the role of microbial exopolysaccharides in dessication tollerance

Sunderland J (2008) 0416455

31

Section II

This section contains my Honours thesis which investigates the

effects that Rhizobial Exopolysaccharides have on desiccation

tolerance of Rhizobia In vitro.


32

Title: The effect of exopolysaccharides on the desiccation

tolerance of rhizobia

Author: Jared P Sunderland

SID: 200416455

TABLE OF CONTENTS

Summary........................................................................................................................... 33

1. Introduction.................................................................................................................. 34

2. Materials and Methods................................................................................................. 38

2.1. Survival of EPS mutants under desiccation stress ................................................. 38

2.2. Isolation and measurement of EPS ........................................................................ 39

2.3. Survival of R. leguminosarum bv. trifolii TA1 with EPS from S. meliloti mutants

....................................................................................................................................... 41

3. Results........................................................................................................................... 43

3.1 Desiccation tolerance of EPS mutants .................................................................... 43

3.2 Improved survival of R. leguminosarum bv. trifolii TA1 with EPS from S. meliloti

mutants.......................................................................................................................... 46

4. Discussion ..................................................................................................................... 47

Acknowledgements........................................................................................................... 51

5. References..................................................................................................................... 52

TABLE OF FIGURES

Table 1 EPS mutant strains of S. meliloti……………………………………………………...39

Table 2: Standard curve preparation for anthrone method testing of EPS concentration.41

Figure 1: Percentage survival of Sinorhizobium mutants according to type of EPS

production………………………………………………………………………………..43

Table 3: S.meliloti strains tested listed in order of decreasing survival at 8 days post

desiccation……………………………………………………………………………….45

Figure 2: Structures of succinoglycan EPS I (a) and (b) and galactoglucan EPS II of S.

meliloti……………………………………………………………………………………………..48


33

Summary Sinorhizobium meliloti, the rhizobial symbiont of Lucerne (Medicago sativa) is a widely

used inoculant in agricultural industry. In comparison to Rhizobium leguminosarum bv.

Trifolii, S. meliloti has much greater survival on pre-inoculated seed. In order to

determine the reason for this difference, and to develop a greater understanding of cell

protection mechanisms, the effects that the exopolysaccharides (EPS) produced by these

strains have on desiccation tolerance was tested. S. meliloti mutants which produced

varying amounts and combinations of the exopolysaccharides succinoglycan (EPSI) and

galactoglucan (EPSII) were both studied for their effect on survival of rhizobia post

desiccation, and it was found that EPSII producing strains had a greater level of survival.

(EPS). To see whether this also had an effect on R. leguminosarum, both EPSI and EPSII

were extracted from broth cultures of the appropriate S. meliloti mutants (Rm1021 and

Rm9001 respectively), and applied to washed R.leguminosarum cells prior to desiccation.

The survival post desiccation was than compared with R. leguminsarum with its natural

EPS and it was found the survival improved with the addition of EPS from S. meliloti.

These results demonstrate that there is a significant relationship between the type of EPS

present in the immediate cell environment, and their desiccation tolerance. It is possible

that with further testing and development, natural EPS produced by S. meliloti may play a

biotechnological role in improving cell survival on pre-innoculated seed.


34

1. Introduction Rhizobial inoculants have been used in conjunction with legume crops since the end of

the 19th

century in order to ensure effective nodulation and optimise the potential for

nitrogen fixation (Deaker, 2004). The beneficial effect of these inoculants depend upon

various factors, including the ability of the inoculant strain to out-compete resident soil

bacteria, the suitability of the inoculant strain for the target crop and survival of the

bacteria throughout inoculant storage ,application and in soil.

Currently, seed inoculation is the most common form of rhizobial inoculation. This has

the benefit of being a relatively low cost procedure compared to some other inoculation

procedures. However, if the seed is not used immediately, the rhizobia are prone to

desiccation. Rhizobial species vary in their tolerance to desiccation (Mary et al. 1994)

and this effects their relative survival on the seed coat. In order to increase the survival

of the rhizobia on the seed, synthetic polymers are often used to coat the inoculated seed

to prevent desiccation. Survival of the two species of rhizobia Sinorhizobium meliloti

and Rhizobium leguminosarum bv. trifolii are particularly relevant as they are commonly

used for pre-inoculation of pasture legume seed lucerne and clover respectively.

Natural responses to desiccation include membrane modification and secretion of

exopolysaccharides (EPS) (Potts, 1994). These responses depend on the type of bacteria

and the surrounding environment. This project investigated the role of EPS in desiccation

tolerance of rhizobia. EPS accumulate at the cell wall surface and have been

demonstrated by Beveridge and Graham (1991) to be responsible for the protection of

cells among other roles such as adhesion and nutrient absorption. Roberson and Firestone


35

(1992) found that when Pseudomonas putida in soil was exposed to desiccating

conditions, high amounts of EPS were produced capable of holding many times their

weight of water. Earlier work by Mugnier and Jung (1985) found that xanthan gum; an

EPS produced by the bacterium Xanthomonas campestris protected spray-dried rhizobia

by modulating water activity and limiting heat conduction.

Sinorhizobium meliloti produces two acidic polysaccharides. The first EPS (EPSI) is

succinoglycan. Succinoglycans are produced by many bacteria including Agrobacterium

tumefaciens, Pseudomonas spp., and Rhizobia. They consist of octasaccharide repeating

units composed of seven glucose and one galactose molecule per monomer with

succinate, pyruvate and acetate substituent’s (Stredansky et al. 1998). Low molecular

weight forms of EPSI are symbiotically active in S. meliloti where they assist in

attachment to host cells and signal growth of the infection thread (Gonzalez et al., 1996).

The second EPS (EPSII) produced by Sinorhizobium meliloti is galactoglucan.

Galactoglucans are produced mainly by the bacteria Pseudomonas and Rhizobium. They

are a class of polymers containing repeating disaccharide units of galactose with a

pyruvate substitution, joined by an alpha 1-3- linkage to a glucose molecule that may, or

may not have an acetyl substitution (Kochetkov et al. 1968). Each monomer is then

joined by a beta 1-3 linkage to the following unit (Skorupska et al. 2000). Galactoglucan

production is naturally occurring when EPSI production is blocked, or in phosphate

limiting environments (Skorupska et al. 2000). Production can also be induced through

the mutation of regulatory genes expR or mucR. Like EPSI, low molecular weight EPSII

can also induce nodulation with the host legume Medicago sativa. However, when grown


36

in phosphate limiting conditions, or when mucR regulatory gene is the site of mutation,

only high molecular weight forms of EPSII are produced which are not symbiotically

active (Skorupska et al. 2006). This suggests that EPSII may have a protective role. Due

to the production of EPSII under environmental stress, it is hypothesised that EPSII

producing mutants will have a greater desiccation tolerance that EPSI and EPS deficient

mutants.

Rhizobium leguminosarum bv. trifolii also produces acidic polysaccharides consisting of

repeating octasaccharide units containing glucose, galactose and glucuronic acid (Philip-

Hollingsworth et al., 1989). It is unknown what role this EPS plays in inferring

desiccation tolerance to R. leguminosarum, however, it has been demonstrated by

Breedveld et al. (1990), that biovars of R. leguminosarum are more osmotically sensitive

than S. meliloti and this may be because of differences in the production patterns of EPS.

Sequencing of the mega-plasmids of Sinorhizobium meliloti, and ongoing identification

of the genes involved in EPS biosynthesis has permitted deliberate production of a series

of mutant strains by Tn5 mutagenesis (Leigh et al., 1987, Keller et al., 1988, Long et al.,

1988, Miller et al., 1988, Zhan and Leigh, 1990, Buendia et al., 1991).

The aims of the research described in this paper are to;

i) observe the effects of EPSI and EPSII on the desiccation tolerance of EPS

mutants of Sinorhizobium meliloti, and,


37

ii) observe the effects of EPSI and EPSII from Sinorhizobium meliloti on the

desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 when

compared to its natural EPS.

It is hypothesised that due to the increased production of EPSII by Sinorhizobium meliloti

when under environmental stress, that EPSII producing mutants and EPSII treated cells

of R. leguminosarum bv. trifolii TA1 will have increased desiccation tolerance when

compared to the alternative treatments.


38

2. Materials and Methods

2.1. Survival of EPS mutants under desiccation stress Twenty EPS mutants of Sinorhizobium meliloti produced and maintained by Dr Graeme

Walker from the Massachusetts Institute of Technology (MIT) were supplied to the

Sydney University Centre for Nitrogen Fixation (SUNfix) for this research project (Table

1). Cultures were grown on YMA plates at 28oC for seven days. Suspensions of cells

were prepared by washing the agar surface with 50 mL of sterile water. Desiccation

tolerance was measured by vacuum-drying cell suspensions to 0.1 Torr on a Dynavac

Freeze Dryer. Aliquots (100 µL) of the cell suspension were transferred to sterile

ampoules and vacuum-dried for 4 h. The ampoules were stored at 15 oC in an air-tight

storage container with silica crystals to prevent rehydration from atmospheric moisture.

The numbers of viable cells were counted in the initial suspensions and at 24 h and eight

days after drying. The counts post drying were done by adding 1 mL of sterile water to

each ampoule and allowing them to soak for 1 minute. The ampoules were than vortexed

for 30 seconds. The contents of the tube were then aspirated through a glass pipette 20

times before being transferred to a 10 mL vial of sterile water. Serial dilutions were then

conducted until a dilution of 710− was reached. The 10 5− , 10 6− and 710− dilutions were

then spread plated using aseptic techniques to YMA plates which were incubated at 28oC

for five days prior to plate counting.


39

2.2. Isolation and measurement of EPS

Strain Mutation Description

Rm1021 Wild type Sm

r, produces SG but no detectable EPSII or

active K antigen

Rm7210 Rm1021 exoY210::Tn5 Unable to produce SG

Rm7225 Rm1021 exoH225::Tn5-233 SG lacks succinyl group and is predominantly

HMW

Rm8341 Rm1021 exoZ341::Tn5 SG lacks acetyl group and is predominantly

LMW

Rm8839 Rm1021 exoZ341::Tn5

exoH225::Tn5-233 SG lacks both succinyl and acetyl modifications

Rm8822 Rm1021 exsH13::Tn5 Lacks a SG glycanase MW distribution slightly

shifted to HMW

Rm8826 Rm1021 exsH13::Tn5

exoK445::Tn5-233

Lacks both SG glycanases MW distribution

highly shifted to HMW

Rm8840

Rm1021 exoH225::Tn5-233

exoK445::Tn5

exoZ341::Tn5-Tp

SG lacks acetyl, succinyl groups and a

glycanase

Rm8831

Rm1021 exoK445::Tn5

exsH13::Tn5-132

exoH225::Tn5-233

SG lacks succinyl group and both glycanases

Rm8838

Rm1021 exoK445::Tn5-233

exsH13::Tn5

exoZ341::Tn5-Tp

SG lacks and acetyl group and both glycanases

Rm8530 Rm1021 expR+

Produces SG and EPS II (from LMW to HMW

forms)

Rm9000 Rm1021 expR+

exoY210::Tn5 Produces EPS II (from LMW to HMW forms)

Rm9023 Rm1021 mucR::Tn5 Produces SG and HMW EPS II

Rm9001 Rm1021 mucR::Tn5

exoY210::Tn5 Produces HMW EPS II

Rm8519 Rm1021 ndvB::Tn5-233 Does not produce periplasmic cyclic glucan

Rm41 Wild type Produces SG, probably EPS II and

symbiotically active K antigen

AK631 Rm41 exoB631 Produces symbiotically active K antigen (not

SG and EPS II)

102F34 Wild type Produces SG, no EPS II

LI1 102F34 ndvA::Tn5-lac Unable to transport cyclic β-(1,2) glucans to

periplasm from cytoplasm

TY7 102F34 ndvB::Tn5 Does not produce periplasmic cyclic β-(1,2)

glucan

Table 1 EPS mutant strains of S. meliloti. The EPS mutant strains of S. meliloti can be

divided into three groups based on the wild types Rm1021, Rm41 and 102F34. SG =

succinoglycan (EPS I); EPS II = galactoglucan; MW = molecular weight; HMW = high

molecular weight; LMW = low molecular weight. Rm 1021 normally makes HMW and

LMW forms of SG in a 1:1 molar ratio. LMW forms of SG and EPS II appear to be

symbiotically active but it is not clear what the roles of HMW forms are. The succinyl

modification makes SG more susceptible to glycanase cleavage whereas the acetyl

modification makes SG less susceptible. The information in this table was supplied by

Graeme Walker from MIT.


40

EPS was collected from the remaining water suspensions of S. meliloti mutants and

precipitated according to the methods from Aman et al.(1981) and Scott (1965). The

suspensions were centrifuged at 7500 rpm for 10 min at 4oC in a Sorvall RC2B

centrifuge. The supernatant was collected and the pellet washed with water and re-

centrifuged. The supernatants from each step were pooled and sodium sulphate added to

a final concentration of 10 mM. The acidic polysaccharide was precipitated by adding

the quaternary ammonium salt cetyltrimethylammonium bromide (cetrimide) to a final

concentration of 0.1% and mixed at 37oC overnight. The resulting precipitate was

collected by centrifugation, rinsed with deionised water, and resuspended in a solution of

10% potassium chloride. These samples were than frozen for later analysis where EPS

was measured using the anthrone method (Trevelyan and Harrison, 1950, Reeve et al.,

1997) and calculated as the picograms glucose equivalent/cell.

The anthrone reagent was prepared fresh by dissolving 1.4g anthrone reagent powder in a

solution of 500 mL conc. Sulphuric acid and 200 mL of distilled water. In order to

provide reference points, a standard curve was prepared using a standard solution of

200g/L glucose in reverse osmosis water. Concentrations used for the standard curve

from this solution contained 0, 20, 40, 60, 80, 100 gµ (See Table 2). 0.5 mL of each of

the EPS extracts and standard curve samples were all layered on top of 2.5 mL of

anthrone solution in 25mm test tubes. The contents of the tubes were mixed using a

vortex. Non absorbent cotton wool was used to plug each tube to reduce evaporation

losses from the samples. These tubes were incubated in a boiling water bath for 10 min.

They were than transferred into ice slurry where they were cooled for 5 min, and then


41

stored on ice until reading. The absorbance for each sample was measured at 620nm and

recorded. A standard curve and the associated equation was generated using Microsoft

Excel™ (p=0.013), and the concentration in the samples was then calculated using this

equation.

Table 2: Standard curve preparation for anthrone method testing of EPS

concentration

Glucose ( gµ /mL) 0 20 40 60 80 100

Glucose Standard used (mL) 0 0.1 0.2 0.3 0.4 0.5

Water used (mL) 0.5 0.4 0.3 0.2 0.1 0

Total Volume (mL) 0.5

Anthrone Volume 2.5

2.3. Survival of R. leguminosarum bv. trifolii TA1 with EPS from S.

meliloti mutants

Broth cultures of R. leguminosarum bv. trifolii TA1 and S. meliloti strains Rm1021, and

Rm9001 were grown for seven days using Sucrose-Glucose Broth and acidic EPS was

collected as described in section 2.2. The broths were centrifuged and the supernatant

collected. The cell pellet was washed with sterile water and re-centrifuged and the

supernatants were pooled from each centrifugation step. Sodium sulphate and cetrimide

were added and the solutions were mixed at 37oC overnight. The precipitated EPS was

collected by centrifugation and resuspended in a solution of 10% potassium chloride.


42

The suspensions were dialysed for several days with 3 changes of water. EPS was then

measured using the anthrone method as described above in 2.2(Trevelyan and Harrison,

1950, Reeve et al., 1997) and the suspensions were diluted to obtain equal concentrations

of glucose equivalents. R. leguminosarum bv. trifolii TA1 broth cultures were then grown

for seven days before being centrifuged at 7500 rpm for 10 min in a Sorvall RC2B

centrifuge. The supernatants were discarded and the pellets were resuspended in sterile

water. These suspensions were again re-centrifuged for 10 min at 7500 rpm. The pellets

were then re-suspended in 10 mL of the dialysed and diluted EPS solutions. Desiccation

tolerance was measured by vacuum-drying cell suspensions to 0.1 Torr as described in

section 2.1. Aliquots (100 µL) of the cell suspension were transferred to sterile ampoules

and vacuum-dried for 4 h. The ampoules were stored at 15 oC in a air-tight storage

container with silica crystals to prevent rehydration from atmospheric moisture. The

numbers of viable cells were counted in the initial suspensions and at 24 h and eight days

after drying as described in section 2.1.


43

3. Results

3.1 Desiccation tolerance of EPS mutants

The survival of EPS mutants after storage at low relative humidity and 15°C varied

significantly according to strain and storage time (P = 0.037). The percentage of viable

cells after 1 day and 8 days, in relation to initial suspension, is presented in Figure 1

according to EPS type. Table 3 presents the strains listed in a descending order of

survival over the test period, and their related EPS type. Counts of viable cells at 1 day

give an indication of survival during the drying stage, and counts at 8 days indicate

survival of dried cells during storage. The order of survival, from greatest to least, after 8

days did not follow the same order as that at 1 day suggesting that some strains were

better able to survive the storage conditions than the drying step and vice versa.

Interestingly, there was no statistical co-relation between amount of EPS produced and

desiccation tolerance with the exception of during storage between days 1-8.

Percentage of viable cells post desiccation

of Sinorhizobium mutants

0

5

10

15

20

25

30

0 5 10

Days post desiccation

Pe

rce

nta

ge

re

co

ve

ry (

%)

EPS1

EPS2

Neither EPS

Figure 1: Percentage

survival of

Sinorhizobium mutants

according to type of

EPS production.

(EPS1=Succinoglycan,

EPS2=Galactoglucan,

neither= mutant did not

specifically produce

either EPS1 or EPS2


44

Results were analysed using GenStat by Regression analysis in order to determine the

relationship between loss of viable cells and total EPS production. It was found that there

was a relationship between EPS production and survival from day 1 to day 8 (P=0.046),

although not from pre-desiccation to day 1 (p=0.37), or from pre-desiccation to day 8

(p=0.32). This suggests that EPS production has greater effect on survival during storage

than it does protection during initial desiccation. However, three out of the four highest

surviving strains produced less than 0.2 pg/cell of EPS, and Rm8831, which was the third

lowest survivor produced in excess of 2 pg/cell in one replicate and averaged 1.013

pg/cell over all replicates.

From Table 3, it can be seen that five of the top 7 surviving mutants are capable of EPSII

production, whereas the 13 strains with the highest death rates did not produce EPS II,

with production in these mutants consisting of predominantly EPSI, or in some strains, an

absence of EPS production. The survival of Rm7210, a mutant which lacks production of

EPSI, EPSII and K-antigen, was the highest (48%, p=0.001) over the total time period

suggesting that no EPS production may be optimal. It may be that in the absence of these

extracellular products Rm7210 was able to secrete cyclic β-glucans to protect the cell

(Deaker, 2008 Pers. Comm.), although this has not been tested for, and would require

further investigation. Comparatively, AK631 which produces symbiotically active K-

antigen had a much lower level of survival (11.3%, p=0.023), suggesting that K-antigen

production may place increased stress on the cells during and post desiccation, or that K-

antigen suppresses excretion of cyclic β-glucans.


45

Strain EPS Type

Rm7210 Neither

Rm41 EPSI, Probably EPSII and K antigen

Rm9001 HMW EPSII

Rm8840 Modified EPSI

Rm8530 Both EPS

Rm9023 EPSI and HMW EPSII

Rm9000 EPSII

Rm1021 EPSI


Rm7225 HMW EPSI

AK631 Neither EPS, only K-antigen


102F34 EPSI

LI1 No EPS

TY7 No EPS

Rm8822 EPSI

Rm8826 EPSI

Rm8831 EPSI

Rm8519 No EPS

Rm8341 LMW EPSI

Table 3: S.meliloti strains tested listed in order of decreasing survival at 8 days post

desiccation. Exopolysaccharide (EPS) types include succinoglycan (EPSI) and

galactoglucan (EPSII) and symbiotically active K-antigen


46

3.2 Improved survival of R. leguminosarum bv. trifolii TA1 with EPS

from S. meliloti mutants

Crude extracts of EPS from S. meliloti mutant strains Rm1021 and Rm9001 were

collected by centrifugation and the supernatants applied to TA1 before vacuum-drying.

The two mutants were selected as sources of EPS I (Rm1021) and EPS II (Rm9001)

based on their description (Table 1) and the fact that they were the greatest survivors for

each type of EPS (Table 3). Cells of R. leguminosarum bv. trifolii TA1 had greater

survival at both the 1 day and 8 day counts when they were treated with EPS from S.

meliloti, in comparison to the natural EPS of TA1 (p=0.015). This suggests that EPSI and

EPSII may both play a protective role in protection during desiccation, as well as survival

post desiccation. After suspending TA1 in its own EPS, viability dropped rapidly in the in

the first day, with recovery being only 7.1% of cells from initial solution (p=0.03).

Survival at 8 days was only 3.2%, showing the poor tolerance of this strain to desiccation.

In contrast, the numbers of viable cells of TA1 suspended in S. meliloti EPS gave

recovery percentages at 1 day post desiccation of 23.3% and 28.6% for EPSI and EPSII

respectively. Interestingly, EPSI treated TA1 recovery at 8 days post desiccation was

21.4% (1.9% decrease 1 day post desiccation), compared to EPSII treated cells having a

recovery rate of 24.7% (3.9% decrease from 1 day post desiccation.). The difference in

survival of TA1 between EPSI and EPSII treatments was not statistically significant

(p=0.23), although both were significantly higher in desiccation tolerance than TA1 in its

own EPS (p=0.013, p=0.018 for EPS I and II respectively).


47

4. Discussion The relationship between EPS production in laboratory culture, and desiccation tolerance

has been studied in several papers including Bushby and Marshall, (1977a and 1977b);

Hartel and Alexander, (1986), all of which found either no relationship or an inverse

correlation between EPS production and ability to survive. The work conducted in this

experiment however, investigated the role of EPS in desiccation of cells on surfaces

where there is considerably less available water than is present in dried soil. It is possible

that EPS plays additional roles in soil by holding water and reducing osmotic stress of

cells, but when cells are exposed to air with a low relative humidity, they are subject to

desiccation. Under conditions of low relative humidity, the EPS of the cell must offer

some protection by mechanisms such as the physico-chemical properties of the polymer,

as supported by the increased survival with EPSII over EPSI in S. meliloti and of S.

meliloti EPS over R. leguminosarum EPS.

The demonstrated superiority of EPS II over EPSI in protecting desiccated cells of S.

meliloti, may be explained by their different structures. (See Figure 2) EPSII consists of

repeating disaccharide units of Galactose with a pyruvate substitution, joined by a alpha

1-3 linkage to a glucose molecule that may, or may not have a acetyl substitution (See

figure 2) (Kochetkov et al. 1968). This is then joined by a beta 1-3 linkage to the

following unit (Zevenhuizen, 1997, Skorupska et al. 2000) The alpha-1-3 and beta-1-3

linkages would result in flexible, helical molecules that may adapt readily to changes in

cell shape and pressure differences according to hygrostatic pressure of the wet, or

desiccated cells. This is supported by research conducted by Murrell (1988).

Comparatively, the beta linkages present in both EPSI and EPS from R. leguminosarum


48

bv. trifolii TA1, are more like cellulose fibres (Sutherland, 2001). This may reduce ability

of the EPS to shrink/swell and change shape according to cell tugor leaving open chnnels

to allow diffusion of air. The mode by which desiccated cells are protected may be the

reduction of oxygen transmission through the polymer films. It is possible, as suggested

by Gontard et al. (1996) that tight packing of polymer cotes restricts oxygen transmission

which may protect cells from the detrimental effect of unrestricted oxygen on dry cells

(which has been demonstrated by Deaker, Unpublished data.)

The view that EPS structure may be heavily influential on the survival of cells is further

demonstrated by the fact that the EPSI produced by the better surviving Rm8840 lacks

Figure 2: Structures of succinoglycan EPS I (a) and (b) and galactoglucan EPS II of S. meliloti.

(Succ=succinyl, Ac=acetate, Glc= glucose, Gal= galactose)


49

both succinyl and acetyl modifications. The relative protection afforded by this EPS may

be due to a reduction in the rate of water loss. It is clear that modification in chemical

structure can affect molecular physico-chemical properties as demonstrated by Hart et al.

(1999) with the removal of acetyl groups in xanthan gum increasing water binding and

thus reducing diffusion. Similarly, the difference in structures of EPSI and EPS from R.

leguminosarum may explain the variation in survival of desiccated TA1 cells. The R.

leguminosarum EPS has been deomonstrated to contain methoxy, acetyl, pyruvate acetyl,

succinyl and 3-hydroxybutyryl groups compared with acetyl, succinyl and pyruvyl

substitution groups of EPSI (Philip-Hollingsworth et al., 1989, Reuber and Walker,

1993). These different substituent’s may affect intermolecular bonding and the general

physico-chemical structures of the polymer.

It has been desmonstrated here that the lack of production of cyclic beta-glucans is

detrimental to desiccation tolerance of S. meliloti. Breedveld and Miller (1994) found that

periplasmic cyclic beta-glucans play a key role in hypoosmotic adaption of rhizobia

which counteracts the turgor pressure from the cell cytoplasm. However, it is possible

that this may not as much affect the cells desiccation tolerance, but rather the tolerance of

the cell to re-wetting procedure for counting employed in this experiment, due to the loss

of ability to withstand the extreme hydrostatic pressure experience on rehydration with

deionised water. This is something that might possibly be counteracted by increasing the

dissolved solids of the re-wetting solution to reduce hydrostatic pressure. The strains

which produced cyclic beta-glucans but were unable to secrete them also had a reduced

survival. This may be due to the reduced ability to adapt to elevated osmotic stress of the


50

drying process. This is further supported by the findings of Breedveld et al. (1990) who

found that S. meliloti mutants deficient in EPSI secreted cyclic beta-glucans at increasing

levels which correlated with increasing osmolarity of the media.

The findings of this research provide potential support for the biotechnological

application of natural EPS production and the use of non-native EPS for protecting

rhizobial inoculants. This may also be applied to non-rhizobial biotechnology

applications where the long term survival of desiccated bacterial cells is important.

However, further research is required in order to investigate the effects that using non-

native EPS have on the ability of rhizobia to quickly and successfully induce effective

nodulation in pre-innoculated seed. This may be of concern as EPS produced by rhizobia

are involved in Rhizobium-legume signalling at the initiation of the infection process.


51

Acknowledgements

I am very grateful to Dr Rosalind Deaker for supervision and support, as well as for

supplying the materials and skills required. I am also grateful to Dr Graeme Walker for

the supply of mutants for testing.


52

5. References

Aman, P., McNeil, M., Franzen, L-E., Darvill, A. G. and Albersheim, P. (1981)

Structural elucidation using HPLC-MS and GLC-MS of the acidic polysaccharide

secreted by Rhizobium meliloti strain 1021. Carbohydrate Research 95: 263-282.

Battisti, L., Lara, J. C., and Leigh, J. A. (1992) Specific oligosaccharide form of the

Rhizobium meliloti exopolysaccharide promotes nodule invasion in alfalfa. Proc Natl

Acad Sci USA 89: 5625-5629.

Becker, A. and Pühler, A. (1988) Production of exopolysaccharides. In The

Rhizobiaceae. Spaink, H. P., Kondorosi, A. and Hooykaas, P.J.J (eds). Dordrecht, The

Netherlands, Kluwer Academic Publishers, pp. 97-118.

Beveridge, T. J., and Graham, L. L. (1991) Surface layers of bacteria. Microbiol Rev 55:

684-705.

Breedveld, M. W., Zevenhuizen, L. P. T. M., and Zehnder, A. J. B. (1990) Osmotically

induced oligo- and polysaccharide synthesis by Rhizobium meliloti SU-47. J Gen

Microbiol 136: 2511-2519.

Breedveld, M. W., Zevenhuizen, L. P. T. M., and Zehnder, A. J. B. (1992) Production of

cyclic β –1, 2 glucans in R. leguminosarum. J Bacteriol 174: 6336-6342.


53

Buendia, A. M., Enenkel, B., Koplin, R., Niehaus, K., Arnold, W., Puhler, A. (1991) The

Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as a UDP-

glucose 4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum

biovar viciae strain TOM. Mol Micrbiol 5: 1519-1530.

Bushby, H. V. A. and Marshall, K. C. (1977a) Water status of rhizobia in relation to their

susceptibilty to desiccation and to their protection by montmorillonite. J Gen Microbiol

99: 19-27.

Bushby, H. V. A. and Marshall, K. C. (1977b) Some factors affecting the survial of root-

nodule bacteria on desiccation. Soil Biol & Biochem 9: 143-147.

Costerton, J. W., Cheng, K-J., Geesey, G. G., Ladd, T., Nickel, J. C., Dasgupta, M., and

Marrie, T. J. (1987) Bacterial biofilms in nature and disease. Ann Rev Microbiol 41:

435-464.

Dazzo, F. B. and Brill, W. J. (1979) Bacterial polysaccharide which binds Rhizobium

trifolii to clover root hairs. J Bact 137: 1362-1373.

Gemell, L. G., Hartley, E. J. and Herridge, D. F. (2005) Point-of-sale evaluation of

preinoculated and custom-inoculated pasture legume seed. Aust J Exp Agric 45: 161-169


54

Glazebrook, J., and Walker, G. C. (1989) A novel exopolysaccharide can function in

place of the calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium

meliloti. Cell 56: 661-672.

Gontard, N., Thibault, R., Cuq, B., and Guilbert, S. (1996) Influence of relative humidity

and film composition on oxygen and carbon dioxide permeabilities of edible films. J

Agric Food Chem 44: 1064-1069.

Gonzalez, J. E., Reuhs, B. L., and Walker, G. C. (1996) Low molecular weight EPS II of

Rhizobium meliloti allows nodule invasion in Medicago sativa. Proc Natl Acad Sci USA

93: 8636-8641.

Hart, T. D., Chamberlain, A. H. L., Lynch, J. M., Newling, B. and McDonald, P. J.

(1999) A stray field magnetic resonance study of water diffusion in bacterial

exopolysaccharides. Enz Microbiol Tech 24: 339-347.

Hartel, P. G., and Alexander, M. (1986) Role of extracellular polysaccharide production

and clays in the desiccation tolerance of cowpea bradyrhizobia. Soil Sci Soc Am J 50:

740-745.

Keller, M., Muller, P., Simon, R., and Pühler, A. (1988) Rhizobium meliloti genes for

exopolysaccharide synthesis and nodule infection located on megaplasmid 2 are actively

transcribed during symbiosis. Mol Plant-Microbe Interact 1: 267-274.


55

Leigh, J. A., and Lee, C. C. (1988) Characterisation of polysaccharides of Rhizobium

meliloti exo mutants that form ineffective nodules. J Bacteriol 170: 3327-3332.

Leigh, J. A., and Walker, G. C. (1994) Exopolysaccharides of Rhizobium: synthesis,

regulation, symbiotic function. Trends Genet 10: 63-67.

Leigh, J. A., Reed, J. W., Hanks, J. F., Hirsch, A. M. and Walker, G. C. (1987)

Rhizobium meliloti mutants that fail to succinylate their Calcofluor-binding

exopolysaccharide are defective in nodule invasion. Cell 51: 579-587.

Lien, L., Fellows, C. M., Copeland, L., Hawkett, B. and Gilbert, R. G. (2002) Water-

binding and oxygen permeability in poly(vinyl alcohol) films. Aust J Chem 55: 507-512

Long, S., Reed, J. W., Himawan, J., and Walker, G. C. (1988) Genetic analysis of a

cluster of genes required for the synthesis of the Calcofluor-binding exopolysaccharide of

Rhizobium meliloti. J Bacteriol 170: 4239-4248.

Martin, M., Lloret, J., Sanchez-Contreras, M., Bonilla, I., and Rivilla, R. (2000) MucR is

necessary for glactoglucan production in Sinorhizobium meliloti EFB1. The Amer

Phytopath Soc 13: 129-135.


56

Mary, P. Ochin, D. and Tailliez, R. (1985) Rates of drying and survival of Rhizobium

meliloti strains during storage at different relative humidities. Appl Env Microbiol 50:

207-211.

Mary, P., Dupuy, N., Dolhem-Biremon, C., Defives, C. and Tailliez, R. (1994)

Differences among Rhizobium meliloti and Bradyrhizobium japonicum strains in

tolerance to desiccation and storage at different relative humidities. Soil Biol Biochem

26: 1125-1132.

Mendrygal, K. E., and Gonzalez, J. E. (2000) Enviromental regulation of

exopolysaccharide production in Sinorhizobium meliloti. J Bacteriol 182: 599-606.

Miller, K. J., Gore, R. S. and Benesi, A. J. (1988) Phosphoglycerol substituents present

on the cyclic β –1,2- glucans of Rhizobium meliloti 1021 are derived from

phosphatidylglycerol. J Bacteriol 170: 4569-4575.

Mugnier, J. and Jung, G. (1985) Survival of bacteria and fungi in relation to water

activity and the solvent properties of water in biopolymer. Appl Env Microbiol 50: 108-

114.

Murrell, W. G. (1988) Bacterial spores: nature's ultimate survival package. In

Microbiology in Action. Murrell, W. G. and Kennedy, I. R. (eds.) Letchworth, England,

Research Studies Limited Press Ltd., pp. 311-346.


57

Osa-Afiana, L. O. and Alexander, M. (1982) Differences among cowpea rhizobia in

tolerance to high temperature and desiccation in soil. Appl Env Micobiol 43: 435-439.

Phillip-Hollingsworth, S., Hollingsworth, R. I. and Dazzo, F. B. (1989) Host-range

related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii

and Rhizobium leguminosarum. J Biol Chem 264: 1461-1466.

Potts, M., (1994) Desiccation tolerance of prokaryotes. Microbiol Rev 58: 755-805.

Reuber, T. L. and Walker, G. C. (1993) Biosynthesis of succinoglycan, a symbiotically

important exopolysaccharide of Rhizobium meliloti. Cell 74: 269-280.

Roberson, E. B. and Firestone, M.K. (1992) Relationship between desiccation and

exopolysaccharide production in a soil Pseudomonas sp. Appl Env Microbiol 58: 1284-

1291.

Scott, J. E. (1965) Fractionation by precipitation with quaternary ammonium salts. In

Methods in Carbohydrate Chemistry: Volume V General Polysaccharides. Whistler, R.

L, BeMiller, J. N. and Wolfrom, M. L. (eds). London, Academic Press, pp38-44.

Sutherland, I. W. (2001) Biofilm exopolysaccharides: a strong and sticky framework.

Microbiol 147: 3-9.


58

Tease, B. E. and Walker, R. W. (1987) Comparative composition of the sheath of the

cyanobacterium Gloethece ATCC 27152 cultured with and without combined nitogen. J

Gen Microbiol 133: 3331-3339.

Urzainqui, A., and Walker, G. C. (1992) Exogenous suppression of the symbiotic

deficiencies of Rhizobium melitoti exo mutants. J Bacteriol 174: 3403-3406.

Zhan, H., and Leigh, J. A. (1990) Two genes that regulate exopolysaccharide production

in Rhizobium meliloti. J Bacteriol 172: 5254-5259.

the role of microbial exopolysaccharides in dessication tollerance

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