Exo--(1-'3)-G1ucan (Curdlan) Biosynthesis by Agrobacterium sp.

ATCC 31749

Robert D. Hancock

Presented for the degree of Doctor of Philosophy

The University of Edinburgh

1995 Declaration

I hereby delare that this thesis was composed by myself and that the work presented within is my own.

Robert D. Hancock

13th January 1995 Acknowledgements

I would like to thank Prof. I.W. Sutherland for his expert guidance, encouragement and advice throughout the course of this study. I would also like to thank Mrs. Lynn Kennedy for discussion and, in particular, technical advice. In addition, I would like to thank the many people who have worked in the laboratory over the past three years and contributed towards the investigative atmosphere.

[would like to thank the SERC (BBRSC) and Zeneca for financial support. In particular

Dr. Ian Shirley at Zeneca for his help in obtaining payment.

Finally, I would like to thank my parents for financial and moral support over a six year university career. Abbreviations

ADP: Adenosine diphosphate

ADTS: 2,2-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid)

ATP: Adenosine triphosphate

cHNO3 : Concentrated nitric acid (69.0-71.0% w/v)

CMP: Cytosine monophosphate

CTP: Cytosine triphosphate

DCW: Dry cell weight dH20: Glass distilled water

DPn: Number average degree of polymerisation

EDTA: Ethylene-diaminetetraacetic acid

EGTA: Ethylene glycol-bis( 3-aminoethyl ether) N,N,N' ,N,-tetraacetic acid

GDP: Guanosine diphosphate

Gly-gly: Glycine glycine h: Hours

KDO: 3-deoxy-D-manno-octulosonic acid

Molar

MES: 2-(N-morpholino) ethanesulfonic acid

MOPS: 3-(N-morpholino) propanesulfonic acid mm Hg: Millimetres of mercury (1 mm Hg= 13 3.32 Pa)

Normal r.p.m.: Revolutions per minute

TDP: Thiamine diphosphate TEMED: N,N,N' ,N' -tetramethylethylenediamine

Tris: Tris (hydroxymethyl)-aminomethane

Iris HC1: Ti-is (hydroxymethyl)-aminomethane hydrochloride

TTP: Thiamine triphosphate sp. gr.: Specific gravity

St. dev.: Standard deviation about the mean

UDP: Uricline diphosphate

UHQ: Ultra filtered

UMP: Uricline monophosphate

UTP: Uridine triphosphate Aims of the Project

Although a considerable amount of information is available regarding the chemistry, theological properties and applications of curdlan (see Chapter 1) few studies have been undertaken regarding the biosynthesis of the polysaccharide.

A number of studies have examined the culture conditions which favour curdlan synthesis by both

Agrobacteriutn sp. ATCC 31749 (e.g. Phillips a al., 1983) and related- bacteria (e.g. Harada et at,

1966). These studies were mainly concerned with conditions of culture which may improve curdlan yield. The initial aim of this project was therefore to re-examine these results and also to attempt to find new methods for the improvement of curdlan yield from Agrobacterium sp. ATCC 31749. In addition, an attempt was made to examine the underlying mechanisms which resulted in improved curdlan yields under certain culture conditions.

Although the very early stages of the curdlan biosynthetic pathway have been examined (Kai et al.,

1993) very little, if any, data is available regarding later events of curdlan biosynthesis by

Agrobacterium sp. ATCC 31749 and a second aim of this project was therefore to examine the biosynthetic mechanism adopted by the bacterium. This will be achieved through examination of the effect of various metabolic inhibitors on curdlan production by Agrobacterium sp. ATCC 31749 and also through structural examination of the native product, a form of curdlan which has not been previously investigated. Abstract

Exo-341--'3)-Glucan (Curdlan) Biosynthesis by Agrobacterium sp. ATCC 31749

Curdlan is a water-insoluble, alkali-soluble (Ogawa et al., 1972), linear 3-(1--'3)-glucan (Harada et at, 1968a) produced as an exopolysaccharide by a number of strains of Agrobacteriurn, Alcaligenes

(Hisamatsu et at, 1977) and Rhizobium (Ghai et al., 1981). The compound has unusual theological properties in that it is capable of forming two types of gel when an aqueous suspension is heated

(Harada, 1974). These properties have led to a considerable degree of commercial interest (Harada et al., 1993).

Although information is now available regarding the physicochemical properties of curdlan, less is known about the biosynthesis of the molecule. The present study describes physiological conditions which promote curdlan synthesis, the effect of metabolic inhibitors on production and the nature of the product of Agrobacterium sp. ATCC 31749, a laboratory derivative of a strain originally isolated from soil (Harada and Yoshimura, 1964).

Nitrogen depletion in the medium was essential for production, and depletion of sulphur or phosphorus in batch culture did not promote curdlan synthesis. Similarly, initiation of curdlan production was observed when bacteria were transferred to nitrogen free medium but not media free of sulphur or phosphorus. A model for the mechanism of nitrogen dependent control is presented.

Agrobacterium sp. ATCC 31749 grew well on a number of monosaccharides and lactose, moderately well on succinate and poorly on glycerol. Good curdlan production was observed from mannose, glucose and galactose with a reasonable curdlan yield obtained from sucrose. Curdlan yields when lactose, maltose or glycerol were supplied were poor and no curdlan was obtained from culture on succinate. The effects of other physiological conditions were examined. Arsenate inhibited neither initiation nor continuation of curdlan biosynthesis when added to the medium in concentrations up to 10mM. 5mM sodium azide inhibited curdlan production but not glucose uptake. A similar effect was observed when the ionophore tetracaine (1mg ml -') was added to the medium. EDTA inhibited curdlan production and glucose uptake whilst EUTA inhibited neither. Chloramphenicol inhibited curdlan production as did rifampicin.

Analysis of the kinetics and degradation products of enzymic hydrolysis revealed a similarity between neutralised gels and gels formed by low temperature heating. Results obtained with native curdlan or curdlan preparations obtained by boiling aqueous suspensions were similar. The implications of these results on curdlan biosynthesis are discussed. Contents

Chapter 1: Occurrence, Chemistry and Structure of Curdlan and

Curdlan-type Polysaccharides. 1.1 Sources of curdlan and curdlan-type polysaccharides 1

1.2 Rheological properties of curdlan 2

1.3 Chemistry, conformation and ultrastructure of curdlan and curdlan gels 5

1.4 Commercial applications of curdlan 15 1.5 Summary 17

Chapter 2: Mechanisms of Biosynthesis and Export of Bacterial Polysaccharides 18

2.1 Entry of carbon substrate 18 2.2 Precursors of exopolysaccharide biosynthesis 21 2.3 The formation of heteropolysaccharide repeat units 26

2.4 Polymerisation and export of bacterial exopolysaccharides 35

2.5 Direct polymerisation of sugar nucleotides into bacterial exopolysaccharides 42

2.6 Models of curdlan biosynthesis by Agrobacterium sp. ATCC 31749 44

Chapter 3: Physiological Influence and Biochemical Control of 45

Exopolysaccharide Biosynthesis

3.1 Effects of changes to culture conditions 45

3.1.1 Effect of carbon substrate 45

3.1.2 Effect of growth phase and rate and nutrient limitation 48

3.1.3 Other changes in culture conditions 52 3.2 Biochemical control of exopolysaccharide biosynthesis 54 3.2.1 Control of substrate uptake 54 3.2.2 Control of sugar nucleotide precursors 55 3.2.3 Control of isoprenoid lipid availability 58 3.2.4 Control of glycosyltransferase activity 60

Chapter 4: Genetic Organisation and Control of Exopolysaccharide Biosynthesis 62

4.1 Organisation of exopolysaccharide genes 62

4.2 Genetic control of exopolysaccharide synthesis 67

Chapter 5: Materials and Methods 72

5.1 Maintenance of organism, culture conditions and product recovery 72

5.1.1 Organism 72

5.1.2 Maintenance of Agrobacterium sp. ATCC 31749 72

5.1.3 Growth media and conditions 72

5.1.4 Growth measurements and calculations 74

5.1.5 Product recovery 75

5.1.6 Preparation of curdlan samples 75

5.2 Analytical Procedures 76

5.2.1 Total carbohydrate 76

5.2.2 Anthrone assay 77

5.2.3 Reducing sugar 77

5.2.4 Glucose 78 5.2.5 Acetyl groups 78

5.2.6 Paper chromatography 79

5.2.7 Sulphate 80

5.2.8 Phosphate 80

5.2.9 Ammonium 81

5.2. 10 Protein 81

5.2.11 pH 82

5:2.12HPLC 82

5.2.13 Viscometry 82

5.3 Cell fractionation and protein concentration 83

5.3.1 Cell fractionation by osmotic shock 83

5.3.2 Cell fractionation by chloroform shock 83

5.3.3 Protein concentration 84

5.3.4 Protein fractionation by PAGE 84

5.4 Polysaccharide modification 86

5.4.1 Carboxymethylation 86

5.4.2 Acetylation 87

5.5 Hydrolysis of curdlan 88

5.5.1 Enzymic hydrolysis 88

5.5.2 Acid hydrolysis 88

Chapter 6: Effect of Physiological Conditions on Curdlan Biosynthesis 89

by Agrobacterium sp. ATCC 31749 6.1 The effect of pH and addition of biological buffers 89

6.2 The effect of nutrient limitation on curdlan production by Agrobacterium sp. 91

ATCC 31749

6.3 The effect of carbon substrate on curdlan production by Agrobacterium sp. 94

ATCC 31749

6.4 The effect of incubation temperature on growth and curdlan production 96

by Agrobacterium sp. ATCC 31749

6.5 The effect of changes in osmotic pressure of the medium on curdlan 98

production by Agrobacterium sp. ATCC 31749

6.6 Discussion 101

Chapter 7: The Effect of Metabolic Inhibitors on Curdlan Production by 102

Agrobacterium sp. ATCC 31749

7.1 Experimental design 102

7.2 The effect of cation chelators 103

7.3 The effect of tetraaine 105

7.4 The effect of sodium azide 107

7.5 The effect of arsenate 103

7.6 The effect of 2-deoxy-D-glucose 103

7.7 The effect of aniline blue 109

7.8 The effect of chloramphenicol 110

7.9 The effect of rifampicin 112

7.10 Discussion 113 Chapter 8: Structure of Curdlan: Enzymic and Acid Hydrolytic Studies 115

8.1 Attempts to isolate -(1-'3)-glucanase activity from Agrobacterium sp. 115

ATCC 31749

8.2 Curdian is a weak inducer of Oerskovia xanthinolytica -(1-3)-g1ucanases 119 8.3 Acid hydrolysis of curdlan In -41 8.4 Enzymic hydrolysis of curdlan 121

Chapter 9: Concluding Discussion 123

9.1 Potential mechanism for curdlan synthesis in response to nitrogen deficiency 123

in Agrobacterium sp. ATCC 31749

9.2 Potential further studies on Agrobacterium sp. ATCC 31749 125

I)-(1--'3)-glucanase

9.3 Crystallinity of native curdlan: potential implications for curdlan biosynthesis 127

9.4 The effect of metabolic inhibitors: further work and implications for models 129

of curdlan biosynthesis

9.5 Potential biological functions of curdlan 131

References 134 Chapter 1 Occurrence, Chemistry and Structure of Curdlan and Curdlan-Type Polysaccharides

1.1 Sources of Curdlan and Curdlan-Type Polysaccharides Polymers of glucose joined primarily by B-(1-'3)-linkages (curdlan-type polysaccharides) have been isolated from a wide variety of natural sources in addition to being synthesized by chemical means from simple organic molecules (Kochetkov and Bochkov, 1969). Molecules of this family have been isolated from higher plants e.g. cotton (Hayashi et al., 1987) in the form of callose; from fungi e.g. Lyophyllum ulmarium (Kazuno et al., 1991); from algae e.g. laminaran from seaweeds (Percival and McDowell, 1967) and paramylon from the unicellular alga Euglena gracilis (Marchessault and Deslandes, 1979). B-(1--3)-glucans have also been found in the cell walls of some yeasts e.g. Saccharomyces cerevisiae (Nakanishi et al., 1976). Unlike curdlan, produced by a number of bacteria, these molecules are frequently found to contain residues other than glucose or linkages other than B-(1-3). The structure of curdlan and a related polysaccharide are illustrated in Figure 1.1. Curdlan is produced by diverse Genera of bacteria. Indeed, the first pure 13-(1--'3)-glucan

(Harada et al., 1993) was isolated by Tolcuya Harada and colleagues from a bacterium then narnedAlcaligenesfaecalis var. myxogenes (Harada et al., 1966), although the structure was not elucidated until later (Harada et al., 1968a). Through the use of the B-(1-'3)-glucan / specific stain aniline blue (Nakanishi et al., 1974), Hisamatsu and colleagues (1977) were able to detect curdlan production by four strains of Agrobacterium radiobacter and one strain of

A. rhizogenes as well as Alcaligenesfaecalis, as were the group of Nakanishi et al. (1976). In addition, the latter were able to detect curdlan production by five strains of Bacillus (Table

1.1). A mutant strain of Rhizobium trifolii has also been observed to produce curdlan (Ghai CH,CH

I-]

1.1a Curdlan. n=approx. 450 (From Harada et aL, 1993)

riO E1 1/3\01 CH2OH OH CH2 C4,QH

riO OH 01

1.lb Schizophyllan. n=approx. 9000 (From Norisuye et aL, 1980)

Figure 1.1

Structure of curdlan and a related polysaccharide. Table 1.1

Some Biological Sources of Curdlan-Type Molecules

Group Oreanism Polymer Residues Linkages References Higher e.g.Cotton Callose Glucose 6-1,3 Hayashi plants Mang bean Glucuronic 6-1,6 etal.,1987 acid Harada, 1974

Algae Seaweeds Laminaran Glucose 6-1,3 Percival and Mannitol 6-1.6 McDowell, 1967 Euglena Paramylon Glucose 6-1,3 Marchessault gracilis and Deslandes, 1979

Fungi Saprolegnia Cell wall Glucose 0-1,3 Bulone PS etat,1990 Lyophyllum Fruit body Glucose 0-1,3 Kazuno uftnariun, PS P4 Galactose 6-1,6 etal., Mannose 1991 PS P5 Glucose B-1,3,6-1,6 Schizophyllum Lentinan Glucose 6-1,3 Misakai commune Schizophyllan 6-1,6 etal., Screlotiwn Sclero- Glucose 6-1,3 1993 glnconicum glucan 6-1,6 Vol variella VVG Glucose 6-1,3 volvacea - 6-1,6

Yeasts Saccharomyces NA NA NA Nakanishi bacilli etal.,1976 Trichasporon NA NA NA pullulans

Bacteria Alcaligenes Curdlan Glucose 0-1,3 Harada faecalis etat,1968a Agrobacterium BPS NA NA Nakanishi sp. 1 strain etal.,1976 A. radiobacter BPS NA NA Hisamatsu 4 strains el al., rhizogenes BPS NA NA 1977 1 strain Bacillus NA NA NA Nakanishi brevis et at, 1976 cereus NA NA NA 1 strain Bacillus sp. NA NA NA I strain Rhizobium sp. Curdlan Glucose 0-1,3 Harada, R. trjfolii Curdlan Glucose 0-1,3 1992 Cellulomonas NA Glucose NA Buller and flavigena Voepel,1990 NA=Data not available, PS=Polysaccharide, EPS=Exopolysaccharide 2

et al., 1981). All of these strains were obtained from stock cultures at the Institute for Fermentation, Osaka, Japan. Recently, a strain of Celluloinonasfiavigena which appears to produce curdlan as an extracellular energy reserve has been isolated (Voepel and Buller, 1990; Buller and Voepel, 1990). Biological sources of curdlan and related molecules are listed in Table 1.1. The bacterium used in the work presented in this thesis was obtained from the American

Type Culture Collection reference number ATCC 31749, presently described as Agrobacterium sp.. This bacterium was derived from strain 10C3, isolated from soil by

Harada and colleagues. The genealogy of ATCC 31749 is described in Table 1.2.

1.2 Rheological Properties of Curdlan Curdlan was first discovered when a succinoglycan-producing strain ofAlcaligenesfaecalis var. inyxogenes; strain 10C3, spontaneously mutated and no longer produced the expected exopolysaccharide but instead produced an insoluble polymer (Harada et al., 1966). Thus one of the most evident rheological features of curdlan is its insolubility in water. As a result of this rheological behaviour early investigators were unable to purify the polysaccharide in the usual manner from culture supernatants, by the addition of dehydrating agents such as ethanol or acetone as with water soluble polymers. This problem was overcome through the observation that curdlan was soluble in 0.5M sodium hydroxide (Harada et al., 1966) and further investigations revealed that curdlan became soluble when the alkali concentration was raised to the range 0. 19M-0.24M or greater (Ogawa et al., 1972). Other investigators also found the polysaccharide to be soluble in dimethyl suiphoxide (Harada et al., 1968a).

Early work established that an aqueous suspension of curdlan in the range 2%-10% (w/v) is capable of forming a gel when boiled, exhibiting a strength similar to that of agar but with Table 1.2

Genealogy of Agrobacterium sp. ATCC 31749

Date Strain Properties Reference

After 1962 Alcaligenes Succinoglycan Harada and faecalis var. production Yoshimura, 1964

inyxogenes 10C3 Growth on 10% Harada et ethylene glycol al., 1965

Spontaneous Mutation

1965 A. faecalis Curdlan Harada 1OC3K production et al., 1966

Chemical mutägenesis

1974 1OC3K-u Uracil Harada Deposited as auxotroph et al., 1993 ATCC 21680 Good curdlan production

Spontaneous mutation

1983 A. faecalis Autotrophic Phillips

ATCC Good curdlan et al., 1983

31749 production 1991 Agrobacterium ATCC 31749 Harada 1992

sp. reclassified C]

a greater degree of elasticity (Harada et al., 1966). This observation led to an intense research effort and the eventual commercial production of curdlan in Japan (see pp. 15-16). It is now known that curdlan is capable of forming two types of gel when an aqueous suspension is heated. As the temperature is raised, the suspension clears to form a sol at 50-

55°C (Fulton and Atkins, 1980) and if it is then cooled forms the low-set gel (Kimura et al.,

1973). If this type of gel was then reheated it melted at 65 0C (Konno and Harada, 1991); however, continued heating led to the production of the high-set gel at 80 0C and above.

Figure 1.2 shows the relationship between heating temperature and gel strength; measured at 300C. Rheological properties and molecular conformation (see pp. 7-15) of the two gel types show considerable differences.

The low-set gel is formed when curdlan is heated above 54 0C in aqueous suspension with a pH below 12 (Ogawa etal., 1972). This type of gel can also be formed when alkaline solutions of curdlan are neutralised; either by dialysis against water (Harada, 1977) or by bubbling carbon dioxide through the solution (Kanzawa et al., 1989). Ogawa and colleagues

(1973) suggested that a degree of polymerisation of around 200 was the minimum required to permit gel formation. Gel strength of the low-set gel was shown to be independent of heating time but was dependent on heating temperature in the range 60-80 0C with a marginal increase in gel strength as the heating temperature was raised (Kimura et al., 1973). This property is illustrated in Figure 1.3. Gel strength has also been shown to be proportional to the curdlan concentration (Harada, 1974). The low-set gel exhibits a low degree of syneresis which increases when the gel is subject to freeze thawing. This treatment; however, increases gel strength (Takahashi and Harada, 1986).

Presumably due to the high degree of interest from the food industry much more attention has been paid to the theological properties of the high-set gel because of its application in foods which are boiled in water during processing or preparation (see pp. 15-16). This type 1,500

1000

-a

-; 500 C-:

0 50 60 70 80 90 100 Heating temperature 'C)

Figure 1.2 The effect of heating temperature on curdlan gel strength. Heating time lOminutes.

Curdlan concentration 3%.

(Adapted from Harada, 1992) 1'

E '-I H lOOt U C . -S

-S

'I. F

Li

C l ' 4 al 75 OC it L - CM 65t m L - o 20 40 50 60 cOO Heating Time (m,nutts)

Figure 1.3 Effect of heating time on curdlan gel strength of gels prepared at various temperatures.

(From Kimura et aL, 1973)

I of curdlan gel is formed when the temperature of an aqueous curdlan suspension is raised above 800C (Nakao et al., 1991). It differs from the low-set gel in a number of rheological parameters. Firstly, the gel formed by heating above 80 0C shows a greater gel strength than that formed at lower temperatures. Gel strength is dependent on heating temperature to a much greater degree than the low-set gel. Furthermore, when the temperature is maintained above 800C, gel strength is also dependent on healing time (Kimura et al., 1973; Nakao et al., 1991; Figure 1.3). High-set gels also exhibit a greater degree of syneresis than low-set gels. Syneresis is increased as heating temperature increases but is reduced by an increased curdlan concentration in the gel (Nakao et al., 1991). The degree of syneresis exhibited by the high-set curdlan gel can also be reduced by the addition of starch or sucrose (Nakao et al., 1991'); although addition of starch to the gel reduced strength. The stability of high-set curdlan gel to freeze thawing was also demonstrated with very little change in gel strength after one cycle of this treatment. However, syneresis was markedly increased by freeze thawing. In addition to low temperature tolerance, the thermo-irreversible high-set gel has been demonstrated to retain its properties at temperatures as high as 150 0C (Adachi et al.,

1990). Finally, high-set curdlan gel shows stability over a wide range of pH. Kimura and colleagues (1973) demonstrated stability in the range 2.9-9.5, with a softer, more elastic gel on the alkaline side, whilst Harada (1974) quotes gel stability in the pH range 2.0-9.5 with a maximum gel strength at pH 2-3. In addition, in the high-set gel the individual curdlan molecules are extremely resistant to acid hydrolysis with no release of oligosaccharide after

30 days in 16% H2SO4 at 320C (Kanzawa et al., 1989) and only 10% of total sugar released when treated with 2N H2SO4 at 1000C for four hours (Harada et al., 1966). / 5

1.3 Chemistry, Conformation and Ultrastrueture of Curdlan and Curdlan Gels

An early report on curdlan first presented evidence that the molecule was composed only

of D-glucose and also that the sugar moieties were connected by B linkages (Harada et al., 1966). It was also demonstrated, through methylation analysis, that the linkage was 1-'3.

The same report proposed a degree of polymerisation of approximately 135 (Harada et al., 1968a). This was later revised and curdlan is now considered typically to consist of

approximately 450 glucose residues (e.g. Harada et al., 1993), although a curdlan supplied by Takeda Chemical Company in the late 1970's was described as having an average degree of polymerisation of 540 (Marchessault and Deslandes, 1979). Thus, at the present time,

evidence suggests curdlan is a 6-(1-3)-D-glucan with an average degree of polymerisation

of 450, i.e. a molecular weight of approximately 73000 Daltons. Much attention has been directed towards elucidation of the conformation and ultrastructure

of curdlan in order to aid understanding of the gelling mechanism and furthermore, due to the homogeneity of the molecule, as part of an attempt to construct general models of molecular

conformation based on knowledge of chemical and structural formulae. A number of methods have been applied ranging from acidic and enzymic hydrolysis, viscosity measurements and

optical rotation to X-ray diffraction, electron microscopy and 13C nuclear magnetic resonance. In the early 1970's Ogawa and colleagues examined the conformational transition of curdlan

and smaller B-(1-'3)-glucan fragments as an alkaline solution was neutralised, by

measurements of optical rotatory dispersion, viscosity, flow birefringence and the ability of

the molecules to form a complex with the dye Congo Red. In the case of the original

molecule (reported to have a DPn of 400) abrupt changes were observed in all these parameters in the range 0.19-0.24M NaOH or 0.17-0.21M LiOH (Ogawa et al., 1972). It was thus suggested that it concentrations of alkali above this range the molecule assumes a disordered and relatively flexible conformation. It was further suggested that the molecule was capable of adopting this disordered conformation due to the ionisation of hydroxyl groups which led to the breakdown of hydrogen bonds between glucose residues within the molecule. It was proposed that at lower concentrations of alkali these hydrogen bonds allowed the molecule to adopt a stiff helical form. This hypothesis was later supported through X-ray diffraction methods in conjunction with mathematical model refinement suggesting that curdlan exists as a helix stabilised by internal 04---05 hydrogen bonds between adjacent glucose residues (Okuyama et al., 1991). Further evidence to support the hypothesis of a disorder to order transition in the 0. 19M-0.24M range of alkali came from

13C nuclear magnetic resonance observations which showed a sharp reduction in line width of 13C signals at an alkali concentration greater than 0.22M; indicative of the random coil conformation (Saito et al., 1977). Work on soluble fractions resulting from partial hydrolysis of curdlan (DPn <25) demonstrated that optical rotation at 400nm was similar to that of the full length molecule at alkali concentrations greater than approximately 0.24M, whilst at lower concentrations of alkali the optical rotation of the former differed considerably from that of the latter thus supporting the earlier conclusion that at high concentrations of alkali the curdlan molecule takes a disordered conformation (Ogawa et al., 1973). It was further demonstrated that optical rotation in 0. 1M NaOH was dependent on degree of polymerisation of the molecule with fractions of DPn <25 exhibiting a constant negative value which gradually rose with increasing DPn to reach a maximum value when the molecule comprised approximately 200 glucose residues and remained constant thereafter (Figure 1.4). It was thus suggested that the proportion of the molecule taking an ordered conformation increases with increasing degree of polymerisation until a maximum proportion of order at a value close to 200. This SES

0 in 1 1:5

Al DR

Figure 1.4

Optical rotation of curdlan fragments at 439nm in O.1M NaOH. Black circles represent soluble fragments, white circles represent gel forming fragments and mixed circles represent insoluble fragments. (From Ogawa et al., 1973). F]

was further supported by the observation that the wavelength of maximum absorption of

Congo Red; a dye believed to interact only with ordered B-(1-.3)-glucans (Wood, 1982),

shifted only when incubated with molecules comprising 25 glucose residues or more (Ogawa

et al., 1973). Thus the first conformational transition that can be observed in the curdlan

molecule appears to occur on going from the soluble (disordered) form to the insoluble (ordered) form.

That curdlan undergoes a conformational transition as alkalinity is reduced is now well established. However, greater controversy exists when one considers the precise

conformation of the insoluble form. Furthermore, although the properties of neutralised and low temperature induced (60°C-80 0C) gels have similar mechanical properties it remains

unclear whether gels formed by these two different methods have similar conformation and ultrastructure.

Evidence for a similarity in conformation and ultrastructure between neutralised and low-set

curdlan gels was presented by Takahashi and colleagues (1986) and through similar methodology by Kanzawa and others (1989). In both cases the resistance of different curdlan preparations to either enzymic or acidic hydrolysis was examined and the preparations were visualised by transmission electron microscopy either before or after hydrolysis. It was observed that both the neutralised and the low-set preparations were completely hydrolysed by the action of zymolase or another B-D-glucanase from Tràmetes san guinea (Takahashi et al., 1986). Furthermore, 32% H2SO4 completely hydrolysed the neutralised preparation after

30 days at 32 0C whilst only 5% of the low-set preparation was resistant to the same treatment (Kanzawa et al., 1989). Curdlan preparations obtained by heating to higher temperatures showed much greater degrees of resistance. Electron micrographs of the neutralised and low-set preparations exhibited a high degree of similarity being composed of microfibrils estimated to be 10-20nm wide and 100-150nm long (Kasai and Harada, 1980). As discussed later, micrographs from high temperature preparations have a quite different appearance. Thus, based on electron microscopy it would appear that the ultrastructure of

the neutralised and low-set preparations is similar whilst hydrolysis data may point to a similarity on the conformational scale.

Saito and colleagues (1977) examined molecular mobility within the low-set gel through the

use of 13C nuclear magnetic resonance. In the gel 70-80% of C1-05 signals and 40% of C6

signals were invisible when compared with a soluble form of curdlan (DPn 13). Furthermore, a downfield shift of the C4 signal was observed. The latter observation supported earlier

theoretical predictions suggesting that the curdlan molecule would exist as a helix stabilised by hydrogen bonding between 04 and 05 of adjacent glucose residues (Sathyanarayana and

Rao, 1971). Based on this hypothesis the former observation was explained in terms of the three dimensional structure of the gel in which it was suggested single helical curdlan

molecules (NIIVIR visible) formed a meshwork held together by multiple helical junction zones (NMR invisible) providing a model for the way in which the low-set gel is held together, namely through triple-helical junction zones. A more detailed examination using a number of NMR techniques led to the hypothesis that curdlan molecules exist in three states in the low-set gel. These were considered to be: a liquid-like state of single helical portions distant from triple helical portions; an intermediate state of single helical molecular portions close to triple helices; and a solid-like state of triple helices (Saito et al., 1990). This hypothesis has been supported through X-ray diffraction observations suggesting the presence of a small amount of triple-helix within the low-set gel, as described below. A further observation that the resonance signal of the DPn 13 molecule could be seen in mixed gel systems led to the suggestion that the gel contained spaces large enough to allow the smaller molecule to move freely within them. X-ray diffraction studies on curdlan treated in a manner that would produce a low-set gel appear to be fairly limited; probably due to difficulties obtaining samples with a high enough degree of crystallinity, with the result that those diffraction patterns that have been produced show many imperfections and correspondingly are difficult to interpret. Those studies that have been undertaken resulted in the presentation of two different hypotheses. The first hypothesis suggests that molecules in the low-set gel exist primarily as single helices; the second as triple helices. These two hypotheses are not necessarily incompatible and differences observed in diffraction patterns may depend on minor differences in methods of preparation of samples. Furthermore, the preparations are not necessarily the high and low- set gels described in the above section based on rheological properties and many of the samples studied were stretched film samples. In this thesis the samples are classified as low- set or high-set gels depending upon whether the samples were heated above 80 0C during their preparation. It should also be noted that in the majority of reports diffraction patterns have been interpreted with the aid of mathematical modelling to exclude unfavourable conformations from consideration and the emphasis placed on different modelling techniques may have an important impact on the final conclusions drawn. Single helical models for low-set preparations have been reported by Takeda and co-workers

(1978) and also by Okuyama and colleagues (1991). The former considered the conformational energy of a number of potential molecular structures and then further refined their models by comparison of calculated and observed diffraction intensities. They concluded that the majority of molecules existed as right handed single helices with seven glucose residues per turn with a minor proportion of the molecules present as 7/1 triple helices. This result was consistent with the 13C nuclear magnetic resonance studies of Saito and colleagues (1977). It was further suggested that the molecular organisation in the low-set gel was as a series of long cylindrical micelles 4nm in diameter with approximately 1 2nm 10

between them (Takeda et al., 1978), however, the micellar diameter has since been revised

and the concensus now appears to favour a diameter of approximately 8nm (e.g. Kasai and

Harada, 1980). Okuyama's group favoured a model based on single helices of six glucose

residues per turn (Okuyama et al., 1991). In this report only right handed helices were

considered as a right handed helix had been established for the high-set form of the gel (e.g.

Deslandes et al., 1980) and it was considered too great a conformational transition to go from

left to right hand helices between the low and high-set gels. Models ranging from five to nine

glucose residues per turn were investigated for single, double and triple helices. The 6/1

single helical model was the most favoured in terms of interatomic contacts and agreement

between observed and calculated diffraction patterns. The single stranded structure was

further supported by the observation that the high-set gel requires greater energy than the

low-set gel to produce (higher temperature). The former is believed to have a triple stranded

structure (Deslandes et al., 1980). Furthermore, the degradation temperature of the low-set

form is about 300C lower than that of the high-set curdlan gel. Another observation that may

support the single helical hypothesis for low-set curdlan gel is the sudden change in optical rotation and other parameters on neutralisation of an alkaline solution investigated by Ogawa

and colleagues (1972). A similar sudden change in the line width of 13C NMR signals was

observed on going from 0. 19M to 0.22M NaOH (Saito et al., 1977). If the low-set gel were composed of triple helices the above results would suggest that the molecules were capable of going instantaneously from a random coil to a triple helix, contrary to thermodynamic theory which would suggest two distinct phases, namely random coil to single helix followed by aggregation to triple helices.

In addition to the study of polysaccharide molecules, Okuyama and colleagues (1991) examined the state of water within the low-set gel structure. By comparison of differential 11

scanning colorimetry curves of low-set curdlan gel at various relative humidities over the

range -200C to +200C with that of the melting curve of ice and also through an examination

of the density and water! content of low-set curdlan across the full range of relative humidities

they concluded that low-set curdlan contained three types of water at relative humidities

greater than 95%. The observation that even at 0% relative humidity low-set curdlan gel still

contains approximately 10% by weight water, led to the suggestion that one type of water

present is crystalline bound water. This result was supported by deuterium oxide exchange

experiments where infra-red spectra demonstrated that not all H20 could be exchanged for

D20 in an alkali gel film (Fulton and Atkins, 1980). When a wet, low-set specimen is dried in air and diffraction patterns are recorded at regular intervals, fairly sharp images recorded initially become more diffuse as the sample dries. It was thus suggested that as water is lost, the molecular arrangement within micelles is disturbed and simultaneously loss of intramicellar water caused individual micelles to come into closer contact (Takeda et al., 1978). This data was supported by the observation that above 95% relative humidity, the differential scanning colorimetry curve for low-set curdlan is similar to that of water (Okuyama et al., 1991); thus in addition to crystalline bound water there also appears to be free water within micelles and between micelles. Thus a second model for the way in which the low-set gel is meshed together would be through the occurrence of molecules in micellar domains, held together through hydrogen bonding involving water (illustrated in Figure 1.5).

Further support for the involvement of hydrogen bonding in low temperature gelation came from the observation that both urea and. ethylene glycol lower the healing temperature required for the formation of the low-set gel (Harada, 1977). Urea breaks hydrogen bonds whilst ethylene glycol accelerates the formation of hydrogen bonds. This perhaps surprising result was explained by invoking a gelling mechanism in which it was suggested that hydrogen bonds between molecules and/or micelles in aqueous suspension are broken, thus allowing

7/1 Helix Structure of 8 lice[ le Ultrostructure of An OrIented Gel (ROOU re.veroture)

Iry 4 4 a

W a a

—

44 dlitaeflcl.IOA

4 4 a 4 4 a 4 4 .4 4 a a4 '4 4 Wiceilt diatter Ca- 30 A a

W

'4w a '4 4 PwMictile •$iatte.• a: ca. 3)A

'4 Water solectau,

1.5a Low-set gel structure (From Kasai and Harada, 1980)

Mce4lfl Carcinirg IriDe Hgces Trtple -dclix

'I H20 4,0 krdlcn

I-1,Q RO

lntertrdeojcrly Bound 'te of Ctyskdlization JO GM r,ctwcxk lcsed Or CLflIOnl

1.5b Triple helices within a 1Sc 3D Gel network micelle

(From Fulton and Atkins, 1980) Figure 1.5

Models for Curdlan Gel Structure 12

the molecules to disperse evenly in solution. On cooling, hydrogen bonds reform allowing gelation of the aqueous system. This hypothesis was later supported by examination of the differential scanning colorimetry curves of aqueous suspensions of curdlan in which a sharp endothermic peak was observed at 56 0C related to the swelling of curdlan (Konno and Harada, 1991). In contrast to single helical models for the low-set curdlan gel, Fulton and Atkins (1980)

proposed a triple helical model. The rather imperfect diffraction diagram they obtained led

to the conclusion that within the triple helix single helices contained somewhere between six and seven glucose residues per turn with water molecules interacting to untwist the calculated

symmetrical relationship of a triplex in which individual helices are based on the 6/1 model. This hypothesis was further supported by deuteration experiments in which not all of the water of crystallisation could be replaced by deuterium oxide. It was suggested that the irreplaceable water was inaccessible, being buried within the core of the triple helical

structure. The suggestion of a triple helical molecular arrangement in the low-set gel has been supported through observations made by electron microscopy. A solution of 0.05% curdlan in 0.3M sodium hydroxide was dialysed against water to produce a gel which was then mechanically dispersed. Structural elements of the gel were then examined by transmission electron microscopy after negative staining. Microfibrils of approximately 20nm diameter were observed which had a fringed appearance. The investigators therefore suggested that a number of triple helical systems were in parallel alignment along the microfibril

(Marchessault and Deslandes, 1979). However, despite the presentation of a hypothesis in which the curdlan molecules are present in the triple helical conformation Fulton and Atkins

(1990) supported the oncept of a micellar arrangement for the gel (Figure 1.5); although 13

they suggested that the gelling mechanism was due to partial dissolution of micelles thus allowing them to reassociate to form junction zones. In contrast to the low-set curdlan gel a proportion of the high-set gel was resistant to

hydrolysis by either 13-(1-'3)-glucanases (Takahashi et al., 1986) or 32% sulphuric acid

(Kanzawa et al., 1989). Furthermore, it was observed that gels heated to 120 0C had a

greater proportion of resistant gel (54-59%) than those heated to 95 0C (34%) and also that gels heated at 120 0C for four hours showed greater resistance (63-66%) than those heated

at the same temperature for thirty minutes (54-59%). This data would suggest the occurrence of some energy requiring conformational transition leading to a structure inaccessible to either enzymes or protons. The high-set gels were examined by transmission electron microscopy before or after hydrolysis. Prior to hydrolysis, 95 0C preparations revealed an arrangement of microfibrijs connected by finer elementary fibrils which were absent after hydrolysis. In the preparation heated to 120 0C, electron dense pseudocrystalline forms were observed bound by fibrils which were absent after hydrolysis. Previous data, hydrolytic resistance and electron micrograph observations (for the data on low-set curdlan see pp. 7-8) led to the suggestion that the low-set gel was formed by hydrogen bonding whilst the high-set gel was formed through hydrophobic interactions (Kanzawa et al., 1989). This hypothesis was supported by differential scanning colorimetry curves for aqueous suspensions of curdlan in which a broad endothermic peak was observed between approximately 80 0C and 1050C (Konno and

Harada, 1991). It was suggested that this peak was due to hydrophobic interactions between curdlan molecules thus forming the high-set gel. An exothermic peak was observed at 120 6C to 1400C and this was possibly due to partial crystallisation of curdlan micelles. Similar experiments were undertaken on a truncated curdlan preparation (DPn 131 cf. 455 for the normal preparation) and the sample heated to 60 0C exhibited 35% resistance to hydrolysis.

Furthermore, electron micrographs of samples heated to 60 0C, 900C or 1200C showed a 14

high degree of similarity. It was thus suggested that the smaller curdlan molecule was able

to form hydrophobic bonds more easily; even at temperatures as low as 60 0C (Takahashi et

al., 1986).

X-ray diffraction studies on high-set curdlan indicate a higher degree of crystallinity than the

low-set form (e.g. Kasai and Harada, 1980; Marchessault et al., 1977). The available

literature appears to present a much more coherent view of the conformation of the 8-(1 —3)-

glucan molecules of the high-set form compared to the low-set; nearly all models being based

on a triple helical arrangement.

An early diffraction pattern of the high-set gel of high quality was produced by Marchessault

and colleagues (1977). Oriented fibres were annealed in a sealed bomb at 143 0C. Two

diffraction patterns could be recorded; one at relative humidities less than 20% and one when

humidities were greater than 20%. Analysis of the diffraction pattern at 0% relative humidity led to the conclusion that the molecules were present as triple stranded helices each helix containing six glucose residues per turn leading to a unit cell containing a total of eighteen glucose residues. The patterns recorded at higher relative humidities were believed to correspond to the crystalline hydrate and revealed disruption of the symmetry of the sixfold triple helix, presumably by the interaction of water molecules. These conclusions were later supported by similar diffraction data obtained by Takeda and colleagues (1978) using a sample annealed at 130 0C. Later work refined the triple helical model through the use of a series of complex mathematical equations describing the statistical likelihood of various conformations as a function of various bonding parameters (Deslandes et al., 1980). It was thus concluded that the triple helix of the dry crystalline form was right handed and stabilised by hydrogen bonding between 02 hydroxyls of the individual glucan chains. A similar refinement was undertaken on the crystalline hydrate form of high-set curdlan (Chuah et al., 15

1983). This supported earlier findings that water caused a disruption of the symmetry of the

six-fold triple helix (Marchessault et al., 1977) and confirmed the previously described right

hand turn of the triple helix (Deslandes et al., 1980). It was further deduced that thirty six

water molecules were involved in the hydrate structure (two per glucose residue) through density measurements. An attempt to define the position of the water molecules within the

triplex proved difficult, with the conclusion that there was a statistical fluctuation of water molecules within the structure, however, it was suggested that most water molecules were located on the periphery of the triplex with 04 and 06 oxygens most frequently hydrogen bonded to water.

1.4 Commercial Applications of Curdlan The 'commercial applications found for curdlan are primarily, although not exclusively, a result of the rheological properties displayed by the substance. It is frequently used as a gelling agent in industry and food processing. Commercial interest in curdlan was first expressed by Takeda Chemical Industries Ltd. in

1968 (Harada, 1992). A subsidiary of the company, Wako Pure Chemical Industries Ltd. tested pilot plant production in 1971 and 1972 and now commercially produce approximately two tonnes per annum (Harada et al., 1993). Curdlan has been found to be both non-toxic and nutritionally inert in humans; as a result the International Research and Development Cooperation in the U.S.A. has found curdlan to be safe (Harada et al., 1993). The majority of applications for curdlan appear to have been found within the food industry where it can be used in foods that require heating in water during production or cooking, either replacing more conventional food additives or allowing the production of new foods.

Addition of curdlan to noodles provided superior elasticity and body, likewise hamburgers containing between 0.3% and 1% curdlan were found to have improved textural properties 16

(Kimura et al., 1973). Curdlan has also been used as a thickener and stabiliser in salad dressings and spreads (Harada, 1977) and as a stabiliser in ice cream (Harada et al., 1993).

In addition to the ability of curdlan to improve textural properties of foods it has been of use as a water holding agent e.g. in sausages (Harada, 1977). New foods produced from curdlan include thin layered flavoured gels, soy milk gels and jelly-fish like gel food (Nakao et al.,

1991): One appeal of curdlan foods may be the non-calorific value of curdlan (Harada,

1977).

In addition to food uses a number of patents have been filed for industrial applications.

Curdlan has been used in place of agar in biological culture media (Hasegawa et al., 1983).

It can also be used as a binding agent in tobacco products (Harada, 1977), or can act as a support for immobilised enzymes (Harada et al., 1993), ceramics or active carbon (Harada,

1992). It can also be used to prevent segregation in the production of concrete (Harada,

1992).

Finally curdlan has been shown to have certain biological activities which may point to its use as a therapeutic agent. Curdlan sulphate has been reported to be effective against the onset of AIDS in HIV positive patients (Harada, 1992). Both curdlan (Sasaki et al., 1978) and the water-soluble curdlan derivative carboxymethylcurdlan (Sasaki et al., 1979) had an inhibitory effect on tumour growth in mice. These therapeutic effects are believed to be due the immunomodulating activity of curdlan.

Finally it should be mentioned that many of the above applications are in the experimental stage and it remains to be seen whether they become widely used. 17

1.5 Summary

Curdlan is a 13-D-(1-'3)-glucan produced by a number of bacteria as an exopolysaccharide.

The molecule produced by Agrobacterium sp. ATCC 31749 has a molecular weight of

approximately 73,000 Da and this value appears to remain constant whenever the culture is

harvested.

The polysaccharide is soluble in alkali and a few other solvents but is insoluble in water.

However, when an aqueous suspension is heated it is capable of forming two types of gel.

The first is formed when a suspension heated above 600C is cooled, exhibits a low degree

of syneresis, has a gel strength independent of heating time and melts when reheated above

650C. Molecular conformation within this gel appears to be primarily of 6/1 single helices

arranged in micelles which maintain their structure through hydrogen bonding.

The second is formed when a suspension of curdlan is heated above 800C, exhibits a higher degree of syneresis, has a higher gel strength than the former gel which is dependent on heating time and is reported to melt in the range 150 0C to 1600C. Molecular conformation within this gel appears to be 6/1 triple helices stabilised by hydrogen bonding. Again there appears to be a micellar arrangement with some pseudocrystalline forms stabilised by hydrophobic interactions.

The above rheological properties have led to a considerable amount of commercial interest in the polysaccharide, particularly from the food industry.

\ lu

Chapter 2 Mechanisms of Biosynthesis and Export of Bacterial Exopolysaccharides

2.1 Entry of Carbon Substrate With a few exceptions e.g. dextran (Sutherland, 1990) bacterial exopolysaccharides are

synthesised within the cell. The first step in exopolysaccharide biosynthesis must therefore

be uptake of carbon substrate. As this process represents a biochemical event essential to the functioning of the cell and is not specific to exopolysaccharide biosynthesis only a brief

discussion of the known mechanisms will be presented here. (Further discussion of carbon substrate uptake effects on exopolysaccharide biosynthesis, see pp.54-55).

For a molecule to enter the cytosolic compartment of a grain negative bacterium it must pass through a considerable array of barriers. The first barrier that a carbohydrate must cross in order to enter the Gram-negative bacterial cell is the outer membrane (Figure 2.1). This layer consists of protein, phospholipid and lipopolysaccharide. Transport across this membrane occurs via proteinaceous pores creating a local hydrophilic environment through which solutes can pass via diffusion (Ames, 1986). These channels show varied degrees of specificity, e.g. ompF porin shows low specificity and will transport oligosaccharides of molecular weight less than 500 Daltons, amino acids, phosphorylated carbohydrates or salts

(Lugtenberg and Van Alphen, 1983). The lamB porin shows specificity for malto- oligosaccharides up to maltoheptaose (Nikaido and Vaara, 1985).

Once into the periplasmic space, a substrate can diffuse through this gel filled cell compartment (Whitfield and Valvano, 1993) and through the porous peptidoglycan layer (Ames, 1986). Slime Polysaccharjde

Capsular Polysaccharide (CPS)

Upopolysaccharide (LPS)

Outer Membrane

Periplasm

Peptidoglycan

Cytoplasmic Membrane

Figure 2.1

Structure of the Gram-negative cell envelope. (From Whitfield and Valvano, 1993) 19

The final barrier to carbohydrate entry into the cytoplasm is the cytoplasmic membrane,

composed of phospholipid and protein (Ames, 1986). This membrane is impermeable to almost every solute and five distinct transport mechanisms have evolved in order to allow

entrance of nutrients to the cell. The first mechanism, depicted in Figure 2.2, differs from the others in that it is not coupled to an energy releasing reaction. This mechanism bears a

similarity to solute translocation across the outer membrane in that transport appears to be via a pore-like mechanism; i.e. transport occurs by translocation from one complexation site

of the porin to the next (Saier, 1985). Other mechanisms of carbohydrate transport across the inner membrane utilise various forms

of energy coupling in order to drive solutes against a concentration gradient. Symport mechanisms (Figures 2.213 and 2.2C) couple the translocation of carbohydrate with that of

an ionic species. In both the lactose proton symport (Kaczorowski and Kaback, 1979) and the melibiose sodium symport (Tsuchiya et al., 1982) one ion is transferred per carbohydrateS molecule. In the case of the hydrogen symport, the proton and sugar bind independently

(Wright et al., 1981) and both must be bound for transport to occur (Kaczorowski and

Kaback, 1979), whilst in the case of the sodium symport, binding of the two enzyme substrates is co-operative (Tokuda and Kaback, 1978). The fourth system of carbohydrate transport across the bacterial membrane is typified by maltose uptake, a primary active transport process involving up to six separate proteins.

Initially, malto-oligosaccharides diffuse across the outer membrane via the LamB (Ames,

1986)/MalL (Saier, 1985) porin. Once into the periplasmic space the malto-oligosaccharide then binds to the maltose binding protein (MalE) via hydrogen bonding (Saier, 1985), leading to a conformational change in the binding protein which allows the protein to interact with the MaIG and MaW proteins located in the cytoplasmic membrane. This protein-protein interaction leads to yet another conformational change in the binding protein allowing release A Facilitated Diffusion Gly (Glycerol)

B Lac Proton Symport (Lactose)

C Mel Sodium Symport NC (Melibiose)

D V Active Transport (Maltose Mat at

E Group iranslocation MU (Mannhtol) PEP

pyruvate

Figure 2.2 Mechanisms of carbohydrate transport into the bacterial cell.

(From Saier, 1985) 20

of the carbohydrate substrate (Szmelcman et al., 1976). Simultaneously, interaction of the binding protein with the proteins of the cytoplasmic membrane (Ma1G, Ma1F) allows exposure

of the substrate binding site of these proteins (Ames, 1986) and the subsequent transport of maltose across the cytoplasmic membrane, probably in a screw-like fashion which would

allow specificity for malto-oligosaccharides of different molecular weights (Saier, 1985). The

cytoplasmic component, MalK, is believed to be involved in energy coupling for the system

possibly via a nucleotide binding site (Dean et al., 1989). The final component of the system (MolA/MaIM) is a periplamic protein of unknown function (Saier, 1985). Recent articles appear to suggest that this protein does not play a role in maltose transport (e.g. Dassa and

Muir, 1993). Similarly, Agrobacterium radiobacter has two periplasmic glucose binding proteins which increase in concentration in parallel with glucose uptake rate when the organism is grown under glucose limitation (Cornish et al., 1988a), suggesting that glucose is taken up by a binding-protein dependent mechanism in this organism. The proteins have

also been shown to be involved in uptake of galactose and xylose (Cornish et al., 1989) and this mechanism of uptake also occurs under nitrogen limitation (Cornish et al., 1988b).

Similar proteins have been observed in A. tutnefaciens (Cornish et al., 1989) and another periplasmic binding protein, specific for lactose, has been identified in A. radiobacter

(Williams et al., 1990) suggesting that binding protein dependent sugar transport may be fairly widespread among Agrobacterium sp. The final mechanism of transport across the cytoplasmic membrane, group translocation, links transport with phosphorylation of the translocated carbohydrate. Group translocation systems have been identified for the transport of a diverse range of carbohydrates such as glucose, fructose, mannitol, N-acetyl glucosamine and 13-glucosides (Dills et al., 1980). The cascade of events for group translocation of glucose are depicted in Figure 2.3. The first Glucose Glucose-6-P-

P S e PEP

Figure 2.3

Components of the glucose group translocation system in Escherichia coli.

(Adapted from Saier, 1985). 21

three components of this system are general components found in the majority of group

translocation events whilst the final two enzymes are specific to glucose group translocation. As can be seen in Figure 2.3, phosphoenol pyruvate is the high-energy phosphate donor with

the released phosphate group being transferred along a series of enzymes each catalysing phosphorylation of the next until the translocating enzyme (11g1c) is primed by phosphorylation of a histidyl residue. This phosphate is then transferred to glucose during translocation across the cytoplasmic membrane resulting in an increase in the intracellular concentration of glucose-6-phosphate (Saier, 1985).

2.2 Precursors of Exopolysaceharide Biosynthesis With the exception of dextrans which have a specific requirement for sucrose (Hestrin,

1962) and a few other polysaccharides such as levans and mutans (Sutherland, 1982) the majority of both homo and heteropolysaccharides synthesised by bacteria can be formed from a wide range of carbon substrates (Sutherland, 1982). It is thus apparent that a certain degree of sugar re-arrangement must occur prior to commitment to exopolysaccharide biosynthesis.

A number of studies have attempted to identify the biochemical pathways through which an exogenous substrate is metabolised prior to entering the pathways of polysaccharide biosynthesis; usually via a nucleotide diphosphate sugar (Sutherland, 1979a). Agrobacterium carbohydrate metabolism was examined. Seven of seven species tested utilised the pentose phosphate pathway. Five of those seven (A. radiobacter, A. tumefaciens, A. rubi, A. rhizogenes and A. tellatum) also utilised the Entner-Duodroff pathway whilst A. pseudotagae and A. gypsophilae utilised the Emden-Meyerhof (glycolytic) pathway (Arthur et aL, 1975). Figure 2.4 illustrates pathways of carbohydrate metabolism in A. tumefaciens.

Curdlan biosynthesis was examined in Agrobacterium sp. ATCC 31749 through the use of specifically labelled 13C glucose in conjunction with NMR spectroscopy of the curdlan

Pentosa Cice

CH,OH :CX1OH 7 CH'OH G;uc:e a as

itH;t3H 2 CHO fjCtOSt° 'C - ructn5e.4 P 'CH k CH'OH séii1op C H Q P '—a. C}l2QP G:ac:se iP C11, OH Eryfluast. P runsseaoiasa GA 3 P Guc:se S P En Oner Daucoro t t 'c=a Iransatcajase 4, COON

CHO •rHGH At S P VucnE! CH.OH c_a \L :' UP ap E ,mrrasa Seantotulose]? GA3P GOr iC.l4QP J t CHH iCH,OP tiOSflOIuCJ1lC! 40P0 Zylulese 5 P Rbuiase 5 p XOPG ,o aloalass 'CHO COON

Cncx - 'CHOP H'GP GA 3 P Pyruvar Ribose 5 P

Figure 2.4

Pathways of carbohydrate metabolism in Agrobacterium tumefaciens.

(From Arthur etaL, 1973) 22

product (Kai et al., 1993). When 1-' 3C-glucose was used as substrate the majority of the

13C label remained on position 1 in the curdlan product indicating direct polymerisation of glucose. This phenomenon was also observed when 2- 13C-glucose or 6- 13r -giucose1 were

used as substrates. When 1-13C-glucose was used as substrate a small but significant amount

of label was observed in the carbon 3 position on curdlan. It was suggested that the glucose had been degraded to 1- 13Cdihythoxyacetone phosphate via the Emden-Meyerhof pathway

followed by rearrangement to 3.1 3C-dihydroxyacetone phosphate and then recombination of trioses to glucose leaving some label in position 3 of the reconstituted glucose. This would

suggest that Agrobacterium sp. ATCC 31749 has pathways of carbohydrate utilisation similar to A. pseudotagae and A. gypsophilae (Arthur et al., 1975). Rearrangement of the 13C label was also observed when 6- 13C-glucose was used as a substrate with a considerable amount of label appearing in position I of the curdlan synthesised from this substrate. This rearrangement was suggested to occur via isomerisation of 3- 13 C-glyceraldehyde phosphate to 1- 13CdihyJroxyacetone phosphate within the glycolytic pathway followed by the formation of glucose via gluconeogenesis. Finally, when 2- 13C-glucose was used as substrate, transfer of label was observed to C-i and C-3 positions in curdlan. A suitable hypothesis was that the position of the label was altered by rearrangements within the pentose phosphate pathway leading to the formation of fructose-6-phosphate and then isomerisation to glucose-6-phosphate prior to entering the pathway of curdlan biosynthesis. These observations led to a hypothesis suggesting five routes to curdlan biosynthesis. These were: direct polymerisation of glucose; recombination of trioses with Ci_C3 rearrangements; recombination of isomerised triose to glucose with transfer of label from C6 to Ci; formation of glucose from fructose-6-phosphate produced via the pentose phosphate cycle; and neogenesis of glucose from fragments formed in various pathways of glycolysis. A 23

quantitative analysis was undertaken to determine the relative input of each route; however, this would depend on culture conditions and direct polymerisation of glucose was the major

route to curdlan biosynthesis under the conditions tested. As has already been suggested, the direct precursors of the monosaccharide residues found

in bacterial polysaccharides are usually nucleotide diphosphate sugars (Sutherland, 1993;

Jarman, 1979) or occasionally nucleotide monophosphate sugars as is the case when 3-deoxy-

D-manno-2-octulosonic acid is incorporated into the core region of bacterial lipopolysaccharides (Sutherland, 1990). These molecules have a number of functions, the foremost being to act as energy rich, activated forms of the monosaccharides to be polymerised into carbohydrate constituents of bacterial lipopolysaccharides, capsular and exopolysaccharides, periplasmic glycans, cell wall polysaccharides and teichoic acids (Sutherland, 1993). In addition primary nucleotide diphosphate sugars, formed by the reaction of a nucleotide triphosphate with a sugar phosphate (or sugar), can act as precursors of secondary nucleotide diphosphate sugars through enzyme catalysed interconversions as described below. These activated sugars also have a role to play in the regulation of carbohydrate anabolism (see pp. 55-58). Evidence for the role of nucleotide diphosphate sugars in polysaccharide biosynthesis has come from kinetic studies of the flux of radioactive intermediates into polysaccharides in particulate enzyme preparations from various bacteria and also through the isolation and biochemical characterisation of mutants unable to synthesise certain polysaccharides.

The biosynthesis of the exopolysaccharide succinoglycan by Rhizobium meliloti has been extensively studied at the genetic level (Glucksmann et al., 1993a; Glucksmann et al., 1993b;

Reuber and Walker, 1993b; Long et al., 1988a). This polysaccharide has been shown to consist of repeat units containing glucose and galactdse in the molar ratio 7:1 (Jansson et al.,

1977) with the structure shown in Figure 2.5. During the course of these investigations S 5 5 "Gl& 'Gic' £G;ct 1GaI1—: 6 $

Glc

I t Gic 3

Gic 13

Pyr ,

0-Acetyl and C-Succinye present at undeterm,ned sites.

Figure 2.5

Structure of succinoglycan.

(From Zhan et aL, 1990) 24

mutants were generated which exhibited various defects in polysaccharide biosynthesis. One

study analysed the level of production and chemical nature of the lipopolysaccharide,

succinoglycan and 13-(1,2)-glucan produced by strains of R. meliloti with mutations in a

number of exo loci (Leigh and Lee, 1988). Whilst most mutants failed to produce

succinoglycan only exoC mutants were incapable of producing B-( 1 ,2)-glucan. In addition

exoC and exoB mutants were observed to produce altered lipopolysaccharide as judged by sodium dodecyl sulphate polyacrylamide gel elctrophoresis. It was suggested that bacteria

with mutations at these two loci were deficient in an early non-specific step of succinoglycan biosynthesis such as the formation of UDP-glucose or UDP-galactose. Confirmation that

exoB was involved in one of these early steps was provided through an examination of

sequeice homology of the R. meliloti exoB gene with that of the UDP-glucose 4-epimerase

encoding galE gene of E. coli and also through direct measurements of enzymic activity in

wild type and exoB mutant cell extracts of R. meliloti (Buendia et al., 1991). In exoB mutant

cells of this bacterium the inability to form succinoglycan and normal lipopolysaccharide is

due to an inability to polymerise galactose, due to an absence of its activated precursor IJDP-

galactose formed by C-4 epimerisation of UDP-glucose. As 8-(1,2)-glucans do not require

galactose they are produced in quantities similar to wild type cells (Leigh and Lee, 1988).

Similar phenotypic defects have been observed in R. leguminosarum (Canter Cremers et al.,

1990) and Erwinia stewartii (Leigh and Coplin, 1992) and again the mutations were observed

to occur in genes encoding IJDP-glucose 4-epimerase. It has also been shown that a mutant

strain of Agrobacterium radiobacter failing to produce exopolysaccharide can be corrected by complementation with the R. meliloti exoB gene (Aird et al., 1991).

ExoC mutants of Agrobacteriutn tumefaciens displayed similar polysaccharide defects to

those of exoC mutants of R. meliloti. The ability of cell extracts of the A. tumefaciens 25

mutants to form UDP-glucose from glucose, glucose-i-phosphate and glucose-6-phosphate

was examined. As expected wild type cells and exoC mutants complemented with wild type

exoC were able to synthesise UDP-glucose from all three substrates; however, extracts from cells with a disrupted exoC gene were only able to form UDP-glucose from glucose-i- phosphate (Uttaro et al., 1990). The evidence therefore supported the hypothesis that exoC encoded for the enzyme phosphoglucomutase and furthermore that the inability of cells provided with glucose to produce polysaccharide was a result of a deficiency in the metabolic pathway leading to UDP-glucose. Similar pleiotropic polysaccharide deficiencies have been

observed in Erwinia stewartii, Xanthomonas campestris, Pseudomonas solanacearum and

Pseudomonas aeruginosa and it has been proposed that the deficiencies result from lesions in pathways to nucleotide diphosphate sugar synthesis (Leigh and Coplin, 1992). Another piece of evidence suggesting a role for phosphorylated sugar nucleotides in polysaccharide biosynthesis in bacteria has come from studies with antimicrobial agents which

specifically and competitively inhibit CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO synthase) e.g. a-C-( 1 ,5-anhydro-8-amino-2,7,8-trideoxy-D- manno-octopyranosyl) carboxylate. The core region of bacterial lipopolysaccharides contains 3-deoxy-D-manno-octulosonic acid (KDO) (Whitfield and Valvano, 1993). Upon addition of the inhibitors, lipopolysaccharide synthesis ceased in Escherichia coli, Salmonella typhimurum (Goldman et al., 1988a), Pseudomonas aeruginosa (Goldman et al., 1988b),

Agrobacterium tumefaciens and Aeromonas salmonicida, (Goldman et al., 1992), thus suggesting that CMY-KDO is the activated precursor of KDO residues in lipopolysaccharide. Another important function of primary nucleotide diphosphate sugars is to act as precursors for secondary nucleotide cliphosphate sugars. Although, in the case of curdlan, this function may appear irrelevant it must be remembered that other polysaccharides produced by

Agrobacterium sp. ATCC 31749 may contain a number of residues other than glucose. Any 26

of these residues in which the donors are secondary sugar nucleotides derived from UDP-

glucose will represent a potential drain on the availability of UDP-glucose for curdlan biosynthesis and so a brief discussion of derivatives of UDP-glucose will be presented.

Table 2.1 lists many of the residues so far identified in bacterial polysaccharides and describes the nucleotide linked donors if known. Twelve D-glucose-derived residues have been identified and fourteen uridine-linked sugars have been observed in bacterial cells. The former class may represent a drain on the availability of UDP-glucose for curdlan biosynthesis whilst the latter may represent a drain on the availability of UTP for UDP-glucose synthesis. UDP-glucose acts as the glucose donor for all bacterial polysaccharides with the exception

Of the storage polymer glycogen for which the glucose donor is ADP-glucose (Romeo et al., 1990). UDP-glucose can also undergo a number of rearrangements to provide donors for other residues. These include C-4 epimerisation to produce UDP-galactose and C-6 oxidation to produce UDP-glucuronic acid (Shibaev, 1986). The biochemical reactions discussed in this section occur in the cytoplasm where under normal conditions, there would be a readily available pool of enzyme substrates.

2.3 The Formation of Heteropolysaccharide Repeat Units In the synthesis of a number of bacterial hetero-exopolysaccharides, the next stage of biosynthesis is the build-up of repeat units of the polysaccharide chains involving transfer of sugars from their nucleotide diphosphate derivatives to a lipid acceptor associated with the cytoplasmic membrane (Troy, 1979). The requirement for the lipid-intermediate stage was first demonstrated in the biosynthesis of lipopolysaccharide (Osbourn and Weiner, 1968) and peptidoglycan (Higashi et al., 1967) and has since been observed in the biosynthesis of capsular polysaccharides (Troy, 1979) and exopolysaccharides (Sutherland, 1993). All Number of different n,onosaccliarjtlrs known

Activated Iorms 1

Biogenetic Total group U U G C A known Unknown 'Total

I. n- Fruci occ-dcriscd a I(Ioses

I)-glIsrvP Series 5 2 - I - 8 4 12

I)-galarlo Series 5 3 - I - 9 I 10 I)- ,p,aunn Series 2 - 3 I - 6 2 8 t-glurn - - - - - Series - 2 2

L-galacla Series - - 2 - - 2 2 4

I.- ;nt',iun Series - I -- - 2 I 3

()tIsc,s 2 I I - - 4 It) 14

Tool - 14 7 6 4 - 31 22 53

2. I)-flhI)oqe-dcrbed aldoces

Total - - - - - - 4 4

3. i)-.Scdoheptnlose-derived aldosec

Total - - - - 2 2 3 5

4. ketoses and 3-deoxyaldiulosonic acids

Total - - - 2 - 2 5 7

Total 14 7 6 6 2 35 34 69

I, Abbreviations denote nucleoside residues of activated forms

Table 2.1

Monosaccharide components described in bacterial polysaccharides and the activated

precursors if known.

(From Shibaev, 1986) 27

bacterial heteropolysaccharides found external to the cell membrane except dextran and

related • polymers, so far examined have been shown to involve a lipid intermediate stage (Sutherland, 1990). This mechanism of biosynthesis is not limited to heteropolysaccharides

and a number of homopolysaccharides including the polysialic acid capsule of Neisseria meningitidis utilise a lipid intermediate stage (Masson and Holbein, 1985).

In the majority of cases where the lipid has been analysed, it has been found to be a C55 polyisoprenyl alcohol derivative, undecaprenol phosphate (Whitfield and Valvano, 1993) sometimes referred to as bactoprenol. Possible roles for the undecaprenol lipid include spatial organisation of the substrates for the glycosyl transferase reactions at the cytoplasmic face of the inner membrane, involvement in the translocation of repeat units to the cell surface during polymerisation and through selective phosphorylation of the lipids, control of polysaccharide biosynthesis (see also pp. 58-60). However, arguments for and against theses roles remain primarily hypothetical although a number of gene sequences for glycosyl transferases have indicated membrane associations for the gene products in Rhizobium meliloti (Glucksman et al., 1993a).

The first stage in the build-up of polysaccharide repeat units involves the transfer of a monosaccharide from its activated nucleotide diphosphate derivative to bactoprenol phosphate. In the majority of cases sugar- i-phosphate is transferred with the concomitant production of lipid pyrophosphate sugar and nucleotide monophosphate (Sutherland, 1990)

This process has been observed to require only one gene product (GumD) in the case of xanthan biosynthesis by Xanthomonas campestris (Vanderslice et al., 1990); however, the addition of the initial galactose to lipid carrier in succinoglycan biosynthesis by Rhizobium meliloti requires two gene products (Reuber and Walker, 1993b). Sugars are then transferred to the non-reducing end of the growing chain in an elongation reaction with the release of nucleotide diphosphates (Sutherland, 1990). Current opinion holds that each transferase

reaction is catalysed by a specific glycosyl transferase.

Much of the pi9neering work providing evidence for the above mechanism in the

biosynthesis of bacterial exopolysaccharides was undertaken in Marcelo Dankert' s laboratory

using particulate enzyme preparations formed through EDTA treatment of whole cells. This

treatment permeabilised cells, thus allowing penetration of substrates whilst maintaining an

environment in which lipid acceptors were endogenously available in the correct spatial

orientation for access by enzymes. As previous studies had demonstrated the role of lipid

acceptors in the biosynthesis of bacterial heteropolysaccharides early work from this

laboratory centred on the biosynthesis of cellulose by Acetobacter xylinum. Later research

has shown that the lipid linked intermediates observed were involved in the biosynthesis of

Another polysaccharide produced by the same bacterium (see below) and that cellulose

biosynthesis does not involve lipid linked intermediates (see pp. 42-44). EDTA treated cells were incubated with UDP-[ 14C]-glucose. Radioactivity was incorporated into several

phases; alkali insoluble, alkali soluble and lipophilic (Garcia et al., 1974). The alkali insoluble

portion was presumed to be cellulose. Incorporation into the lipophilic material had an

absolute requirement for magnesium, an observation that has since been observed in a number

of other species such as Xanthornonas campestris (Jelpi et al., 1981a), Rhizobinin ineliloti

(Tolmasky et al., 1982) and Agrobacteriwn tumefaciens (Staneloni et al., 1984). Standard

analytical methods were used to investigate the nature of these lipophilic products. Three

fractions were observed when the lipophilic phase was subjected to DEAE-cellulose column

chromatography and identified as lipid-pyrophosphate-glucose, lipid-pyrophosphate- cellobiose and lipid-monophosphate-galactose. Kinetic studies were interpreted as suggesting that these compounds acted as intermediates in the production of polysaccharide. Evidence 29

supporting the biochemical analysis of the monophosphate linkage of the latter product was

obtained using [ 32P]-UDP-glucose as substrate. Radioactivity was incorporated only into the former products adding credence to the proposal that polysaccharide repeat units are

frequently initiated by the transfer of sugar phosphate to the lipid acceptor. No radioactive

products were observed when 14C-labelled glucose, glucose-i-phosphate, ADP-glucose, GDP-glucose or TDP-glucose were used as substrates, thus supporting the hypothesis that UDP-glucose is the immediate donor of glucose to most bacterial polysaccharides. The

detergent triton-X inhibited incorporation of radioactivity, emphasising the importance of the correct lipid environment in glycosyltransferase reactions.

Later studies suggested that lipid pyrophosphate cellobiose was capable of acting as an

acceptor for further monosaccharide residues in A. xylinum. As previously observed (Garcia

et al., 1974), radioactive lipid pyrophosphate glucose and lipid pyrophosphate cellobiose

were isolated when UDP-[ 14C]-glucose was used as substrate (Couso et al., 1980). Two possibilities were conceived for the production of the disaccharide derivative; either sequential

addition of glucose to lipid from UDP-glucose or transfer of glucose from one lipid pyrophosphate glucose to a second lipid pyrophosphate glucose. Two experimental approaches supported the former mechanism. In the first, radioactive lipid pyrophosphate

glucose was supplied as an exogenous substrate in either the presence or absence of unlabelled UDP-glucose. When UDP-glucose was present, synthesis of radioactive lipid pyrophosphate cellobiose was observed whilst in the absence of UDP-glucose only radioactive lipid pyrophosphate glucose was isolated. The second method involved a two stage incubation. In the first stage cells were incubated with UDP-[ 14C]-glucose at OC leading mainly to the formation of lipid pyrophosphate glucose. After washing cells were incubated at 300C in the presence or absence of UDP-glucose. When UDP-glucose was present in the second incubation, lipid pyrophosphate cellobiose was formed at the expense 30

of lipid pyrophosphate glucose. In the absence of UDP-glucose no lipid disaccharide derivative could be isolated. A further series of experiments employing labelled or unlabelled

GDP-mannose and UDP-glucose with manipulation of the conditions of incubation showed that the lipid linked cellobiose derivative acted as a precursor of a mannose-containing, lipid- linked trisaccharide identified as lipid-pyrophosphate-glucose-( 1 -'4)-13-glucose-( 1 -3)-B- mannose.

By 1982, the synthesis of a lipid linked heptasaccharide had been demonstrated in EDTA treated A. xylinum cells (Couso et al., 1982). Through manipulation of experimental conditions all but reaction eight (repeat unit polymerisation) of the scheme presented in

Figure 2.6b were demonstrated. It was thus shown that EDTA treated A. xylinum cells were able to form the lipid-linked repeat unit of the exopolysaccharide acetan (Figure 2.6a) in vitro, suggesting that the lipophilic product may be an intermediate in exopolysaccharide biosynthesis. The demonstration of the final reaction, lipid-linked intermediate incorporation into acetan, was achieved through the use of electroporation to permeabiise cells (Semino and Dankert, 1993). The repeat unit was synthesised at 10 0C, a temperature at which no polymer synthesis was observed. In addition to the heptasaccharide repeat unit demonstrated previously (Couso et al., 1982), radioactive acetate could also be incorporated into the cellobiose derivative or larger fragments through the addition of [ 14C]-acetyl coenzyme A, suggesting a role for this molecule as a donor of acetyl groups and also demonstrating acetylation at the repeat unit stage of polysaccharide biosynthesis. Once radioactive lipid intermediates had been accumulated at 10 0C, cells were washed and an upshift of temperature to 300 C resulted in the production of radioactive acetan, suggesting a precursor product relationship. Permethylation analysis of the polysaccharide revealed that some of the side chains lacked the terminal rhamnose. The hexasaccharide repeat unit was thus capable of

CH.CK - 01:0K I - ozr C 01 CHI 9p2 CICHI COCK /

L—fl-o-Ma,i.i —3)-o-GIc-4 I-4)-o-GIc-1 I-

O-Acetvl

2.6a Chemical structure of acetan. (From Semino and Dankert, 1993)

uOP

UOP-GcIJA){ UDP-GIc Man01-3)GLc-GLc -PP - prenot GOP - 4 3 Gtc(al-4)GLcUA-Man-GIc-Gic -PP - prenot I GDP-Man1 GIc(0I - 4) Glc -P,P - prenoi 6 UOP UDP UDP-GC UOP42 Gc(1I-6)Gtc-GIcUA-Man-Gc -Gtc - PR- prenot - GIc Gte - PR- prenot TOP 1Op-Pha UMP 11 Pha(1- 6)GIc-Gtc- GICUA-Man- Gte-Gte - PP - prenot 8 --pot;saeeharide UOPGtc .k.N P,, not

UDP-Gat UDP -P-prenot prenot

2.6b Proposed mechanism of biosynthesis of acetan in Acetobacter xylinum. (From Couso ci at, 1982)

Figure 2.6

The structure and biosynthesis of acetan. 31

acting as a substrate for the polymerising enzyme(s). A mutant strain of A. xylinum capable of producing a truncated acetan in vivo with a repeat unit lacking the last three sugars of the side chain has been isolated (Mac Cormick et al., 1993).

Similar methods to those described above have been employed to study xanthan biosynthesis by Xanthomonas campestris (lelpi et al., 1981a; b). In this organism, EDTA-treated cells were capable of polymerising repeat units to form xanthan (Jelpi et al., 1993). Xanthan gum is composed of a cellulosic backbone with a side chain of glucuronic acid and mannose in the ratio 1:2 attached to every second glucose residue of the backbone (Figure 2.7a). This polymer also has varying degrees of acetylation and pyruvylation (Jansson et al., 1975). The repeat unit was built up sequentially onto polyprenol phosphate by the initial transfer of glucose-i -phosphate from UDP-glucose to produce lipid pyrophosphate glucose followed by the transfer of glucose, mannose from GDP-mannose, glucuronic acid from UDP-glucuronic acid and finally a second mannose residue: When the three nucleotide linked sugar donors were added together, xanthan gum was also synthesised (lelpi et al., 1981a).

Phosphoenolpyruvate was the pyruvate donor from which pyruvate was incorporated into repeat units prior to polymerisation (lelpi et al., 1981b). As in acetan biosynthesis by A. xylinum, acetyl residues were donated by acetyl coenzyme A, also prior to polymerisation

(Jelpi et al., 1983). Polymerisation occurred through the addition of new repeat units at the reducing end of the growing polysaccharide chain as shown in Figure 2.7b (lelpi et al., 1993).

As was the case in A. zylinuin, truncated repeat units were able to act as substrates for the polymerising enzyme(s) with small quantities of cellulose, polytrimer and polytetramer produced, when cells which had accumulated the respective repeat unit were incubated under suitable conditions (lelpi et al., 1993). Mutants of X. campestris capable of producing truncated polymers in vivo, albeit in low quantities, have been genetically engineered (Betlach et al., 1987; Vanderslice et al., 1990). 2.7a Chemical structure of Xanthan

(From lelpi et at, 1981b)

(lOP Gil 61c GOP Man

(lOP f GOP Gil — 2 pill LiDidP-P-GIC-GIc -Man

(lop Gil _OP GIcA

L. pd .;0;d-P•P-GC -Gil Man- GI zanthan GOP Man LipidPP - 01 -'61C'61 -a;;—' 2t iianfl Man I L!P.dP-P41c GkA [ GIcA Man

Lp,d -P-P EGIk Gic Man ?IC, Man

2.7b Proposed mechanism of biosynthesis of xanthan in Xanthomonas campestris. (From lelpi et at, 1993)

Figure 2.7

The structure and biosynthesis of xanthan 32

Lipid bound saccharides have also been identified in Rhizobium meliloti cells treated with

EDTA and incubated with radioactive nucleotide diphosphate sugars. These corresponded to the octasaccharide repeat unit of succinoglycan, an exopolysaccharide produced by this organism. Smaller fragments were also observed, correspdnding to truncated repeat units

(Toirnasky et al., 1982). Succinoglycan contains glucose and galactose in the ratio 7:1 (Jansson et al., 1977; see also Figure 2.5) and problems associated with demonstration of sequential build up of the lipid bound saccharide were partially overcome by using mutants defective in the enzyme UDP-glucose 4-epimerase preventing added UDP-glucose being converted to UDP-galactose (Tolmasky et al., 1982). Certain strains of Agrobacterium tumefaciens also produce succinoglycan and similar lipid intermediates to those found in R. meliloti were observed when EDTA treated cells of A. tumefaciens were incubated with the appropriate sugar donors (Staneloni et al., 1984).

Recently the biochemical methods described above have been used in conjunction with gene sequence analysis in order to obtain an understanding of features of the glycosyl transferase enzymes involved in succinoglycan biosynthesis by Rhizobium meliloti (Glucksmann et al., 1993a). Attachment of the first sugar of the succinoglycan repeat unit requires two proteins, the products of exoF and exoY. ExoY is believed to act as the transferase based on its homology to GumD of Xanthomonas campestris which acts to transfer the initial glucose of the xanthan repeat unit to polyisoprenol phosphate from UDP-glucose (Vanderslice et al.,

1990). ExoY is also homologous to RtbP of Salmonella typhimurium, involved in the transfer of galactose to bactoprenol phosphate in the synthesis of lipopolysaccharide 0- antigenic side-chains (Jiang et al., 1991). ExoF encodes a putative protein of mass 45.8 kDa which contains an N-terminal signal sequence (Muller et al., 1993). The function of ExoF is less well understood although a certain degree of homology has been observed with GumB 33

of X. campestris, believed to be involved in repeat unit polymerisation of xanthan

(Vanderslice et al., 1990) and KpsD of Esherichia coli involved in export of the polysialic acid capsule (Wunder a al., 1994). Hydrophobicity profiles of ExoF suggest that the protein is anchored to the cytoplasmic membrane at its amino terminus (Muller ci al., 1993) whilst alkaline phosphatase fusions to the protein suggest that the bulk of the protein is located periplasmically (Long et al., 1988b). Other transferase reactions in succinoglycan biosynthesis by R. meliloti require only one enzyme per sugar transferred. From sequence data, the predicted mass of these proteins range from the 26kDa ExoW (Glucksmann et al., 1993a), believed to be involved in the incorporation of the seventh sugar to the repeat unit (Reuber and Walker 1993b), to the

44kDa ExoL involved in transfer of the third sugar to the repeat unit (Becker et al., 1993). Two types of spatial organisation have been suggested for these enzymes with hydrophobicity plots predicting the products of exoL, exoO and exoV, responsible for the addition of the third, fifth and sixth sugars respectively (Becker ci al., 1993; Reuber and Walker 1993b) to be located cytoplasmically, whilst other transferase enzymes are postulated to be anchored to the cytoplasmic membrane by their respective carboxyl termini (Glucksmann ci al., 1993a). No glycosyl transferase has yet been identified for the addition of the final sugar to the succinoglycan repeat unit in this organism and the possibility remains that one of the sugar transferases so far identified may have a dual role (Glucksmann et al., 1993a). The remaining loci required for the production of fully substituted succinoglycan repeat units in R. meiiioti have also been sequenced (Buendia et al., 1991; Becker ci' at, 1993;

Glucksmann ci' al., 1993b). ExoZ was responsible for the transfer of acetyl groups to the repeat unit from acetyl coenzyme A (Reuber and Walker 1993a). This protein has a predicted mass of 34kDa and hydrophobicity data reveal the protein to have several putative transmembrane regions (Buendia ci al., 1991). Similarly ExoH responsible for the addition kyj

of succinate from succinyl coenzyme A (Leigh et al., 1987) showed a total of eleven hydrophobic domains and was consistent with the structure of transmembrane proteins

(Becker et al., 1993). lii contrast ExoV responsible for the addition of pyruvate (Reuber and

Walker 1993b) had a hydrophobicity profile characteristic of a cytoplasmic protein

(Glucksmann et al., 1993b).

From the above results it was postulated that interactions may occur between ExoZ and the membrane-anchored ExoM responsible for the addition of the fourth sugar adjacent to a newly acetylated glucose residue in position three of the succinoglycan repeat unit

(Glucksmann et al., 1993a) whilst ExoH (addition of fifth sugar) may interact with ExoW

(succinylation of fifth sugar) (Glucksmann et al., 1993b).

The importance of spatial relationships and physicochemical conditions was futher emphasised by studies with the uncoupler 2,4-dinitrophenol on 0-antigen synthesis in

S.typhimurium (Marino et al., 1991). This compound inhibited in vivo synthesis of 0 polysaccharide chains and the initiation reaction involving the transfer of galactose-1- phosphate to undecaprenol phosphate was blocked. There was no inhibitory effect in vitro when isolated membrane fractions were used as enzyme preparations. Secondary effects of a dissipation of proton motive force were examined and under the conditions tested there was little effect on intracellular ATP levels or UDP-galactose:UMP ratios. Futhermore, the initiation reaction was essentially isoenergetic and was driven by coupling to the subsequent elongation reactions which were not inhibited. The requirement for proton motive force was thus indirect. 35

2.4 Polymerisation and Export of Bacterial Exopolysaccharides

The later stages in bacterial exopolysaccharide biosynthesis are the least well understood events. Experimental difficulties caused by the requirement for the correct spatial arrangement of the various components involved and the correct physicochemical environment, prevent the use of simple in vitro assays. However, immuno-electron microscopy gave early indications as to the sites of export and more recently, genetic techniques have allowed dissection of the various processes involved.

Polymerisation of polysaccharide repeat units has now been studied in a number of systems.

In Xanthomonas campestris, chain growth has been shown to occur by the addition of new repeat units to the reducing end of the growing chain as shown in Figure 2.7b (lelpi et al.,

1993). Similarly 0-antigen chains of Escherichia call and Salmonella enterica grow by the addition of new repeat units to the reducing end of the polysaccharide chain (Bastin et al.,

1993). In all cases studied, polysaccharide chains composed of repeat units built up on lipid carriers are formed by the transfer of polymeric chain fragments to the non-reducing end of monomeric repeat units (Shibaev, 1986).

The production of mutants, through the use of transposon insertions, has identified at least three potential genes involved in polymerisation and/or export of succinoglycan in Rhizobium meliloti. Reuber and Walker (1993b) examined lipid-linked intermediates of succinoglycan in various R. meliloti mutants. Strains with mutations in exoP, exoQ or exoT exhibited similar profiles to wild-type strains when lipid-linked intermediates were subjected to gel filtration chromatography, unlike mutants with defective glycosyltransferase genes. The exoQ mutant, however, lacked material thought to be a repeat unit dimer in wild-type strains. It was thus suggested that the exoQ gene product was involved in repeat unit polymerisation whilst ExoP and ExoT were involved in translocation of the polysaccharide to the cell surface. All three genes have been fully sequenced and deduced primary and secondary structures of the respective proteins are consistent with their proposed functions. Thus ExoQ was predicted to span the membrane multiple times (Muller et al., 1993) as was ExoT (Glucksmann et al., 1993b). In addition ExoT has been shown to share 23% homology with GurnJ, proposed to have a role in xanthan export in Xanthomonas cainpestris (Betlach et al., 1987) and 21% homology with NdvA required for extracellular expression of B-(1,2)-glucan in R. meliloti

(Stanfield et al., 1988). ExoP is a putative 84kDa protein with three potential integral membrane regions as judged by hydrophobicity data. TnphoA fusions suggested that much of the remainder of the protein was periplasmically orientated (Glucksmann et al., 1993b). Although the putative protein lacked honiology to other known proteins, regions of similarity to nucleotide-binding regions of ATP-binding cassette membrane transporters were observed

(Glucksmann et al., 1993b). Perhaps the fullest model of polysaccharide repeat unit polymerisation presented to date is that of Bastin et al. (1993) concerned with 0-antigens of Iipopolysaccharides. Initial work examined the expression of the Escherichia coli 075 rJb gene cluster involved in 0-antigen biosynthesis (Batchelor et al., 1991). Three phenotypes were observed when different DNA fragments were mobilised into an E. coli strain which did not express O-polysaccharide. The recipient strain expressed either normal 075 lipopolysaccharide with a characteristic bi-modal molecular weight pattern, lipopolysaccharide consisting only of monomeric 0-antigen chains (polymerase defective mutants) or lipopolysaccharide in which bi-modal molecular weight distribution was not observed and only higher molecular weight species were present when analysed by polyacrylanñde gel electrophoresis. Further characterisation of the latter mutant indicated that the wild-type lipopolysaccharide distribution could be restored by complementation with a 1.75kb DNA fragment found adjacent to rfb in E. coli 075. This 37

gene was postulated to control 0-antigen chain length and was thus named rol-regulator of

U-length. Both the E. coli 075 and Salmonella lyphimurium LT2 rol genes were subsequently sequenced and predicted to code for proteins of 325 and 327 amino acids respectively with 72% homology between the two proteins. It was further observed that the

S. typhimurium rol gene was able to regulate 0-polysaccharide chain length in E. coli 075 lacking the gene, leading to the suggestion that the mechanism of action of rol was independent of 0-antigen structure (Batchelor et al., 1992). Bastin et al. (1993) identified a similar gene capable of regulating 0-antigen length in E. coli and also S. enterica, which they termed cld-chain length determinant. Similarly they demonstrated that the cld gene of

E. coli K12 acted on a range of 0-antigens and an E. coli 0111 strain expressed lipopolysaccharide of different molecular weight, dependent on the source of complementing cld. A statistical analysis indicated that in the absence of cld, there was a constant probability of transfer of a growing 0-antigen chain to lipid A, with concomitant termination of elongation. In the presence of cld this probability of transfer was reduced for shorter chains and increased for longer chains. A model was proposed for 0-antigen chain polymerisation in which the polymerase enzyme was envisaged as having two sites (Figure 2.8a). It was suggested that the R site receives new monomeric undecaprenol-linked repeat units (UPP-

01). Once this site is occupied the D site donates additional polymeric 0-polysaccharide repeat units (UPP-On) to extend the chain length by one unit (UPPO n+ l), after which the extended chain is returned to the D site. Alternatively the polymerase may interact with a ligase enzyme leading to the ligation of 0-antigen to lipid A core. In the absence of CId the latter reaction was envisaged as having a constant probability of occurring, with 0-antigen chain length having no influence on the likelihood of the polymerase and ligase enzymes interacting. Cld was thought to modulate the probability of the ligase, reaction through interaction with the polymerase enzyme (Figure 2.8b). It was proposed that CId existed in

19 9 , n-I n-2 I, n-I n.I n-I i-I n-1 P oP n PP rWi

E RK Uqw mc R 0 q o

polvmerimtion ligation

2.8a Proposed mechanism of synthesis of lipopolysaccharide in Escherichia coil.

El 71 ,

2.8b Proposed interactions of enzymes involved in E. coil LPS biosynthesis.

Figure 2.8

Model for 0-antigen polymerase and CId action in E.coli. (From Bastin et all, 1993) two distinct states; the E conformation favouring extension of the 0-antigen chain and the T state favouring interaction of polymerase with ligase and chain termination. Finally it was suggested that Cid acted as a molecular clock switching from the E state to the T state after a defined period of time thus favouring ligation of longer 0-antigen chains. Immuno-electron microscopy, particularly in conjunction with the use of metabolic inhibitors and more recently with mutated strains, has provided a method for the study of export of bacterial extracellular polysaccharides. Early work established that E. coli has points of adhesion between inner and outer membranes (Bayer, 1968): Cells were plasmolysed and embedded in resin and ultrathin sections were stained and visualised by scanning electron microscopy. In cells of Escherichia coli strain B, most of the inner membrane had come away from the cell wall; however, in a few places the inner membrane appeared to maintain a connection with the cell wall, the diameter of the connections being approximately 20-30nm

(Bayer, 1979). It was estimated that 200-400 of these adhesion zones existed per cell (Bayer, 1968). These zones of adhesion have since been demonstrated in a number of Gram negative enterobacteria and fresh water bacteria; e.g. E. coli strains B, Cl, K12, K29 and 1(26,

Salmonella lyphimurium, S. anatum, Caulobacter and Shigella (Bayer, 1979). Ferritin linked monoclonal antibodies have been used to stain newly formed lipopolysaccharide and capsular polysaccharide in certain bacteria. Bayer and Thurow (1977) studied synthesis of the K29 capsule in a temperature sensitive E. coli mutant. Cells were grown at 46 0C, a temperature at which capsular polysaccharide was not expressed. The temperature was shifted down to,

250C, a capsule permissive temperature, and polysaccharide was observed on the exterior of the cell by the binding of ferritin-linked anti-K29 antibody after approximately 20 minutes, initially as tufts. Plasmolysis of the cells revealed that these emerging tufts were frequently observed over zones of adhesion, suggesting a possible role for these morphological 39

structures in polysaccharide export. Recently Kroncke et at (1990b) demonstrated capsular antigen within these zones of adhesion. Similarly Muhlradt et al. (1973) demonstrated that newly-synthesised lipopolysaccharide was exported from adhesion zones in Salmonella lyphimurium. It is interesting to note that a recently recognised family of membrane fusion proteins function in conjunction with cytoplasmic membrane transporters of the ATP-binding cassette family (Dinh et al., 1994), a group of proteins that has been implicated in polysaccharide transport (see below), thus potentially providing a molecular basis for the observed zones of adhesion in Gram negative bacteria.

Metabolic inhibitors have been used in conjunction with immuno-electron microscopy to examine the energy requirements of polysaccharide translocation. Group II bacterial capsules are not synthesised at temperatures below 20 0C (Boulnois and Jann, 1989) and thus effects of inhibitors on both initiation and continuation of capsule biosynthesis can be studied.

Kroncke et al. (1990b) examined capsule biosynthesis and expression in Escherichia coli. In uninhibited cells, capsular polysaccharide initially appeared as tufts on the cell surface approximately 15 minutes after cells were shifted to a capsule permissive temperature and covered the entire cell after approximately 50 minutes. Similar time scales were observed for capsule expression in E. coli Kl using phage binding assays to detect capsule expression (Whitfield et al., 1984). Chloramphenicol, an inhibitor of protein synthesis (Gale et al., 1981), prevented surface expression of capsular polysaccharide when added prior to or shortly after shift to capsule permissive temperature but had no inhibitory effect if added 15 minutes after shift to permissive temperature (Kroncke et al., 1990b). Similar results were obtained by Whitfield a al. (1984); however, this group observed a reduced rate of capsular polysaccharide expression when chloramphenicol was added at times up to 45 minutes after temperature shift. Both groups reported reversibility of inhibition after washing of cells.

Results were interpreted to suggest either that initiation of capsule biosynthesis requires protein synthesis (Kroncke et al., 1990b) or that continuous protein synthesis was required for capsule biosynthesis, possibly to replace peptides of short half-life (Whitfield et al., 1984).

Additionally, Kroncke et al. (1990b) observed that chloramphenicol concentrations lower than 2mM resulted in biosynthesis of capsular polysaccharide that could be localised to the cytoplasmic compartment of the cell, but that above 2mM, capsular polysaccharide was not synthesised. The membrane de-energising agents carbonyl cyanide-m-chlorophenyl hydrazone

(CCCP) and azide inhibited surface expression of capsules in a non-time dependant manner; i.e. whether added prior to or after shift to permissive temperature. In the presence of CCCP, or azide added 15 minutes after temperature upshift, capsular polysaccharide was observed in the cytoplasm. Transport of polysaccharide was thus proposed to be an energy requiring process. These results also demonstrated that capsular polysaccharide is assembled at the cytoplasmic face of the inner membrane (Kroncke et al., 1990b). Recently immuno-electron microscopy has been extended to study capsular polysaccharide expression in E. coli mutants. Three distinct regions have been observed in the gene cluster

(kps) required for synthesis and surface expression of group II capsules in this organism

(Boulnois and Jann, 1989). Kroncke et al. (1990a) examined expression of the KS capsule in recombinant E. coli harbouring genetic defects in regions 1, 2 or 3 of the kps gene cluster. Capsular polysaccharide was visualised under the electron microscope through staining with ferritin linked anti-KS antibody. Mutants in region 2 did not bind antibody suggesting a defect in biosynthetic enzymes. Region 3 mutants expressed capsular polysaccharide in the cytoplasm whilst region 1 mutants bound anti-KS antibody at the cell membrane. In addition capsular polysaccharide could be extracted from lysates of region 3 mutants and the periplasm of region 1 mutants. Region 1 mutants had a lipid substituted capsular polysaccharide whilst lipid could not be identified in association with polysaccharide from cells harbouring a 41

mutation in region 3. It was thus suggested that region 3 was involved in either transport of polysaccharide across the cytoplasmic membrane or alternatively that this region was involved in lipid substitution of polysaccharide which was a prerequisite for transport. Region 1 was postulated to be involved in transport across the outer membrane.

The E. coli kps gene cluster has now been more fully analysed. Region 2 contains genes unique for each particular capsule whilst region 1 and region 3 genes appear to have a stronger degree of homology between different strains and encode similar functions in different strains (Boulnois and Roberts, 1990). Region 1 contains at least five loci (Wunder et al., 1994) and appears to be involved in transport of capsular polysaccharide across the outer membrane (Boulnois and Jann, 1989). Recently one of the region 1 genes, kpsD, has been sequenced (Wunder et al., 1994). A putative signal sequence suggested a periplasmic location for the protein, a result supported by Western blot analysis using KpsD antiserum.

KpsD mutants were unable to transport capsular polysaccharide across the outer membrane and it was thus suggested that KpsD may function as a periplasmic binding protein.

The entirety of the E. coli KS region 3 of the kps gene cluster has been sequenced and two open reading frames organised as a single transcriptional unit were identified encoding for the proteins KpsM and KpsT (Smith et al., 1990). KpsM had six potential membrane spanning domains as shown by hydrophobicity data and was assigned a potential role as a carrier or pore transporter across the inner membrane. KpsT had potential adenine nucleotide binding sequences and was suggested to be involved in coupling ATP hydrolysis to membrane transport. Similar genes were found in' encapsulated Haemophilus influenzae (Kroll et al., 1990). 42

2.5 Direct Polymerisation of Sugar Nucleotides into Bacterial Exopolysaccharides

A number of bacterial homopolysaccharides have been shown to be synthesised without the requirement for build-up on undecaprenol derivatives, thus providing an alternative mechanism for the biosynthesis of these molecules. The early stages of biosynthesis are similar to those employed by cells synthesising heteropolysaccharides. Thus there may be a cycling of sugars through various metabolic pathways followed by activation through the formation of sugar nucleotide derivatives, as described in Chapter 2.2. In the final stages however, sugars are directly polymerised into polysaccharide chains with the release of nucleotide diphosphates, as has been demonstrated for B-( 1 ,2)-glucan synthesis in Rhizobiwn and Agrobacterium (Zorreguieta and Ugalde, 1986) and cellulose synthesis in Acetobacter xylinum and Agrobacterium tumefaciens (Thelen and Delmer, 1986). This type of mechanism has also been observed in the biosynthesis of 13-

(l-'3)-glucans in yeast (Shematek et al., 1980), fungi (Bulone et al., 1990) and higher plants (Hayashi et al., 1987). Of all the exopolysaccharides produced without the requirement for lipid intermediates, cellulose biosynthesis by Acetobacter xylinum is the most extensively studied, probably due to its applicability as a model for cellulose synthesis in higher organisms. A. xylinum synthesises the polysaccharide by direct transfer of glucose from UDP-glucose to the growing cellulose chain, catalysed by UDP-glucose: 1,4-B-D-glucan 4-13-D-glucosyltransferase located in the inner membrane (Bureau and Brown, 1987). Enzyme activity required Mg2+ (Ross et at, 1991), had a temperature optimum of 30 0C (Aloni et at, 1983; Bureau and Brown, 1987) and pH optimum in the range 7.5 to 8.5 (Ross et at, 1991). Analysis of the genetic region encoding for cellulose synthesis in A. xylinum has been undertaken and four open reading frames were revealed (Wong et al., 1990). A single gene, bcsB, was suggested to encode for rX

the catalytic subunit of cellulose synthase which had a predicted mass of 85.3 kDa. A potential N-terminal signal sequence was observed similar to that of secreted proteins, strengthened by the observation that the bcsB gene product stained as two bands of approximately 91 kDa and 67 kDa on SDS-PAGE gels. The first 18 amino acids of the 91 kDa product were sequenced and corresponded to the deduced bcsB amino acid sequence starting at codon 25, similarly the 67 kDa protein displayed an amino acid sequence as deduced from codons 196-206 thus suggesting that the 67 kDa protein was a proteolytic fragment of the 91 kDa protein. Further evidence to support the proposal that the enzyme is membrane bound came from the observation that the amino acid sequence was consistent with a transmembrane helix at the C-terminus and also the presence of potential B-sheets reminiscent of porin structures. Although bcsB encodes for a protein of low molecular weight the solubilised cellulose synthase complex has been shown to have a molecular weight of approximately 420 kDa by gel filtration chromatography suggesting the presence of a hetero- oligomeric multienzyme complex (Ross et al., 1991) probably containing regulatory components as will be discussed (p. 61). In another strain of A. xylinum Saxena et iii. (1994) found that the equivalent of the bcsB gene was fused with the equivalent of the bcsA gene to give the gene acsAfi which encoded for a polypeptide of 168 kDa carrying both substrate and activator binding sites. This protein was predicted to contain eleven transmembrane segments and was described as an integral membrane protein. This prediction was supported by analysis of alkaline phosphatase activity in strains carrying acsAB::phoA fusions. Although strains with mutations in the acsA, B or C genes were unable to produce cellulose in vivo, they were still capable of cellulose synthesis in vitro due to the presence of a second gene encoding cellulose synthase activity (acsAIT), showing similarity to the acsAB fragment. Very few biochemical studies have been undertaken on cellulose export; however, direct observation has provided data concerning these events. Electron microscopy has revealed a EEl

row of pores along the surface of A. xylinum (Ross et al., 1991) with corresponding particles along the inner surface of the outer membrane (Brown et al., 1976) from which the cellulose fibrils appear to be extruded in a co-operative process to form a cellulosic ribbon extending from individual bacteria. Recent genetic studies have suggested a role for AcsC in forming pores from which the bacterial cellulose is extruded (Saxena et al., 1994). AcsC showed sequence similarity to VirBlO of Agrobacterium tumefaciens, which may form a pore spanning both membranes to allow transfer of T-DNA to plant cells.

2.6 Models of Curdlan Biosynthesis by Agrobacterium sp. ATCC 31749 Two general models of curdlan biosynthesis can thus be proposed. In the first, one can envisage build-up of oligomers of glucose joined by 13-(1--'3) linkages on undecaprenol derivatives followed by polymerisation and export in a manner similar to that described for heteropolysaccharide biosynthesis (Figure 2.9). Alternatively, a single synthase enzyme may be responsible for the direct polymerisation of UDP-glucose to curdlan in a manner similar to that observed for cellulose biosynthesis in Acetobacter xylinuzn or cell wall synthesis by yeast (Shematek et al., 1980). INITIATION ELONGATION glucose glucose 7A, ATP t PI Iu-P gtu-P UTP j 2ATP

UDP•çlu UMP LI PID-PP-gI UC UTP

ATP

LIPID-;)

t LPS and P LIPID-PP-gIu 2 PtPlldoglycan Synthesis LIPID-pp

W OP glu 'curdlan' LIPID-PP glu, D to

Figure 2.9 Potential model for curdlan biosynthesis by Agrobacterium sp. ATCC 31749.

(From Phillips and Lawford, 1983) Chapter 3 Physiological Influence and Biochemical Control of Exopolysaccharide Biosynthesis

3.1 Effects of Changes to Culture Conditions Studies have been undertaken on both Gram positive and Gram negative bacteria concerning the effects of alteration of various parameters of culture upon exopolysaccharide production. In particular, physiological studies have been used to optimize polysaccharide yield for the commercialisation of bacterial polysaccharides. Generalisations are difficult to make and those conditions found to favour exopolysaccharide synthesis in one species cannot necessarily be assumed to favour polysaccharide synthesis in another.

3.1.1 Effect of Carbon Substrate The carbon source from which bacterial polysaccharides are synthesized generally has little effect on exopolysaccharide composition but frequently affects polysaccharide yield. Thus

Eagon (1956) observed no effect of carbon source on the composition of a polymannan produced by Pseudomonasfluorescens OSU 64; similarly a more complex polysaccharide produced by Pseudonionas sp. NCIB 11264 had a monosaccharide composition independent of carbon source (Williams and Wimpenny, 1977) and even the more variable polysaccharide alginic acid from two species of Pseudomonas showed very little variation in the ratio of D- mannuronic to L-guluronic acid when grown on either glucose or fructose (Conti et at,

1994). In contrast carbon substrate affected the composition of exopolysaccharides produced by Kiebsiella sp. K32 and Acinetobacter calcoaceticus BD4 (Bryan et at, 1986); however, it was not determined whether this was due to a variation in the monosaccharide composition of individual polysaccharides or whether more than one polysaccharide was produced, the Me

relative proportions of which varied depending on the carbon substrate provided. One finding perhaps supporting the latter hypothesis was that the ratios of xanthan to a polyglucorhamnan produced by Xanthornonas juglandis were dependent on the limiting nutrient in the culture medium (Evans et al., 1979).

In contrast to exopolysaccharide composition, polysaccharide yield can vary widely depending on the choice of carbon substrate. Harada and Yoshimura (1964) observed succinoglycan production by Alcaligenes faecalis 10C3 from hexoses, pentoses, polyols, deoxysugars, disaccharides and polysaccharides the conversion efficiency being greatest from sucrose (40%) and lowest from rhanmose (20%) with slight exopolysaccharide production from arabinose, lactose and ethylene glycol. A similar situation was encountered for curdlan production by a mutant of strain 10C3 (Harada et al., 1966). The highest conversion efficiency was observed from glucose (43%) and the lowest from cellobiose (17%). Although galactose had been observed to provide good yields of succinoglycan in strain 100, very little curdlan was produced from this substrate by the mutant strain. The media used were slightly different although in both cases the pH was similar and this appeared to be a critical parameter in exopolysaccharide production. A similarly wide range of carbon substrates were observed to support exopolysaccharide production by Pseudomonas sp. NCIB 11264

(Williams and Wimpenny, 1977) with reasonable yields obtained from mono-, di- and trisaccharides and organic acids. Polysaccharide yield was generally higher from hexoses than pentoses, with the exception of xylose which gave better yields than galactose or mannose.

The effect of mixed carbon substrates on xanthan yield in 96 hour batch culture by

Xanthomonas catnpestris was examined (Souw and Demain, 1979). Xanthan production in • media containing 2% (w/v) glucose could be improved by the addition of 1% sucrose, 0.5% fructose or 0.5% xylose to the medium. An increase in the concentration of the latter two 47

substrates was inhibitory to xanthan production. Similarly the addition of low concentrations of pyruvate, succinate or a-ketoglutarate to a 2% glucose medium stimulated xanthan production. The greatest yield of xanthan was observed in media containing 4% sucrose with low concentrations (0.3-1%) of pyruvate, a-ketoglutarate or succinate. Finally, although the majority of studies on exopolysaccharide biosynthesis from various carbon sources have been primarily empirical, systematic studies have been undertaken in an attempt to link the redox state of the carbon substrate with the efficiency of conversion to exopolysaccharide. These studies indicate the maximum efficiency at which carbon substrate can be converted to exopolysaccharide, providing an upper limit for yield improvement.

Linton et al. (1987a) measured a number of parameters for Agrobacterium radiobacter growth and exopolysaccharide production in continuous culture. Theoretical calculations from measured values suggested that in nitrogen-limited continuous culture, approximately

90% of total cell ATP turnover was associated with exopolysaccharide synthesis. It was concluded that exopolysaccharide production was either a consequence of ATP overproduction, whereby synthesis of exopolysaccharide dissipated excess ATP, or that exopolysaccharide production imposed the rate of ATP turnover on the cells. The observations that exopolysaccharides containing oxidised residues such as uronic or other organic acids (e.g. acetate, pyruvate) can provide much of the ATP required for their own synthesis (Jarman and Pace, 1984) . may favour the second conclusion as exopolysaccharide production would be an inefficient mechanism of ATP dissipation. Further work by Linton and colleagues (1987b) found a correlation between the redox state of the supplied carbon substrate and the rate of exopolysaccharide production in Agrobacterium radiobacter. Thus, a high specific rate of exopolysaccharide production was observed from glucose where the net ATP balance for the production of each exopolysaccharide repeat unit was approximately

-10 whilst virtually no exopolysaccharide was produced from ethanol with a net ATP balance rii

of approximately +50. The respiration rate of the bacteria when grown on ethanol was higher than when they were grown on glucose. In addition, a mutant strain unable to produce exopolysaccharide had a similar ATP turnover rate to the wild type bacteria. It was thus concluded that the organism had some mechanism in addition to exopolysaccharide production to allow ATP turnover.

3.1.2 Effect of Growth Phase and Rate and Nutrient Limitation

Three patterns of exopolysaccharide expression have been observed in batch cultured bacteria: those in which exopolysaccharides are synthesized in the stationary phase after growth has ceased due to nutrient limitation e.g. Agrnbacterium sp. ATCC 31749 (Phillips et al., 1983), Cellulomonas flavigena (Voepel and Buller, 1990); those in which exopolysaccharide biosynthesis is associated with growth e.g. Pseudomonas fluorescens (Conti et al., 1994), Xanthomonas campestris (Tail et al., 1986); and finally, those in which exopolysaccharide synthesis continues at a steady rate throughout the growth cycle e.g.

Kiebsiella sp. K32,Acinetobacter calcoaceticus (Bryan et al., 1986). In some cultures, the quantity or molecular weight of polysaccharides have been observed to decline as the culture ages due to enzymes in the culture broth capable of hydrolysing the product. Conti et al.

(1994) observed a decrease in the molecular weight of alginate produced by Pseudomonas fluorescens as batch cultures aged. The organism possessed an alginate lyase, although the majority of activity was intracellular regardless of the age of the culture. However, it was observed that the molecular weight decrease was accompanied by an increase in acetylation of the polymer and furthermore that the alginate lyase activity associated with the bacterium had a higher specificity for deacetylated alginate. These results may therefore suggest a role for the enzyme in depolymerization through degradation of under acetylated areas of the polysaccharide. Similarly Orts et al. (1987) observed a reduction in the rate of curdlan production by Agrobacterium sp: ATCC 31749 in high shear fermentation vessels approximately 50 hours after the cells had entered stationary phase. Culture supernatants from cells grown under various conditions were incubated with commercial samples of curdlan for 70 hours. Samples were taken at the beggining and the end of the culture period and a set volume of alkali was added to dissolve the curdlan to allow viscosity measurements to be undertaken. The viscosity of polysaccharide solutions is dependent on the molecular weight of the polymer (Mitchell, 1979). In all cases a slight reduction in viscosity of the curdlan solutions was observed. In those cultures with the highest degree of cell lysis, the viscosity of solutions of curdlan fell further after incubation in culture supernatant, than in those cultures with a lower degree of cell lysis, or control flasks. It was thus suggested that cell lysis in the high shear conditions within the fermentor released an enzyme capable of curdlan hydrolysis, thus causing the apparent decrease in the rate of curdlan production.

Further evidence to support the hypothesis, was the observation of a constant rate of curdlan production over 90 hours post-stationary phase, when the bacteria were grown in a low shear vibro-mixed fermentor.

The stage at which polysaccharide is harvested in batch culture and the dilution rate in continuous culture can have an effect on the yield and physical properties even in the absence of polysaccharide lyases and this may relate to the availability of isoprenoid lipids at different growth rates (e.g. Sutherland, 1979a; see pp. 58-60). Tait et al. (1986) found that the consistency index of xanthan from Xanthomonas campestris was minimal during exponential growth and the extent of acylation was reduced during this period of growth. Similarly, exopolysaccharide isolated from continuous cultures at high dilution rates had a higher acetyl and lower pyruvyl content than that isolated at lower rates of dilution. Exopolysaccharide yield was unaffected by dilution rate in the range 0.03-0.06 h' Evans et al. (1979) found 50

little variation in exopolysaccharide yield from X. juglandis over the dilution rate range 0.03- 0.05 irs' and physical measurements suggested that polysaccharide isolated from cultures under different rates of dilution was of similar composition. In contrast, Phillips et al. (1983) found that Agrobacterium sp. ATCC 31749 was only capable of curdlan production at dilution rates below 0.1 iws' in nitrogen-limited culture and that under these conditions exopolysaccharide production was about a quarter of that found in batch culture. A two- stage continuous process was, however, more successful for increasing exopolysaccharide yield (Lawford et al., 1982). A similar situation was encountered in Pseudomonas sp. NCJB 11264 where exopolysaccharide production increased with decreasing dilution rate, although the cell population remained approximately the same at the dilution rates tested (Williams and Wimpenny, 1978). This phenomenon has also been demonstrated in biofilms of

Staphylococcus epidermis where decreased growth increased exopolysaccharide yield although exopolysaccharide yield was low under all growth conditions in the planktonic state (Evans et al., 1994). One factor which is frequently found to promote polysaccharide synthesis is excess carbon substrate in the presence of some other essential nutrient limitation. Polysaccharide production was thus stimulated in Aerobacter aerogenes by limiting amounts of phosphate, ammonium or sulphate (Duguid and Wilkinson, 1953). In species where exopolysaccharide production is associated with the stationary phase of growth subsequent to the uptake of an essential nutrient (e.g nitrogen-limited cultures of Agrobacterium sp. ATCC 31749), exopolysaccharide biosynthesis may represent an overflow mechanism for energy and substrate dissipation under conditions of continued carbon uptake (Linton, 1990). Although there are theoretical and experimental observations to support this hypothesis (pp. 47-48) this may not be a universal phenomenon. Parsons and Dugan (1971) observed that glucose 51

absorbed by Zoogloea ramigera was initially channelled into synthesis of the storage compound poly-13-hydroxybutyrate. Once external carbon source had been effectively reduced to zero, exopolysaccharide was formed at the expense of poly-B-hydroxybutyrate.

The stimulating effect of high carbon to essential nutrient ratios is not however, confined to those bacteria which synthesise exopolysaccharides in the stationary phase.

A number of studies have been undertaken on the effect of nutrient limitation on exopolysaccharide production by Xanthomonas campestris. Tait et al. (1986) found that optimal exopolysaccharide production was promoted by nitrogen limitation in batch culture and that deficiencies in magnesium, sulphur, iron or phosphorus promoted xanthan production in descending degrees of efficacy. Xanthan was also produced when X. campestris was carbon limited. Although this provided the lowest xanthan yield efficiency of conversion was highest at 64%. Similar results were obtained in continuous culture by Davidson (1978) who additionally found that potassium deficiency stimulated xanthan production. The proportion of pyruvate in the polymer was dependent on nutrient limitation with sulphur limitation yielding a polymer with 8.5% and phosphate limitation yielding only 0.9%. Levels of acetate in the polymer were more consistent. Similar experiments were undertaken with X. juglandis

(Evans et al., 1979). Limitation in sulphur, nitrogen or phosphorus all yielded high quantities of exopolysaccharide. Under certain nutrient limitations there was a tendency of cultures to mutate to non-exopolysaccharide producing strains although the nutrient limitation providing the best polysaccharide yield also had the lowest rates of culture variation. Culture variation was also a problem in continuous culture of alginate-producing strains of Pseudomonas mendocina under phosphate limitation (Sengha et al., 1989). X. juglandis produced polymers other than xanthan under certain conditions. In sulphur-limited cultures, which provided the highest total polysaccharide yield, the ratio of xanthan to a second polysaccharide was lowest while in carbon limited cultures yieldingn only a third of the polysaccharide in sulphur-limited 52

cultures, the reverse was true. A third, poorly soluble polysaccharide tentatively identified as cellulose was produced in potassium limited cultures. The nitrogen source also has an effect on polysaccharide yield in X. campestris (Souw and Demain, 1979) with NH4NO3, NaNO3 and a number of amino acids stimulating xanthan production to a greater extent than

(NI-L4)2SO4 when provided in equivalent amounts. High carbon:nitrogen ratios also favoured exopolysaccharide production in Chromobacterium violaceum (Corpe, 1964) and Zoogloea ramigera (Norberg and Enfors, 1982) whilst in Zoogloea strain MP6 limiting quantities of both carbon and nitrogen rather than high carbon to nitrogen ratios favoured exopolysaccharide production (Unz and Farrah, 1976).

3.1.3 Other Changes in Culture Conditions

pH appears to be a critical factor in the production of exopolysaccharides and the optimum pH for polysaccharide production is frequently close to that observed for growth. Harada and colleagues (1965) found that addition of 1% CaCO3 to a yeast extract medium doubled the amount of succinoglycan produced by Alcaligenesfaecalis 10C3. It was postulated that. this was due to the higher final pH (6.4 cf. 5.8) when CaCO3 was added. Curdlan production by a mutant of strain 10C3 was also pH dependent with curdlan found in media having a final pH in the range 5.5-7.0 but not media with a final pH lower than 5.0 (Harada et al., 1966).

Continuous culture was used to show that the optimum pH for growth and curdlan production by Agrobacterium sp. ATCC 31749 were 7.0 and 5.9 respectively (Phillips et al.,

1983). Similarly, polysaccharide production by Aerobacter aerogenes (Duguid and Wilkinson, 1953) and Pseudomonas sp. NCIB 11264 (Williams and Wimpenny, 1978) was favoured by culture close to neutrality. 53

Growth at sub-optimal temperatures may favour exopolysaccharide production as has been observed for polysaccharide biosynthesis on nitrogen rich media by Aerobacter aerogenes (Duguid and Wilkinson, 1953). This may be due to increased availability of isoprenoid lipids for exopolysaccharide synthesis (Sutherland, 1979b; 1982; pp.58-60). Conversely a number of bacteria yield the greatest amount of polysaccharide at optimum growth temperatures e.g.

Pseudomonas sp. NCIB 11264 (Williams and Wimpenny, 1978). In Rhizobium trtfolii TA- 1, growth at temperatures in excess of 250C, the optimal temperature for growth, stimulated the production of cyclic B-( 1 ,2)-glucans at the expense of exopolysaccharide and capsular polysaccharide (Breedveld et al., 1990b). The effect of oxygenation appears to vary depending upon the bacterial species examined.

Facultatively anaerobic enteric organisms such as Aerobacter aerogenes (Duguid and Wilkinson, 1953) require oxygen for exopoiysaccharide production. Similarly Pseudomonas mendocina produced no alginate under oxygen limitation; however, the conversion efficiency of glucose to exopolysaccharide was at similar levels under dissolved oxygen tensions ranging from 10-122 mmHg (Sengha et al., 1989). This is in contrast to results obtained for alginate biosynthesis by Azotobacter vinelandii in which increased levels of oxygenation were accompanied by increased respiration rates with the result that most of the supplieli carbon substrate was oxidised to CO2 at the highest dissolved oxygen tensions (Jarman, 1979).

Similarly, increased aeration in shaken cultures of Acetobacter sp. reduced cellulose biosynthesis despite growth remaining constant (Dudman, 1960). Recent electron micrographs have shown that shaken cultures of A. xylinum have the particles proposed to be the sites of cellulose synthesis arranged in a random pattern. In static cultures a linear arrangement is observed and this may be significant in the control of cellulose synthesis

(Saxena et al., 1994). In curdlan producing organisms, the polymer represents a barrier to oxygen transfer to the cells and high dissolved oxygen tension is required for maximal rates 54

of polysaccharide synthesis (Lawford and Rousseau, 1990) as is exemplified by the 30% increase in the specific rate of curdlan production by batch cultures of Agrobacterium sp.

ATCC 31749 when fermenter impeller speed is increased from 600 to 850 r.p.m. (Phillips et al., 1983). Finally, alterations in osmotic pressure influenced patterns of exopolysaccharide expression.

Rhizobium meliloti SU-47 increased production of succinoglycan in media containing 0.2M NaCI or other ionic or non-ionic osmolytes. In media of lower osmotic pressure, cyclic B-

(1,2)-glucans and succinoglycan repeating units were synthesised (Breedveld et al., 1990a). Zevenhuizen and Faleschini (199 1) observed that increased concentrations of NaCl in the culture medium stimulated the production of high molecular weight succinoglycan by R. ineliloti YE-2SL whilst suppressing the expression of succinoglycan repeat units and it was suggested that this effect may be due to a stimulation of the repeat unit polymerase. Recently an endo-B-1 ,3- 1 ,4-glucanase has been identified as part of the exopolysaccharide synthesising operon in R. meliloti (Becker et al., 1993) and this enzyme may function in the production of low molecular weight succinoglycan (Glucksmann et al., 1993b). Thus sodium chloride might also inhibit the glucanase.

3.2 Biochemical Control of Exopolysaccharide Biosynthesis

3.2.1 Control of Substrate Uptake

As described in Chapter 2. 1, Agrobacterium radiobacter and A. tumefaciens take up a number of carbon substrates via periplasmic binding protein-dependent mechanisms (Cornish et al., 1988a; 1989; Williams et al., 1990). Growth in continuous culture under glucose limitation led to the emergence of two novel strains of A. radiobacter, AR 18 and AR9, which 55

hyperproduced either glucose binding protein 1 (GBP1) or GBP2 respectively. When these strains were introduced to ammonium-limited media, strain AR9 produced succinoglycan at a similar rate to the wild type strain and a significant repression of the glucose uptake system was observed. Strain AR 18, however, produced approximately one and a half times as much succinoglycan as the wild type and glucose uptake by this strain exceeded that of wild type strains (Cornish et al., 1988b). This suggested that GBP1 was less repressed by transfer of bacteria to ammonium limited media, a conclusion supported by the observation that a forty fold excess of galactose in the medium, a competitive inhibitor of GBP1 (Cornish et al., 1988a), inhibited glucose uptake by approximately 50% in strain AR9 but by approximately 95% in strain ARI8. These results further suggested that substrate uptake was the limiting step in succinoglycan production from glucose. A strain of similar phenotype to strain AR18 was produced by growth in a galactose limited chemostat (strain AR18a) (Cornish et al., 1989) and this strain also showed increased glucose uptake and succinoglycan production when grown under ammonium limitation. However, when AR18a was grown under ammonium limitation with galactose as the carbon substrate, despite the fact that galactose uptake was greater than in wild type strains, succinoglycan production continued at similar rates to wild type suggesting that other factors were limiting in succinoglycan production from galactose. These results suggest that exopolysaccharide production can be improved by an increase in substrate uptake rates; however, exopolysaccharide-specific controls are likely to have an important role.

3.2.2 Control of Sugar Nucleotide Precursors

Once carbon substrate has entered the cell, the next stage at which biochemical control may operate is in control of the availability of nucleotide pyrophosphate sugars. This control 56

mechanism is fairly non-specific as these precursors are shared by pathways leading to the formation of a number of bacterial polysaccharides. Colanic acid, an exopolysaccharide expressed by several species of Enterobacteriacae contains glucose, galactose, fucose and glucuronic acid. Grant et al. (1970) observed a correlation between the levels of UDP-glucuronic acid, GDP-fucose and colanic acid expression in several strains of Escherichia coli. No similar correlation was observed with the levels of UDP-glucose and UDP-galactose. The levels of UDP-glucose dehydrogenase (catalysing the formation of UDP-glucuronic acid from UDP-glucose), phosphomannose isomerase and GDP-fucose synthase (involved in the synthesis of GDP-fucose) were lower in non-mucoid strains. In certain strains, colanic acid synthesis could be induced by growth on p-fluorophenylalanine and under these conditions the levels of GDP-fucose and UDP- glucuronic acid were similar to those in constitutively mucoid strains. The high levels of

UDP-galactose and UDP-glucose in all strains was suggested to be due to the involvement of these precursors in the synthesis of other polysaccharides. Thus the levels of sugar precursors may exert an effect on exopolysaccharide biosynthesis and this would represent a possible point of biochemical control. Three mechanisms of regulation have been identified in exopolysaccharide-synthesising bacteria. Firstly, the finding that levels of enzymes involved in the synthesis of sugar nucleotides can vary depending upon exopolysaccharide expression (e.g. Grant et al., 1970) would suggest genetic control. Secondly, a number of studies observed feedback inhibition by the product of a particular biosynthetic pathway on one of the enzymes involved in synthesis of the end product. This mechanism has been demonstrated in a number of bacteria containing either mannose (type 1) or fucose (type 2) or both sugars (type 3) in their exopolysaccharides (Kornfeld and Ginsburg, 1966). The biosynthetic pathway to GDP- 57

rnannose and GDP-fiicose is presented in Figure Ma. GDP-mannose pyrophosphorylase was strongly inhibited by GDP-fucose in type 2 bacteria, whilst the enzyme was strongly inhibited by GDP-mannose in type 1 and type 3 bacteria. GDP-mannose inhibition was competitive in Salmonella zyphimurium (type 1) but GDP-fticose inhibition in Aerobacter aero genes (type 2) was not competitive. This suggested that a separate binding site existed for GDP-fucose on the enzyme, emphasising the concerted manner of control. Similarly, GDP-mannose hydro-lyase exhibited different kinetics of inhibition dependent upon the bacterial type studied. The enzyme from Salmonella hvittingfoss (type 3) was competitively inhibited by

GDP-fucose whilst that from A. aerogenes (type 2) was inhibited by GDP-fucose in a co- operative manner, the extent of inhibition increasing with increasing concentrations of GDP- mannose. As illustrated in Figure 3. lb the overall conclusion from these observations was that those bacteria having only one of the two sugar residues in their polysaccharides were able to control the activities of the enzymes involved in precursor synthesis dependent upon the cellular concentration of the end product of the synthetic pathway whilst those bacteria that had both mannose and fucose in their polysaccharides controlled the activities of the enzymes leading to their formation independently. Similarly TDP-rhamnose inhibited TDP- glucose pyrophosphorylase (the first enzyme in the biosynthetic pathway) isolated from

Pseudomonas aeruginosa via a binding site distinct from the active site of the enzyme (Melo and Glaser, 1965). This inhibition could be reversed by high concentrations of TTP, suggesting that the energy balance of the cell was also an important factor. Inhibition of

TDP-glucose pyrophosphorylase by TDP-rhamnose was also observed in an enzyme preparation from Salmonella anatum (Bernstein and Robbins, 1965). UDP-glucose also inhibited this enzyme and TDP-glucose inhibited UDP-glucose pyrophosphorylase, demonstrating inhibition by products across biosynthetic pathways. This may be of advantage GDP-mannose pyrophosphorylase

Mannose-1 -phosphate-->GDP-mannose–*–*GDP-fficose

GDP-mannose hydro-lyase

3.la Pathway of biosynthesis of GDP-mannose and GDP-fucose in bacteria.

(1) Men-i-P -Mcr, pcyscccflcriCe

2) Mcn--P 'tMenP-FJC —poiyscccrcflde

"P-Men -iC?p-FC _..eQyscCCflcr1de

3.1b Feedback control sites in GDP-mannose and GDP-fucose biosynthesis.

(From Kornfeld and Ginsburg, 1966).

Figure 3 Control of biosynthesis of GDP-mannose and GDP-fucose. to an organism where polysaccharide biosynthesis is blocked at a later stage leading to the accumulation of nucleotide linked sugars. Another mechanism by which bacteria may control the supply of sugar precursors is through nucleotide sugar pyrophosphate hydrolases. These enzymes are released from osmotically shocked Escherichia coli although it is postulated that they are membrane-associated and capable of hydrolysis of intracellular sugar nucleotide pools (Ward and Glaser, 1968). A mutant was isolated in which the UDP-sugar bydrolase(s) was postulated to be cytoplasmic rather than periplasmic. The half life of UDP-sugar pools in the mutant was estimated to be approximately half that of wild type (Ward and Glaser, 1969); however, the amount of polymeric glucose within the mutant was reduced with respect to the wild type strain. It was calculated that a much smaller fraction of UDP-sugar turnover was due to polysaccharide synthesis in the mutant than in the wild type, suggesting that hydrolysis of sugar precursors may represent another aspect of exopolysaccharide biosynthetic control.

3.2.3 Control of Isoprenoid Lipid Availability A number of indirect observations have led to the suggestion that the availability of isoprenoid lipid carriers may have an important role in the control of exopOlysaccharide biosynthesis. The most obvious example is that of bacteria failing to produce exopolysaccharide until the late logarithmic and stationary phases of growth or at low dilution (and hence specific growth) rates in continuous culture e.g. Agrobacterium sp ATCC 31749

(Phillips et al., 1983). Similarly the production of greater amounts of exopolysaccharide at sub-optimal growth temperatures by some bacteria e.g. Aerobacter aerogenes (Duguid and Wilkinson, 1953) may represent an increased availability of isoprenoid lipids for exopolysaccharide synthesis at lower growth rates where less lipid is perhaps required for the 59

synthesis of wall and lipopolysaccharides. Another physiological observation suggesting limited amounts of lipid carrier is that of lower molecular weight exopolysaccharide productiOn by certain bacteria during logarithmic growth e.g. Kiebsiella sp. K32 (Bryan et al., 1986). If a model for exopolysaccharide biosynthesis similar to that described by Bastin et al. (1993) (pp. 36-38) for 0-antigen synthesis is accepted, then the polymerase enzyme will only polymerise repeat units for a set time before the exopolysaceharide is released. When cell growth is rapid, leading to a potential limitation in the availability of lipid for repeat unit formation, fewer repeat units may be polymerised within the set time period of the polymerase enzyme prior to polysaccharide release, thus leading to a polymer of lower molecular weight. In addition to these physiological observations, a number of mutants have been isolated with altered patterns of polysaccharide expression which may be related to alteration of isoprenoid lipid availability. Norval and Sutherland (1969) isolated mutants of Kiebsiella aerogenes unable to produce exopolysaccharide at low temperatures but with normal exopolysaccharide expression at 370C. Lipopolysaccharide was reduced but not eliminated when the bacteria were incubated at 200C. Levels of enzymes involved in polysaccharide biosynthesis were comparable between wild type and mutant strains at both temperatures. It was thus suggested that there was a lesion in the pathway of isoprenoid lipid biosynthesis which only manifested itself at low temperatures (Sutherland, 1975). As a small amount of lipopolysaccharide was produced at low temperatures, a hierarchy of polysaccharide synthesis was suggested with cell wall polysaccharides having priority over lipopolysaccharides which in turn had priority over exopolysaccharides. Increased exopolysaccharide biosynthesis was observed in bacitracin resistant mutants of K. aerogenes (Sutherland, 1975). Bacitracin prevents dephosphorylation of isoprenoid lipid pyrophosphate (Siewert and Strominger, 1967) effectively removing the compound from available lipid pools and it was thus suggested that bacitracin resistant mutants had higher levels of isoprenoid lipids. The results suggested that lipid availability was a limiting factor in exopolysaccharide biosynthesis in the wild type strain. Having established that the availability of isoprenoid lipids may represent a limiting factor in exopolysaccharide biosynthesis, bacteria could control polymer biosynthesis through the availability of lipids. The active form of the molecule is the lipid phosphate and a number of enzymes have been identified which may control the levels of this molecule (Figure 3.2). A membrane-associated isoprenoid alcohol phosphokinase has been identified in Staphylococcus aureus (Higashi et al., 1970) as has an isoprenoid alcohol .phosphatase (Willoughby et al., 1972) thus providing the bacterium with the potential ability to control the interconversion of bactoprenol and bactoprenol phosphate. Finally, polymer release yields bactoprenol pyrophosphate and an isoprenyl pyrophosphate phosphatase has been observed in

Micrococcus lysodeikticus capable of returning the pyrophosphate derivative to the active monophosphate form (Goldman and Strominger, 1972).

3.2.4 Control of Glycosyltransferase Activity Very few studies have been undertaken concerning this aspect of control probably due to the requirement for membrane bound complexes for activity preventing examination of individual transferases. Sutherland and Norval (1970) observed an inhibitory effect of UMP on the transfer of the initial glucose of the Kiebsiella aerogenes exopolysaccharide repeat unit to lipid acceptor in membrane preparations. UDP was also inhibitory, but to a lesser extent than UMP. Strength of inhibition was reversed in the transfer of subsequent sugars with UDP revealing a greater degree of inhibition than UMP. A feedback inhibition mechanism therefore appeared to be operating. JPP- C55- Isoprenyl pyrophosphate I P - C 5 - Isoprenyl phosphate

IRA - C 55- Isoprenoid alcohol

Isopentenyl pyrophosphate + Farnesyl pyrophosphate

I PP olymer - Mucopeptide C55 IPP LPS or Te,choic ac:d Phosphatase Exopolysacohoride 'P

C55IPA" Phosphatase Intracellular and Phosphokinos ,j] membrane - bound precursors ATP

IPA

Figure 3.2 Regulation of carrier lipids. (From Sutherland, 1979). 61

One transferase enzyme which has received a great deal of attention concerning its regulation is the cellulose synthase of Acetobacter xylinuin. Early attempts to obtain high levels of cellulose synthesis by solubilised protein proved difficult (e.g. Glaser, 1958).

Enzyme solubilised in the presence of digitonin could be stimulated to produce cellulose at high rates in the presence of a protein co-factor and GTP (Aloni et al., 1983). Further work

(Ross et al., 1985) elucidated the function of the protein co-factor (later termed diguanylate cyclase) which was to convert GTP to an unusual nucleotide later identified as bis-(3'-5')- cyclic diguanylic acid (c-di-GMP) (Ross et al., 1987) which was found to activate cellulose synthase. Two membrane bound phosphodiesterases degraded c-di-GMP to GMP; the first enzyme was inhibited by calcium. A model was proposed for the control of cellulose synthesis (Figure 3.3) in which the diguanyl ate cyclase and phosphodiesterase enzymes were closely associated with cellulose synthase and controlled the level of c-di-GMP through the inhibitory action of calcium on the first phosphodiesterase. Evidence for a similar regulatory mechanism for cellulose synthesis by Agrobacterium tumefaciens has since been presented

(Amikam and Benziman, 1989).

Finally, the rate of crystallisation of the energetically unfavourable cellulose I microfibrils in vivo may limit the rate of cellulose synthesis in A. xylinum (Benziman et al., 1980).

Calcofluor interferes with the crystallisation of cellulose I and its addition to media in which

A. xylinum was producing cellulose increased the rate of production. However, Saxena et al. (1994) observed a random arrangement of cellulose extrusion pores in cells not synthesising the polymer and suggested that the random crystallisation of cellulose II may effectively block the pores through which the molecule is extruded. This may in turn lead to an alteration of the internal levels of c-di-GMP, which could reduce cellulose biosynthesis.

No mechanism for this potential control was presented. I,4-/3- glucan

Non- V., memorom Diquonylate 'activated Igctrvated 2P1 cyclase 2P I cellulose I cellulose synthoSe synfriase PPr// PGoG 2 5GMP ZGTP pppGpG 7 I - QGQG_1 2 I

Mq

UDP—glucose UDP

PDE A-Phosphodiesterase A PDE B-Phosphodiesterase B p.51 triphosphate dimer

IT-Cyclic diguanylic acid Ill-Inactive open dimer

Figure 3.3 Control of cellulose synthesis in Acetobacter xylinum. (From Ross et aL, 1987). 62

[*tm siti t! Genetic Organisation and Control of Exopolysaccharide Biosynthesis

4.1 Organisation of Exopolysaccharide Genes Clustering of genetic loci involved in exopolysaccharide biosynthesis is fairly widespread and has been observed in the plant symbiont Rhizobium meliloti (Hynes et at., 1986) and pathogen Xanthomonas campestris (Harding et at., 1987; Vanderslice et at., 1990), in the human pathogens Escherichia coli (Boulnois et at, 1987; Roberts et al., 1988), Haemophilus influenzae (Kroll et al., 1989) and Neisseria meningitidis (Frosch et al., 1991) and the opportunistic human pathogen Pseudomonas aeruginosa (Ohman and Chakrabarty, 1981) and also in the flocculating bacterium Zoogloea ramigera (Easson et at., 1987).

The genetics of succinoglycan biosynthesis by Rhizobium metitoti has been extensively studied. Initial investigations suggested that genes required for succinoglycan biosynthesis were located on the second symbiotic megaplasmid (Hynes et at, 1986) clustered within 22kb of DNA (Long a at, 1988a). It is now evident that all but four of the known R. meliloti exo genes are located within this cluster (Finan et at, 1986; Leigh et at, 1987; Long et at,

1988a; Than and Leigh, 1990; Buendia et at, 1991; Reuber a at, 1991; Glucksmann et at,

1993a; 1993b; Reuber and Walker, 1993b; Muller a al., 1993). ExoC, exoD (Finan ci at,

1986), exoR and exoS (Doherty a at., 1988) reside on the chromosome (Table 4.1). As can be observed in Figure 4.1 a many of the genes within the exo cluster are arranged as polycistronic operons. Of those genes arranged as single transcriptional units, exoX is a regulatory gene (Than and Leigh, 1990) whilst exoB represents a region of DNA coding for the only UDP-glucose 4-epimerase present in this species (Buendia et al., 1991). Of the Table 4.1 Genes Involved in Succinoglycan Biosynthesis by Rhizobium rneliloti Gene Location Gene Product Reference excA P glucosyl- Finanetat, 1986 transferase 1 Reuber and Walker, 1993b exoB P UDP-glucose Finan et al., 1986 4-epimerase Buendia et al., 1991 exoC C phospho- Finan et at, 1986 glucomutase Glucksmann et al., 1993

exoD C ? Finanetal., 1986 exoF P Required to charge Finan et al., 1986 lipid pyrophosphate. Reuber and Walker, 1993b Potential regulation Zhan and Leigh, 1990 exoH P Succinyl transferase Leigh et al. 1987

exol P ? Glucksrnannetal., 1993b exoK P endo-B-1,3- Long et al., 1988a 1,4-glucanase Becker et al., 1993 exoL P glucosyl- Long et al., 1988a transferase 2 Reuber and Walker 1993b exoM P glucosyl- Long et al., 1988a transferase 3 Reuber and Walker 1993b exoN P UDP-glucose pyro Long et al., 1988a phosphorylase Glucksmann et at, 1993b exoO P glucosyl- Reuber and Walker, 1993b transferase 4 exoP P transport? Long et al., 1988a Reuber and Walker, 1993b exoQ P polymerase? Long et al., 1988a Reuber and Walker, 1993b exoR C transcriptional Doherty et at, 1988 regulator Reed et al., 1991 exoS C transciptional Doherty et al., 1988 regulator exoT P transport? Reuberetal., 1991 Glucksmann et al., 1993b Table 4.1 (Continued) Genes Involved in Succinoglycan Biosynthesis by Rhizobium meliloti

Gene Location Gene Product Reference exoU P glucosyl- Glucksmann et al., 1993a transferase 5 exoV P pyruvate ketalase Glucksmann et al., 1993b exoW P glucosyl- Glucksrnann et al., 1993a transferase 6 exoX P negative Zhan and Leigh, 1990 regulator Muller et al., 1993 exoY P galactosyl- Muller et al., 1993 transferase exoZ P acetyl Buendia et al., 1991 transferase Reuber and Walker, 1993a

P=megaplasmid 2 C=chromasome fl r I-, I r1 U V F 0 13 P N OM A L IC H I T WV 2 kb_H

Boxes represent genes, arrows indicate operons 4.1a The exo gene cluster of Rhizobium meliloti

(From Glucksmann et aL, 1993a)

Pei? Pal? I Pal Acy 7 III V Exp? IV Ket II

i_U ....E I I s_G ,....H 1._il J 1r< I i....LI.....M 1 1.41 1.5 1 4.7 1 2.2 1 3.5 1 1.351 1.01

I-V: Glycosyltransferases I-V Acy: Acetyltransferase Ket: Pyruvate ketalase Pol: Polymerase

Exp: Export

Numbers beneath the gene designations represent the size of BamHl restriction fragments

4.1b The gum gene cluster of Xanthomonas capestris

(From Vanderslice et aL, 1990)

Figure 4.1 Two EPS gene clusters 63

chromosomal genes exoC codes for phosphoglucomutase (Glucksmann et al., 1993a) required for the interconversion of glucose- i-phosphate and glucose-6-phosphate, whilst exoR and exoS represent regulatory operons (Doherty et al., 1988). An operonic organisation has also been proposed for some Pseudomonas aeruginosa alginate genes (Chitnis and Ohman, 1993). A number of studies have suggested that control of mucoidy in this organism is through transcriptional activation of algD (Deretic et al.,

1987a; 1987b), part of the gene cluster located at 35 minutes on the P. aeruginosa chromosome (Darzins et al., 1985). However, Tatnell et al. (1994) observed that overexpression of A1gD alone did not significantly increase alginate production in this organism, suggesting that genes downstream of algD are also expressed via the algD promoter. Similarly, the four genes required for cellulose synthesis by Acetobacter xylinum occur as an operon (Wong et al., 1990; Saxena et al., 1994). In Agrobacterium tuinefaciens the genes required for cellulose synthesis are clustered on the chromosome (Robertson et al., 1988); however, nothing is known about the operonic organisation of the genes.

Although the genetics of exopolysaccharide biosynthesis has been studied to a lesser extent in species of Rhizobium other than R. meliloti, similarities in the genetic organisation have been observed. Chen et al. (1988) isolated thirty exopolysaccharide mutants of Rhizobium sp. NGR234. Twenty-eight of these mutants could be complemented with 46kb of contiguous DNA from a wild type strain and twenty-six were complemented by a 15kb fragment covering four complementation groups. An additional complementation group was mutated outside this gene cluster. These data suggest that exo genes within this organism are clustered in a similar manner to R. meliloti. Zhan et al. (1990) produced data which confirmed the degree of similarity between Rhizobium sp. NGR234 and R. meliloti through cross species complementation and hybridisation experiments. The two organisms produce similar but distinct exopolysac.charides (Figure 4.2). R. meliloti exoA, B, F, L, M and P ------$ —GIc 1--4GIc' 8 GIc .2 _iG;cL_SGlcL_3 GaI' 4 S I GcA 3 a

• GIcA 4 a

Pyr ( )Gal_a or3—OAc

4.2a Structure of Rhizobium sp. NGR234 exopolysaccharide

5 3 5 —GIc 1 'G'' G' Gal 8 5

GIc

I i Gic 3 5

,1 3 Ic

yr _GIcI

0-Acervi and C-Suctnyl present at uncetervmned sites.

4.2b Structure of R. meliloti succinoglycan

Figure 42 Structures of Rhizobium polysaccharides (From Zhan et aL, 1990) riI

mutants could be complemented with DNA from the Rhizobiuni sp. NGR234 gene cluster, whilst mutations in exoC, D, J, K or H were not complemented and exoQ mutants were incompletely complemented. As can be observed in Table 4.1 the genes in R. meliloti which could be complemented would probably have equivalent counterparts in Rhizobium sp.

NGR234. In R. melilot4 exoC and exoD are unlinked with the rest of the biosynthetic locus and a lack of complementation by DNA from the exo cluster of Rhizobium sp. NGR234 may represent a similar organisation in the latter organism. ExoK and exoH encode for endo-fi-

1,3-1 ,4-glucanase (Becker et al., 1993) and succinyltransferase (Leigh et al., 1987) respectively and these enzymes may not have counterparts in Rhizobi urn sp. NGR234 (Figure

4.2). In R. meliloti ExoQ is probably the repeat unit polymerase (Reuber and Walker, 1993b) and the partial complementation by Rhizobium sp. N0R234 probably represents the reduced efficiency with which the polymerase from the latter organism is able to utilise the succinoglycan substrate. Similar experiments in which R. meliloti DNA was used to complement Rhizobium sp. N0R234 demonstrated that the exoY and exoC loci in the latter organism were functionally equivalent to exoF and exoB; respectively, of R. meliloti.

Similarly, R. meliloti exoB DNA was able to complement R. legurninosarurn exoB mutants

(Canter-Cremers et al., 1990). The exoD complementation group of Rhizobi urn sp. NGR234 was equivalent to the exoL.41v1 fragment of R. meliloti. In addition to the observed functional equivalence, directions of transcription of a number of genes within the two organisms were the same. One difference was the reversed gene order of R. meliloti exoM and exoA with respect to the corresponding genes within the Rhizobi urn sp. NGR234 exoD operon. In R. phaseoli two genes involved in control of exopolysaccharide synthesis, psi (Borthakur et al., 1985) and psr (Borthakur and Johnston, 1987), were remote from other exopolysaccharide genes. A similar organisation is encountered in R. meliloti when one considers exoR and exoS 65

(Doherty et al., 1988) and similarities in the control of exopolysaccharide synthesis are observed as discussed in Chapter 4.2. • Cross genus complementation has been observed between Rhizobium rneliloti and the closely related Agrobacteriuin tumefaciens (Cangelosi et at, 1987; Stanfield et al., 1988) and A. radiobacter (Aird et al., 1991). Cangelosi et al. (1987) obtained three types of succinoglycan mutant of A. tumefaciens as observed phenotypically. The first class revealed less fluorescence than wild type on agar plates containing calcofluor and were complemented by DNA of R. ineliloti complementation group G, later shown to be an insertion in the intergenic region between exoX and exoY (Muller et al., 1993). Type 2 mutants produced B- glucans but failed to synthesise succinoglycan and were complemented by R. meliloti exoA,

B, D or F genes. The gene products are specific for succinoglycan biosynthesis (Reuber and

Walker, 1993b; Buendia et al., 1991). Type 3 mutants failed to synthesise either succinoglycan or B-glucans and were all complemented by the R. meliloti exoC gene. In both

R. meliloti (Glucksmann a al., 1993a) and A. tutnefaciens (Uttaro et al., 1990) exoC encodes phosphoglucomutase. Similarly R. yneliloti exoB, C or D mutants could be complemented with A. tumefaciens DNA; however, R. meliloti mutated in exoA or F could not be complemented. Aird et al. (1991) obtained a number of A. radiobacter exopolysaccharide mutants through chemical mutagenesis with nitrosoguanidine. Fragments of wild type DNA approximately 30kb in size were used to complement the mutants and it was observed that complementing DNA was located in five different parts of the bacterial genome suggesting a less ordered structure of loci involved in exopolysaccharide synthesis in A. radiobacter than observed in R. meliloti. However, some mutants were only unable to produce exopolysaccharides when grown on dicarboxylic acids and all of these mutants were corrected by complementation with one segment of wild type DNA suggesting that some of the mutated genes were involved in dicarboxylic acid metabolism. This result emphasised the possibility Ml

that some of the exopolysaccharide-negative mutants may have mutations in genes not directly involved in exopolysaccharide synthesis. The exoB gene of R. meliloti, coding for UDP-glucose 4-epimerase (Buendia et al., 1991), was able to correct one A. radiobacter mutant; however, no other mutants were corrected with a variety of R. meliloti exo genes. In addition to succinoglycan, both Rhizobium meliloti and Agrobacterium tumefaciens are capable of synthesising 13-(1,2)-glucans (York et al., 1980; Gorin et al., 1961). Production of these polysaccharides requires two genes in both organisms, ndvA and ndvB in R. meliloti

(Dylan et al., 1986) and chvA and chvB in A.tumefaciens (Douglas et al., 1985). These genes are able to cross complement between the two species (Dylan et al., 1986). Widespread loci have also been observed for the synthesis of xanthan by Xanthomonas campestris. Harding et al. (1987) obtained mutants which could be corrected by a contiguous

13kb stretch of wild type DNA. Later studies (Vanderslice ci' al., 1990) elucidated that this cluster of twelve genes covered 16kb of DNA involved in the synthesis, polymerisation and export of xanthan gum (Figure 4. lb). In addition to this gene cluster, mutations in at least two other areas of the X. campestris genome are able to disrupt exopolysaccharide synthesis (Thome et aL, 1987). One of these, constituting 27kb, also affects synthesis of a number of extracellular enzymes and it was suggested that this cluster of up to seven genes may be required for regulation (Thorne ci' al., 1987).

Although none of the genes required for curdlan production by Agrobacterium sp. ATCC 31749, or for succinoglycan production by its parent strain, 10C3, (Table 1.2) have been identified, data is available regarding spontaneous and chemical mutagenesis of the organism.

When initially isolated from soil, the organism produced succinoglycan from a number of carbon sources (Harada and Yoshimura, 1964). A spontaneous mutant was obtained which no longer produced succinoglycan but instead produced curdlan with reasonable efficiency 67

on a number of carbon sources. The mutant arose by repeated transfers to medium containing ethylene glycol as the sole carbon source (Harada a at., 1966). The spontaneous mutation rate was quantified at 0.01 after growth for sixty days on agar plates and these mutants were stable for at least a year (Amemura a at., 1977). A similar ability to mutate to curdlan production has been observed in Rhizobiutn trfo1ii (Ghai et al., 1981). Two possibilities are likely regarding the spontaneous mutation to curdlan producing strains. Firstly, the genes required for succinoglycan production may be unstable as has been observed for the region encoding exopolysaccharide production by Zoogloea ramigera (Easson a al., 1987). A small quantity of curdlan was produced by strain 10C3 (Harada et at., 1968b) and the possibility exists that more of the carbon source is directed to curdlan production if a mutation prevents the organism producing succinoglycan. The second mechanism could involve a mutation in control genes leading to less succinoglycan and more curdlan production. This mechanism could have produced the four groups of mutants isolated by chemical mutagenesis of strain

10C3 by Harada and colleagues (1968b). In the parent strain and one group of mutants, much succinoglycan and little curdlan was produced. The second group of mutants produced roughly equal amounts of the two polymers whilst other groups produced either curdlan or succinoglycan. Finally, mutagenic agents reported to eliminate plasmids did not affect production of either curdlan or succinoglycan (Amemura et al., 1977)suggesting that the required genetic information is chromosomal.

4.2 Genetic Control of Exopolysaccharide Synthesis Many bacterial responses to environmental stimuli are controlled via two component regulatory systems. These include responses to medium osmorality, nitrogen limitation, phosphate limitation and responses to toxic compounds. A sensor protein receives an environmental stimulus leading to interaction with a regulator protein which then affects levels 101.1

of transcription of relevant genes. High degrees of homology have been observed between sensor proteins and regulator proteins from different organisms (Ronson et al., 1987).

Evidence has now accumulated to suggest that this type of system also operates in the control of exopolysaccharide synthesis by a number of bacteria.

Osbourn et al. (1990) synthesised oligonucleotide probes corresponding to highly conserved regions of both sensor and regulator proteins involved in two component responses to environmental stimuli. Subsequent to restriction enzyme digestion of Xanthomonas campestris DNA the probes bound to two separate regions which were then sequenced. One of the regions contained an open reading frame which had extensive homology with genes encoding regulator proteins. Adjacent to this, a second open reading frame was transcribed in the opposite direction and coded for a protein having homology with sensor proteins of two component systems. Mutations in the putative regulatory gene resulted in decreased xanthan production suggesting that the encoded protein acted as a transcriptional activator of exopolysaccharide synthesis genes. Other unconnected phenotypical changes were observed raising the possibility that the protein controlled a number of responses to environmental change. A second area of DNA with positive regulatory functions has been observed in X. campestris (Tang et al., 1991). Transposon mutagenesis revealed that up to seven genes may be mutated within the cluster of 27kb, mutation in any one of which results not only in inability to synthesise exopolysaccharide but also amylase, endoglucanase, polygalacturonate lyase and protease. Hence the genes were designated rpf-regulation of pathogenicity factors. Furthermore, because mutation in any one of the genes led to the same xanthair, enzyme- phenotype it was suggested that the genes worked in series rather than in parallel. One of the genes, rpfC, was sequenced. The N-terminal region of the putative protein had homology to sensor proteins and the C-terminal region had homology to regulator proteins of the two component environmental sensing system. Between these two regions was an area coding for approximately 30 amino acids which did not share homology to any proteins involved in two component systems and it was suggested that the gene encoded a protein that was cleaved post-translationally in the 30 amino acid linker region. In contrast, a gene involved in negative regulation of enzyme and exopolysaccharide synthesis has been observed in X. campestris (Tang et at, 1990). When a 2.7kb fragment of X. cainpestris DNA was introduced into wild type strains on a high copy number plasmid exopolysaccharide and enzyme synthesis was inhibited. Conversely if the gene was mutated exopolysaccharide and enzymes were overexpressed. Double mutants which carried mutations in both the negative regulator and the rpf gene cluster produced low quantities of enzymes and xanthan indicating that overproduction in the negative regulator mutant was dependent on the rpf gene products. A similar gene dosage effect has been observed when the copy number of psdA is increased in Agrobacteriurn tuinefaciens (Kamoun et al., 1989). However in this case only exopolysaccharide production was examined and it is not clear whether the gene controls other biosynthetic pathways.

The genetic control of exopolysaccharide production has been examined in a number of species of Rhizobium. In R. ineliloti exoR and exoS act as negative regulatory genes. Mutations in either gene led to an increased amount of succinoglycan. In the case of exoS mutants more succinoglycan was produced in media lacking NH 4+ than in media containing the ion; however, the quantity of succinoglycan produced was greater than that produced by wild type in comparable media. ExoR mutants produced extremely high quantities of succinoglycan regardless of the presence or absence of NFI 4+ (Doherty et al., 1988). The exoR gene product may therefore be part of a two component environmental sensing system.

ExoR or exoS mutants carrying exoF or exoP::TnPhoA fusions had increased alkaline phosphatase activity, suggesting that ExoR and ExoS act at the levels of transcription or FA

translation. These data also demonstrate that the proteins act on more than one operon. A more detailed analysis of exoR mutants revealed that mRNA levels of exoA, F, H, K, L, M,

N, P, Q, X and Y were higher in the mutants than in wild type organisms; however, exoB mRNA concentrations were comparable in mutant and wild type organisms (Reed et al.,

1991). This data indicated that ExoR acted at the level of transcription and furthermore that at least three operons were affected by the protein.

A second system of genetic control in R. meliloti is that involving the exoX and exoF genes. In exoX mutants succinoglycan production is increased and conversely, multiple copies of intact exoX repressed succinoglycan production (Zhan and Leigh, 1990). However, repression of succinoglycan synthesis could be overcome by increasing the copy number of exoF and the ratio of the two genes correlated with succinoglycan production. Thus when exoX and exoF were present in equal numbers, succinoglycan was produced at the same rate as in wild type cells; if the exoX copy number exceeded that of exoF, succinoglycan synthesis was repressed and if there were more copies of exoF than exoX succinoglycan synthesis was enhanced. At least one copy of exoF was required for succinoglycan synthesis even in an exoX mutant background. It was suggested that ExoX acts as a negative regulator by binding to the biosynthetic apparatus in the bacterial membrane. ExoF was postulated to modulate the activity of ExoX by binding to the protein and preventing it from interacting with its site of action. Similarly, in Rhizobium sp. N0R234 polysaccharide biosynthesis was inhibited when exoX was present in greater copy number than exov(Gray et al., 1990). The two genes were sequenced and ExoY had a potential hydrophobic stretch which could act as a membrane spanning domain or allow interaction with hydrophobic regions of other proteins.

ExoX had a similar hydrophobicity plot to Psi from R. phaseoli, a gene product which inhibits exopolysaccharide synthesis in R. phaseoli when the gene is present in multiple copies 71

(Borthakur et al., 1985). Multiple copies of psi introduced into R. ieguminosarum were able to repress synthesis of exopolysaccharide in this organism. In R. phaseoli the product of another gene, psr, repressed transcription of psi (Borthakur and Johnston, 1987). In conclusion the genetics of bacterial exopolysaccharide synthesis, particularly among bacteria interacting with plants, have varying degrees of common organisation and similar strategies for control of exopolysaccharide biosynthesis. Hence information obtained from other plant-interacting bacteria may provide clues as to the strategies adopted for the control of curdlan production by Agrobacterium sp. ATCC 31749. 72

Chapter 5 Materials and Methods

5.1 Maintainence of Organism, Culture Conditions and Product Recovery

5.1.1 Organism

The organism used in this study was Agrobacterium sp. ATCC 31749. Genealogy of the organism is described in Table 1.2.

Oerskovia xanthinolytica was obtained from the I.C.M.B. culture collection, University of Edinburgh.

5.1.2 Maintainence of Agrobacterium sp. ATCC 31749

The bacterium was maintained on high nitrogen MSM (see below) agar plates (15g l Oxoid agar no.3) at 40C, after two days incubation at 30 0C. The organism was transferred at one month intervals. In order to visualise curdlan production the organism was grown on low nitrogen MSM (see below) supplemented with 0.05g 11 aniline blue (Hisamatsu et al., 1977; see Plate 5.1). Back-up cultures were maintained on agar slants at room temperature and in addition lyophilised cultures were maintained under vacuum (Gherna, 1981).

5.1.3 Growth Media and Conditions The organism was routinely grown in a chemically defined mineral salts medium, as described by Phillips et al. (1983). The salts mixture was (g 1 1/mM) K1112PO4 (1.742/12.8), K2HPO4 (0.488/2.8), NH4C1 (4.012/75), Na2SO4 (1.633/11.5), citric acid (0.210/1.0),

FeC13 (0.015/0.09), CaC12.6H20 (0.022/0.1) and MgCl2.6H2O (0.254/1.25). The medium Plate 5.1

The Appearance of Agrobacterium sp. ATCC 31749 on MSM/Aniline Blue Agar

Bacteria were streak plated onto low nitrogen MSM agar containing 0.005% (w/v) aniline blue. Where a thick bacterial lawn is present the available nitrogen has been utilised resulting in curdlan production (see pp. 91-94). Curdlan specifically interacts with aniline blue resulting in the coloration of colonies. 73

was routinely supplemented with 50niM MOPS and the pH adjusted to 7.0 (pp .92-93). This medium is described as high nitrogen MSM. Glucose was routinely supplied as the carbon source at 10 g f' to provide a carbon deficient medium (Phillips et al., 1983). In order to induce curdlan production the NH4CI concentration was reduced to 20mM (1.07g 15 and the glucose concentration was increased to at least 20g f to provide a nitrogen deficient medium (Phillips a at, 1983). This medium is described as iow nitrogen MSM. Sterilisation was by autoclaving at 17 p.s.i. and 120 0C for 20 mm; carbon sources were autoclaved separately. Growth was routinely in Erlenmeyer flasks (lOOmJ medium in 250m1 flasks or 750m1 medium in 21 flasks) at 300C with rotary shaking at 200 r.p.m. For growth and curdlan production in fermenters seed cultures were grown in low nitrogen

MSM overnight. A 1.81 Bioengineering fermentor vessel containing 1.21 low nitrogen MSM without MOPS, but with the addition of lOOpl antifoam was inoculated with lOOmi seed culture. The temperature was maintained at 30 0C. The pH was automatically maintained at

7.0 by the addition of 4N KOH or HC1, unless otherwise stated. A dual standard impeller maintained mixing by rotation at 650 r.p.m. The initial glucose concentration was approximately 20g f'. Once growth had ceased, the impeller speed was increased to 800 r.p.m., the pH reduced to 6.0 and the glucose concentration increased to approximately 50g 1'. Sterile air was passed through the medium throughout the fermentation. Sampling was achieved by aseptic transfer.

Oerskovia xanthinolytica was cultured in a mineral salts medium. The composition of the medium was (g ['1mM) Na2HPO4(25.20/177.52), KH2PO4 (3.00/22.04), K2SO4 (1.00/5.74),

NaCl (1.00/17.11), MgSO 71-JO (0.20/0.81), CaCl2 (0.02/0.18), PeSO 4 (0.001/0.007), casein hydrolysate (1.00). The pH of the medium was adjusted to 7.0 prior to autoclaving. To

1 obtain curdlan-degrading enzymes, 0.133g f dried active baking yeast was added as a source 74

of 13-glucan, prior to autoclaving. The carbon substrate was ig 1 - 1 glucose. Growth was in

Erlenmeyer flasks as described for Agrobacteriutn sp. ATCC 31749.

5.1.4 Growth Measurements and Calculations The growth of the organism was routinely estimated by measurement of the optical density of cultures at 660nm. This method was considered appropriate because curdlan production does not commence until growth of Agrobacterium sp. ATCC 31749 has ceased (Phillips et al., 1983; Chapter 6.2). In order to obtain doubling times natural log of the optical density was plotted against time and the equation: lnOD660=at + b where: lnOD660=10g optical density 660nm t=time a and b=constants was obtained by regression analysis using the Minitab statistical package. The doubling time was calculated as the reciprocal of a. Similarly glucose consumption rates were calculated from the equation:

Glc=at + b where: Glc=glucose remaining (mg ml") a=glucose consumption rate (mg ml" h") and curdlan production from the equation: Curd=at + b where: Curd=curdlan present (mg ml") a=curdlan production rate (mg ml" hd) 75

5.1.5 Product Recovery

Curdlan was routinely harvested according to the method of Phillips et al. (1983) as shown in Figure 5.1. Cell mass and curdlan mass were obtained after lyophilisation.

In addition a microassay was developed to allow for the chemical estimation of product masses. imi of culture was transferred to a 1.5m1 eppendorf tube. The culture was microfuged for 5 minutes and the supernatant retained for glucose estimation (p. 78). The pellet was washed three times in distilled water, resuspended in imi of 0.5N NaOH and microfuged for 5 minutes to pellet the cells. Curdlan was then estimated in the supernatant by the phenol sulphuric acid assay (Dubois et al., 1956; p. 76).

5.1.6 Preparation of Curdlan Samples

Neutralised curdlan gels were obtained as described in Figure 5.1. Low-set curdlan gels were obtained by heating aqueous suspensions of dried neutralised gels at 60 0C for 30 minutes after which time the turbidity of suspensions was observed to clear. The solutions were cooled to room temperature to allow the gels to set, homogenised and lyophilised.

High-set curdlan gels were obtained by heating aqueous suspensions of neutralised gels in boiling water baths for one hour after which time the suspensions had gelled. The gels were cooled to room temperature, homogenised and lyophilised.

Native curdlan was prepared without dissolution in sodium hydroxide. One litre cultures were centrifuged (10 mm, 10000 r.p.m.), precipitates were resuspended in a small volume of distilled water and dialysed against three changes of distilled water with 48 hours between changes. Samples were then lyophilised. After lyophilisation samples were resuspended in lOOml 500mM Tris pH 7.5 containing 0.2 mg mi1 protease type XIV (Sigma) and incubated at 370C for 48 hours. The samples were then washed in distilled water three times and finally resuspended in distilled water and lyophilised. Weighed subsamples were resuspended in Figure 5.1

Flow Chart of Harvesting Procedure of Agrobacteriuin sp. ATCC 31749 Cultures

Culture

Add equal volume IN NaOH

Neutralise with IN HC1

Centrifuge 10000 r.p.m., 10 minutes

It, Pellet Supernatant

Resuspend 0.5N NaOH

Centrifuge 10000 r.p.m., 10 minutes.

Pellet, cells Supernatant

Freeze dry Neutralise with IN HC1

Centrifuge 10000 r.p.m.

/

Supernatant, discard Pellet,Wash with dH20

Curdlan, freeze dry 76

0.5M NaOH, microfliged for 5 minutes and total supernatant carbohydrate was estimated by the phenol sulphuric acid assay (p. 76). All samples contained at least 90% (w/w) alkali soluble carbohydrate. Similarly, weighed samples were resuspended in distilled water, microfuged and supernatant carbohydrate estimated. No water soluble carbohydrate was

•detected. Protein was also estimated with the BioRad dye reagent (p. 81) and all samples contained less than 2.5% (wlw) protein. Finally, samples were hydrolysed in 0.5N H2SO4 at 1000C for 18 hours and monosaccharide components were analysed by descending paper chromatography (p. 79) with butanol:pyridine:water (6:4:3) as the solvent system. The only detectable monosaccharide was glucose.

5.2 Analytical Procedures

5.2.1 Total Carbohydrate

Total carbohydrate was determined using a microassay of the phenol sulphuric acid method

(Dubois et al., 1956).

Reagents:H2504 sp. gravity 1.84

5% (wlv) aqueous phenol To 200iil of carbohydrate solution in acid washed glass tubes, 200pl of aqueous phenol was added. The tubes were mixed and imI of 1-12SO4 was added. The tubes were again mixed and left to stand for 10 minutes. The absorbance at 490nm was measured and carbohydrate was estimated from a standard curve prepared using known concentrations of glucose

(Analar). Blank tubes were prepared using 200pi distilled water. The method was adapted for the estimation of curdlan by replacing distilled water with 0.5N

NaOH as the solvent. A separate standard curve was prepared using 0.5N NaOH as a blank 77

and glucose (Analar) in 0.5N NaOH as standards. As can be observed in Figure 5.2 the use of NaOH as the solvent caused only a marginal decrease in the sensitivity of the assay.

5.2.2 Anthrone Assay In order to detect cellular carbohydrate without interference from cell wall amino sugars, a microassay of the modified method of Roe (1955) was used. Reagent: 0.05% (w/v) anthrone, 1% (w/v) thiourea in 66% (v/v)

To i0øpi sugar solution in acid washed glass tubes, lml of anthrone reagent was added.

The tubes were mixed and placed in a boiling water bath for 15 minutes. The tubes were brought to room temperature in a second water bath and left in the dark for 30 minutes.

Absorbance was measured at 620nm and carbohydrate estimated from a standard curve using known quantities of glucose (Analar). Distilled water was used as a substrate blank.

5.2.3 Reducing Sugar

Reducing sugar was estimated with a microassay of the method of Park and Johnson (1949).

Reagents: A- 0.05% (w/v) KFe(CN)6

0.53% (w/v) Na2CO3 + 0.065% (w/v) KCN 0.15% (wlv) FeNH4(SO4)2.12H20 + 0.1% (v/v) Triton X100 in 0.05N H2SO4

To 200j.tl sugar solution in acid washed glass tubes, 200pI solution B and 200jil solution A were added. The tubes were mixed and placed in a boiling water bath for 15 minutes. The tubes were removed from the water bath and allowed to cool to room temperature. lml of solution C was added and the tubes were mixed gently and left for 15 minutes. Absorbance was measured at 690nm and reducing sugar estimated from a standard curve prepared using Figure 5.2. Effect of 0.5N NaOH as.. Solvent on Phenol Sulphuric Acid Assay

O.D. 490nm 2

£ •

1

0.5

5 10 15 20 25 30 ug Glucose

Sample solvent i - Water 0.05N NaOH Zncwn quantities of glucose (Anctar) were dissolved in either dH20 or O.5N NaGH. Assays were undertaken as described (p. 76) and optical density was ,-lotted against glucose mass, a'N20 or O.5N NaOH were used as blanks vA

known concentrations of glucose (Analar). Blank tubes were prepared by treating distilled water in the same manner as standards and unknowns.

5.2.4 Glucose Glucose was determined by the glucose oxidase assay. This assay works on the principle:

D-glucose + 1120 + 02 gluconate + 11202 Glucose oxidase

H202 + Reduced ADTS -, 21120+ Oxidised ADTS Peroxidase the oxidised ADTS is a strongly coloured compound.

Reagents: Glucose oxidase (Boehringer) (1mg ml -' )

Peroxidase (Sigma) (2mg m1 1 ) ADTS (Sigma) (10mg nit') Freshly prepared

500mM Tris buffer pH 7.0

All reagents were dissolved in 500niM Tris buffer pH 7.0. 50p1 glucose oxidase, lOpJ peroxidase, 15011 ADTS and 4501i1 buffer were mixed in a 1.5m1 eppendorf tube. 100111 glucose solution was added and the tubes were mixed again and incubated at 370C for one hour in a water bath. The absorbance was measured against a water blank and the amount of glucose estimated from a standard curve using known concentrations of glucose (Analar).

5.2.5 Acetyl Groups

A microassay of the method described by Kabat and Mayer (1961) was used for the determination of acetyl groups. 79

Reagents: 2M Hydroxylamine hydrochloride

3.5M Sodium hydroxide

Hydrochloric acid sp. gr. 1.18

:0.37M FeC13.6H20 in 0. IN HG!

To 200utl test solution 400l of a freshly mixed (1:1) solution of hydroxylamine hydrochloride and sodium hydroxide were added. The tubes were mixed and held at room temperature for 2 minutes. 200p1 hydrochloric acid were added and the tubes were again mixed. Finally, 200iil of ferric chloride were added, the tubes were mixed and the absorbance measured at 540nm. The acetyl content was estimated from a standard curve constructed from various dilutions of 0.004M acetylcholine chloride in 0.001M sodium acetate buffer pH

4.5. Substrate blanks contained 200i1 distilled water.

5.2.6 Paper Chromatography

Descending paper chromatography was undertaken on Whatman no. 1 Chr paper using solvent systems as described in the text. Standard carbohydrate solutions were at 1 P of 0. 1M solutions, test solutions were applied as described in the text. Chromatograms were developed by a modification of the method of Trevelyan et al. (1950). They were first passed through a saturated solution of silver nitrate in acetone followed by 2% (w/v) sodium hydroxide in methanol to reveal the location of sugars. Finally, the chromatograms were fixed in 5% (w/v) aqueous sodium thiosulphate. Chromatograms were air dried between each developing solution. FIc

5.2.7 Sulphate Sulphate determination was by the method of Terho and Hartiala (1971). In principle Ba 2 forms a strongly coloured compound when reacted with rhodizonate. The presence of S04 2 causes precipitation of Ba2+ as BaSO4 thus reducing the intensity of the colour. Reagents: BaC12 buffer- lOrni 2M acetic acid 2m1 5mM BaC12

8m1 20mM NaHCO3 Made up to lOOml in ethanol K rhodizonate solution- 5mg K rhodizonate 100mg L-ascorbic acid

In lOOml 80% ethanol

To 0.5ml of sulphate solution in acid washed glass tubes 2.0ml of absolute ethanol was added followed by l.Oml of Bad2 buffer and 1.5ml of potassium rhodizonate solution. The tubes were mixed and left in the dark for ten minutes. Simultaneously a water blank received the same treatment. Absorbance of the water blank and sulphate solutions were then measured at 520nm. The difference in absorbance between the water blank and sulphate solutions was calculated (AA520)and the sulphate concentration estimated from a standard curve plotting AA520 against sulphate mass for solutions of known Na2SO4 concentration.

5.2.8 Phosphate.

Phosphate was determined using a microassay of the method reported by Rand et al. (1975).

Reagents: SN H2SO4

K(SbO)C4H404. 112H20 2.743g1' in dH20

(NH4)6Mo7024.4H20 40g1' in dH20 0.1M Ascorbic Acid

SOml sulphuric acid, 5m1 potassium antimonyl tartrate solution, 1 5m1 ammonium molybdate

solution and 30m1 ascorbic acid were mixed to form a combined reagent just prior to

determination of phosphate. To imi of phosphate solution, 160p1 of combined reagent was added. Tubes were mixed

and left to stand for ten minutes. Absorbance was measured at 880nm and phosphate was estimated from a standard curve prepared using known concentrations of KH2PO4. Distilled

water was used as a reagent blank.

5.2.9 Ammonium Ammonium was determined by microassay of the phenate method (Weatherburn, 1967).

Reagents: Hypochlorous acid- 40m1 dH20, lOm! 5% NaHCI03 pH 6.5-7.0. 3 mlvi Manganous sulphate

1 Phenate- 25g l NaOH + lOOg phenol To lml ammonium solution in a 1 .Sml eppendorf tube, Spl manganous sulphate solution was

added and the tubes were mixed. 50j11 hypochlorous acid and 60p1 phenate were added and the tubes were again mixed and allowed to stand for ten minutes. Absorbance was measured

at 630nm against a similarly treated sample of distilled water and ammonium was estimated from a standard curve prepared using known concentrations of NI-14C1.

5.2. 10 Protein

Protein was estimated using the microassay procedure with the BioRad dye reagent as

described by the manufacturers. To 800iil protein solution, 200ji1 dye reagent were added and the tubes were mixed. The tubes were allowed to stand for 5 minutes and the absorbance

/ measured at 595mm The amount of protein was estimated from a standard curve constructed using known quantities of IgG. Water was used as a substrate blank.

5.2. 10 pH pH was determined using a PHM-1 l0-130C probe with a Horiba F-8L pH meter.

5.2.11 HPLC High performance size exclusion chromatography was undertaken using Toya Soda (Japan)

G4000PW or G6000PW columns. UHQ water was used as the mobile phase at a flow rate of 0.25m1 mind controlled by a Gilson model 303 pump. Samples were dissolved in UHQ water and filtered through 0.45nm filters prior to injection of 20pl via a rheodyne. Sample detection was through a Knauer differential refractometer and traces and analysis were obtained using a Shimadzu C-RIB chromatopac. Molecular weights of samples were estimated by comparison of retention time against standard curves constructed using dextrans of known molecular weight.

5.2.12 Viscometry Solution viscosity was determined using a Brookfield DV-11 cone and plate digital viscometer. The cone spindle employed was model CP-41 (cone angle 30, radius 2.4cm) operating to induce a shear rate of 12o s 1 . 2m] samples were subjected to shear for 60 seconds at 25 0C prior to recording viscosity. The machine was recalibrated prior to each use with a standard solution of known viscosity which displayed Newtonian flow characteristics. *1

5.3 Cell Fractionation and Protein Concentration

5.3.1 Cell Fractionation by Osmotic Shock

The method of Osborn et al. (1972) was used to obtain the periplasmic fraction of bacterial cells. Cells were harvested as described in Figure 5.1 and washed once in high nitrogen

MSM. Cells were then resuspended in 1150 culture volume of cold (40C) 750mM sucrose in 10mM Tris pH 7.8. Lysozyme was added to a final concentration of l00ig ml' and the solution was stirred in an ice bath for 2 minutes. Stirring was continued whilst two volumes of cold (40C) 1.5mM EDTA pH 7.5 was added over a period of 10 minutes. Spheroplasts were harvested by centrifugation (10 mm, 10000 r.p.m.). The supernatant (periplasmic fraction) was dialysed against appropriate buffer (see text) in the cold, overnight and ultracentrifuged (100000g, 4 hrs) to remove outer membrane. The sphaeroplasts were resuspended in a small volume of appropriate buffer (see text) and lysed in a French pressure cell to obtain the cytoplasmic fraction. Cell membrane was removed by ultracehtrifugation.

5.3.2 Cell Fractionation by Chloroform Shock

For smaller cell quantities the method of Ames et al. (1984) was suitable for cell fractionation. Cells were harvested and washed in fresh culture medium. The second supernatant was poured off and cells were resuspended in the residual culture medium.

1/100th culture volume of chloroform was added and tubes were mixed and left to stand for

15 minutes at room temperature. 1/10th culture volume of 0.01M Tris pH 8.0 was added and the spheroplasts were harvested by centrifugation (10000 r.p.m., 10 minutes). The periplasmic and cytoplasmic fractions were obtained as described (Chapter 5.3.1). [ff11

5.3.3 Protein Concentration All procedures were undertaken at 40C. To obtain culture supernatant protein, cells and curdlan were removed from the medium by centrifugation (10000 r.p.m., 1 hi) without the addition of NaOH. Supernatant volume was reduced by tangential flow filtration and supernatant protein was resuspended in 200m1 of appropriate buffer (see text). The protein was then dialysed against buffer and the volume reduced further by dialysis against polyethylene glycol (6000 grade). Once a suitable protein concentration had been achieved

Ind aliquots were stored at -20 0C. Periplasmic and cytoplasmic protein was similarly concentrated by dialysis against polyethylene glycol after dialysis against buffer and stored in lml aliquots at -20 0C.

5.3.4 Protein Fractionation by PAGE

The method of Laemmli (1970) was used. Stock solutions: 30% (w/v) Acrylamide + 0.8% (w/v) bisacrylamide

l.5MTris pH 8.8 0.5M Tris pH 6.8 NNNN- tetramethylethylenediamine (TEMED) (100%)

10% (w/v) ammonium persulphate Electrode buffer, Tris base 3g f' + glycine 14.4 g 1-i

2X sample buffer, 0.02M Tris pH 8.8 + 20% (w/v) glycerol + 0.004% (w/v) bromophenol blue

For reducing gels 0.4% (w/v) SDS was added to the 1.5M and 0.5M Tris buffers, 0.1%

(w/v) SDS was added to the electrode buffer and 2% (wfv) SDS was added to the 2X sample buffer. Gels were prepared between glass plates separated by 1mm spacers. FtIi

10% Acrylamide gel: 7.5m1 1.5M Tris pH 8.8 lOmi Acrylamide stock

12.5m1 distilled water 3°M' TEMED

30iil ammonium persuiphate

15% Acrylamide gel: 7.5m] 1.5M Tris pH 8.8

15 ml Acrylamide stock 4.2 ml distilled water 3.Oml glycerol (100%) 3Opl TEMED

30iil ammonium persulphate

4.5% Stacking gel: 5.0ml 0.5M Tris pH 6.8

3.0m1 Aciylamide stock 12.0inl distilled water

3Opl TEMED

30il ammonium persuiphate

All gels were degassed prior to the addition of ammonium persulphate. 10% and 15% acrylamide gels were poured and overlaid with water saturated butanol. Once the gels had set the butanol was removed and the gel top washed with distilled water prior to overlay with

4.5% stacking gel. After the stacking gel had set, gels were placed into a gel tank and running buffer was added. The gels were then pre-electophoresed at lOmA for one hour. Samples were mixed with sample buffer (1:1). For reducing gels (containing SDS) samples were boiled in buffer for five minutes. The samples were then loaded into the gel wells and the gels were run at lOmA until samples were observed to enter the separating gels. The current was then increased to 20mA and the gels run until the sample front was approximately

1cm from the bottom of the gel. The gels were developed using the silver stain method (BioRad bulletin 1089).

Reagents: Fixative 1, 40% (v/v) methanol + 10% (v/v) acetic acid

Fixative 2, 10% (v/v) ethanol + 5% (v/v) acetic acid

1 Oxidiser, lOg K2Cr207 + 0.2ml 1-i cHNO3 Silver stain, 2.Og l l AgNO3 Developer, 30g 1' Na2CO3 + 0.5m1 1-i

formaldehyde

Stop, 0.5% (v/v) acetic acid

Gels were removed from between the glass plates and gently shaken in fixative 1 for 30 minutes. They were then shaken in two changes of fixative 2 for 15 minutes each. Gels were then transferred to oxidiser for five minutes and washed once in distilled water. The gels were then exposed to silver stain for five minutes, briefly washed in distilled water and developed to the required intensity in three changes of developer.

5.4 Polysaccharide Modification

5.4.1 Carboxymethylation

Curdlan was carboxymethylated according to the methods of Sasaki et al. (1979). Method A: 3g of curdlan were dispersed into 80m1 isopropyl alcohol and stirred for 30 minutes. 8m1 30% (w/v) NaOH were added over a period of 60 minutes and the mixture was stirred for a further 90 minutes. 3.6g monochioracetic acid was added and stirring was continued for 5 hours at 659C. The carboxymethylated product was collected by centrifugation, redissolved in distilled water and neutralised with Amberlite IR-4 10 resin

(HCO3- form). The product was then extensively dialysed against distilled water prior to lyophilisation. This method gave a product named CMC A with a degree of substitution of

0.47 (Sasaki et al., 1979) Method B: 1.5g of curdlan was stirred in 40m1 of isopropyl alcohol for 30 minutes. Four

Imi aliquots of 30% (w/v) NaOH were added at 15 minute intervals and the solution was stirred for a further 90 minutes. Three 0.6g portions of monochloracetic acid were added at

10 minute intervals and the solution was then warmed to 50 0C and stirred for 2.5 hours. The product was recovered as above. This method gave a degree of substitution of 0.98 (Sasaki et al., 1979) and the product was termed CMC B.

5.4.2 Acetylation Acetylation was undertaken according to the procedure of Skjak-Braek et al. (1986). 100mg of curdian was dispersed in lOOml of a 1:1 mixture of pyridine and acetic acid anhydride. The reaction mixture was stirred at 22 0C for 16 hours. Two volumes of acetone were added and the resulting precipitate was removed by centrifugation and resuspended in distilled water. The polymer was then extensively dialysed against distilled water and lyophilised. After lyophilisation the dried material was resuspended in distilled water and any insoluble polymer was removed by centrifugation. iIi] Mi

5.5 Hydrolysis of Curdlan

5.5.1 Enzymic Hydrolysis 2mg ml' curdlan and 0.1mg ml-, or 1mg ml-i enzyme (see text) were incubated at 30oC in

50mIvI potassium phosphate buffer pH 7.0. The rate of hydrolysis was examined by estimation of the increase in reducing sugar (p. 77). The amount of reducing sugar released was corrected by subtraction of the reducing sugar found in enzyme blanks (2mg ml' curdlan in buffer) and substrate blanks (enzyme in buffer) from that observed in experimental tubes. To examine the degradation products of complete hydrolysis the reactions were continued until the amount of reducing sugar present became constant. Any remaining solid material was removed by centrifugation and washed thrice in distilled water. The washes were combined with the original supernatant and both the supernatant and the precipitate were lyophilised. After lyophilisation the precipitate was dissolved in 0.5M NaOH whilst the supernatant was redissolved in distiled water. The amount of carbohydrate was determined for each fraction (p. 76). For the water soluble fraction the proportion of glucose was also estimated (p. 78) as was the average degree of polymerisation (total carbohydrate/reducing sugar).

5.5.2 Acid Hydrolysis

Acid hydrolysis of 100mg curdlan samples was undertaken in 2.5m1 H2SO4 in sealed glass ampules using the acid concentrations, times and temperatures described in the text. After hydrolysis, any remaining solids were removed by centrifugation and washed thrice in distilled water. The supernatant and washes were mixed and neutralised with Amberlite IR-410 resin (HCO3 form). The two fractions were lyophilised and analysed as described above. Chapter 6 Effect of Physiological Conditions on Curdlan Biosynthesis by Agrobacterium sp. ATCC 31749

6.1 The Effect of pH and Addition of Biological Buffers

Initial investigations of curdlan biosynthesis by Agrobacterium sp. ATCC 31749 revealed that little, if any curdlan was produced when bacteria were grown in Erlenmeyer flasks containing low nitrogen MSM. However, Phillips et al. (1983) .reported high yields of curdlan when the bacterium was grown in the same medium in pH-controlled fermenters. The possibility therefore existed that without control, the pH of the medium might have fallen low enough to inhibit curdlan biosynthesis by the bacterium. ! To test this hypothesis, various biological buffers were added to low nitrogen media and growth, curdlan production and medium pH were monitored over a period of 48 hours in triplicate flasks. As can be observed in Figure 6.1 growth rates during the exponential period were similar in all cases with doubling times of approximately 3.3 hours. In both control flasks (MSM without added buffer) and flasks containing buffer the pH remained above 5.1 during the period of measurement of exponential growth. However, bacteria grew to a lower optical density in medium lacking additional buffers suggesting that the rate of increase of acidity in medium alone was sufficient to inhibit growth due to low pH prior to limitation by another factor.

Final curdlan concentration was low in both low nitrogen MSM and low nitrogen MSM supplemented with 50mtvI Tris. In both media the final pH was below 4; In the two media where the final pH remained higher (MSM supplemented with either 50mM HEPES or 50mM

MOPS) the final curdlan concentration was greater, with 2.78 mgml' present in medium containing 50mM MOPS. Although similar quantities of curdlan were produced in media M,W1 (Jill)' MSM + 50mM 'Iris

O.D. 661mm p1.' 0.!) 660nni pH H:

0.1 0.1

I I 1 0.02' I I I I '2 0,011 1 ' 12 0 5 10 1520 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45

Iin IglonIII'IIS 45/-Ill mg/ **l Time (hrs) FIII.I igloco*eI-12 52.1-1.72 tog/mI Time (hrs) Palau40e 310 11-Il 07 h's Td'3 33*/009 h,' PIonl •- — ' I25,/-O.OImtlml Optical density ± pH Optical density ±pH

MSM + 50mM HEPES MSM + 50mM MOPS

O.D. 660nns pH_ 0,D.660nm .- pH It

0.] 0.1

I 1 1 0.01' ' 11 2 0.01' '2 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45

Final igi*co'ei' 5-75-1-0 20 m1 ml Time (h Fl) Final '5 lucosei-.30 '/1.45 tog/mi Time (hi's) Td +3.30*/-0.04 hr, Final *1, d tn co*rtnIr.IIon' 240*1-040 mg/mI Tda.M* .o.n hia Fm.lcIIrdi.n co*c.*l,.,mno-2.70n /-037 mg/mI —Optical density ±pl-1 Optical density ±pH Figure 6.1

- The Effect of Buffers on Medium pH and Growth of Agrobacterium sp. ATCC 31749

Triplicate flasks (250 ml) containing 100 ml low nitrogen MSM + 2% (w/v) glucose and the buffer described were inoculated from an overnight culture (high nitrogen MSM). Optical density and pH were recorded at the times indicated. After 48 hours the glucose concentration in each flask was estimated (p. 78) and the cultures harvested. Final curdlan concentrations were estimated after Ivoohilisation. Final 1chicose1final pls,-)-r r,nnr.enrrntinn .1- qt dev Tdtlniihiina time ./_ ct dew supplemented with either MOPS or HEPES the final .pH in the MOPS-supplemented media was 5.45 compared with 4.3 in HEPES-supplemented media.

To further examine the effects of pH on curdlan production by Agrobacteriuni sp. ATCC 31749, bacteria were grown to nitrogen depletion in batch culture, in a fermenter with the pH

maintained at either 5.0 or 7.0. Curdlan production was reduced in media at pH 5.0

compared with that at pH 7.0 (Figure 6.2). Regression analysis of the data points indicated

in Figure 6.2 revealed that bacteria grown at pH 5.0 had a faster initial rate of glucose consumption than those grown at pH 7.0. This result suggests an even greater difference in the efficiency of conversion of glucose to curdlan between bacteria grown at pH 5.0 and those

grown at pH 7.0. In addition, whilst bacteria grown at pH 7.0 had a constant rate of glucose consumption over the entire incubation period, there was a marked decrease in glucose

consumption after 150 hours when bacteria were maintained at pH 5.0. These results may represent responses to environmental stress leading to a deregulation of metabolic control and subsequent loss of cell viability when bacteria were cultured at pH 5.0.

In order to minimise differences due to loss of cell viability in long term culture, curdlan

production was examined over five hours in nitrogen free media at a number of different pH

values. To minimise pH changes, buffer concentrations within the media were increased to

100mM. Curdlan production was similar over this time period in media o,pH 5.0, 6.0 or 7.0 whilst at pH 4.0, 8.0 or 9.0 curdlan production was severely inhibited (Figure 6.3). This is in agreement with the results of Harada et al. (1966) who observed curdlan production by a mutant of strain 10C3 at pH 5.5-7.0 but not at pH 4.2.

As a result of the above experiments, MSM was subsequently supplemented with 50mM

MOPS and the pH raised to 7.0 with 4N KOH prior to inoculation. As can be observed in

Figure 6. 1, the pH of the medium becomes stable at approximately 5.5. The glucose uptake Figure 6.2. Curdlan Production by Agrobacterium sp. ATCC 31749 in Media of Different pH

Growth, Substrate Utilisation and Growth, Substrate Utilisation and curdlan Production by ATCC 31749 at pH 7 Curdlan Production by ATCC 31749 at pH 5

O.D. 660 nm Substrate/Product (mg/ml) O.D. 660nm Substrate/Product (mg/m])

+ -1- + 4+ . 4- I 40

30 1!]

• ~ • .4- r + F .x% 4-f. 44- '4-1- • "C- A

+ >1 - 'I- —a-

ri 0 20 40 60 80 100 120 140 160 180 200 220 240 0 20 40 60 80 100 120 140160180200220 Time (hrs) Time (hrs)

O.D.660 Glucose Curdlan O.D.660nm 4- Glucose Curdlan

Bacteria were inoculated and cultured in a 1.8 I Bioengineering fermenter vessel as described (p. 73). Samples were taken at the times indicated and optical density was measured until the cultures reached stationary phase (p. 74). Glucose (p. 78) and curdlan (p. 75) concentrations in the media were also estimated. Additional glucose was added at the times indicated by arrows. Horizontal bars delineate the data used to estimate initial glucose uptake rates (p. 74). Figure 6.3: The Effect of pH on Curdlan Production by ATCC 31749

Curdian (mg/ml)

LII

10

0.

0 1 2 3 4 5 Time (hrs)

Medium pH 4.0 ±5.0 *6.0 -°- 7.0 *8.0 +9.0 Buffer Used Tris HCI Tris HCI MES MOPS Gly-Gly Tris base

Overnight cultures (high nitrogen MSM) were divided into six, harvested and washed in nitrogen-free MSM of appropriate p1-I. The bacteria were resuspended in nitrogen- free MSM of appropriate pH and 1% (w/v) glucose was added at t=O. Curdlan concentration was then esflmated (p. 75) at hourly intervals. 91

data from Figure 6.2 suggests that bacteria are viable in media of pH 5.0 for approximately 140 hours whilst curdlan production at this pH is not severely inhibited (Figure 6.3).

6.2 The Effect of Nutrient Limitation on Curdlan Production by Agrabacterium sp. ATCC 31749

As discussed in Chapter 3, nutrient limitation has frequently been observed to promote

exopolysaccharide synthesis in a number of bacteria, for example Xanthomonas campestris (Tait et al., 1986). Experiments were therefore devised to study the effect of changes in

medium composition on curdlan production by Agrobacterium sp. ATCC 31749.

Overnight cultures, grown under carbon limiting conditions (Phillips et al., 1983), were aseptically centrifuged and washed in appropriate media. Cells were then resuspended and incubated in medium lacking either nitrogen, phosphorous or sulphur. Samples were taken

at one hour intervals and assayed for the quantity of curdlan present (p. 75). As can be observed in Figure 6.4a, lack of nitrogen in the medium promoted curdlan production whilst absence of phosphorus or sulphur had no effect, with curdlan production remaining low and similar to that of control cells in full MSM. In addition, Figure 6.4b demonstrates that absence of nitrogen and sulphur from the medium did not stimulate the production of curdlan any further than the absence of nitrogen alone. Similarly, Phillips et al. (1983) observed curdlan production in the post-stationary phase of nitrogen depleted batch cultures, however no data was available regarding depletion of other nutrients. In addition, a curdlan-producing strain of Cellulomonasfiavigena was observed to synthesise large quantities of curdlan when initial medium concentrations of NH4CI were low (Buller and Voepel, 1990).

It has been suggested that stimulation of exopolysaccharide synthsis by nutrient limitation may be caused by an increased availability of isoprenoid lipids after cessation of growth (Sutherland, 1975). Experiments were therefore conducted to examine whether growth Figure 6.4. The Effect of Transfer to Nutrient Free Media on Curdlan Production by Agrohacterium sp. ATCC 31749

6.4a. Curdlan Production by ATCC 31749 6.4b. Effect of Multiple Nutrient in Nutrient Deficient Media Deficiency on Curdlan Production

Curdlan (mg/mi) Curdlan (mg/mi)

0 1 2 3 4 5 6 0 1 2 3 4 5 Time (hrs) Time (hrs)

Absent Nutrient Absent Nutrient None ± Nitrogen Sulphur -°- Phosphorus None ± Nitrogen, Sulphur * Nitrogen -°- Sulphur

Overnight cultures (high nitrogen MSM) were divided into four and washed in modified MSM lacking the appropriate nutrient. Bacteria were resuspended in the appropriate medium and 1% (w/v) glucose was added at t=O. Curdlari concentration in the medium was estimated athourly intervals (p.75). 92

ceased upon transfer of Agrobacterium sp ATCC 31749 to nutrient-free media. As can be

observed in Figure 6.5a transfer of bacteria to ammonium-free media led to an immediate

cessation of growth (as measured by O.D. at 660nm) with a concomitant stimulation of

curdlan production. Addition of nitrogen to nitrogen-free medium stimulated growth but

inhibited exopolysaccharide production. In contrast, transfer to phosphate- or sulphate-free

medium had little effect on growth (Figures 6.5b and 6.5c), suggesting that cellular reserves

were sufficient to allow growth to continue over the period of the experiment. The absence

of these nutrients did not raise curdlan production above the level in control flasks containing

complete MSM. These results might thus support the hypothesis that curdlan production is

stimulated, in the absence of nitrogen, due to an increased availability of isoprenoid lipid

caused by the cessation of growth and therefore wall synthesis.

In order to examine the effect of phosphate and sulphate limitation further, periods of culture

were increased. Triplicate flasks containing different initial concentrations of either phosphate

or sulphate, with 4% glucose as the carbon source, were inoculated with equivalent cell

numbers and incubated at 300C, with shaking, for seven days. The flasks were harvested and the final cell mass, curdlan concentration and phosphate or sulphate concentrations in the medium were analysed. The results are presented in Figure 6.6 and Table 6.1.

As can be observed (Table 6.1) the final culture pH remained close to 6 in all flasks. The bacterium was able to produce curdlan under appropriate conditions at this pH (Figure 6.3).

It was observed that nutrient uptake correlated with initial nutrient concentration, the amount of nutrient absorbed by the cells being lowest in media with the lowest initial nutrient concentration. In addition, final dry cell weight increased as the amount of nutrient absorbed increased (Figure 6.7). In the case of phosphate there was an almost linear relationship between the amount of nutrient absorbed and final cell mass. The relationship was less Figure 6.5a. Growth and Curdlan Production by ATCC 31749 in Ammonium Free Medium

Curdian (mg/ml) O.D. 660nm 0.

11 31

Eli .2

11

[II El

'-I U 0 1 2 3 4 5 6 Time (hrs)

Time NH4 Added NoNH4 ±NJ-J4Obss *NH43brs XQDnoNH4 +O.D. Ohrs O.D. 3hrs

Overnight cultures (high nitrogen MSM) were washed in nitrogen-free MSM and resuspended in nitrogen-free medium with 1% (w/v) glucose. NH 4CI (75mM) was added back to the media at the times indicated. Figure 6.5b. Growth and Curdlan Production by ATCC 31749 in Phosphate Free Medium

Curdian (mg/ml) O.D. 660nm

0.25 L

1.6 _- A -- r 0.15 1.2

01 0.8

-

0.05 0.4

[ii 1 11 1 2 3 4 5 a Time (hrs)

Time PO4 Added No PO4 + PO4 Ohrs PO4 3hrs O.D. no PO4 O.D. Ohrs O.D. 3hrs

Overnight cultures (high nitrogen MSM) were washed in phosphate-free MSM and resuspended in phosphate-free medium with 1% (w/v) glucose. KH 2PO4 (12.8mM) and K2HPO4 (2.9niM) were added back to the media at the times indicated. • . Figure 6.5c Growth and Curdlan Production by ATCC 31749 in Sulphate Free Medium

Curdlan (mg/ml) U_Z&MflUTI [I!

Li rel

LII .2

[I [Si

LI! 591

b0 1 2 3 4 5 6 Time (hrs)

Time SO4 Added No SO4 ± SO4 Ohrs SO4 3hrs O.D. No SO4 ± O.D. Ohrs O.D. 3hrs

Overnight cultures (high nitrogen MSM) were washed in sulphate-free MSM and resuspended in sulphate-free medium with 1% (w/v) glucose. Na 2SO4 (11.5mM) was added back to the media at the times indicated. Figure 6.6 The Effect of Altering Medium Composition on Curdlan Production by Agrobacterium sp. ATCC 31749

The Effect of Varying Phosphate The Effect of Varying Sulphate Concentration on Curdlan Production Concentration on Curdlan Production

Cell/Curdlan Mass (mg/nil) Phosphate Concentration (mM) Cell/Curdlan Mass (mg/nil) Sulphate Concentration mM I 2. 25 'I

1. 1.

10 1 10

Eu

8.7 28.7 I.48.7 68.7 88.7 108.7 128.7... 148.7... Th27. % Phosphate in Standard Medium % Sulphate in Standard Medium

Biomass ± Initial [PO4] Curdlan Final [PO4] Biomass + Initial [804] * Curdlan Final [804]

Triplicate flasks containing different initial concentrations of phosphate or sulphate (expressed as % in standard medium) were inoculated and incubated at 30 degrees centigrade for 7 days. Flasks were harvested and cell mass and curdlan mass were estimated after lyophffisation. Initial nutrient concentrations were known, final nutrient concentrations were estimated in culture supernatants after removal of cells and curdlan by centrifugation (pp. 80-81). Biomass=final cell mass. Curdlan=fmnal curdlan mass. Initial [PO4/S041=initial nutrient concentration. Final [PO4/8041=final nutrient concentration. Table 6.1 The Efect of Nutrient Limitation on Curdlan Production by ATCC 31749

Nutrient [Initial] [Final] n(Nutrient) DCW Curdlan Final pH mlvi mM niMples mg/ml mg/nil

P042- 15.6 5.92+1-0.15 9.68 2.40+1-0.13 0.02+1-0.01 5.89+1-0.03

10.92 4.33+1-0.68 6.59 2.24+/-0.06 0 6.01+/-0.04

7.8. 3.62+/-0.07 4.18 2.14+/-0.07 0 5.96+/-0.07

3.12 1.47+1-0.36 1.65 2.00+1-0.03 0 5.91+1-0.04

1.56 0.21+1-0.03 1.35 1.99+1-0.05 0 5.95+/-0.03

SO4 2- 20 13.83+1-0.03 6.17 2.85+/-0.08 0.06+1-0.05 5.91+1-0.04 10 5.41+1-0.03 4.59 2.74+1-0.02 0.01+1-0.01 5.94+/-0.02

5 1.84+1-0.06 3.16 2.53+/-0.04 0 5.92+1-0.04

3 1.05+1-0.00 1.95 2.41+1-0.13 0.02+1-0.02 6.06+1-0.06

1 0.74+1-0.07 0.26 2.37+1-0.21 0 6.10+1-0.10 Nutrient [Initial] [Final] n(Nutrient) DCW Curdlan Final pH

mM mm mMoles mg/ml mg/ml

NH4 100 ND ND 2.02+1-0.02 0.02+1-0.00 5.26+1-0.11

75 ND ND 2.02+1-0.02 0.03+1-0.01 5.18+1-0.03

50 ND ND 1.84+1-0.03 0.02+1-0.01 5.37+1-0.15

30 ND ND 1.86+1-0.09 0.46+1-0.23 5.95+/-0.01

20 ND ND 1.83+1-0.16 13.44+1-0.39 6.54+1-0.01

10 ND ND 1.16+1-0.09 7.36+1-0.08 6.75+1-0.03

NO3 - 100 ND ND 1.19+1-0.05 0.04+1-0.08 7.53+1-0.01 75 ND ND 1.19+1-0.07 0.09+1-0.02 7.49+1-0.02

50 ND ND 1.25+1-0.02 0.05+1-0.02 7.48+1-0.01

30 ND ND 1.37+1-0.11 0.06+1-0.02 7.48+1-0.02

20 ND ND 1.78+1-0.05 4.68+1-1.14 7.68+1-0.01 10 ND ND 1.62+1-0.09 6.74+1-0.26 7.36+1-0.01 []=concentration / n(nutrient)=amount of nutrient absorbed +1- St. Dcv.

DCW=dry cell weight +1- St. Dev. ND=Not determined Figure 6.7. The Influence of Absorbed Nutrient on ATCC 31749 Cell Mass

Dry Cell Weight (mg/ml) wt

21

2.5

2.3

2.1

1.9l 0 1 2 3 4 5 6 7 8 9 10 Amount of Nutrient Absorbed (mMoles)

Nutrient Sulphate ± Phosphate

Data were that obtained from the experiments presented in Figure 6.6 and Table 6.1. The x-axis describes the total amount of nutrient absorbed in each flask and the y-axis describes the final bietnass in the flasks. Observe the relationship between absorbed nutrient and final biomass. 93

straightforward in the case of sulphate; however, linearity was still approached. These data

would suggest that at the lower initial concentrations of nutrient, the cells experienced

nutrient deficiency in either phosphate or sulphate. However, curdlan production was

minimal under all conditions tested and thus bactoprenol availability may therefore not be the controlling factor in curdlan synthesis by this organism.

In contrast, cells grown in medium with low initial concentrations of ammonium produced

large quantities of curdlan (Table 6.1). Attempts to assay the final concentrations of

ammonium in the media by the phenate method were unsuccessful due to the formation of interfering colour. This method was suitable for the assay of ammonium in the medium used

in fermenters and it was therefore likely that MOPS caused the interference. However, a lower dry cell weight was observed in flasks containing low initial ammonium concentrations when compared to the cell mass supported by media with high initial ammonium

concentrations (Table 6.1; Figure 6.8). Although the highest quantities of curdlan were

obtained from flasks with an initial ammonium concentration of 20mM, the ratio of curdlan mass to cell mass was similar in flasks with either lOniM or 20mM as the initial ammonium concentration. A small amount of curdlan was synthesised in flasks with an initial ammonium concentration of 30mlvI and this concentration may therefore represent the transition from nitrogen limited growth to growth limitation by another factor.

Similarly, when nitrate (KNO3) was used as the nitrogen source good curdlan yields were obtained when the initial nitrate concentration was 10mM or 20mM (Table 6.1). However, in this case an initial nitrate concentration of lOmIVI provided the greatest curdlan yield and also the highest ratio of curdlan mass to cell mass. The yield of curdlan was less than when equivalent concentrations of ammonium were used as the nitrogen source. There was no correlation between dry cell weight and initial nitrate concentration. An initial concentration of 20m1v1 nitrate provided the highest cell yield with a decline in cell yield on raising or

Figure 6.8 The Effect of Altering Nitrogen Source on Curdlan Production by Agrobacterium sp. ATCC 31749

The Effect of Varying Ammonium The Effect of Varying Nitrate Concentration on Curdian Production ConcentratiOn on Curdlan Production curaian:rnoma,. 3loman/CurdIafl (mg/mi) Curdiqn:Biomasu Bioman/Curdlan (Mg/ml) 1 4. 14

1: a I: 12 ii 10 /\ 8 -8 8 + 6 6 • N 4 • H' 2

I' 10 20 30 40 60 60 70 80 90 1 lOU 0 LU 20 30 40 60 60 70 80 90 100 0 Initial 1N114C1J (MM) • Initial (1NO3) (mid)

- Biomass -- Curdlan a'-- Curdlan:BIOmUU - Biomass -4--- Curdian - Curdian:BlomaJ$

Triplicate flasks containing various intial nitrogen concentrations (either as NH4CI or KNO3) and 4% (w/v) glucose were Inoculated and incubated at 30 degrees centigrade for one week. Flasks were harvested and final cell mass and curdlan mass was estimated after lyophhlisation The ratio of curdlan to biomass was also calculated- Initall ( )=Initial nutrient concentration

- lowering the initial concentration of nitrate. These results were interpreted to suggest that

high nitrate concentrations were inhibitory to growth of Agrobacterium sp. ATCC 31749. In summary, absence of nitrogen from the medium has an immediate stimulatory effect on

the production of curdlan by Agrobacterium sp. ATCC 31749 (Figure 6.4) which is accompanied by entry into the stationary phase of growth (Figure 6.5a). Similarly, low initial concentrations of ammonium in growth media leads to a relatively low cell mass but

production of large quantities of curdlan (Figure 6.8). Previous work supports this data,

batch fermented Agrobacterium sp. ATCC 31749 produced curdlan after growth had ceased

due to the utilisation of available nitrogen within the medium (Phillips et al., 1983). In contrast, transfer of bacteria to media lacking either phosphate or sulphate did not prevent

growth or stimulate curdlan production over a period of six hours (Figures 6.5b and 6.5c).

However, when Agrobacterium sp. ATCC 31749 was cultured for seven days in media of reduced initial phosphate or sulphate lower final cell masses were observed. In addition, a lower quantity of nutrient was absorbed by the cells (Figure 6.6). These data suggest that growth was limited by the respective nutrient. Curdlan was not produced in significant quantities in media of reduced phosphate or sulphate concentration. It therefore seems likely that curdlan production is specifically stimulated by the absence of nitrogen. The selective advantage and potential biochemical control of this specificity is discussed in Chapter 9.

6.3 The Effect of Carbon Substrate on Curdlan Production by Agrobacterium sp. ATCC 31749.

Table 6.2 provides results on the effect of carbon substrate on curdlan production by

Agrobacterium sp. ATCC 31749.. The organism was able to grow on a number of monosaccharides, disaccharides, carboxylic acids and glycerol. The use of glucose as the Table 6.2

The Effect of Carbon Substrate on Curdlan Production by Agrobacterium sp.ATCC 31749

Substrate Final pH DCW (mgnml) Curdlan (mgmL') Ratio

Sucrose 5.72+/-0.19 2.01+1-0.20 15.45+1-1.02 7.69

Lactose 5.92+1-0.01 2.11+1-0.22 4.80+1-0.63 2.27

Glucose 5.91+/-0.02 2.63+1-0.28 16.94+/-0.98 6.44

Galactose 5.61+/-0.18 2.02+1-0.07 1.46+1-0.14 0.72

Maltose 5.81+/-0.02 2.14+1-0.22 10.84+/-0.46 5.07

Mannose 5.98+1-0.04 2.01+/-0.26 3.12+1-0.04 1.55

Glycerol 4.58+1-0.24 1.01+/-0.05 0.00+/-0.00 0

Succjnate 9.09+1-0.03 2.06+1-0.04 0.24+1-0.03 0.11

Citrate 7.62+/-0.11 1.21+1-0.14 0.27+1-0.01 0.22

Pyruvate 9.27+/-0.11 1.76+1-0.23 0.00+1-0.00 0

Triplicate flasks (250m1) containing low nitrogen MSM with 4% (w/v) carbon substrate

(lOOnil) were inoculated from overnight cultures (high nitrogen MSM). Flasks were incubated at 300C with reciprocal shaking (200 r.p.m.) for seven days. pH=Final culture pH +1- St. Dev. D.C.W.=Dry cell weight +1- St.Dev.

Curdlan=Final curdlan concentration +1- St. Dev. Ratio=Ratio of curdlan mass to cell mass 95

carbon substrate provided the highest cell yield and good cell yields were obtained when

maltose, lactose, succinate, galactose, mannose or sucrose were the carbon substrates. Pyruvate and glycerol provided slightly lower cell yields whilst the lowest cell yield was

obtained from citrate. Similarly, good cell yields were reported for strain 100 from glucose,

galactose, mannose, maltose and sucrose. However, this strain grew poorly on lactose producing only 0.60mgmf' dry cell weight (Harada et al., 1965). One interesting feature was

the variation in final pH of cultures grown on different carbon sources. Non-carbohydrate

carbon sources were neutralised prior to autoclaving. The final culture pH in most cases was in the range 5.5-6.0; however, when salts of carboxylic acids were provided as carbon substrate the final culture pH tended towards alkalinity (Table 6.2). The final pH of cultures

supplied with glycerol also differed considerably from that of cultures and the low final pH may have inhibited growth (Figure 6.1).

Curdlan yield was greatest when Agrobacteriuni sp. ATCC 31749 cultures were supplied

with glucose, although sucrose grown cultures provided a yield similar to that obtained from glucose fed cultures and the ratio of curdlan to cells was higher from sucrose. Maltose also provided a curdlan yield considerably greater than that when cultures were supplied with the remaining substrates. Harada a aL (1966) reported similar results for curdlan production by a mutant of strain 10C3; however, greater curdlan production was observed from mannose than maltose. The provision of lactose or mannose as the carbon substrate resulted in reasonable curdlan yields from Agrobacterium sp. ATCC 31749 while that from galactose was poor in agreement with the results of Harada and colleagues (1966) for a curdlan producing mutant of strain 10C3. Very little curdlan was obtained from succinate or citrate and no curdlan was detected when bacteria were grown on pyruvate or glycerol. Two differences in exopolysaccharide biosynthesis between Agrobacterium sp. strain ATCC 31749 and strain IQC3 were that the former produced little exopolysaccharide from galactose in Eel

contrast to strain 10C3 and also that while strain lOC3 produced little polymer from lactose strain ATCC 31749 was able to produce high yields of curdlan from this substrate.

The above results suggest a degree of versatility in the ability of Agrobacterium sp. ATCC 31749 to utilise carbon substrates. The capacity of the organism to interconvert various

carbon substrates to glucose- i-phosphate is also implied. Kai et al. (1993) demonstrated at least five mechanisms by which carbon fragments could be converted to glucose- i-phosphate

by Agrobacterium sp. ATCC 31749 prior to polymerisation into curdlan.

6.4 The Effect of Incubation Temperature on Growth and Curdlan Production by Agrobacterium sp. ATCC 31749

A number of investigators have reported increased exopolysaccharide production by bacteria when they were grown at sub-optimal temperatures (e.g. Duguid and Wilkinson, 1953). In

addition, it was observed that certain bacteria produce greater amounts of exopolysaccharide when grown at low dilution (and hence specific growth) rates in continuous culture e.g.

Pseudomonas sp. NCIB 11264 (Williams and Wimpenny, 1978). Experiments were therefore designed to examine the effect of incubation temperature on doubling times and curdlan production by Agrobacterium sp. ATCC 31749. As earlier work in cabinet incubators revealed a degree of temperature fluctuation, the bacteria were incubated in shaking water baths.

The optimal growth temperature was 30 0C (Table 6.3), the bacteria having a doubling time of 3.57 hours. This temperature was quoted as being optimum for growth by Phillips et al.

(1983); however, no data were presented. Growth at 5 0C higher or lower than the optimum temperature had little effect on doubling times with only a marginal increase observed.

However, when the organism was incubated at 200C or 400C, growth was severely inhibited, Table 6.3

The Effect of Incubation Temperature on Curdlan Production by Agrobacteriuin sp.

ATCC 31749

Temperature (°C) Td (hrs) D.C.W. (mgmll) Curdlan (mgmP1)

20 8.99+1-0.66 1.97+/-0.02 4.40+1-0.26

25 3.85+/-0.14 1.68+/-0.01 6.55+/-0.49

30 3.57+1-0.18 2.05+1-0.05 21.92+1-2.51

35 3.92+/-0.12 2.14+/-0.04 17.16+/-1.04

40 9.67+1-0.54 1.17+1-0.15 0.80+1-0.45

Triplicate flasks containing low nitrogen MSM with 4% (w/v) glucose were inoculated from overnight cultures (high nitrogen MSM). Flasks were incubated at the appropriate temperature in linear shaking water baths at 108 r.p.m. (216 strokes per minute) for seven days.

Td=Doubling time +1- St. Dev.

D.C.W.=Final dry cell weight +1- St. Dev.

Curdlan=Final curdlan concentration -i-I- St. Dev. 97

with doubling times in excess of nine hours. As can be observed (Table 6.3) the organism

appears to be less well adapted to growth at temperatures in excess of the optimum, with

doubling times when incubated at 35 0C or 400C being greater than when the organism was incubated at 25 0C or 200C respectively. However, the variation was minimal and could be

accounted for statiscally. Harada et al. (1965) suggested that the optimal temperature for

growth of strain 100 was 28 0C. Similarly, the results presented in Table 6.3 may suggest

an optimal growth temperature below 30 0C for Agrobacterium sp. ATCC 31749. The optimal growth temperature of strain 10C3 was defined as the temperature which resulted in

the highest cell mass after either 3 or 5 days incubation (Harada et al., 1965). This method may have been misleading as little correlation was observed between final dry cell weight and

doubling time for ATCC 31749 (Table 6.3). In addition, curdlan production was observed

at all temperatures. As shown in Chapter 6.2, curdlan production occurs as a result of nitrogen limitation, suggesting that cells were growth-limited by nitrogen at all temperatures. The differences in final dry cell weight may therefore represent differing nitrogen requirements

at different temperatures or alternatively, cell death and lysis at the extremes of temperature. Curdlan production was maximal at the optimum growth temperature (Table 6.3).

Incubation at 350C also resulted in good curdlan production. Curdlan production was low at 25 0C and 200C whilst very little was produced when bacteria were incubated at 40 0C. These results may reflect the efficiency of enzymes involved in curdlan biosynthesis at the different temperatures. As demonstrated (Chapter 6.2) curdlan production does not commence until growth has ceased and therefore the presumed increased availability of isoprenoid lipids when the doubling time of the organism was reduced was unlikely to play an important role. In contrast, succinoglycan production by Alcaligenesfaecalis strain 10C3 was observed to increase at sub-optimal incubation temperatures (Harada et al., 1965). 6.5 The Effect of Changes in Osmotic Pressure of the Medium on Curdlan Production

by Agrobacterium sp. ATCC 31749

Agrobacterium sp. ATCC 31749 was derived from a succinoglycan-producing strain (Table 1.2). hi addition to curdlan and succinoglycan biosynthesis, related strains produce 13-(1,2)-

glucans under appropriate conditions (Harada and Harada, In press). Harada et al. (1968) defined four groups of Alcaligenes faecalis (now Agrobacterium sp.) dependent on their

ability to synthesise exopolysaccharides. The first group, which included strain 10C3, produced much succinoglycan and a little curdlan. Group 2 strains produced similar

proportions of the two polysaccharides whilst group 3 produced curdlan only and group 4 succinoglycan only. Spontaneous mutation occurred from group 1 to group 2 strains

(Amemura et al., 1977). Although Agrobacterium sp ATCC 31749 belongs to group 3 the

phenotypic similarity of the four groups raises the possibility that Agrobacterium sp. ATCC 31749 has cryptic genes for succinoglycan biosynthesis.

Species of Rhizobium produce a similar spectrum of polysaccharides to Agrobacterium

(Zevenhuizen, 1987). Changes in the osmotic pressure of the culture medium influenced the relative amounts of various polysaccharides synthesised by R. meliloti (Breedveld et al., 1990a; Zevenhuizen and Faleschini, 1991). To study the potential effects of osmotic pressure on Agrobacteriwn sp. ATCC 31749 cultures were incubated in fermenter vessels in either low nitrogen MSM or low nitrogen MSM supplemented with increasing quantities of sodium chloride. The incubation was undertaken as described in Chapter 5.1.3. all

Glucose consumption and curdlan production were inhibited by the addition of NaCl to the medium (Table 6.4). In addition doubling time was increased, suggesting that the organism

was metabolically stressed by the addition of NaCl.

Table 6.4

The Effect of Addition of NaCl to the Medium on Curdlan Production by Agrobacteriutn

sp. ATCC 31749

[NaC1] Td Glucose Curdlan

M hrs mg mV hr-' mg; mt'hf'

O 3.67 0.148 0.111

0.3 12.08 0.039 0.016

0.6 21.16 0.033 0.001

[NaCl]=Sodium chloride concentration Td=Doubling time

Glucose=Average glucose consumption rate

Curdlan=Average curdlan production rate

Bacteria were cultured and analyses undertaken as described in Figure 6.9. Doubling time, glucose consumption and curdlan production were estimated as described (p. 74). 100

As can be observed in Figure 6.9, curdlan production did not commence until the nitrogen in the medium had been absorbed. This result supports the results presented in Chapter 6.2

and is in agreement with the results of Phillips et al. (1983). In low nitrogen MSM curdlan

production continued at a constant rate for approximately 180 hours and then decreased to a slightly lower rate for the remainder of the incubation (Figure 6.10). A similar phenomenon

was observed by other investigators (Phillips et al., 1983; Orts et al., 1987) and it was suggested that the lower rate of production was caused by cell death and lysis with the possible release of B-( 1-. 3)-glucanases (see also Chapter 8.1). However, glucose was

consumed at a constant rate (Figure 6.10) suggesting the presence of a steady cell population.

The presence of water soluble carbohydrate was determined by subtraction of the amount of glucose present in the medium (Chapter 5.2.3) from the amount of total water soluble carbohydrate (Chapter 5.2.1). As can be observed (Figure 6.10), curdlan was the only

extracellular carbohydrate produced by the organism when grown in low nitrogen MSM.

Similar incubation kinetics were observed when cells were grown in medium supplemented

with 0.3M NaCl (Figure 6.10); however, the metabolism of the bacteria was slowed as evidenced by doubling time, glucose consumption and curdlan production rates (Table 6.4).

The only extracellular carbohydrate detected was curdlan. The slower, second rate of curdlan production began after approximately 100 hours accompanied by a decrease in the rate of glucose consumption (Figure 6.10) suggesting cell lysis might be the causal mechanism.

In contrast, cells grown in medium supplemented with 0.6M NaCl produced very little curdlan although high quantities of water-soluble carbohydrate were detected (Figure 6.10).

Visual inspection of the flow characteristics of the culture did not indicate increased viscosity.

This material was not examined further and may represent the contents of lysed cells. Figure 6.9

The Dependence on Nitrogen Depletion for Curdlan Production by ..4grobacterium sp. A'IEC 31749

OM NaCl 0.3M NaC1

0.0. 660 m mg(SubstratefFroduct)/mJ O.D. 660nm mg (Substrate/ProducO/mi .50 :1 .0 50

+

4- 40 40 + -+

30 -30 17 i/1. H -20

10 -10

_ t x I I I I I I 1yIMnI 0. 1 1x IflI —I__i-- t-X I I 1)- 0 5 10 15 20 25 30 35 40 45 50 55 60 0 5 10 15 20 25 30 35 40 45 50 55 60 1' Time (Firs) Time (Firs)

0.D.660 +Glucose .1 NI13*100 Curdlan O.D. 660 + Glucose NH3100 '' Curdlan

1.8 I Bioengineering fermenter vessels containing low nitrogen MSM with or without added NaCl were inoculated from overnight cultures and cultures were incubated as described (p. 73). Samples were taken at the times indicated and the absorbance at 660nm (0.D. 660nm), glucose concentration (p. 78), curdlan concentration (p. 75) and ammonium concentration (p. 81) in the medium were estimated. Ammonium is shown at lOOx the calculated concentration. X-axis arrows indicate times of addition of further glucose. U.3M NaCl Figure 6.10

O.D. 660mn mg (Substrate/ProducQ/inl The Effect of NaCI on Curdlan Production by Agrobacterium sp. ATCC 31759 10 50

OM NaC1 .4 •40 + .4. r -30 O.D. 660 rim mg(Substrate/Product)/ml

in 20

4

- 10

- x_ -94-*- - xx 1.1 0 0 100 200 300 400 Time (hrs)

O . D. 660 ± Glucose -°- ci-io aq -'4- Curdlan

06M NaC1

O.a660om mg(Substrate/Product)/,nl 10 50

N- -I.

I.. / ¼ - +- 1- I 0.1 0 / I 0 100 200 300 400 - Time (hrs)

O.D.660 Glucose ' - CHO aq Curdlan / a °sJ

- .- -- Culture and analyses were as described (see Figure 6.9). Aqueous carbohydrate in the medium was estimated by subtraction A -,,.lAn 70n - nn of glucose (p.78) from total aqueous carbohydrate (pp. 76-77) after removal of cells and curdlan by centrifugation. X-axis Time (hrs) arrows represent times of addition of further glucose. '4- O. D. ~ Glucose CFlOaq curdlan 101

6.6 Discussion

Initial investigations of curdlan production by Agrobacterium sp. ATCC 31749 were disapointing with little, if any, polysaccharide synthesis. The aim of much of the work

presented in this Chapter was therefore to optimise conditions for curdlan production.

Optimum curdlan production was obtained in media with a low ammonium content, buffered to maintain a pH close to neutrality and with excess glucose as the carbon source.

Curdlan biosynthesis has been described as secondary metabolism because it does not

commence until growth has ceased (Phillips a al., 1983). The results presented in Chapter 6.2 are in agreement with this result; however, it was also apparent that curdlan production

was tightly and specifically regulated as the organism only produced curdlan in nitrogen depleted medium. In particular, it is consistent with the results to suggest that curdlan

biosynthesis by Agrobacterium sp. ATCC 31749 is not limited by the availability of isoprenoid lipids. Growth at sub-optimal temperatures increased the doubling time of the organism but failed to enhance curdlan production (Table 6.3). Similarly, increasing the

osmotic pressure of the medium increased doubling times but failed to enhance curdlan production. These culture conditions may have a detrimental effect on the curdlan biosynthetic pathway. However, curdlan production was possible in medium containing 0.3M

NaC1 but did not commence until all the nitrogen had been absorbed (Figure 6.9) despite the fact that the doubling time of cells was approximately four times that of cells grown in medium without NaCl (Table 6.4). It would therefore appear that the cells sense the absence of nitrogen and respond by commencing curdlan production. A potential biochemical mechanism for this phenomenon is described in Chapter 9. 102

Chapter 7 The Effect of Metabolic Inhibitors on Curdlan Production by Agrobacterium sp. ATCC 31749

7.1 Experimental Design

Chapter 6.2 describes the necessity for the absence of nitrogen for the synthesis of curdlan

byAgrobacterium sp. ATCC 31749. The experiments presented in this Chapter utilise this

observation in order to allow examination of the effect of metabolic inhibitors on both the

initiation of curdlan biosynthesis and on the continued production of the polymer. In addition,

the absence of nitrogen leads to a cessation of bacterial growth (Figure 6.5a) thus allowing

a direct comparison of curdlan biosynthesis and removing the potential for experimental error

due to an increase in biomass of control cells.

Overnight cultures were grown in high nitrogen MSM to mid-log phase. They were divided

into three, aseptically centrifuged and washed in nitrogen-free MSM in the absence of carbon

substrate. In the portion to be used to investigate the effect of inhibitors on curdlan synthesis

initiation, the wash contained added inhibitor. Finally the cells were resuspended in three

separate flasks containing nitrogen-free MSM with 1% (w/v) glucose and inhibitors were

added at the times indicated.

Samples were taken at hourly intervals, microfractionated as described in Chapter 5.1.5 and analysed as described. The experiments were repeated at least three times to ensure that the apparent effects of inhibitors were not caused by experimental error. 103

7.2 The Effect of Cation Chelators

The effects of 100mM EDTA or EGTA on curdlan biosynthesis by Agrobacteriuni sp.

ATCC 31749 were examined. EDTA acts as a general chelator of a number of metal ions whilst EGTA specifically binds calcium. The chelators have two physiological effects; firstly, they cause a reduction in the availability of certain ions by sequestering them from the

medium. Secondly, due to their ability to bind cations in the outer membrane of the cell they cause a destabilisation of the membrane.

EGTA had no effect on curdlan production rates or glucose uptake (Figure 7.1). Other

investigators have suggested a role for Ca2+ in the export of bacterial polysaccharides (e.g. K. Jann, Unpublished observations). In contrast, EDTA caused immediate and complete

inhibition of curdlan expression by Agrobacteriuin sp. ATCC 31749. In addition, glucose uptake was 50% inhibited (Table 7.1).

Table 7.1 The Effect of Cation Chelators on Curdlan Production and Glucose Consumption by

Agrnbacrerium sp. ATCC 31749

Treatment Time Added Glucose Curdlan

EGTA 100mM Not Added 0.179 0.041

0 his 0.186 0.052

3 his 0.034

EDTA 100mM Not Added 0.183 0.036

Ohrs 0.089 NS

Time added=time inhibitor added to flask. Curdlan=curdlan production rate (mg ml -'hr-') Glucose=Glucose consumption rate (mg ml-'hr-t). NS=no significant correlation. Figure 7.1 The Effect of Cation Chelators on Curdlan Biosynthesis by Agrobacterium sp. ATCC 31749

The Effect of 100mM EDTA on Curdlan The Effect of 100mM EGTA on Curdlan Expression by ATCC 31749 Production by ATCC 31749

Curdlan (mg/mi) Glucose (mg/ni Curdlan(mg/ml) Glucose (mg/ml) 0.4

X...

0.2

0.1 4

3 U 1 2 3 4 5 6 0 1 2 3 4 5 6 Time (hrs) Time (hrs) Time EDTA Added Time EGTA Added Curdlan Control ± Curdlan Ohrs )If Curdlan 3hrs Curdlan Control ± Curdlan Ohrs * Curdlan 3hrs X Glucose Control Glucose Ohrs ° Glucose Control X Glucose Ohrs

Experiments were undertaken as described (p. 102). Control=no chelator added. Ohrs=chelator added at t=0. 3hrs=chelator added at t=3. Curdlan=curdlan concentration. Glucose=glucose concentration. 104

Inhibition of glucose uptake may have occurred via two mechanisms. Sequesterisation of

ions in the culture medium may have resulted in a deficiency of enzyme co-factors in the cells. However the cells were not growing and internal supplies were therefore likely to be sufficient. The second potential mechanism was that of outer membrane damage. One

possible effect of this could be the loss of a periplasmic glucose binding protein, as glucose

uptake by Agrobacterium radiobacter was shown to be via periplasmic binding proteins

(Cornish et al., 1988a; 1988b).

Whatever the mechanism causing reduced glucose uptake it was possibly not the sole mechanism for the inhibition of curdlan biosynthesis. As glucose uptake continued in EDTA treated cells it was possible that enough glucose was absorbed to allow for metabolic turnover

with excess remaining to be channelled into curdlan biosynthesis. Curdlan biosynthesis was

unlikely to be affected by cation chelators as the presumed site of synthesis is at the inner

membrane, however, essential divalent cations could still be abstracted. The ability of cells to continue to absorb glucose suggested that cellular processes were not curtailed. A second

potential mechanism preventing the expression of curdlan would be an inability to export the polysaccharide. A number of studies have suggested that bacterial polysaccharides expressed

on the cell surface are exported via zones of adhesion between the inner and outer membrane

(Muhlradt et at, 1973; Bayer and Thurow, 1977; Kroncke et at, 1990b). Similarly, electron micrograph evidence suggested a close association between inner and outer membrane at sites of cellulose synthesis in Acetobacter xylinu,n and these site of synthesis were easily damaged (Brown a al., 1976). Furthermore, genetic evidence demonstrated that one of the genes in the cellulose synthase operon (acsC) had homology to genes encoding proteins that are components of membrane channels (Saxena et al., 1994). It was therefore possible that the ability of EDTA to chelate ions in the outer membrane may have led to the incorrect 105

functioning of an outer membrane polysaccharide transporter. If curdlan was transported

through zones of adhesion, leakage from the periplasmic space across the outer membrane

would not be expected to occur and hence curdlan would not be detected in the medium.

Recent studies have revealed the importance of the relative positions of the proposed sites of

cellulose synthesis for production of the polymer by Acetobacter xylinutn (Saxena et al.,

1994). If an analogous mode of curdlan biosynthesis is considered for Agrobacterium sp. ATCC 31749 the membrane damaging effect of EDTA may alter the positions of synthetic

sites and abolish synthesis. An alternative mechanism for curdlan transport could be via

periplasniic binding proteins similar to KpsD of Escherichia coli (Wunder et al., 1994). The likely membrane leakiness, as suggested by the glucose uptake data, could cause a significant proportion of the proposed periplasmic binding protein to be lost. A mechanism of transport

similar to that involved in maltose uptake by E. coli could be envisaged. A charged cytoplasmic membrane component (i.e. with substrate bound) could release its substrate only

upon interaction with the periplasmic binding protein, leading to transfer from the cytoplasmic

membrane protein. Hence, in the absence of the binding protein, curdian would not be

released free into the periplasm and would thus not be detected in the culture medium due to periplasmic leakage.

7.3 The Effect of Tetracaine

Tetracaine acts as an ionophore (Dame and Shapiro, 1976) resulting in the loss of ion gradients across the cell membrane. Addition of this compound to the medium caused an immediate cessation of curdlan production with a reduction in the rate of synthesis from 0.038mg mi'hr-i to 0.007mg ml-ihr-,. However, it had very little effect on glucose uptake, the rates of uptake being roughly equal with control cells absorbing 0.318mg ml-' and tetracaine treated cells absorbing 0.325 mg m1111r-1 (Figure 7.2). As tetracaine would be Figure 7.2. The Effect of 1mg/mi Tetracaine on Curdian Production by ATCC 31749

Curdian (mg/ml) Glucose (mg/ml) no

31

31

0 1 2 3 4 5 6 Time (hrs)

Time Added Curdian Control ± Curdlan Ohrs Curdian 3hrs X Glucose Control • Glucose Ohrs

Experiments were undertaken as describrd (p. 102). Curdian=curdlan concentration. Glucose=giucose concentration. Control=no tetracaine added. Ohrs=tetracaine added t--O. 3hrs=tetracaine added t=3. expected to destroy the electrochemical gradient across the cell membrane, symport or antiport mechanisms of glucose uptake would be expected to be inhibited. The data therefore

may suggest that these potential mechanisms of glucose uptake by Agrobacterium sp. ATCC 31749 are of little, if any importance. The ability of tetracaine-treated to cells to continue to absorb glucose also suggested that membrane damage was not so extensive as to result in cell death.

Inhibition of curdlan expression could have occurred at a number of points in the

biochemical pathway. For the majority of bacterial extracellular polysaccharides investigated,

biosynthesis has been localised to the cytoplasmic face of the inner membrane e.g.

Escherichia coli group II capsular polysaccharides (Kroncke et al., 1990b). Genetic studies have revealed that many of the enzymes involved in exopolysaccharide biosynthesis are

localised to the cytoplasmic membrane e.g. ExoP and ExoQ of Rhizobium meliloti (Glucksmann et al., 1993b; Muller et al., 1993) and cellulose synthase of Acetobacter

xylinum (Bureau and Brown, 1987). Thus depolarisation of the cell membrane of Agrobacterium sp. ATCC 31749 may lead to conformational changes in certain of the biosynthetic enzymes requited for the production of curdlan. Similarly, membrane depolarisation may result in an inability of enzymes to interact with their substrates or co-

factors. Changes in the electrochemical gradient across bacterial cell membranes have been shown to influence membrane fluidity (Lelkes, 1979) leading to changes in the activity of membrane associated enzymes. Marino et al. (199 1) presented evidence to suggest that the initiation reaction of 0 antigen synthesis by Salmonella typhimurium was dependent on maintainence of proton motive force across the cytoplasmic membrane. Similarly, the addition of tetracaine to non-growing cells of a Klebsiella aerogenes strain resulted in the synthesis of exopolysaccharide with a reduced viscosity (Russell, 1993) indicative of lower 107

molecular weight. Thus in K aero genes membrane depolarisation may result in the incorrect function of the exopolysaccharide repeat unit polymerase.

In Agrobacterium sp. ATCC 31749 glucose uptake was unaffected by the addition of tetracaine. Glucose uptake enzymes are located in the cell membrane and therefore the

observed inhibition of curdlan production may have been caused by mechanisms more specific

than general effects on membrane fluidity. One possibility is an effect on enzymes involved in the transport of curdlan. Proton motive force was demonstrated to be a requirement for

transport of Escherichia coli group II capsular polysaccharide across the cytoplasmic membrane (Kroncke a al., 1990b) and also for the transport of core lipopolysaccharide by Salmonella typhimurium (Marino a al., 1985; McGrath and Osborn, 1991).

7.4 The Effect of Sodium Azide Azide (N3-) blocks the electron transfer chain by binding to the haem group of cytochrome

a3 (Stryer, 1988b). This would be expected to result in a dissipation of proton motive force within an organism exposed to azide. Figure 7.3 demonstrates the effect of the addition of

azide to non-growing, nitrogen limited cultures of Agrobacterium sp. ATCC 31749. As can be observed, azide had a strong inhibitory effect on both the initiation and the continuation

of curdlan synthesis. In those cells exposed to SmrvI azide at time zero the curdlan production rate was reduced from 0.029 mg ml-' h' to 0.006mg ml'hl This effect was not due to a general metabolic poisoning as separate experiments revealed that azide-treated cells absorbed glucose at a similar rate to control cells (control cells 0.198mg ml 1h1, azide treated cells

0.256mg mlh-').

These results suggest a requirement for proton motive force in curdlan biosynthesis or export and support the observations of the effect of tetracaine. Azide may be expected to Figure 7.3 The Effect of 5mM Azide on Curdlan Production by ATCC 31749

Curdian (mg/ml) [IL

0.25 S

0.2

0.15

It

0.05

1 2 3 4 Time (hrs) Experiments were undertaken as described (p.102). None=no azide added. Ohrs=azide added Time Azide Added at t=O. 3hrs=azide added at t=3. • None (control) ± 0 hrs * 3 hrs have a less damaging effect on the cell membrane than tetacaine as its mode of action is a chemical rather than physical dissipation of proton motive force.

7.5 The Effect of Arsenate

Arsenate acts as an analogue of phosphate and is able to replace the high energy phosphate group in certain acyl phosphate derivatives: As a result of this property, the addition of

arsenate results in the uncoupling of oxidation from phosphorylation (Stryer, 1988a). In

Salmonella lyphimurium, lOmJvI arsenate reduced the intracellular ATP pool but had little effect on proton motive force across the cell membrane (Marino et al., 1985).

As can be observed in Figure 7.4, 10mM arsenate had no effect on either the initiation or

continuation of curdlan synthesis by Agrobacterium sp. ATCC 31749. In contrast, bacteria inoculated onto MSM agar plates containing 10mM arsenate exhibited no growth after 48

hours at 300C (plate 7.1) suggesting that bacteria were not resistant to arsenate. The substrate conversion efficiencies (mg curdlan produced/mg glucose absorbed) for all three

treatments were c. 20% (data not shown), significantly lower than the 50% conversion

efficiency observed by Phillips et al. (1983) in batch fermenters, or the theoretical maximum yield of 74% (Phillips and Lawford, 1983). The data were therefore interpreted to suggest that over the period of the experiment, sufficient ATP could be generated from proton motive

force to drive the observed levels of curdlan biosynthesis.

7.6 The Effect of 2-Deoxy-D-Glucose

2-Deoxy-D-glucose (2-DO) is an analogue of glucose and in mammalian cells is converted to GDP-2-DG. The latter compound acts as a competitive inhibitor with UDP-glucose in the formation of lipid linked sugars (Hubbard and Ivatt, 1981). As can be observed in Figure 7.5 Figure 7.4. The Effect of 10mM Arsenate on Curdlan Production by ATCC 31749

Curdlan (mg/ml) 0.25

IL

In

0.1

0.05

1 2 3 4 5

Experiments were undertaken as described (p. 102). Time (hrs) Control=no arsenate added. Ohrs=arsenate added at t=o Time Arsenate Added 3hrs=arsenate added at t=3. Control ±Ohrs *3hrs Plate 7.1

Growth of Agrobacterium sp. ATCC 31749 on MSM Agar Supplemeted with 10mM

Arsenate

7.1-a MSM Agar, 1% (w/v) glucose

7.lb MSM Agar, i 1;C" (w/v) glucose, 10mM arsenate

Plates were inoculated from a loop and incubated at 30°C for 48hrs. The Effect of 2-De6xy-D-Glucose on Curdlan Production by ATCC 31749

Curdlan (mg/ml) Ely'

0..

[II

0.

0 1 2 3 4 5 Time (hrs) Experiments were undertaken as described (p. 102). O=no carbon substrate. Glc=glucose. 2-DG=2-deoxy-D-glucose. %=% (w/v). Carbon Substrate 0.1% Glucose ± 0.1% 2-DG *01%Glc + 0.f%2-DG 0.1%G1c+1%2-DG *0 Figure 7.5 109

the provision of 2-DO as sole carbon substrate to Agrobacterium sp. ATCC 31749 resulted in complete inhibition of curdlan biosynthesis. Similarly, the absence of carbon substrate

resulted in a lack of curdlan biosynthesis. This result suggested that in addition to lack. of nitrogen the presence of excess carbon substrate was a requirement for curdlan biosynthesis.

The cells used in this experiment were harvested in the logarithmic phase of growth when they would not be expected to have accumulated large intracellular energy reserves. The rate of

curdilan production was similar when cells were provided with either glucose only (0.067mg mi4hr-t) or equivalent amounts of glucose and 2-DO (0.065mg ml-ihr-i). However when the

cells were piovided with a ten times excess of 2-DO over glucose curdlan biosynthesis was inhibited by approximately 80% reducing the rate of curdlan production to 0.0 12mg m1''

This observation suggested that 2-DO or its metabolic products compete with the natural substrate of one of the enzymes involved in curdlan biosynthesis but was not a suitable

substrate for polymerisation into curdlan.

7.7 The Effect of Aniline Blue Aniline blue is a curdlan binding dye. The formation of colour complexes between aniline

blue and curdlan was stable and was not decolorised by repeated washes in water or methanol

(Nakanishi et al., 1974) suggesting a strong interaction.

Figure 7.6 demonstrates the effect of the addition of 0.005% (w/v) aniline blue to the medium on curdlan biosynthesis by Agrobacteriuni sp. ATCC 31749. As can be observed,

aniline blue had an inhibitory effect on curdlan biosynthesis. In longer term culture this inhibitory effect was not observed (Figure 7.7). Similar experiments were undertaken with

Acetobacterxylinum in which the addition of calcofluor to the medium resulted in an increase in the rate of cellulose synthesis (Benziman et al., 1980). It was suggested that calcofluor

binding prevented crystallisation of cellulose I microfibrils and it was proposed that Figure 76. The Effect of Aniline Blue on Curdlan Production by ATCC 31749

Curdlan (mg/ml) Glucose (mg/ml) 0.1 z

0.1. .5

[In .5

[SI

0.0: .5

0 1 2 3 . . 4 5 .6' Experiments were undertaken as described (p. 102). Time (hrs) Control=no aniline blue added.Ohrs=anilirie blue added at t=O Curdlan=curdl an concentration. Glucose=glucose concentration Time AB Added 3hrs=aniline blue at t=3. Curdlan Control ± Curdlan 0 hrs Curdlan 3 hrs

X Glucose Control • Glucose 0 hrs A- Glucose 3 hrs Figure 7.7. Long Term Culture of ATCC 31749 in the Presence of Aniline Blue

Curdlan (mg/ml)

VA

1.5

ii

0.5

o 44ccC__L±_J_ o io 20 30 40 50 60 70 80 90 100 110 120 Triplicate flasks (lOOm! low nitrogen MSM + 2% w/v glucose in 250m1 flasks) were Time (hrs) inoculated from an overnight culture (high nitrogen MSM). Aniline blue flasks had 0005% (w/v) aniline blue added to the medium. Curdlan concentration was Treatment estimated chemically (p75) Control Aniline Blue 110

crystallisation was the rate limiting step in cellulose synthesis. Results presented in Chapter

8 suggest that native curdlan may also have a certain degree of crystallinity. Through the

mechanism of binding of aniline blue to curdlan, the crystallisation of curdlan molecules may be prevented as they are extruded by the bacterium. The possibility is therefore raised that

the energy of crystallisation in some way contributes to export of the polysaccharide. The lack of observable inhibition in long term culture may be a result of steric inhibition by curdlan

fibrils extending from bacterial cells preventing the high molecular weight dye interacting with curdlan fibrils prior to their crystallisation.

7.8 The Effect of Chioramphenicol

Chloramphenicol inhibits the peptidyltransferase reaction (Gale et al., 1981), its mode of action therefore being to prevent translation of mRNA. Chloramphenicol (lOOpg nt')

inhibited curdlan synthesis when added prior to or after transfer of Agrobacterium sp. ATCC 31749 to nitrogen-free medium (Figure 7.8). Furthermore, the use of the anthrone reagent to assay cellular carbohydrate suggested that levels of intracellular carbohydrate were similar

between control and treated cells at around 0.35mg (mgDCW 1 . This result suggested that addition of chioramphenicol to cultures of Agrobacterium sp. ATCC 31749 did not result in a decrease of the levels of enzymes involved in general carbohydrate metabolism. As

discussed in Chapter 6.2 the initiation of curdlan biosynthesis requires the absence of nitrogen. The observation that the addition of chloramphenicol prevented synthesis of curdlan prior to transfer to permissive medium suggests a requirement for protein synthesis for the initiation of curdlan synthesis. This has implications concerning the control of curdlan biosynthesis, as discussed in Chapter 9. Similarly initiation of biosynthesis of the Escherichia coli Ki, KS or

K12 capsules required protein synthesis (Whitfield et al., 1984; Kroncke et al., 1990b). Figure 7.8.The Effect of Chioramphenicol on Curdlan Production by ATCC 31749

Curdlan (mg/ml) 0.3

0.25

0.2

0.15

0.1

0.05

1 2 3 4 5 Experiments were undertaken as described (p. 102) Control=no chioramphenicol added. Time (hrs) Ohrs=lOOug/ml chioramphenicol added att=O. 3hrs= lOOug/ml chloramphenicol added at t=3 Time Added Control -1- Ohrs )lE3hrs 111

However, if protein synthesis was inhibited after capsule initiation had begun, the capsules

continued to be synthesised. In Agrobacterium sp. ATCC 31749 this was not the case and the addition of chioramphenicol at any time after curdlan synthesis had begun, resulted in

inhibition of polymer synthesis. The latter observation suggested a rapid turnover of certain proteins involved in curdlan biosynthesis. In order to attempt to isolate the polypeptide(s) required for curdlan biosynthesis, overnight

cultures of Agrobacterium sp. ATCC 31749 (high nitrogen MSM) were aseptically

transferred to nitrogen-free medium in either the presence or absence of lOOgg ml ' chloramphenicol. After a second overnight incubation the cells were fractionated and cell protein was concentrated as described in Chapter 5.3. The buffer used for dialysis was 10mM

Tris pH 6.8. Cell fractions were run on various concentrations of polyacrylamide gels, both reducing and non-reducing and no differences in the protein profiles were observed between control cells and those treated with chloramphenicol.

During the course of studies on antibiotic sensitivity, a number of chloramphenicol resistant

mutants arose. MSM agar plates overlaid with antibiotic discs were stored at 4 0C after 48

hours incubation at 300C. Initially, the area around the chloramphenicol impregnated arm of antibiotic discs was free of bacteria. However, after about one month at 4 0C small colonies began to emerge in the zone of clearing. One colony was picked at random and streak plated onto MSM agar containing I0ig ml' chloramphenicol. The bacteria grew well on this

medium and a second colony was picked at random, grown overnight in high nitrogen MSM

and transferred to nitrogen-free MSM to analyse the effect of chioramphenicol on these

mutants. As can be observed in Figure 7.9 chloramphenicol inhibited curdlan biosynthesis by approximately 25% (rate of curdlan production in control flasks 0.043mg ml'l1, in chloramphenicol containing flasks 0.031 1'hr-O. However, when resistant mutants were plated onto low nitrogen MSM with 0.005% (w/v) aniline blue they required a longer period Figure 7.9 The Effect of Chioramphenicol on Curdlan - Production by ATCC 31749 Chi Res Mutants

Curdian mg/ml

(

0.

(

0.

(

0.

0 1 2 3 4 5 6 Time (hrs)

Time Added Control + Ohrs *3 hrs Experiments were undertaken as described (p. 102). Mutants were isolated as described (p. III). Chi res=chloramphenicol resistance. Control=no chioramphenicol added. Ohrs=lOOpg ml-' chioramphenicol added t=0. 3hrs=100j.tg m1' chioramphenicol added t=3. 112

of incubation to absorb the dye to the same extent as wild type cells. This result would suggest that the resistant mutants were slower growing than wild type, therefore requiring more time to absorb the available nitrogen and begin curdlan biosynthesis. Similar results were observed with tetracycline resistant mutants isolated in a similar fashion.

7.9 The Effect of Rifampicin

Like chloramphenicol, rifampicin is an inhibitor of protein synthesis. However, unlike chloramphenicol, which blocks translation, rifampicin is an inhibitor of transcription preventing the synthesis of mRNA via inhibition of bacterial RNA polymerase (Gale et al., 1981).

As can be observed in Figure 7.10 rifampicin has an inhibitory effect on curdlan production by Agrobacterium sp. ATCC 31749. Inhibition of curdlan biosynthesis was not immediate and the rate of curdlan synthesis remained close to that of control cells for up to an hour after addition of the antibiotic. Two possibilities were therefore raised. Firstly, mRNA encoding proteins required for curdlan synthesis may be present prior to the addition of rifampicin.

Upon addition of the antibiotic, preformed mRNA could continue to be translated until degradation of the nucleic acid occurred. The chloramphenicol data suggests that at least one of the proteins required for curdlan biosynthesis is highly unstable and the implication was therefore that the mRNA was stable for approximately one hour. Alternatively, rifampicin may take some time to exert its action. Even when added to the nitrogen-free cell wash, the inhibitory effect of rifampicin was not observed for an hour. Thus the bacteria were able to respond to the absence of nitrogen and transcribe the genes required for curdlan biosynthesis prior to inhibition of transcription by rifampicin. - Figure 7.10. The Effect of Rifampicin (lOOug/ml) on Curdlan Production by ATCC 31749

Curdlan (mg/ml) fl7

0.

0.

0.

0.

0.

0.

0 1 2 3 4 5 •6 Time (hrs)

Time Added Control ±0 hrs 3 hrs

Experiments were undertaken as described (p. 102). Control=no rifampicin added. Ohrs=lOOpg rn! 1 rifampicin added t=O. 3hrs=100pg ml -' rifampicin added t=3. 113

7.10 Discussion

The data presented in this Chapter provide preliminary work concerning the requirements and energetics associated with curdlan biosynthesis by Agrobacteriuni sp. ATCC 31749. Although a number of possibilities arise definitive answers will require further work.

Suggestions concerning potential further work are outlined in Chapter 9.

Despite the inconclusive nature of the work, a number of hypotheses can be presented based on the work undertaken here and by analogy with more detailed studies undertaken on other species. The requirement for membrane integrity and the associated electrochemical gradient is clearly demonstrated by the effects of EDTA, teracaine and azide. Specifically, maintainence of proton motive force is a requirement for curdlan biosynthesis. In other species the proton gradient was shown to be important to create the correct environment for interaction of the various enzymes, substrates and co-factors involved in polysaccharide biosynthesis (Marino et al., 1991) and also for the transport of polysaccharide across the inner membrane (Kroncke et al., 1990b). Arsenate had no effect on curdlan biosynthesis despite the ability of arsenate to inhibit growth of Agrobacterium sp. ATCC 31749. Considering the high ATP requirement for polysaccharide biosynthesis this result was unexpected. A number of proteins involved in bacterial exopolysaccharide translocation share homology with nucleotide binding sites e.g.

Escherichia coli KpsT (Smith et al., 1990) suggesting a requirement for ATP hydrolysis in polysaccharide translocation.

The effect of protein synthesis inhibitors on polysaccharide production was unexpected. The observation that continuous protein synthesis was required for polysaccharide production implies that positive regulation of curdlan synthesis has an important role. Furthermore, the rapid cessation of polysaccharide production in the absence of protein synthesis suggests that the organism is able to respond to environmental changes rapidly and this may have 114

implications concerning the pathogenic lifestyle of Agrobacterium. These implications are discussed further in Chapter 9. 115

Chapter 8 Structure of Curdlan: Enzymic and Acid Hydrolytic Studies

8.1 Attempts to Isolate 8-(1-3)-Glucanase Activity from Agrobacterium sp. ATCC 31749

Few bacteria producing a particular exopolysaccharide also yield enzymes capable of degrading the polymer (Sutherland, 1990). However, a number of exceptions do exist. The alginate synthesising species Azotobacter vinelandii, A. chroococum, Pseudomonas fluorescens and P. putida all have alginate lyase activity (Kennedy et al., 1992; Conti et al.,

1994). Recently, Rhizobiunt meliloti was found to have a gene encoding an enzyme capable of degrading succinoglycan as part of the polymer's biosynthetic operon (Becker et al.,

1993). A recent isolate of Cellulomonasfiavigena synthesises curdlan in the presence of excess carbon substrate; the same bacteria effect its degradation during periods of starvation

(Voepel and Buller, 1990). Orts et al. (1987) provided evidence for weak curdlan degrading activity in the supernatant of Agrobacterium sp. ATCC 31749 cultures and a more recent article has also reported 13-(1-'3)-glucanase activity from curdlan producing species of

Agrobacteriurn (Harada and Harada, In press).

Initially, the method of Orts et al. (1987) was used to test for curdlan degrading activity.

Triplicate flasks containing either high nitrogen MSM and 1% (w/v) glucose (C-limited), low nitrogen MSM and 3% (wlv) glucose (N-limited) or high nitrogen MSM with 0.1% (wlv) glucose and 1% (w/v) curdlan (curdlan as C-source) were inoculated and incubated as described (Chapter 5.1.3) for one week. The cells were then removed by centrifugation and the supernatant placed in fresh flasks. 1% (w/v) curdlan and 0.04% (w/v) sodium azide were added and the flasks were incubated with shaking at 30 0C. Subsamples were taken at the 116

times indicated (Figure 8.1) and sufficient 10M NaOH was added to bring the curdlan- supernatant mixture to 0.5M NaOH. The viscosity of the solutions were then measured as described (p. 82). Curdlan suspended in fresh MSM was used as a control. Figure 8.1 shows that there was no appreciable change in viscosity of the alkaline solutions for up to 100 hours.

In contrast, Orts et al. (1987) reported a 32% decrease in the viscosity of similarly treated curdlan suspensions in C-limited culture supernatants after 70 hours incubation, a 10% decrease in N-limited culture supernatants and a 29% viscosity decrease in culture supernatants where curdlan was the carbon source. The results presented here would suggest that Agrobacterium sp. ATCC 31749 does not release an endo-B-(I - 3)-glucanase into the culture medium, as breaks in the glucan chain would drastically reduce the molecular weight of curdlan and hence viscosity of alkaline solutions. The possibility remains that an exo- glucanase was present.

In order to try to clarify the disparity of results between the work presented here and that of Orts et al. (1987) another strategy was attempted. Again, culture supernatants had curdlan and azide added but the incubation time was increased to 240 hours. After incubation, the contents of the flasks were placed into dialysis sacks and dialysed against three x2 litre changes of distilled water. Both the diffusate and retentate were collected and lyophilised.

The difibsate was resuspended in distilled water and the retentate was resuspended in an equal volume of 0.5M NaOH. The amount of carbohydrate in each fraction was estimated by the phenol-sulphuric acid assay and in addition, the amount of glucose in the retentate was estimated to ensure that the results were not severely affected by the glucose added to the culture medium. Table 8.1 shows that incubation of curdlan in culture supernatant resulted in more dialysable carbohydrate than incubation of curdlan in fresh MSM. Furthermore, the proportion of glucose in the difflisable material was extremely low in all cases suggesting that Degradation of Curdlan by ATCC 31749 Supernatañt Degradation of Curdlan by ATCC 31749 Supernatant Control N-limited Cultures

Intrinsic Viscosity (cps) Intrinsic Viscosity (cps) 3

2.5

2 r 1.

.5 1.5 .

10 0 . 24 53 98.5 24 53 98.5 Time (hrs) Time (hrs)

Degradation of Curdlan by ATCC 31749 Supernatant Degradation of Curdlan by ATCC 31749 Supernatant C-limited cultures Curdlan as C-Source

- Intrinsic Viscosity (cps) Intrinsic Viscosity (cps)

.5

0 24 53 98.5 0 24 53 98.5 Time (hrs) Time (hrs) Figure 8.1 Viscosity of Curdlan After Incubation in ATCC 31749 Culture Supernatant

Curdlan was added to the appropriate culture supernatant (p. 115) in triplicate flasks, 0.04% (w/v) azide was added and the mixtures were homogenised and incubated at 30°C. 2m1 samples were removed at the times indicated and lOOpI IOM NaOH added to dissolve the curdlan. Sample viscosity was measured as described (p. 82). Curdlan incubated in fresh low nitrogen MSM was used as control. Data is presented as the mean of three flasks +/- st. dev. Table 8.1

Examination of Carbohydrate Fractions of Curdlan after Incubation in Agrobacterium sp.

ATCC 31749 Culture Supernatant

Culture Fraction % Total CHO % Glucose in CHOd

Control Diffusate 1.18+/-0.09 1.11+1-0.18

Retentate 98.82

C-limited Diffusate 5.80+1-0.49 0.30+1-0.04

Retentate 94.20

N-limited Diffusate 7.71+/-1.70 0.32+/-0.01

Retentate 92.29

Curdlan Diffusate 3.55+1-0.36 0.49+/-0.02

Retentate 96.45

1% (wlv) curdlan was incubated in culture supernatants of Agrobacterium sp. ATCC 31749 grown in medium that was either C-limited, N-limited or had curdlan supplied as the carbon source for 240 hours at 30°C. Control flasks contained fresh MSM. The culture supernatants and curdlan were then transferred to dialysis sacks and dialysed against three 21 changes of distilled water. Diffusates and retentates were collected and lyophilised prior to analysis. CHO=Carbohydrate

CHOd=Diffusate carbohydrate

Numbers are expressed i-I- st. dev. 117

the observed results were not due to residual glucose supplied as the carbon substrate. These results suggest that Agrobacterium sp. ATCC 31749 may have curdlan-degrading activity; however, another possibility is that the cliffusable carbohydrate may be derived from lysed cells.

In order to observe directly any changes in the molecular weight of insoluble curdlan fractions after incubation in culture supernatant, the curdlan was collected and acetylated as described in Chapter 5.4.2 in order to produce a soluble form of the polymer. The soluble acetyl derivative was then analysed by high performance size exclusion chromatography (p. 82, Figure 8.3b). As can be observed in Figure 8.2, curdlan incubated in the supernatant of nitrogen limited cultures or those supplied with curdlan as the carbon source was of a lower molecular weight than curdlan incubated in the supernatant of carbon limited cultures or fresh

MSM. Similarly, curdlan incubated in the supernatant of nitrogen limited cultures yielded the greatest amount of dialysable carbohydrate. However, these results should be treated with some caution as the amount of soluble carbohydrate retrieved after acetylation was extremely low and the degree of acetylation was estimated to be approximately one acetyl group per five glucose residues. In addition, the calculated molecular weight of curdlan incubated in fresh

MSM or carbon limited culture supernatant was much higher than previously reported values

(e.g. Harada et al., 1993).

To overcome the difficulty of acetylating curdlan, an alternative approach was adopted in which the molecule was rendered soluble by carboxymethylation (p. 86-87). Curdlan was harvested from cultures ofAgrobacterium sp. ATCC 31749 (low nitrogen MSM) at different limes and carboxymethylated in order to determine whether the molecular weight of the polymer decreased as the culture aged. However, as can be seen in Figure 8.3a HPLC traces suggested a high degree of polydispersity within the carboxymethylcurdlan samples, in contrast to acetylcurdlan (Figure 8.3b). Two potential reasons for the apparent polydispersity Figure 8.2. Molecular Weights of Acetylcurd.lan After Incubation in Culture Supernatant

Molecular Weight (kDaltons) % of Total Material 250 —1100

200

150 rsi

100

50 20

0 Control Curdlan C Limited N Limited Culture Conditions

S HMW Product S LMW Product S %HMW E % LMW

1% (w/v) curdlan and 0.04% (w/v) azide were added to culture supernatants and incubated at 30°C for 240 hours. Insoluble curdlan was collected by centrifugation, washed and lyophilised. After lyophilisation the material was acetylated (p. 87) and analysed by HPLC (p. 82). Contfol flasks contained fresh low nitrogen MSM. HMW product=high molecular weight product. LMW prbduct=low molecular product. %HMW/LMW=% of area under curve of FIPLC trace. Figure 8.3 a

HPLC Traces of Carboxymethylcurdlan

L-i ;cii-• 3 - ....3L.j

Both traces are of the same sample. 10mg ml -1 carboxymethylcurdlan obtained from a nitrogen limited culture after 148 hours and modified as described (p. 86, method B). The column used was a G6000PW column with UHQ water as the eluent, flow rate

0.25mlmin 1 . Note the polydispersity; variation of elution time of samples was also a problem. Figure 8.3b

HPLC Traces of Acetylcurdlan

ü@. @0. 00. Ü@.

'Jr

00.00.00.00.

Both traces are of the same sample 10 mg rn! 1 acetylcurdlan modified as described (p. 87) after incubation for 240 hours in nitrogen-limited culture supernatant. The column used was a G6000PW column with water as the eluent, flow rate 0.25 nil min'. Note that only two major peaks were observed. 118

of carboxymethylcurdilan were the high viscosity of even low concentrations of carboxymethylcurdlan, and the possibility of different molecules of curdlan being variably carboxymethylated.

To record curdlan degradative activity more directly, nitrogen limited cells of

Agrobacterium sp. ATCC 31749 were fractionated as described (p. 83) and cytoplasmic, periplasmic and supernatant proteins were concentrated in SOmIvI potassium phosphate buffer pH 7.0. Neutralised curdlan was incubated with the protein fractions (p. 88) and the release of reducing sugar was recorded. As can be observed in Figure 8.4 no reducing sugar was released. For comparison, the release of reducing sugar when curdlan was incubated with Oerskovia xanthinolytica supernatant protein is also presented. The latter organism expresses enzymes capable of hydrolysing B-(1-3)-glucans (Jeffries and Macmillan, 1981). The dialysis experiments and HPLC work presented above suggests that Agrobacterium sp.

ATCC 31749 has weak 8-(l-'3)-glucanase activity; however criticisms can be raised against both experiments. In addition, the lack of any change in viscosity when curdlan was incubated in culture supernatants and the inability to detect any release of reducing sugar when curdlan was incubated with Agrobacterium sp. ATCC 31749 protein concentrates, suggest that the bacterium is unable to degrade its own polysaccharide product. These results are contrary to the report of Orts et al. (1987). Harada and Harada (In press) also reported B-(1-'3)-glucanase activity from curdlan producing species of Agrobacterium; however, no data was presented. The possibility remains that Agrobacterium sp. ATCC 31749 has weak

B-(1-'3)-glucanase activity. If the organism does contain a curdlan degrading enzyme it may be involved in biosynthesis. A biosynthetic role has been suggested for the succinoglycan depolymerising ExoK of Rhizobium meliloti (Glucksmann et al., 1993b). Figure 8.4. Degradation of Low-Set Curdlan by Nitrogen Limited ATCC 31749 Enzyme Preparations

Reducing Sugar (mg/ml) 1 .''

ii Eli

'I 0.

Time (hrs)

Cell Fraction Supernatant ± Cytoplasm * Periplasm -°- Ox Supernatant

2 m nil' neutralised curdlan was incubated with I mg m1' protein at 30°C. Samples were taken at the times indicated and reducing sugar estimated as described (pp. 77-78). Protein was obtained from nitrogen deficient cultures of Agrobacterluin sp. ATCC 31749. Ox supernatant=protein from the supernatant of Oerskovia xant/ünolytica cultures containing -glucan as the carbon source. 119

8.2 Curdlan is a Weak Inducer of Oerskovia xanthinolytica 0-(1-'3)-Glucanases

Oerskovia xanthinolyticcf produces four inducible D-glucanases capable of degrading laminaran. Three of the enzymes are also capable of lysing viable yeast cells and yeast glucan

(Jeffries and Macmillan, 1981). As an initial study of curdlan structure Oerskovia xanthinolytica was grown in three different media in order to examine the ability of curdlan to induce 8-(1-'3)-glucanases in the organism. All media contained the salts mixture and casein hydrolysate described in Chapter 5.1.3. YE medium was additionally supplied with I f' yeast extract and 20g -1 glucose, YC medium contained 0. 133g'l dried baking yeast

(autoclaved) and ig ç 1 glucose and CN medium contained 0.133g 1-i neutralised curdlan and lg f glucose. The cultures were inoculated and incubated for one week, after which supernatant protein was concentrated and used as a crude enzyme preparation to examine activity upon neutralised curdlan preparations.

As can be seen in Figure 8.5, only minimal activity was observed when 0. xanthinolytica was cultured in YE medium and this may represent the constitutive expression of enzymes.

The level of activity was marginally higher when the organism was grown in CN medium but was still much lower than the activity observed when the organism was cultured on YC medium. In addition, transfer of the organism from YE agar to CN agar resulted in the appearance of zones of clearing around colonies in which gelled curdlan was digested resulting in a loss of graininess around colonies (data not shown).

Jeffries and Macmillan (198 1) examined the specificity of the four enzymes induced by yeast cells in 0. xanthinolytica. Enzyme I was an endo-glucanase which preferentially cleaved B-

(1-4)-linkages adjacent to 13-( 1-'3)-linkages and cleaved 8-(1-6)-linkages adjacent to 13-

(1-'3)-linkages. Enzyme II was classified as an endo-B-(1-'3)-glucanase. Enzymes ifia and ilIb were endo-13-(1-'3)-glucanases requiring substrates of•DP>=14. Furthermore, the four enzymes acted synergistically on yeast glucan. The above results may therefore be interpreted Figure 8.5 Release of Reducing Sugar from Curdlan by 0. xanthinolytica Enzyme Preparations

mg reducing sugar released/ml/mg protein 12

0.1

0.1 0..

2mg/mi curdlan was incubated with lmg/ml Time (hrs) supernatant protein at 30 degrees centigrade. Release of reducing sugar was estimated as described (p. 77). Enzyme preparations were Enzyme Preparation obtained from Oerskovia xanthineolytica culture supernatants. YE=yeast extract medium. YC=yeast cell medium. CN=curdlan medium. (p. YE ±y -*CN 118). 120

to suggest that some but not all of the 0. xanthinolytica enzymes were induced by curdlan. Linkages other than B-(1-'3) maybe required for full enzyme induction. Thus the above data supports previous chemical analysis that curdlan contains only B-(l-'3)-linkages (Harada et al., 1966).

8.3 Acid Hydrolysis of Curdlan

Initial attempts to study curdlan structure by hydrolysis were made using H2SO4 as the hydrolytic agent. Samples of neutralised curdlan were subjected to hydrolysis in various acid strengths at 100°C. M can be observed in Figure 8.6, initial rates of hydrolysis were similar, whatever acid concentration was used. However, when 0.05N or 0.5N H2SO4 was used, the rate of hydrolysis began to slow after approximately five hours. In contrast, when IN H2SO4 was used, hydrolysis continued at a steady rate until all the polymer had been rendered soluble. Previous work has demonstrated that neutralised curdlan preparations were of low crystallinity and non-resistant to acid hydrolysis (Kanzawa et al., 1989). The data presented for hydrolysis by IN or 0.5N H2SO4 support the previous conclusions of Kanzawa and colleagues. In addition, the slowing of the rate of hydrolysis of neutralised curdlan after approximatelt 10 hours by 0.5N H2SO4 and the absence of complete hydrolysis by 0.05N

H2SO4 (Figure 8.6) may suggest a degree of molecular heterogeneity in the neutralised preparation. This data may therefore support the model presented by Saito and colleagues

(1977; 1990) in which the low-set curdlan gel was considered to consist of molecules existing in three states (see p. 8). Figure 8.6 0 Curdlan Recovery after Hydrolysis at 100 C

% Remaining H20 Insoluble 10C

on

20

We! 5 10 1520 25 30 35 40 45 50

100mg curdlan and 2.5mI acid were placed in sealed glass ampules and incubated for the Hydrolysis Time (hrs) times Indicated. Soluble and Insoluble carbohydrate was recovered as dcacrlbed (p. 881 and the quantifies of each estimated chemically (p. 76). insoluble carbohydrate Acid Strength exprsaed as % of total. 0.05N H2SO4 ± 0.5N H2SO4 * iN H2SO4 121

8.4 Enzymic Hydrolysis of Curdlan

Table 8.2 shows the specific activity of three of the enzymes used in the investigation of curdlan structure. Cellulase (B-(1-'4; 1-'3)-glucanhydrolase) exhibited the lowest activity

against all curdlan preparations. Lysing enzymes (containing yeast glucanase, protease and

cell lytic activities) showed similar activity against the neutralised and native preparations with slightly higher activity when high-set curdlan was the substrate; the reason for this is unclear.

Laminarinase (B-(1-'3)-glucanase) had the highest activity against all preparations. This data

supports the assertion that curdlan consists primarily of 13-(1--3) linkages (Harada et al., 1966). The specific activity when native and high-set preparations were substrates for

laminarinase was about 50% lower than that when neutralised curdlan was the substrate. High-set curdlan shows a high degree of crystallinity in portions of the molecule (Takahashi

et al., 1986) which are resistant to enzymic hydrolysis and the lower rate of degradation by

laminarinase may be a reflection of this property. The data imply that native curdlan may have resistant portions. The activity of driselase (containing laminarinase, xylanase and cellulase activities) was also examined; however, the action patterns of this enzyme were more

complex, with a rapid initial release of reducing sugars which then slowed (Figure 8.7). Similarly, a rapid initial release of reducing sugar was observed with all enzymes when

carboxymethylcurdlan was the substrate (approximately 0.3mg , r-'mg protein-i) followed by a slower second release (Figure 8.8), with the exception of cellulase for which

carboxymethylcurdlan was a poor substrate. The high initial release may be a result of the solubility of carboxymethylcurdlan allowing easier access by the enzymes. It may be that the

- carboxymethyl substitution interferes with the ability of the enzyme to hydrolyse the 8-(l -3)-

linkages. If there were blocks of undersubstituted residues these may be hydrolysed first.

The second slower rate of hydrolysis may be caused by the remaining fragments having a

greater degree of substitution. Table 8.2

Enzyme Activity on Different Curdlan Preparations

Enzyme Substrate Activity

Lysing Enzymes Native .0.056

Neutralised 0.051

High-set 0.068

Cellulase Native 0.021

Neutralised 0.018

High-set 0.019

Lanulnarinase Native 0.109

Neutralised 0.177

High-set 0.111

Substrates were obtained as described (pp. 75-76). Triplicate tubes containing 2mg ml -' curdlan and 1mg -' protein were incubated as described (p. 88). Samples were taken at half hourly intervals and reducing sugar estimated (pp. 77-78). Corrections were made for the non-specific release of reducing sugar by running enzyme and substrate blanks alongside the assay tubes. Enzyme activities are expressed as mg reducing sugar released h -' (mg protein)*

Lysing enzymes were reported by the manufacturer to contain yeast glucanase, protease and cell lytic activity. Cellulase was described as -(1--'4; 1-3)-glucanhydrolase and lanulnarase was described as 3-(1--'3)-g1ucanase. Figure 8.7. The Activity of Driselase (1mg/mi) Against Curdian Preparations

Reducing Sugar (mg/ml) 0.4

0.3

0.2

0.1

1 2 3 4 5 6 7 8 2mg/mi curdlan was incubated with Time (hrs) 1mg/mi driselase as described (p. 87). Samples were taken at the times indicated and reducing sugar Curdlan Preparation estimated as described (p. 77). a Neutralised ± High-set Figure 8.8.Degradation of Carboxymethyl- curdians by Glucanases (1mg/mi Enzyme)

Reducing Sugar (mg/ml)

I'

0. 6-

0. 0.

'I 11

0 1 2 3 4 5 6 7 8 Time (hrs)

Enzyme, Substrate Lysing Enzyme, CMC A + Lysing Enzyme, CMC B * Laminarinase, CMC A ••• Laminarinase, CMC B

2 m ml' carboxymethylcurdlan was incubated with I mg ml' enzyme as described (p. 88). Samples were taken at the times indicated and reducing sugar estimated as described (pp. 77-78). cc A and CMC B represent carboxymethylcurdlan prepared by method A or B (p. 87) respectively. 122

In a further series of experiments enzymic hydrolysis was allowed to continue to completion, judged by no further release of reducing sugar. Samples were harvested and analysed for proportion of soluble carbohydrate, glucose in soluble carbohydrate and average degree of polymerisation of soluble carbohydrate. Results are presented in Table 8.3. As can be observed the degree of polymerisation of the carboxymethylcurdlans remained higher than that of other substrates supporting the hypothesis that the substitution reduces the ability of the substrate to interact with enzymes. Similar proportions of carbohydrate remained insoluble when either native or high-set curdlan were substrates. Lower amounts of insoluble carbohydrate were recovered when neutralised or 60 0C preparations were substrates with very small proportions of insoluble carbohydrate when lysing enzymes or laminarase were the hydrolytic enzymes. This was in agreement with the results of Takahashi et al. (1986) who found neutralised and 60 0C preparations of curdlan unresistant to zymolase and supports their hypothesis that the molecular organisation of the two preparations was similar. Takahashi et al. (1986) found curdlan preparations heated above 950C to be resistant to zymolase. Similarly, the work presented here shows that curdlan preparations heated to 100 0C for one hour were resistant to all three enzymes tested. Takahashi et al. (1986) interpreted their results to suggest that high-set preparations had a degree of crystallinity and this hypothesis was supported by electron micrographs and also acid hydrolysis (Kanzawa et al., 1.989). The observation that native preparations show a similar degree of resistance to high-set preparations (Table 8.3) supports the hypothesis that native curdlan has a certain degree of crystallinity. In addition this data supports the interpretation of the effect of aniline blue on curdlan biosynthesis byAgrobacterium sp. ATCC 31749 (p. 109-110). Table 8.3 Curdlan Degradation Products after Complete Hydrolysis by Various Enzymes

Enzyme Substrate Insol CHO Glucose DP

Cellulase Native 70.68+1-1.85 0 3.87+1-0.46

Neutral 28.73+1-4.68 79.04+/-1.12 1.11+/-0.03

600C 30.87+/-3.19 99.16+/-0.83 1.16+/-0.23

High-set 50.97+/-4.14 97.48+/-3.56 1.56+1-0.32

CMC A 3.03+1-1.24 7.27+1-0.32 5.76+/-0.09

CMC B 0.87+1-0.60 0.47+1-0.39 12.15+1-1.02

Laminarinase Native 10.40+1-1.32 39.68+1-7.82 1.26+/-0.08

Neutral 0.69+1-0.27 76.40+/-1.15 0.97+/-0.02

600C 0.84+/-0.15 84.8+/-2.14 1.12+/-0.07

High-set 10.84+1-0.95 63.83+1-7.27 1.12+1-0.10

CMC A 0.74+1-0.05 12.04+1-0.89 1.75+1-0.09

CMC B 0.33+1-0.00 7.174-1.51 1.45+1-0.05

Lysing Native 21.51+/-0.36 81.36+/-2.08 1.12+/-0.03 enzymes Neutral 1.59+1-0.31 89.28+1-2.51 1.11+1-0.02

600C 1.46+/-1.75 88.32+/-14.69 1.03+/-0.04

High-set 20.37+/-1.81 89.50+1-1.79 1.09+/-0.02

CMC A 0.48+1-0.13 8.66+1-3.38 2.60+1-0.14

CMC B 0.41+1-0.12 0.42+1-0.07 3.28+1-0.17

Experiments were undertaken in triplicate. 2mg substrates and 1mg enzyme were incubated in 50mM phosphate buffer pH 7.0 at 30 0C until no further reducing sugar was released. Samples were then harvested, washed and soluble and insoluble fractions were lyophilised.

Soluble fractions were resuspended in distilled water and insoluble fractions in 0.5M NaOH prior to analysis. All entries are expressed as % of total +1- st. dcv. Cellulase was described by the manufacturers as 13-(1-4; 1-'3)-glucanhydrolase, laminarase was described as glucanase and lysing enzymes were said to contain yeast glucanase, protease and cell lytic activities.

Curdlan preparations were as described (pp. 75-76), CMC A and CMC B were carboxymethylcurdlans prepared by method A or method B respectively (pp. 86-87).

Insol CHO= % of total carbohydrate as insoluble carbohydrate

Glucose= % of soluble carbohydrate as glucose

DP= Average degree of polymerisation of soluble fraction calculated as amount of reducing sugar/amount of total soluble carbohydrate. 123

PHi

Concluding Discussion

93 Potential Mechanisms for Curdlan Synthesis in Response to Nitrogen Deficiency in

Agrobacterium sp. ATCC 31749

The work presented in Chapter 6.2 clearly demonstrates the requirement for nitrogen deficiency to allow the synthesis of curdlan by Agrobacteriutn sp. ATCC 31749. Thus, curdlan production is controlled by environmental factors. As discussed in the introduction (pp. 67-70), a certain amount of evidence has accumulated to suggest that other bacterial species capable of interaction with plants have polysaccharide production controlled by environmental factors e.g. Xanthomonas campestris (Osbourn et al., 1990), Rhizobiutn meliloti (Doherty et al., 1988). In particular, mutations in the R. meliloti exoR gene led to

a deregulation of control of succinoglycan biosynthesis dependent on concentrations of NH 4 within the growth medium (Doherty et al., 1988). Futhermore, mutations in exoR resulted in increased transcription of other exo genes (Reed et al., 1991).

As discussed in Chapter 4, Agrobacterium sp. and Rhizobium sp. share a considerable degree of similarity concerning patterns of exopolysaccharide expression and organisation of exopolysaccharide genes. A model for curdlan biosynthesis by Agrobacterium sp. ATCC

31749 could be proposed in which control is achieved via a two component environmental sensing system (Ronson et at, 1987). Bueno et at (1985) proposed that glnL (ntrB) was the sensor protein encoding gene of a two component system responding to nitrogen deficiency.

It was proposed that high cellular ratios of 2-ketoglutarate to glutamine were detected by the protein, a condition that would be expected under nitrogen deficiency. A similar protein may be expected in Agrobacterium sp. ATCC 31749. If excess 2-ketoglutarate were present 124

within the cell, this may lead to a conformation change within the protein, either directly via a 2-ketoglutarate binding site or indirectly via a second enzyme which may be involved in modification of the sensor protein (e.g. phosphorylation/dephosphorylation). The activated sensor protein may then interact with a regulator protein, induce a conformational change and cause the regulator to exert its effect. Regulator proteins of two component systems frequently act as transcriptional activators (Ronson et al., 1987). A model for potential control of curdlan biosynthesis is outlined in Scheme 9.1 (p. 125). Scheme 9.1 is one of the simpler models that could be envisaged and the possibility remains that control is more complex than outlined above. Figure 9.1 outlines the proposed model for the regulation of capsular polysaccharide synthesis in the plant pathogen Erwinia sp. RcsC is proposed as the sensor protein in a two component regulatory system. Upon recognition of an unidentified signal RcsC is believed to activate RcsB by phosphorylation allowing the latter to dimerise and transcriptionally activate cps structural genes (Leigh and Coplin, 1992). In addition, phosphorylated or unphosphorylated RcsB is able to form a heterodimer with RcsA which is also capable of transcriptionally activating cps structural genes. However, concentrations of RcsA are usually limited through degradation by Lon protease.

The observation that the addition of chloramphenicol to cultures of Agrobacterium sp.

ATCC 31749 actively synthesising curdlan resulted in an immediate cessation of curdlan production (pp. 110-111), may suggest that post-translational control of curdlan synthesis also has a role. In a mechanism similar to that of Lon protease digestion of RcsA,

Agrobacterium sp. ATCC 31749 may possess an enzyme capable of degrading one of the proteins required for curdlan biosynthesis. This type of mechanism of control would provide the organism with a more rapid method to cease curdlan production. Expression of the CAPSULAR POLYSACCUARIDE FlcsC SIGNAL? PERIPLASM

CYTOPLASM N sP /

Figure 9.1

Control of exopolysaccharide biosynthesis in Erwinia sp.

(From Leigh and Coplin, 1992).

RcsC receives an unknown signal and causes the phosphorylation of RcsB.

Phosphorylated RcsB is able to form a dimer which acts as a transcriptional activator of cps genes. In addition, phosphorylated or unphosphorylated RcsB can form a heterodimer with RcsA and this dimer can also transcriptionally activate cps genes.

Concentrations of RcsA are controlled by the degradative Lon protease. 125

protease would have to mediated via different conditions to those which control biosynthesis. One potential complication is the observed delay in cessation of curdlan biosynthesis when rifampicicin was added to actively synthesising cultures (pp. 112-113). This observation may suggest that transcribed mRNAs have a certain degree of stability. Hence new protein would be translated to replace that degraded if a mechanism to prevent translation were not present. The possible requirement for a rapid response to environmental factors regarding curdlan biosynthesis in Agrobacteriutn sp. ATCC 31749 is discussed in Chapter 9.5.

Scheme 9.1 Potential Mechanism of Control of Curdlan Biosynthesis in Agrobacterium sp. ATCC

31749 Determined by Nitrogen Availability

Nitrogen deficiency

Increased cellular 2-ketoglutarate

Conformational change in sensor protein

Activation of regulator protein

Increased transcription of curdlan biosynthetic genes

9.2 Potential Further Studies on Agrobacterium sp. ATCC 31749 13-(1-3)-Glucanase

The differences between the work presented in this thesis and previous reports (e.g. Orts et at, 1987) concerning the presence of 3-(1--'3)-glucanase activity in Agrobacterium sp. ATCC

31749 require further examination. Phillips et al. (1983) observed two rates of curdlan production over 90 hours post-stationary incubation of batch cultures of Agrobacterium sp. 126

ATCC 31749. Curdlan production was higher in the first 50 hours than in the following 40 hours. Orts a at (1987) suggested that the observed difference was caused by cell lysis and the release of 3-(1-3)-g1ucanases. However, an alternative explanation could relate to the observed control of curdlan biosynthesis dependent on nitrogen availability. If cells were lysed, nitrogenous compounds may be released. These compounds could then be absorbed by viable cells leading to a shift in the ratio of 2-ketoglutarate to glutamine within viable cells and concommitant reduction in rates of curdlan synthesis. One way to test this hypothesis could be to grow cells in a chemostat to steady state under differing concentrations of nitrogen and to examine the relationship between intracellular levels of 2-ketoglutarate and curdlan production rates.

The result obtained by Orts and colleagues concerning degradation of curdlan when incubated in supernatants of Agrobacterium sp. ATCC 31749 cultures, as measured by changes in viscosity (see pp. 115-116), do however, provide stronger evidence for 13-(1'3)- glucanase activity within the organism. These results also suggest that the activity is extremely low and therefore sensitive methods would be required in order to examine any potential enzyme(s). One method would be direct observation of a decrease in molecular weight of curdlan when incubated with cellular protein. This was attempted in this thesis via derivatisation of curdlan to produce a soluble molecule which could be analysed by size exclusion chromatography. Howver, the method chosen for acetylation was inefficient and carboxymethyl derivatives were highly viscous and appeared polydisperse. A method has been described for the production of tri-acetylated cellulose (Husemann and Siefert, 1969) 127 and application of this to curdlan may prove more succesful. Alternatively, if a gene encoding potential fl-(1-'3)-glucanase activity activity could be isolated it could be overexpressed in order to study the enzyme. Potential functions for 3-(1-'3)-glucanase in

Agrobacterium sp. ATCC 31749 could include the release of glucan chains from the organism after synthesis or alternatively to produce low molecular weight forms of the polysaccharide to act as signal molecules for plant interactions. The latter function has been proposed for the exoK gene product of Rhizobium meliloti (Becker et al., 1993).

9.3 Crystallinity of Native Curdlan: Potential Implications for Curdlan Biosynthesis

The work presented describing the resistance of various curdlan preparations to enzymes (pp.

121-122) suggests that the native preparation has a high degree of crystallinity similar to the high-set form. Takahashi et al. (1986) similarly observed resistance of high-set preparations to endo-3-(1-'3)-g1ucanases and suggested that the mechanism of resistance was caused by hydrophobic interactions between individual curdlan molecules. If the mechanism of resistance by native curdlan was due to the same interaction, a number of implications regarding export of the molecule follow. Konno and Harada (1991) studied differential scanning colorimetry curves of aqueous suspensions of curdlan as they were heated. They observed a small endothermic heat flow in the temperature range 80°C- 110°C suggesting that high-set gel formation (i.e. hydrophobic interaction) was an energy requiring process. Thus, the results presented in this thesis would suggest that an energetically unfavourable form of curdlan is the preferred biosynthetic product. Similarly, Ace tobacter xylinum synthesises the energetically unfavourable, crystalline cellulose I (Benziman et at., 1980). Electron 128

microscopy revealed that cellulose was extruded from a linear array of pores in A. xylinuin, individual fibres coming together in an increasing hierachy to form a cellulosic ribbon which trails away from the bacterial cell (Brown et al., 1976). It has recently been suggested that the ability of cellulose to crystallise as the cellulose I product may depend on a linear arrangement of the extrusion pores (Saxena et al., 1994) and a strain of A. rylinum which produced the energetically more favourable cellulose II was observed to have a more diffuse arrangement of extrusion pores (Kuga et al., 1993). The results presented in this thesis may therefore suggest that curdlan is extruded from specififc sites on the cell surface in a co- operative process. Electron microscopy could be used to examine this possibility further.

The effect of aniline blue on the rate of curdlan production by Agrobacterium sp. ATCC

31749 (pp. 109-1 10)could be due to a number of factors. Similar experiments undertaken with Acetobacter xylinum revealed that addition of calcofluor to the medium resulted in increased rates of cellulose synthesis (Benziman et al., 1980) and it was concluded that glucose polymerisation and cellulose crystallisation were coupled processes, crystallisation being the rate limiting step. The observation that the addition of aniline blue to cultures of

Agrobacterium sp. ATCC 31749 depressed the rate of curdlan synthesis may be due to interference in the possible spatial organisation of extrusion pores on the bacterial surface.

Saxena et al. (1994) suggested that as the cellulose ribbon was extruded from cells of

Acetobacter xylinum its rigidity helped to maintain the correct organisation of the extrusion pores. Converesely, if the cellulose did not extrude correctly (i.e. as cellulose II), it may block the pores. Thus, if aniline blue interfered with crystallisation of curdlan extruded from 129

Agrobacterium sp. ATCC 31749 extending fibres may interfere with each other resulting in

the blockage of extrusion pores. Alternatively, crystallisation of curdlan may place a tension on extruding curdlan fibres. If the molecules are extruded from closely associated pores hydrophobic interactions at the tips of the extruded fibres may pull individual fibres toward each other thus causing a tension at the opposite ends of the fibres, closer to the bacterium.

Again, electron microscopy may help to clarify the effect of aniline blue.

9.4 The Effect of Metabolic Inhibitors: Further Work and Implications for Models of

Curdlan Biosynthesis

The addition of 100mM EDTA to cultures of Agrobacteriuin sp. ATCC 31749 inhibited glucose uptake (pp. 103-105) whilst agents likely to affect the elctrochemical gradient across the cell membrane (tetracaine, azide) had no effect on glucose uptake (pp. 105-108). This data raises the possibility that glucose binding proteins are important in glucose transport in this organism. Methods similar to those adopted by Cornish et at. (1988a; 1989) could be used to further examine this possibility. Inability of cells to absorb glucose after osmotic shock may indicate the involvement of glucose binding proteins in uptake. Comparison of periplasmic protein profiles (on polyacrilamide gels) when the organism is grown under conditions of glucose limitation and glucose excess could give an indication as to likely candidates for glucose binding proteins. These proteins could then be purified using standard, non-denaturing techniques and their ability to bind glucose estimated by equlibrium dialysis. 130

EDTA, tetracaiñe and azide were all observed to inhibit curdlan synthesis (pp. 103-108); however, it was not possible to elucidate at which point in the biochemical pathway the inhibitors had their effect and further work is required. The use of radioactive carbon substrates, followed by extraction and purification of lipophilic material may aid in identification of lipid-linked repeat units as has proved successful in other organisms e.g.

Xanthomonas campestris (Jelpi et al., 1993), Agrobacterium tumefaciens (Staneloni et al.,

1984), Rhizobium meliloti (Tolmasky a al., 1982). Such methods would reveal whether transfer to lipid acceptor molecules was blocked and through control experiments whether a lipid-linked intermediate stage was required for curdlan biosynthesis. Furhtermore, by cell fraction and curdlan isolation one may be able to ascertain whether the molecule is assembled intracellularly and whether the addition of inhibitors blocked transposition of the polysaccharide across the cytoplasmic or outer bacterial membrane. Suitable kinetics were developed which allowed the use of radioactive substrates to determine the site of assembly and effect of inhibitors on lipopolysaccharide synthesis in Salmonella typhimurium (Marino a al., 1985; 1991). This work could again be supported by electron microscopy to directly visualise curdlan inside cells if suitable antibodies could be raised. This has been successfully attempted in the study of Escherichia coli capsule synthesis (e.g. Kroncke et al., 1990a;

1990b). Certain of the E. coli capsules are poorly immunogenic due to their similarity to mammalian polysaccharides, however, it was still possible to obtain antibodies to the capsules.

Therefore it should be possible to obtain anti-curdlan inmunoglobulins. 131

The work presented in this thesis did not favour either the direct polymerisation model or the lipid-linked intermediate model (p. 44, Figure 2.9) for curdlan biosynthesis by

Agrobacterium sp. ATCC 31749. Bacitracin inhibits dephosphorylation of isoprenylpyrophosphate (Siewert and Strominger, 1967) and attempts were made to study the effect of bacitracin on curdlan biosynthesis by the organism; however, the bacterium was able to grow on MSM agar containing up to 1mg ml' bacitracin suggesting resistance to the antibiotic. A relatively simple method has been described for the gel-electrophoretic separation and detection of UDP-glucose: (1-3)- and (1-'4)-glucan synthases (Thelen and

Delmer, 1986) and this may be a valuable tool for elucidation of the strategy adopted by

Agrobacterium sp. ATCC 31749 for curdlan biosynthesis.

9.5 Potential Biological Functions of Curdlan

A number of potential roles for curdlan may be postulated. General functions of the polysaccharide may include resistance to desiccation with polysaccharide on the cell surface preventing loss of water due to capilhiary attraction. In addition, considering the environment from which the parent strain of Agrobacterium sp. ATCC 31749 was isolated (p. 2), curdlan may aid in the attachment of the bacterium to soil particles preventing removal by water runoff. Static cultures of Acetobacter xylinum produce a thick cellulose pellicle which can trap metabolically released gasses causing the obligatively aerobic organism to rise to the surface of the culture medium (Ross et al., 1991). Like cellulose, curdlan is water insoluble and appears to have a certain degree of crystallinity, as synthesised (pp. 121-123). The 132

possibility was therefore raised that curdlan may perform a similar function in Agrobacterium

sp. ATCC 31749 although the evolutionary benefits of this function are dubious. Roux bottles containing low nitrogen MSM and 2% (w/v) glucose were therefore inoculated and incubated at 30°C, however, no structures were observed similar to a cellulosic pellicle and although a few colonies were observed at the surface of the medium, the majority of bacteria remained submerged.

Possibly the most likely function of curdlan is in plant-microbe interactions. Bacterial polysaccharides e.g. Rhizobium ineliloti succinoglycan (Leigh et al., 1987; Muller et al.,

1988) and related compounds e.g. Rhizobial lipo-oligosaccharides (Price and Carlson, In press) are important recognition molecules between plants and microbes and may have other roles in maintaining symbiotic or pathogenic relationships. In Agrobacteriuin tumefaciens cultures addition of the plant extract Soytone increased the rate of cellulose synthesis. In addition, the early site-specific stage of A. tumefaciens attachment to plant host involves cellulose fibrils of bacterial origin covering the surface of plant cells (Matthyesse, 1983).

Curdlan may have a similar role in Agrobacteriuin sp. ATCC 31749. Experiments were undertaken to examine the expression of succinoglycan biosynthetic genes in Rhizobium meliloti within nodules of alfalfa (Reuber et al., 1991). It was observed that the genes were primarily expressed in the early symbiotic or invasion zone of nodules but not the late symbiotic zone suggesting that transient exopolysaccharide expression was an important factor in nodule formation. The observation that curdlan biosynthesis is under strict and rapid 133

control inAgrobacteriuin sp. ATCC 31749 (PP. 91-94) may therefore support the hypothesis that the polymer is involved in plant-bacterial interactions. 134

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