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Section II
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tolerance of rhizobia
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
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Summary
Sinorhizobium meliloti, the rhizobial symbiont of Lucerne (Medicago sativa) is a widely
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
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
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
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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
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
and Rhizobium leguminosarum bv. trifolii are particularly relevant as they are commonly
used for pre-inoculation of pasture legume seed lucerne and clover respectively.
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
cells among other roles such as adhesion and nutrient absorption. Roberson and Firestone
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(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
Sinorhizobium meliloti produces two acidic polysaccharides. The first EPS (EPSI) is
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).
Galactoglucans are produced mainly by the bacteria Pseudomonas and Rhizobium. They
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
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
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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
producing mutants will have a greater desiccation tolerance that EPSI and EPS deficient
mutants.
repeating octasaccharide units containing glucose, galactose and glucuronic acid (Philip-
Hollingsworth et al., 1989). It is unknown what role this EPS plays in inferring
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.
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).
i) observe the effects of EPSI and EPSII on the desiccation tolerance of EPS
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ii) observe the effects of EPSI and EPSII from Sinorhizobium meliloti on the
when under environmental stress, that EPSII producing mutants and EPSII treated cells
of R. leguminosarum bv. trifolii TA1 will have increased desiccation tolerance when
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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
Freeze Dryer. Aliquots (100 µL) of the cell suspension were transferred to sterile
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 10−7 was reached. The 10 −5 , 10 −6 and 10−7 dilutions were
then spread plated using aseptic techniques to YMA plates which were incubated at 28oC
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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; and
2.2. Isolation LMW = low molecular
measurement weight. Rm 1021 normally makes HMW and
of EPS
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. 39
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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
concentration of 0.1% and mixed at 37oC overnight. The resulting precipitate was
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.,
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 µ g (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
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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.
concentration
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
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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
section 2.1. Aliquots (100 µL) of the cell suspension were transferred to sterile ampoules
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
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3. Results
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.
25 according to type of
20 EPS production.
EPS1 (EPS1=Succinoglycan,
15 EPS2 EPS2=Galactoglucan,
Neither EPS neither= mutant did not
10
specifically produce
5
either EPS1 or EPS2
0
0 5 10
Days post desiccation
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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),
(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
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,
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
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-
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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
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Crude extracts of EPS from S. meliloti mutant strains Rm1021 and Rm9001 were
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
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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
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
as supported by the increased survival with EPSII over EPSI in S. meliloti and of S.
meliloti, may be explained by their different structures. (See Figure 2) EPSII consists of
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
Comparatively, the beta linkages present in both EPSI and EPS from R. leguminosarum
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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
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
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)
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
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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
(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
1993). These different substituent’s may affect intermolecular bonding and the general
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
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
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
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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
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
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
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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
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5. References
Aman, P., McNeil, M., Franzen, L-E., Darvill, A. G. and Albersheim, P. (1981)
Battisti, L., Lara, J. C., and Leigh, J. A. (1992) Specific oligosaccharide form of the
Rhizobiaceae. Spaink, H. P., Kondorosi, A. and Hooykaas, P.J.J (eds). Dordrecht, The
Beveridge, T. J., and Graham, L. L. (1991) Surface layers of bacteria. Microbiol Rev 55:
684-705.
52
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Buendia, A. M., Enenkel, B., Koplin, R., Niehaus, K., Arnold, W., Puhler, A. (1991) The
99: 19-27.
Bushby, H. V. A. and Marshall, K. C. (1977b) Some factors affecting the survial of root-
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.
preinoculated and custom-inoculated pasture legume seed. Aust J Exp Agric 45: 161-169
53
Sunderland J (2008) 0416455
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
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.
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
54
Sunderland J (2008) 0416455
meliloti exo mutants that form ineffective nodules. J Bacteriol 170: 3327-3332.
Leigh, J. A., Reed, J. W., Hanks, J. F., Hirsch, A. M. and Walker, G. C. (1987)
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
Martin, M., Lloret, J., Sanchez-Contreras, M., Bonilla, I., and Rivilla, R. (2000) MucR is
55
Sunderland J (2008) 0416455
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)
tolerance to desiccation and storage at different relative humidities. Soil Biol Biochem
26: 1125-1132.
on the cyclic β –1,2- glucans of Rhizobium meliloti 1021 are derived from
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.
56
Sunderland J (2008) 0416455
tolerance to high temperature and desiccation in soil. Appl Env Micobiol 43: 435-439.
Potts, M., (1994) Desiccation tolerance of prokaryotes. Microbiol Rev 58: 755-805.
exopolysaccharide production in a soil Pseudomonas sp. Appl Env Microbiol 58: 1284-
1291.
57
Sunderland J (2008) 0416455
cyanobacterium Gloethece ATCC 27152 cultured with and without combined nitogen. J
Zhan, H., and Leigh, J. A. (1990) Two genes that regulate exopolysaccharide production
58