms are cultured statically. The pellicle forms from the organization of cellulose fibrils synthesized and secreted by the bacterium. Many strains oxidize ethanol to acetic acid. They are ubiquitous and commonly found on decaying fruits and vegetables, vinegar, fermented beverages, sugar cane juice, garden soil, etc. Other bacteria that produce cellulose in lesser quantity than A . xylinum include Sarcina ventriculi, which is the sole Grampositive, cellulose producer.6 Also, a number of bacterial species have been found to produce exocellular cellulose fibrils when isolated from activated sewage sludge.’ The cellulose fibrils were believed to account for floc formation. The specific genera isolated included Pseudomonas, Achrombacrer, Alcaligenes, Aerobacter, Rhizobium, Agrobacterium, and Azotobacter. Cellulose fibril formation apparently plays an important part in the infection of plants by Agrobacterium tumefaciens by aiding in attachment of bacteria to plant tissue eventually leading to gall formation.8 A more detailed discussion of the possible role(s) of cellulose for the bacteria that produce it is presented in the ecology section of the review.

111. BACTERIAL GROWTH CHARACTERISTICS, REQUIREMENTS, AND IDENTIFICATION OF CELLULOSE The undefined medium that is characteristically used to cul-

Table l Survey of Bacterial Cellulose Producers Organism (genera) Acetobacter

Extracellular pellicle’ Cellulose ribbons

Agrobacterium

Short extracellular fibrils

Rhizobium

Short extracellular fibrils

Pseudomonas

No distinct fibrils

Sarcina

Amorphous celluloseb

a

436

Cellulose produced

Cellulose assay

Biological Role

Cellulose synthase X-ray diffraction Alkali insolubility Fluorescent brightener Degradation by cellulase X-ray diffraction Alkali insolubility Fluorescent brightener Degradation by cellulase Same criteria as Agrobacterium Alkali insolubility Degradation by cellulase Alkali insolubility

To hold in aerobic environment To colonize natural substrates

Attach to plant tissue

Attach to host plants Flocculation in wastewater

Unknown

Cellulose I - highly crystalline, metastable microfibrils, native cell~lose.~ Cellulose 11 - crystalline allomorph of Cellulose I with antiparallel glucan chains, rare in nature.”

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Microbiology the microfibrillar product is cellulose rather than other noncellulosic polysaccharides.l3 Fibrils of cellulose are extremely strong, and insolubility in alkali (2% NaOH) has therefore been used as a criterion for presence of cellulose. However, other glucose-based polymers (e.g., p-I ,2-D-glUCan,p-l,3-D-ghlcan) are also insoluble in base and therefore one should be careful in using this as the sole criterion for demonstration of cellulose production. Also, the crystalline structure of the polymer is resistant to treatment by a combination of acetic and nitric acid. Cellulose gives a characteristic diffraction pattern upon X-ray crystallography, which is the ultimate method demonstrating its presence. Several inhibitors have been identified that interfere with cellulose synthesis.l4 These include coumarin and dichlorobenzonitrile, which apparently disrupt glycosylation in the synthesis process. In addition, substances called fluorescent brighteners, e.g., Calcofluor White ST or Tinopal LPW, have been used to confim cellulose production. When these stilbene derivatives react with the glucan chains of cellulose by hydrogen bonding, they prevent normal microfibril formation and assembly of cellulosic ribbons by A. ql i n ~ r nDespite .~ the disruption of normal ribbon formation, the binding of these substances causes the bands of cellulose to fluoresce brightly when viewed under ultraviolet light. It is possible to incorporate the fluorescent brighteners into the solid medium. Colonies of A . xylinum and other cellulose-synthesizing bacteria, which are actively synthesizing cellulose fluoresce, while cellulose-negative strains do not. Unfortunately, these brighteners bind to other exopolysaccharides, so additional tests must be done to c o n f m that cellulose has indeed been synthesized by the bacteria.

ture Acetobacter qlinum to maximize both growth and cellulose production was developed by Hestrh and Schramm (Table 2)." Other carbon sources have been shown to be suitable for growth, but they have not been as useful for production of cellulose. A . xylinum grows well over a temperature range of 25 to 30"C, but its temperature optimum is 30°C. The pH of the undefined medium is 6. Historically, to maximize cellulose production, A. xylinum has been cultured statically allowing for the formation of a pellicle composed of cellulose holding within it bacterial cells floating on the surface of the medium. If cultures are aerated by shaking, bacteria grow faster, but produce less cellulose. Rotary shaking results in production of round balls of cellulose rather than the flat, sheet-like pellicle of static cultivation. Doubling time for A. xylinurn held in static culture is between 8 and 10 h, while in aerated culture (by shaking), the organism doubles every 4 to 6 h. When A. xyh u m is cultured on solid medium, colonies that are formed by cellulose-producing strains have a dry, wrinkled appearance. This contrasts to celluloseless strains (cellulose-negative mutants), which appear as smooth, spreading colonies that are usually two or three times the size of cellulose-synthesizing strains after a comparable period of growth.

Table 2 Undefined and Synthetic Media for Cultivation of Acetobacter xylinum Undefined" Glucose Yeastextract Peptone

Synthetic'*

Sodium

2.0% 0.5% 0.5% 0.27%

Glucose NH.,Cl Citric acid N%HPO,

1.0% 0.1% 0.115% 0.33%

phosphate Citric acid

0.115%

KCI MgSO, * 7H,O Nicotinamide

0.01% 0.025% 7.5 mg/I

IV. ULTRASTRUCTURE AND MODEL FOR CELLULOSE SYNTHESIS The ultrastructure of the cellulose synthesis apparatus is best understood in A. xylinurn. Transmission electron microscopy and freeze-etching have revealed the presence of a row of pores on the longitudinal axis of cells. These particles or pores may contain or be composed of the last enzyme(s) involved in cellulose synthesis, and glucan chains that eventually form into the composite ribbon of cellulose may be secreted through these pores.9 Figure 2 is a depiction of this hypothetical model. Each cell that is actively synthesizing cellulose produces a cellulosic ribbon ranging in width from 40 to 60 nm, which appears to be parallel to the longitudinal axis of the bacterial cell. Although A. xylinum is nonmotile, it can be pushed by the formation of the ribbon of cellulose at a rate of 2 pdmin. The ribbon of cellulose is composed of microfibrils, which are the form in which cellulose is secreted through extrusion sites in the outer membrane of the bacterium. These microfibrils appear to be about 1.5 nm wide and aggregate into 3 to 4 nm microfibrils via crystallization of adjacent glucan chains. Finally, these microfibrils band together to form the larger cellulosic

To be able to manipulate growth and know exact chemical constituents, we have developed a synthetic minimal medium for A. xylinurn that permits good growth (Table 2, Figure 1) and maintains cellulose production.'2 The synthetic minimal medium is useful for genetic manipulation of A. qlinum because it can be used to isolate auxotmphic mutants. A culture grown in minimal medium with 1% glucose added produced about half as much cellulose as one in the undefined medium, at a slightly slower growth rate. If any of the ingredients of the medium were omitted, no growth occurred. The synthetic medium was used successfully to isolate a number of amino acid- and nucleotide-requiring auxotrophs after mutagenesis of A . xylinum 'by N-methyl-N'-nitro-N-nitrosoguanidine. To insure that the biosynthetic product made by A. xylinurn is cellulose, a number of different techniques are employed. The techniques vary in complexity and accuracy, and it may be necessary to rely on a number of methods to be sure that 1991

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I

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to’

e

- M a 8 Yedlum (Undellned) Ylnlmel Olucosa w - YInImal 211) Olusoae - Ylnlnel 0.6% .=-a - Ylnlmal 0.2611) O I U C O ~ ~ c.l

I

--+ -

‘111) +

A--+

OIUCOS.

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.’*

and Bundle Initiation LPS envelop.

flGURE 2.

Possible model of cellulose assembly. Glucan chain aggregation

requires multienzyme complexes followed by crystallization of microfibrils

through extrusion pores. Ribbons of cellulose form at the cell ~urface.~

ribbon. Figure 3 shows the presence of the extrusion pores on the side of an A. xylinum cell, a typical ribbon secreted by a cell, and a more highly magnified view of individual ribbon^.^ Bureau and Brown” have demonstrated that cellulose synthesis occurs in the cytoplasmic membrane of A. xylinum rather than in the outer membrane as had been previously thought. Final crystallization of microfibrils may still occur in or near the outer membrane and be dependent upon the ordered nature of the extrusion pores that are seen in freeze fracture preparations of cells. The enzymes involved in the terminal stages of cellulose fibril biogenesis are cell-associated, probably within either inner or outer membranes of the Gram-negative bacterium (see biochemistry section). It is not completely understood how the individual glucan molecules polymerize and assemble into the microfibrillar bundles of cellulose. Much of what is known

438

about these processes comes from studies employing fluorescent brighteners and cellulosic derivatives that alter ribbon formation. Fluorescent brighteners such as Calcofluor bind to 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 bundles of microfibrils so that individual microfibrils remain separated, never forming into highly organized fibrils. More recently, Kai and Kitamura” and Haigler and Chanzy’* have used techniques of X-ray and electron diffraction to study the effects of fluorescent brighteners upon cellulose structure in A. xylinum. Kai and Kitamura reported that brighteners do not effect the extrusion of monomolecular layers of cellulose I from cells. At high concentrations of brightener, a diffraction pattern resembling neither cellulose I nor II appeared. As brightener concentration was lowered, the cellulose I pattern returned. Haigler and Chanzy, using different washing methods than Kai and Kitamura to prepare cellulose samples prior to diffraction techniques, did not observe altered cellulose. They speculate that the harsh treatment with alkali and alcohol used by Kai and Kitamura might have introduced some form of crystallinity. Haigler and Chanzy’s results also showed that cellulose I could be reformed from amorphous cellulosic material that is produced after dye treatment. In the final analysis, when polymerization and crystallization processes are disrupted, normal microfibrillar ribbons of cellulose cannot form. Strong magnetic field strength has also been shown to alter

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Microbiology

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 polymerization 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 are unknown at present.19 The overall model to account for cellulose ribbon assembly has been described as "hierarchical" by a number of investigators. First, small glucan chains aggregate by a self-assembly mechanism into the 3 to 4 nm microfibrils. This event is followed by banding of the microfibrils into bundles, which then form into the complete ribbon. The high degree of organization of the extrusion pores on the surface of the outer membrane facilitates the coordination of the assembly process. An even higher degree of organization of cellulose fibrils can be observed when whole, washed pellicles are viewed under scanning electron microscopy. Structures resembling tunnels, which would indicate a considerable degree of regularity in the organization of microfibrils, appear." These structures provide an even higher level of fibril orientation than ribbon formation described above and argue that synthesis of cellulose by A. xylinum is not occumng randomly. The diameter of tunnels is about 7 pm, with sufficient room for bacteria to move through them.

V. BIOCHEMISTRY OF CELLULOSE SYNTHESIS Even though cellulose is a relatively simple polymer, we still do not completely understand the biochemistry of its synthesis. Difficulties in both development of an in v i m assay for cellulose synthesis and purification of the enzymes involved in this process (e.g., cellulose synthase) have hindered biochemical analyses. These problems are being overcome through use of combined genetic and biochemical techniques with the organism A. xylinum, and a clearer picture of the biochemical steps in cellulose synthesis is beginning to emerge. In addition to glucose, other substrates such as hexoses, hexonates, pyruvate, glycerol, and dihydroxyacetone can be used in the biosynthesis of cellulose.z'-23Many of these substrates are metabolically associated with either the pentose or citric acid cycles, indicating that cellulose synthesis may be tied to oxidative carbohydrate metabolism." However, since cellulose formation in Acetobucter can occur independently of

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Critical Reviews In protein synthesis,25it does not appear to be an indispensable feature of carbohydrate metabolism. Knowledge regarding the proposed biochemical pathway leading from exogenous glucose to cellulose has come from two sources: (1) studies on the enzymes involved in glucosecarbohydrate 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. xylinum.14*24*26*27 Within this group of enzymes, it has been proposed that glucokinase, phosphoglucomutase, and UDF'Gpyrophosphorylase are most likely to be involved in the pathway leading to cellulose synthesis (Figure 4). Glucokinase Glucose- - - - - - -

>

'OO

.

h

Cellulose

Glucose

,

\

Water soluble

*A-A

Phosphoglucomutase Glucose-6-phosphate-- -- -- - -- - - - - - >

A

Alkali rolubk

--

Chloroform soluble

A

4 -

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 carbohydrate via the pentose and citric acid cycles. Phosphorylation 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 provided, whereas fructokinase activity, like that of other sugarmetabolizing 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, phosphorylated glucose in the form of glucose-6-phosphate (G6P) is enzymatically converted to glucose-1-phosphate (G1P) by phosphoglucomutase.24 UDPG-pyrophophorylase is then responsible for the synthesis of UDP-glucose from GlP and UTP.= Even though all four nucleoside sugars may act as precursors for cellulose synthesis, there is no evidence for any other nucleoside diphosphoglucose pyrophosphorylase in A. xylin~m.'~ This observation is also consistent with UDP-glucose acting as primary precursor for cellulose synthesis. This proposed pathway has been substantiated through the use of isotopic-labeling studies in which the flow of carbon from I4Cglucose to celrulose has been examined.28After pulse-labeling A. xylinum cells, the incorporation of label into glucose-phosphate and UDP-glucose was determined. The results of this study are presented in Figure 5 and support the roles of G6P, G1P, and UDP-glucose as precursors for cellulose synthesis.

440

1

5 . - 15

30

45

60

Time (minutes) FIGURE 5. The time course for a pulse-labeling experiment during which cellulose is synthesized from "C-glucose by wild-type Acetobocter xylinum cells. Incorporation of the label into both cellular fractions (e.g., chloroform-,water-, and alkali-soluble fractions) as well as specific cellular components (e.g., UDP-glucose) is demonstrated."

Labeled G6P, GlP, and UDP-glucose appear shortly after cells are exposed to L4C-glucoseand their subsequent decrease is linked to an increase in the incorporation of I4C into cellulose, e.g., an alkali insoluble cell fraction.

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Microbiology UDP-glucose is the substrate for cellulose synthase (E.C. 2.4.1.12, UDP-Glucose: 1,4-~-~-glucan4~-D-,olucosyltransferase) that is believed to be the major enzyme involved in the last step(s) of cellulose biosynthesis. The development of both an in vitro assay and a purification protocol for cellulose synthase 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 UDPglucose by a particulate fraction from A . xylinum. Unfortunately, the rate of synthesis was 90% homogeneity) of cellulose synthase from detergent solubilized membranes. A 96-fold purification was obtained after two rounds of a cellulose entrapment purification protocol. This involves adding substrate and enzyme activators to a detergent-solubilized membrane preparation and trapping the enzyme by centrifugation of the synthesized cellulose product. Their preparations contain two major polypeptide products of 83 and 93 kDa. The 83 kDa polypeptide has been demonstrated to be a glycoprotein by lectin affiiity chromatography, Schiff periodate, and fluoroscein isothiocyanate-Concanavalin A staining. Column chromatography results were consistent with cellulose synthase being a multimeric enzyme. Lin and Brown suggest that the 93 kDa polypeptidechain may represent a second subunit of the multimeric complex. More recently, Lin et al.33have demonstrated by photoaffinity labeling with an analog of UDP-glucose that the 83 kDa subunit is the catalytic subunit for cellulose synthase. In a membranous or detergent-solubilized extract this analog, [(P-32P)-5N3-UDPglucose], would photoincorporate into both 57 and 83 kDa polypeptides. Further analysis using competition assays with UDP-glucose have corroborated that the active site for the enzyme is contained in the 83 kDa polypeptide. They suggest that the 57 kDa polypeptide is phosphoglucomutase,which has been labeled due to contamination of the photoprobe preparation with trace levels of 32P-Glucose-1-Phosphate. As mentioned earlier, cellulose synthase activity in in vitro preparations could be enhanced by the addition of guanosine nucleotides or analogs of guanosine. The work of Ross et al." has further demonstrated that the specific cellulose synthase activator is-bis - (3' * 5 ' ) - cyclic diguanylic acid depicted in Figure 6 . They suggested that cellulose synthase activity is controlled by a multicomponent regulatory system that includes (1) diguanylic cyclase, the enzyme involved in the synthesis of the cyclic diguanylic activator and (2) the activities of two

0

\

/O'

/p\

0/p\

0-

FIGURE 6. The structure of the diguanylic acid activator of cellulose synthase.y

phosphodiesterases, which are involved in the degradation of the activator. One phosphodiesterase is inhibited by Cat+ and hydrolyzes the opening of the cyclic nucleotide and the second, which is not affected by Ca2+, converts the dinucleotide into 5' GMF'. According to this model, the rate of polymerization of UDP-glucose into cellulose is dependent on the intracellular levels of diguanylic activator and the level of Ca2+. The interactions of these components is summarized in the model have recently presented in Figure 7. Amikan and Ben~iman'~ shown the possible involvement of the same activator in the modification/regulation of cellulose synthesis in Agrobacterium tumefaciens.This observation suggests that both organisms may utilize a similar regulatory mechanism, even though the final cellulose product produced (bundles and simple flocs in Agrobacterium vs. complex ribbons produced by Acetobacter) and the rate of cellulose synthesis are markedly different.

VI. GENETICS OF CELLULOSE SYNTHESIS Studies of the genes and gene products involved in cellulose synthesis have been canied out in both Acetobacter xylinum and Agrobacterium tumefaciens. The development of these model systems should provide not only an understanding of the genetics of cellulose biosynthesis in bacterial systems, but also an insight into this process in higher plants. The genetic analysis of cellulose biosynthesis in Acetobacter qdinum has included the isolation of mutants that affect cellulose production, characterization of indigenous plasmid species, and the cloning of genes involved in this-process. A number of researchers, beginning with Schramm and H e ~ t r i n , ~ ~

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Cellulose fibril

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 -) mutants. Both spontaneous and mutagen-induced variants have been isolated. Many reports have indicated that apparent spontaneous celluloseless mutants arise at a high rate when wildtype cells are grown in aerated liquid The frequency with which these mutants occur also increases with the age of the culture. The spontaneous mutants have a mucoid appearance on solid agar like true cel- mutants, but the majority revert back to wild type when grown statically in broth culture. The most likely explanation for such behavior is that these are not true genetic mutants, but instead represent alterations in the phenotypic expression of genetic sequences involved in cellulose synthesis. These phenotypic mutants may be due to changes in gene expression, e.g., the repression of cellulose synthesis genes during growth under aerated conditions, alterations in the cell walkell membrane components induced by the culture conditions, or other nongenetic events. A variety of mutagens have been used in an effort to induce mutations in Acetobacter xylinuh. Nitrosoguanidine, ethyl methane sulfonate, and nitrous acid are very effective mutagens for A . xylinum, whereas hydroxylamine and ultraviolet light are relatively ineffective. 12.37*39 Cellulose-negative mutants can be identified by altered colony morphology (mucoid vs. rough), fluorescence on agar plates containing tinopal or calcofluor, and/or inability to produce a cellulose pellicle in liquid culture. In addition to cel- mutants, cellulose overproducer mutants have also been isolated. These strains produce cellulose with the same characteristics as wild type, but at five to six times the wild-type level resulting in a thickened pellicle (Table 3). There has been little characterization of the overproducer strains to date. Further study of these mutants as well as cel- mutants

442

Table 3 Characteristics of Acetobacter xylinum Strains

I

Characteristic Colony morphology Fluorescence with Tinopal Pellicle production Relative cellulose production (wet weight) a

Wild type

Cel-negative

Small, rough Bright

Large, mucoid Dull

Small, rough Bright

Pellicle

No pellicle 0 . m . 1'

Thick pellicle

1 .o

Overproducer

5.0-6.0

The trace amounts of cellulose produced in these pellicledeficient cultures are thought to be cellulose II.

will determine if these genetic changes represent mutations in the structural or regulatory genes involved in cellulose biosynthesis or pleiotropic mutations, e.g., those affecting the lipopolysaccharide composition of the cell membrane. The existence of indigenous plasmids in A. xylinum was first described by Valla et al?' They examined the plasmid content of a wild-type strain (ATCC 10245) and 13 cel- mutants derived from the wild type and showed that A. xylinum has a complex pattern of plasmids ranging in size from 16 to 300 kb. A significant number of the cel- mutants (8/13) had identifiable changes in plasmid content. These changes included both the loss of specific plasmids as well as apparent rearrangements of plasmid sequences. A further analysis of celand transposon (Tn l)-induced mutants confirmed the complex nature of the Acetobacter plasmid ~ o n t e n t . ~This ' series of mutants showed changes in plasmid content, changes in plasmid copy number, and often rearrangements between plasmid

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Microbiology and chromosomal sequences. Valla et al. were unsuccessful in attempts to cure A . xylinum of any of these indigenous plasmid species. Their results were consistent with both plasmid genes being involved in cellulose synthesis and the existence of essential genes on Acerobucfer plasmids. We have recently analyzed the plasmid content of another wild-type strain of A . xylinum (ATCC 23769).42In contrast to A . xylinum ATCC 10245, this wild-type strain has only a single, low copy number plasmid of approximately 58 kb. Comparison of the plasmid content of this strain with a series of nitrosoguanidine-induced,cellulose-negative mutants showed no gross differences in plasmid content (Figure 8). Restriction digestion analysis with a variety of enzymes revealed only a few minor differences in these plasmid species. We were unable to cure this plasmid with a variety of known plasmid curing agents, e.g., ethidium bromide, naladixic acid, mitomycin C, and sodium dodecyl sulfate. Our results suggest cellulose synthesis is not necessarily linked to the presence or absence of plasmids in strain ATCC 23769. have also examined the plasmid content Saxena and of a variety ofA. xylinum wild-type strains. The various strains had remarkably different plasmid content, with 0 to 10 plasmids found per strain, and sizes ranging from 2 to 230 kb. Southern blot analysis revealed little sequence homology between these

WT

cel-4

different plasmid species. Because some wild-type, celluloseproducing strains contained no plasmid sequences, they also concluded that plasmid genes probably have little involvement in cellulose synthesis. Acefobucter can act as both a recipient and donor for the ’ conjugative transfer of broad host range plasmids from incompatibility groups P and Q.4345 While plasmid sequences can be transferred between Escherichiu coli and A. xylinum or between two A . xylinurn strains, there is no evidence of the mobilization of chromosomal genes during conjugation. The conjugative transfer of plasmids has been useful in the introduction of transposons such as Tn 1 and Tn 5,”~” which can then be used to tag and isolate specific genes. In addition, conjugation has provided a means to introduce cloned DNA sequences into Acerobucferand, by complementation of known mutants, has led to the identification of the UDPG-pyrophosphorylase gene.* Transformation methodologies described for other Gram-negative organisms have not proved successful for A . ~ylinum.~’.~ However, a transformationprocedure and cloning vectors for the related species A . uceri have been described,49 and the possibility exists that a variation on this method may be successful with A . xylinum. As an alternative to the above methodologies, we have recently demonstrated that high voltage electroporationS0can be used to transform A.

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 cellulosenegative (ce1-4, cel-10, cel-13, cel-15, and cel-20) rn~tants.’~

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Critical Reviews In xylinum with broad host range plasmids (pUCD2 and pRK 248). Transformation efficiencies of approximately lo7 colonies per microgram of plasmid DNA can be achieved with electroporation. Therefore, as genetic exchange mechanisms are further developed and improved, our understanding of the genetics of A. xylinum should increase. Recently, two genes encoding enzymes involved in cellulose biosynthesis, UDPG-pyrophosphorylaa and the catalytic subunit of cellulose synthase,” have been cloned. The UDPGpyrophosphorylase gene was isolated from a partial library of Acerobacrer DNA by its ability to complement both cel- mutants of A. xylinum and a galU mutant of E. coli. Cellulosenegative mutants that received recombinant plasmid pVK lOO(240) were able to synthesize cellulose at the wild-type rate and had near wild-type levels of UDF’G-pyrophosphorylase. Assays for diguanylate cyclase, phosphodiesterase A, phosphoglucomutase, UDPG-pyrophosphorylase, and cellulose synthase showed that the three cel- mutants that could be complemented with pVK lOO(240) were deficient in only UDPG-pyrophosphorylase activity. The 2.8 kb Hind III fragment cloned in pVK lOO(240) also complemented an E. coli galU mutant containing a structural gene mutation in UDPGpyrophosphorylase. Valla et al.& have suggested celA, as the designation for this gene. Saxena et aLS1used a different strategy to isolate the gene for the catalytic subunit of cellulose synthase. They used a synthetic oligonucleotide probe homologous to the amino terminus of this 83 kDa polypeptide to ident@ and clone a 9.5 kB Hind III fragment of Acerobacter DNA. Sequence analysis has shown that this DNA fragment contains an open reading frame sufficient to code for a 80 kDa polypeptide. In addition, the amino terminal protein sequence deduced from the nucleic acid sequence provides a match to the 83 kDa subunit, indicating that in all likelihood it is the catalytic subunit for cellulose synthase. The genetic analysis of Acerobacter has provided a number of interesting observations including (1) the existence of a complex pattern of plasmids; (2) the utility of chemical and transposon mutagenesis to induce cel- mutants; (3) the apparent genetic instability in this organism, e.g., the high rate of induction and reversion of spontaneous mutants, chromosomal and plasmid reakangements; and (4) the use of recombinant DNA technology to isolate genes involved in cellulose synthesis. The ability to clone the genes involved in cellulose biosynthesis will not only provide a better understanding of this process but also lead to potential commercialization of microbial cellulose production. Because Agrobacterium tumefaciens can cause disease in a wide variety of plants, it has been the object of study and genetic analysis for quite some time.52Much attention has been paid to the genetic interactions between bacterium and plant cell in the development of crown gall tumors. One aspect of this interaction is the specific attachment of bacterium to plant

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host cell. Much of what is known concerning cellulose synthesis in this organism centers around the role of cellulose in this A variety of mutants have been isolated that affect the ability of Agrobacrerium to attach to plant cells. Included in this group afe mutants deficient in cellulose produ~tion~.’~ as well as those still capable of cellulose ~ynthesis.~’ Further analysis of these mutants has led to the proposal that certain low molecular weight glucans are important in the initial attachment of bacterium to plant cell, whereas the synthesis of cellulose fibrils promotes both the anchorage of the bacterium to the plant cell surface and the formation of bacterial aggregates. Genetic analysis has indicated that many genedgene products are involved in cellulose synthe~is~.”.’~ and that the majority of genes map to chromosomal sites. In fact, many genes are clustered, suggesting that a specific region of the Agrobacrerium chromosome is concerned with the interaction of the bacterium with the plant host cell. What remains to be determined is if the clustering of genes is necessary for the coordinate expression of these sequences and if other cellulose-synthesizing organisms have a similar genetic organization. In fact, recent work by Wong et al.” suggests that at least some of the genes involved in cellulose synthesis in Acerobacter xylinum are also organized in an operon (bcs, bacterial cellulose synthesis). These genetic sequences were localized to a segment of chromosomal DNA that could complement . cellulose-negative strains deficient in cellulose synthase. The operon contains at le& four protein-coding regions (bcsA, bcsB, bcsC, and bcsD), with the catalytic subunit of cellulose synthase encoded by the bcsB region. The functions of the other genetic sequences are not known, but their expression (along with bcsB) appears to be necessary for maximal cellulose synthesis in vivo.

VII. ECOLOGICAL ASPECTS OF CELLULOSE PRODUCING BACTERIA Because A. xylinum was observed to produce a pellicle during Static culture, and since it was considered to be a strict aerobe, investigators proposed that the role of the pellicle was to hold the organisms in an aerobic environment to facilitate their ability to obtain ~ x y g e n . ~ ~ Could . ’ ~ there be other ecological roles for cellulose secreted by A. xylinum? Undried cellulosic pellicles retain water well. Thus, one hypothetical role for cellulose could be moisture retention in the microenvironment to allow for bacterial growth and prevent dessication. Also, our experiments cultivating A . xylinum in candle jars, 5% carbon dioxide gassed enclosures, and Biobags show that A. xyxylinum can grow microaerophilically while continuing to synthesize cellulose,39 so the argument that the pellicle is vital for aerobic growth is no longer as compelling. Laboratory experiments also indicated that pellicles could pro-

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Microbiology tect bacteria from the killing effects of ultraviolet light. This protective effect could be of considerable importance to the organism as it grows on decaying fruits just as it could be holding water for the biochemical degradation of substrates for nutrition. Since A. xylinum is found primarily on decaying fruits, could it be possible that cellulose pellicle synthesis plays a role in successful colonization of a particular habitat? To answer this question, a series of experiments were performed in which pieces of apple were inoculated with various strains of cellulose-producingand celluloselessA . xylinum, and assays were done to determine density of various organisms growing on the apples. Celluloseless and uninoculated controls tended to become dry over the course of the experiment. Molds, fungi, and other bacteria tended to predominate on the pieces of apples that had been inoculated with the cellulose-negative strain. When pellicles from a wild-type and a cellulose-over-producing strain completely covered the pieces of apple, A. xylinum predominated over other microbes growing on the fruit. These results indicate that the ability to synthesize a cellulosic pellicle may allow A. xylinum to colonize food sources more effectively than strains of A. xylinum that do not make cellulose. This colonization ability may be important in the microenvironment as A . xylinum seeks nutrients in competition with other decomposing organisms such as other bacteria and fungi.” Cellulose fibrils have also been implicated in the infection process of plant tissue by Agrobacterium tumefaciens, which is another example of the need of a microorganism to use an exopolymer to hold to a particular substrate.8 Cellulose fibrils anchor bacteria to plant surfaces. Cellulose-negative mutants of Agrobacterium produced by transposon mutagenesis secrete cellulose fibrils so no bacterial aggregates form on plants even though the bacteria are still virulent. The ability of cellulosenegative mutants to form tumors at the site of inoculation was decreased if the site was rinsed with water. The absence of cellulose fibrils apparently reduced the ability of the bacteria to anchor and eventually infect plant tissue. Cellulose fibril formation to enhance flocculation in activated sludge permits the aggregated growth of a variety of Gram-negative bacteria. The ability to form a floc could allow for enhanced microbial growth by giving cells a surface upon which to cling while they are participating in the process of wastewater decomposition.’ To be successful in the environment, many microorganisms have evolved efficient mechanisms to colonize surfaces whether it be the bottom of a ship, the surface of teeth, or decaying fruit. Colonization typically has involved production of exopolysaccharides that aid in attachment, act to prevent drying, pH changes, and effects of toxic molecules upon the colonizer. Cellulose is a highly organized exopolysaccharide, which may Play a role-similar to exopolymers secreted by other bacteria.

VIII. SUMMARY AND CONCLUSIONS Acetobacter, Agrobacterium, Pseudomonas, and Rhizobium

are four genera of Gram-negative bacteria that synthesize cellulose. Sarcina is the sole genus of Gram-positive bacteria to do so. The quality or kind of cellulose synthesized varies among the various microbes from the long microfibrils that form into pellicles for Acetobacter to the short cellulosic fibrils of Agrobacterium and Rhizobium, which permit bacterial attachment to plant tissue. Pseudomonas and Sarcina secrete cellulosethat, while crystalline in form, is more amorphous in structure. Cellulose producers are found among both prokaryotes and eukaryotes; thus one assumes a possible evolutionary relationship in the biosynthetic processes of bacterial and higher organisms. Brownlo has speculated that Sarcina was probably the first cellulose producer. With the evolution of aerobiosis, came the other bacterial cellulose producers that are Gramnegative. The genetic information for cellulose synthesis by eukaryotes probably came via an endosymbiotic mechanism from prokaryotes. When nucleic acid andlor protein sequence information becomes available for cellulose synthases of bacteria and higher organisms, the relationships among these various cellulose synthesizers will become clearer. Cellulose fibrils of Acetobacter xylinum are the most highly organized of those produced by all currently identified bacteria. The fibrils are synthesized at or near the cytoplasmic membrane probably from UDP-glucose via cellulose synthase that requires a cyclic diguanylic acid activator. The molecules of glucose that comprise the fibrils are polymerized as they pass through the various membranes of the Gram-negative cell, possibly with final extrusion through pores in the outer membrane. The fibrils have the capacity to form into a highly ordered and complex structure that under scanning electron microscopy resembles tunnels. The function of this complex extracellular matrix may be as simple as to hold the bacteria in the aerobic environment or it may serve a diversity of functions such as protection from ultraviolet light, retention of moisture, and colonization of substrates. This past decade has seen an explosion of information about the biology of bacterially synthesized cellulose, but there is still much that we do not know. In the future, we should know more about evolutionary relationships between prokaryotic and eukaryotic cellulose biogenesis. Further ecological analyses are needed to understand the role(s) of cellulose for the bacteria in their natural habitats. A better understanding of the actual synthesis process as it occurs in the cell membrane may eventually permit the large scale in vitro synthesis of cellulose and provide potentially an important alternative source of this natural polymer. Detailed genetic analyses will increase our knowledge of the basic mechanisms of cellulose synthesis and may eventually lead to many commercial applications of bacterial cellulose.

ACKNOWLEGMENTS The authors wish to acknowledge the editorial assistance of Barbara Randolph-Anderson and Janne Cannon and the sec1991

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Critical Reviews In retarial help of Pat McCarron. They also appreciate the willingness of authors to provide permission for use of some of the figures. Some of the authors’ research was supported by the Research Council of the University of North Carolina at Greensboro.

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Microbiology D. and Benziman, M., Cyclic diguanylicacid and cellulose synthesis in Agrobucrerium hunefoiens, 1. Bacteriol.. 171,6649, 1989. 36. Schramm, M. and Hestrin, S., Factors affecting production of cellulose at the aidliquid interface of a culture of Acetobacrer xylinum. J . Gen. Microbiol.. 11, 123, 1954. 37. Vatla, S. and Kjosbakken, J., Cellulose-negative nutants of Acerobacter xylinum. J . Gen. Microbiol., 128, 1401, 1982. 38. Smith,R., CharacteriZatiOaof a Plasmid of Acerobacrer xylinum (ATCC 23769): Cellulose Producing Wild Type and Non-Cellulose Roducing Mutant Strains, M.A. thesis, University of North Carolina at Greensboro, Greensboro, 1990. 39. Williams, W. S. and Cannon, R. E.,Alternativeenvironmentalroles for cellulose produced by Acetobacrer xylinwn. Appl. Environ. Microbiol., 55, 2448, 1989. 40. Valla, S., Coucheron, D. H., and Kjosbakken, J., Acerobacrer rylinum contains several plasmids: evidence for their involvement in cellulose formation, Arch. Microbiol., 34, 9, 1983. 41. Vaila, S., Coucheron, D.H., and Kjosbakken, J., The plasmids of Acetobacter xylinwn and their interaction with the host chromosome, Mol. Gen. Genet., 208, 76, 1987. 42. Smitb, R., Cannon, R., and Anderson, S., Characterization of a plasmid from Acetobucrer xylinwn ATCC 23769: cellulose-producing and cellulose-negative mutants, Absn. Annu. Meet. Am. Soc. Microbiol.. p. 181, 1990. 43. Saxena, I. M. and Brown, R. M., Jr., Cellulose biosynthesis in Acerobacrer xylinum: a genetic approach, in Cellulose and Wood Chemistry and Technology, Schuerch, C., Ed., John Wiley & Sons, New York, 1989, 537. 44. Valla, S., Coucheron, D. H., and Kjosbakken, J., Conjugative transfers of the naturally occurring plasmids of Acerobucrer xylinum by Inc-P-plasmid mediated mobilization, 1. Bacreriol.. 165,336,1986. 45. Keesler, G. A., Jr., Genetic Studies of Acetobacter xylinum, M.A. thesis, University of North Carolina at Greensboro, Greensboro, 1985. 46. Valla, S., Coucheron, D. H., Fjaervik, E., Kjosbakken, J., Weinhouse, H., Ross,P., Amikam, D.,and Benziman, M., Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG-pyrophosphorylase structural gene, MoZ. Gen. Genet.. 217.26, 1989. 47. Cohen, S. N., Chang, A. C. Y., and Hsu, L., Non-chromosomal antibiotic resistance in bacteria: genetic transformationof Escherichia coli by R factor DNA, Proc. Nutl. Acad. Sci. U.S.A., 69,2110, 1972. 48. Memck, M. J., Gibbm, J. R., and Postgate, J. R., A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Excherichiu coli, J. Gen. Microbiol.. 133, 2053, 1987. 49. Fukaya, M.,Tayama, K., Okumura, H.,Masai, H.,Hozumi, T., and Beppu, T.,Improved transformationmethod for Acerobucrer with plasmid DNA, Agric. Biol. Chem., 49, 2091, 1985. 50. Dower, W. J., Miller, J. F., and Ragsdale, W. W., High efficiency transformation of E. coli by high voltage electroporation,Nucleic Acids Res., 16, 6127, 1988. 51. Saxena, I. M., Lin, F. C., and Brown, R. M., Jr., Cloning and sequencing of the cellulose synthase catalytic-subunit gene of Acetobacterxylinum. Abstr. Annu. Meet. Am. SOC.Microbiol., p. 277, 1990. 52. Nester, E. W., Gordon, M. P., Amasino, R. M., and Yanofsky, M. F., Crown gall: a molecular and physiological analysis, Annu. Rev. Plum Physiol., 35, 387, 1984. 53. Matthysse, A. G., Holmes, K. V., and Gurlitz, R. H., Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells, 1. Bacteriol.. 145, 583, 1981. 54. Tomshow, M. F., Karlinsky, J. E.,Marks, J. R., and Hurlbert, R. E., Identification of a new virulence locus in Agrobucrerium tumefaciens that affects plysaccharide composition and plant cell attachment, J. Eucreriol., 169, 3209, 1987. 55. h u g l a , C. J., Halperin, W., and Nester, E. W., Agrobacterium

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