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Isolation of Agrobacterium sp., strain from the Azolla leaf cavity
FEMS MicrobiologyLetzers70 (1990) 55-60 Published by Elsevier
55
FEMSLE 04043
Isolation of Agrobacterium sp., strain from the Azolla leaf cavity J a c e k Plazinski, R o n a Taylor, W i l l i a m Shaw, L y n n Croft, Barry G. R o l f e a n d Brian E.S. G u n n i n g Research Schoolof BiologicalSciences. AustralianNational University, CanberraCity, Australia
Received 11 Janaal'y 1990 Revisionreceived6 March 1990 Accepted7 March 1990 Key words: Azolla; Anabaena; Symbiosis; Agrobacterium; Plasmid; D N A hybridization
1. S U M M A R Y
2. I N T R O D U C T I O N
We have isolated a bacterial strain from surface sterilized fronds of the aquatic fern Azolla filiculoides and infer that it occurs in leaf cavities, along with the nitrogen-fixing cyanobacterium Anabaena azollae. This strain grew slowly on rich media, appeared to be Gram-negative, and was identified using a bacteriological multitest system as Agrobacterium sp. This strain, designated AFSR-1, could grow in the presence of 400 ppm of ammonium sulphate. D N A / D N A hybridization analysis showed that a 16 S ribosomal R N A gene is located in the AFSR-I strain on an identical D N A restriction fragment as in a strain of Agrobacterium tumefaciens. In addition, it was found that strain AFSR-1 contained D N A regions homologous to the nodulation genes nodABC and nodL of Rhizobium trifoliL AFSR-1 contains a cryptic plasmid, and the genetic transfer of a broad-host-range plasmid pLAFR3 to strain AFSR-1 was achieved.
Azolla, a genus of floating aquatic ferns established by Lamarck in 1873, is found on the surfaces of fresh water ecosystems in both temperate and tropical regions. All Azolla species contain, as a symbiont, a nitrogen-fixing cyanobacterium Anabaena azollae Strasburger [1]. A symbiotic Anabaena lives in algal packets localized in special leaf cavities of Azolla where the host and symbiont exchange metabolites [2,3]. This symbiotic association has been of interest to agronomists and phytologists for years because of its agronomic importance. The peoples of Asia have long recognized the benefits of growing Azolla for fish feeding, weed control, and particularly, as an alternative source of nitrogen fertilizer in rice paddies [3,4]. Gram-negative eubacteria such as Pseudomonas, Azotobacter [5,6] Alcaligenes, Caulobacter [7] or Arthrobacter [8] have suggested to be associated with the Anabaena in Azoila leaf cavities. It is not clear how specific these associations are, but Wallace and Gates [9] have characterized in detail an Arthrobacter isolated from leaf cavities of four different Azolla species. In this paper we report the isolation and characterization of an Agrobacterium strain from the
Correspondence to: Dr. J. Plazinski, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra City, ACT 2601, Australia.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
56
leaf cavities of Azolla filiculoides Lam. Part of this work was described elsewhere [10].
3. MATERIALS AND METHODS
3.1. Azolla culture and growth conditions A strain of Azolla filiculoides Lam. was collected in Slack's Creek in the Snowy Mountains Region of New South Wales, Australia, and cultured under laboratory conditions as previously described [11]. Two cultures of Anabaena-free ferns, Azolla filiculoides 136 and A. pinnata 97 were kindly provided by the National Azolla Research Centre, Fuzhou, China.
3.2. Isolation of bacteria from leaf cavities Entire fronds were surface sterilized according to the method of Plazinski and Rolfe [12]. The 10th leaf from the tip of the frond was dissected and rolled over three agar plates which contained either nutrient agar, NA [13], tryptone-yeast extract, TY [14] or BMM medium [15]. Only when the "roll test" proved negative on all plates the dissected Azolla leaves which were stored in the protoplast dilution buffer [16], were further squashed in 0.1 ml of protoplast dilutic,n buffe~ and the resultant cell suspension was plated onto TY, BMM and NA plates to examine the total population of extracted microflora. Single colony isolates were purified using routine bacteriological procedures.
3.3. Bacterial strains and plasmids An Agrobacterium tumefaciens strain C58 (a gift from Dr P. Hooykaas), and an Arthrobacter strain ATCC8010 (purchased from the American Type Culture Collection) were used for comparative experiments. A symbiotic strain of Anabaena azollae was isolated from surface sterilized fronds of Azolla microphyUa 431 as described elsewhere
[n]. A plasmid vector, pLAFR3, which is a combination of the broad host range plasmid pRK290 [17] and the polylinker of rap8 [18,19] was used for genetic transfer experiments in the triparentai matings with a helper plasmid pRK2013 [17].
3. 4. Bacteriological identification and growth tests An identification system API 20 NE (Montalieu Vercieu, France) designed for characterization of non-enteric, Gram-negative rods was used. This standardized micromethod combines 8 conventional tests and 12 assimilation tests for identification of Gram-negative bacteria not belonging to the Enterobacteriaceae family. The following reactions were tested: reduction of nitrates to nitrites or nitrogen, indol production and acidification. The presence of enzymes such as urease, ~-glucosidase, ar£inlne dihydrolase, protease and ~8galactosidase was also analysed. Other tests included assimilation of: glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenyl-acetate. Production of 3-ketolactose was determined by the method of Bernaerts and De Ley [20]. Growth rates were monitored in BMM medium containing 100, 200 and 400 ppm of ammonium sulphate.
3.5. DNA isolation procedures Bacterial total DNAs were isolated [21] and plasmids visualized [22] by methods described elsewhere. Total DNAs were digested and separated in 0.8~ agarose gels as described by Maniatiset al. [23]. Restriction endonucleases were used as recommended by the manufacturer.
3.6. DNA probes and hybridizations The following DNA clones were used as hybridization probes: a 0.2 kb Clal DNA fragment containing a nodL gene from Rhizobium trifolii strain ANUS43 (a gift from Dr J. Weinman); a 2.08 kb Pstl/EcoRl DNA clone containing a nodABC from Rhizobium trifolii ANUS43 [24] (a gift from Dr M. Djordjevic), and a 1.7 kb Kpnl/ Pstl DNA fragment containing a 16 S ribosomal RNA gene from Anacystis nidulans [25] which was a gift from Dr M. Sugiura. Isolation of DNA fragments from agarose gels, preparation of hybridization probes and D l q A / DNA hybridization procedures were done according to the methods described in Maniatis et aL [23].
12
4. RESULTS A N D DISCUSSION
3 Kb
We succeeded in isolating one type of bacterium from surface sterilized squashed fronds of Azoila filiculoides. For ~'hese experiments more than 20 different fronds in four independent tests were used. Since the surface sterilized fronds appeared to be bacteria-free we presumed that the isolated microorganism must have come from the Azolla leaf cavities. In confirmation, when samples of two Anabaena-free Azolla ferns were used for plating experiments, no bacteria was isolated from the squashed fronds. The isolated bacteria grew slowly as aerobic, Gram-negative, short rods on TY, NA (3 days) and BMM (5 days) plates. In order to characterize the isolated bacteria an identification system AP! 20NE was used. Tested bacteria appeared to have an urease, ~8-glucosidase and ,8-galactosidase activity. All the other conventional tests were negative. The assimilation tests revealed that the isolated bacteria were able to assimilate all substrates except caprate, adipate and phenyl-acetate. Running 3 independent tests of AP! 20NE system identical results were obtained, showing unequivocally that the isolated bacterium was an Agrobacterium strain. This strains was designated AFSR-1. Several other laboratories have already reported isolation of bacteria such as Pseudomonas [5], Caulobacter [7] or Arthrobacter [8,9] from various Azolla species. Because our results contradict those previously reported, we attempted to characterize further the AFSR-1 strain. The common biochemical test for the production of 3-ketolactose was employed. This test is routinely used for characterization of the Agrobaeterium strains [20]. The AFSR-I strain did not produce a 3-ketolactose, therefore it should be placed into the biovar 2 cluster of agrobacteria as suggested by Kersters and DeLey [26]. Since the applicability of small subunit ribosomal R N A (16 S RNA) sequences for bacterial classification is now well accepted [27-30], we used this method to characterize the AFSR-] strain. Fig. 1 shows results of hybridization of the r R N A gene probe to total DNAs isolated from the AFSR-1 and three other bacterial strains. AFSR-1 and Agrobacterium tumefaciens C58 strains con-
44.7
lID
| ~"
Q
a
~
~ 0.55
Fig. 1. Autoradiogram of 32P-labelled rRNA gene probe hybridized to ~otal DNAs extracted from: (1) Agrobacterium radiobacter AFSR-I; (2) Arthrobacter strain ATCCS010; (3) symbiotic Anabaena azollae isolated from Azolla microphylla fern. and (4) Agrobacteriumtumefaciens strain C58. All DNA samples were digested with EcoRl. Sizesate shown in kilobase pairs (kb).
tain D N A fragments which hybridize to the probe and are identical in electrophoretic mobility. Hybridizing fragments from Arthrobacter are of different size, indicating that the AFSR-1 is more closely related to A. tumefaciens than to Arthrobacter. Therefore, the rRNA hybridization analysis could not detect small taxonomic differences between AFSR-1 and C58 strains, but detected gross differences, such as those between Arthrobacter and agrobacteria. It has been suggested that bacteria in the Azolla cavities protect Anabaena azollae by diminishing intracavity ammonium accumulation [31]. In order to check this hypothesis the AFSR-1 strain was grown in the presence of different concentrations of ammonium sulphate. The experiment showed that the AFSR-I strain was able to grow normally
A
B
C
pD-
ch~"
Fig. 2. Plasmid visualizationand DNA/DNA hybridization in the Agrobacterium radiobacter strain AFSR-I. (A) Horizontal gel electrophoresisof AgrobacteriumradiobacterAFSR-I lysate showing the presence of a 100 Mdal indigenous plasmid (p). (B) Corresponding autoradiogram showing hybridization of AFSR-1 plasmid DNA with a Rhizobium nodL probe. (C) Plasmid profile of the AFSR-I postconjugant. The presence of the native plasmid (p) as well as the pLAFR3 (pL-3) vector was demonstrated by using a modified Eckhardt method [21]. ch, chromosomal DNA. Details concerning DNA probes are presented in MATERIALSANDMETHODS.
in medium containing 400 ppm. Thus the AFSR-1 strain could indeed utilize excess N H ~ nitrogen inside the cavity and thereby could protect synthesis of the Anabaena azollae nitrogenase complex. Using an in-gel lysis electrophoretic method [22] we found that the AFSR-1 strain possesses an indigenous plasmid of molecular weight of 100 M D a (Fig. 2A). Several heterologous D N A probes were used to seek homologous regions on the AFSR-1 plasmid DNA. One D N A probe, the Rhizobium nodL [32], hybridized to the AFSR-1 plasmid (Fig. 2B). The nodL gene, which has homology to the acetyl transferase of the E. coli lacA gene [32,33], is required for the host-specific nodulation of red clover [32]. Another Rhizobium D N A clone, a nodABC operon, hybridized weakly to the digested chromosomal D N A of the AFSR-I strain (Fig, 3B). The products of the Rhizobium nodABC operon are involved in synthesizing a low molecular weight factor which evokes early plant responses such as root hair deformations and plant
cell mitosis during the Rhizobium-legume symbiotic interactions [34]. Positive hybridization of the~e Rhizobium D N A sequences to the AFSR-1 gehome has important implications in studying the evolutionary origins of these genes. Moreover, if such genes are functional in the AFSR-1 strain, they may effect the development of the AzollaAnabaena symbiosis or help the bacterium to persist in the Azolla cavity. Further studies are needed to determine whether this bacterium has any effect on the Azolla-Anabaena symbiosis. It is possible that a bacterium which lives in Azolla leaf cavities could be exploited as a vehicle for introducing genes and gene products into the host plant. We explored this by attempting to transfer a broad host range plasmid p L A F R 3 [18,19] into the AFSR-1 strain. The transfer was done by triparentai mating using a helper plasmid pRK2013 [17]. Putative postconjugants were screened on B M M plates containing 4 / t g / m l of tetracycline. The tetracycline resistant postconjugants, which were purified three times on the same medium, appeared at a low frequency of 1 - 2 × 10-6 per recipient cells. The postconjugants
A
B kb ,i~' -,,7.2 ....
~'~4.3
Fig, 3, Hybridization of the Rhizobium nodABC gene probe to the genomic DNA of AFSR-I. (A) AFSR-I genomic DNA digested with Hindlll and separated in a 0.8?0 agarose gel; (B) Corresponding autoradiogram of 32P-labelled Rhizobium nodABC DNA clone hybridized to the total DNA of AFSR-1. Sizes are given in kilobase pairs (kb). Details concerning DNA probes are presented in MATERIALSAHDMETHODS.
59 tested were found to contain b o t h the indigenous a n d the p L A F R 3 plasmid (Fig. 2C), as s h o w n by the in-gel lysis electrophoretic method. The successful plasmid transfer into the AFSR-1 strain indicates that it should be possible to deliver cloned genes into this strain, a n d potentially to deliver gene p r o d u c t s (e.g., for insecticide production) to Azolla cavities.
ACKNOWLEDGEMENTS W e are grateful to D r P. H e b b a r for his help with the muititest system, to Drs. M. Sugit~,a, M. Djordjevic and J. W e i n m a n for the D N A probes, a n d to Professor Liu C h u n g - C h u for providing Anabaena-free Azolla cultures. W.S. wishes to t h a n k Prof. B. G u n n i n g and Dr. S. L e t h a m for the o p p o r t u n i t y to w o r k in the Plant Cell Biology G r o u p , RSBS, at A N U , and he also thanks the N o r t h e r n Territory University Council for study leave and financial s u p p o r t . This work and o u r collaboration with the N a t i o n a l Azoila Research C e n t r e in F u z h o u w a s s u p p o r t e d by the Australian C e n t r e for International Agricultural Research, project 8501.
REFERENCES [1| Strasburger, E. (1873) Jena: Hermann Dabis Verlag, 1, p. 86. [2| Ashton, P.J., and Walmsley, R.D. (1976) Endeavour 35, 39-43. [31 Moore, A.W. (1%9) Bot. Rev. 35, 17-34. 14] Lumpkin, T.A., and Phicknett, D.L. (1980) Econom. Bot. 34, 111-153. [5] Bottomley, W.B. (1920) Ann. Bot. 39. 353-365. 161 Peters, G.A., Mayne, B.C. (1974) Plant Physiol. 53, 813824. [71 Newton, J.W,, and Herman, A.I. (1979) Arch. Microbiot. 120,161-165. [81 Gates, J.E., Fisher, R.W., and Candler, R.A. (1980) Arch. Microbiol. 127,163-165. [91 Wallace, W.H., and Gates, J.E. (1986) Appl. Environ. Microbiol. 52, 425-429. [10] Plazinski, J. (1990) in Molecular Biology of Symbiotic
Nitrogen Fixation (GresshofL P.M. ed.), pp. 51-75. CRC Press inc., Boca Raton, Florida. [11] Plazinski, J., Zheng" Q., Taylor, R., Rolfe, B.G., and Gunning. B.E.S. (1989) FEMS Microbiol. Lett. 65, 199204. [12] Plazinksi, J., and Rolfe, B.G. (1985) Appl. Environ. Microbiol. 49, 984-989. [13l Bani, D., Barberio, C., Bazzical,~o. M., Favilli, F., GalIori, E., and Polsinelfi, M. (1980l 2. Gen. Microbiol. 119, 239-244. [14l I/cringer. J.E. (1974) J. Gen. Microbiol. 84,188-18~y. [15] Rolfe, B.G.. and Gresshoff, P.M. (1980) Aust. J. Biol. Sci. 33, 491-504. |16~ Gresshoff, P.M., Skomicki, M.L., Eadie, J.F., and Rolfe, B.G. (1977) Plant Sci. Left. ,0, 299-304. [17] Ditta, G., Stanfield, S., Corbin, D., and Helinski, D.R. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7347-7351. [18] Friedman, A.M., Long, S.R., Brown, S.E., Buikema, W.J., and Ausub¢l, F.M. (1982) Gene 18, 289-296. [19] Vieira. J., and Messing, J. (1982) Gene 19, 259-268. [201 Bernaens, M.J., and De Ley, J. (1963) Nature 197, 406407. [21] Schofield. P.R., Djordjevic, M.A., Rolfe, B.G., Shine, J., and Watson, J.M. (1983) Mol. Gen. Genet. 192, 459-465. [22] Plazinski, J., Cen, Y.H.. and Rolf¢, B.G. (1985) Appl. Environ. Microbiol. 49,1001-1003. [23] Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. [24] Schofield. P.R., and Watson, J.M. (1986) Nucleic Acid Res. 14, 2891-2903. [251 Tomioka, N., and Sugiura, M. (1983) Mol. Gen, Genet. 191, 46-50. [26] Kersters, K., and De Ley, J. (1984) in Bergey's Manual of Systematic Bacteriology, Vol. 1, pp. 244-254, Williams and Wilkins, Baltimore/London. [27] Grimont, F., Grimont, P.A.D. (1980) Annales de I'lnstitut Pasteur/Microbiologie 137 B, 165-175. [28] Saunders. N.A.. Harrison, T.G., Kachwalla, N., and Taylor, A.G. (1988) J. Gen. Microbiol. 134, 2363-2374. [29] De Buyser. M.L., Mm'van, A., Grimont, F., and El Solh, N. (1989) J. Ge,n., Mic,obiol. 136, 989-999. [301 Lowson. P.A., Gharbia, S.E.. Shah, H.N., and Clark, D.R. (1989) FEMS Microbiol. Len. 65, 41-46. [31] Okoronkwo. N., and Van Hove, C. (1987) Microbios 49, 39-45. [32] Weinman, J.J., Djordjevic, M.A., Sargent, L.C., Dazzo, F.B.. and Rolfe, B.G. (1988) in Molecular Genetics of Plant-Microbe Interactions (Palacios, R., Verma, D.P.S., eds.) pp. 33-34, APS Press, St. Paul, Minnesota. [33] Downie. J.A. (1989) Mol, Microbiol. 3, 1649-1652. [34] Schmidt, J., Wingender, R., John, M., Wieneke, U., Schell. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8578-8582.
55
FEMSLE 04043
Isolation of Agrobacterium sp., strain from the Azolla leaf cavity J a c e k Plazinski, R o n a Taylor, W i l l i a m Shaw, L y n n Croft, Barry G. R o l f e a n d Brian E.S. G u n n i n g Research Schoolof BiologicalSciences. AustralianNational University, CanberraCity, Australia
Received 11 Janaal'y 1990 Revisionreceived6 March 1990 Accepted7 March 1990 Key words: Azolla; Anabaena; Symbiosis; Agrobacterium; Plasmid; D N A hybridization
1. S U M M A R Y
2. I N T R O D U C T I O N
We have isolated a bacterial strain from surface sterilized fronds of the aquatic fern Azolla filiculoides and infer that it occurs in leaf cavities, along with the nitrogen-fixing cyanobacterium Anabaena azollae. This strain grew slowly on rich media, appeared to be Gram-negative, and was identified using a bacteriological multitest system as Agrobacterium sp. This strain, designated AFSR-1, could grow in the presence of 400 ppm of ammonium sulphate. D N A / D N A hybridization analysis showed that a 16 S ribosomal R N A gene is located in the AFSR-I strain on an identical D N A restriction fragment as in a strain of Agrobacterium tumefaciens. In addition, it was found that strain AFSR-1 contained D N A regions homologous to the nodulation genes nodABC and nodL of Rhizobium trifoliL AFSR-1 contains a cryptic plasmid, and the genetic transfer of a broad-host-range plasmid pLAFR3 to strain AFSR-1 was achieved.
Azolla, a genus of floating aquatic ferns established by Lamarck in 1873, is found on the surfaces of fresh water ecosystems in both temperate and tropical regions. All Azolla species contain, as a symbiont, a nitrogen-fixing cyanobacterium Anabaena azollae Strasburger [1]. A symbiotic Anabaena lives in algal packets localized in special leaf cavities of Azolla where the host and symbiont exchange metabolites [2,3]. This symbiotic association has been of interest to agronomists and phytologists for years because of its agronomic importance. The peoples of Asia have long recognized the benefits of growing Azolla for fish feeding, weed control, and particularly, as an alternative source of nitrogen fertilizer in rice paddies [3,4]. Gram-negative eubacteria such as Pseudomonas, Azotobacter [5,6] Alcaligenes, Caulobacter [7] or Arthrobacter [8] have suggested to be associated with the Anabaena in Azoila leaf cavities. It is not clear how specific these associations are, but Wallace and Gates [9] have characterized in detail an Arthrobacter isolated from leaf cavities of four different Azolla species. In this paper we report the isolation and characterization of an Agrobacterium strain from the
Correspondence to: Dr. J. Plazinski, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra City, ACT 2601, Australia.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
56
leaf cavities of Azolla filiculoides Lam. Part of this work was described elsewhere [10].
3. MATERIALS AND METHODS
3.1. Azolla culture and growth conditions A strain of Azolla filiculoides Lam. was collected in Slack's Creek in the Snowy Mountains Region of New South Wales, Australia, and cultured under laboratory conditions as previously described [11]. Two cultures of Anabaena-free ferns, Azolla filiculoides 136 and A. pinnata 97 were kindly provided by the National Azolla Research Centre, Fuzhou, China.
3.2. Isolation of bacteria from leaf cavities Entire fronds were surface sterilized according to the method of Plazinski and Rolfe [12]. The 10th leaf from the tip of the frond was dissected and rolled over three agar plates which contained either nutrient agar, NA [13], tryptone-yeast extract, TY [14] or BMM medium [15]. Only when the "roll test" proved negative on all plates the dissected Azolla leaves which were stored in the protoplast dilution buffer [16], were further squashed in 0.1 ml of protoplast dilutic,n buffe~ and the resultant cell suspension was plated onto TY, BMM and NA plates to examine the total population of extracted microflora. Single colony isolates were purified using routine bacteriological procedures.
3.3. Bacterial strains and plasmids An Agrobacterium tumefaciens strain C58 (a gift from Dr P. Hooykaas), and an Arthrobacter strain ATCC8010 (purchased from the American Type Culture Collection) were used for comparative experiments. A symbiotic strain of Anabaena azollae was isolated from surface sterilized fronds of Azolla microphyUa 431 as described elsewhere
[n]. A plasmid vector, pLAFR3, which is a combination of the broad host range plasmid pRK290 [17] and the polylinker of rap8 [18,19] was used for genetic transfer experiments in the triparentai matings with a helper plasmid pRK2013 [17].
3. 4. Bacteriological identification and growth tests An identification system API 20 NE (Montalieu Vercieu, France) designed for characterization of non-enteric, Gram-negative rods was used. This standardized micromethod combines 8 conventional tests and 12 assimilation tests for identification of Gram-negative bacteria not belonging to the Enterobacteriaceae family. The following reactions were tested: reduction of nitrates to nitrites or nitrogen, indol production and acidification. The presence of enzymes such as urease, ~-glucosidase, ar£inlne dihydrolase, protease and ~8galactosidase was also analysed. Other tests included assimilation of: glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenyl-acetate. Production of 3-ketolactose was determined by the method of Bernaerts and De Ley [20]. Growth rates were monitored in BMM medium containing 100, 200 and 400 ppm of ammonium sulphate.
3.5. DNA isolation procedures Bacterial total DNAs were isolated [21] and plasmids visualized [22] by methods described elsewhere. Total DNAs were digested and separated in 0.8~ agarose gels as described by Maniatiset al. [23]. Restriction endonucleases were used as recommended by the manufacturer.
3.6. DNA probes and hybridizations The following DNA clones were used as hybridization probes: a 0.2 kb Clal DNA fragment containing a nodL gene from Rhizobium trifolii strain ANUS43 (a gift from Dr J. Weinman); a 2.08 kb Pstl/EcoRl DNA clone containing a nodABC from Rhizobium trifolii ANUS43 [24] (a gift from Dr M. Djordjevic), and a 1.7 kb Kpnl/ Pstl DNA fragment containing a 16 S ribosomal RNA gene from Anacystis nidulans [25] which was a gift from Dr M. Sugiura. Isolation of DNA fragments from agarose gels, preparation of hybridization probes and D l q A / DNA hybridization procedures were done according to the methods described in Maniatis et aL [23].
12
4. RESULTS A N D DISCUSSION
3 Kb
We succeeded in isolating one type of bacterium from surface sterilized squashed fronds of Azoila filiculoides. For ~'hese experiments more than 20 different fronds in four independent tests were used. Since the surface sterilized fronds appeared to be bacteria-free we presumed that the isolated microorganism must have come from the Azolla leaf cavities. In confirmation, when samples of two Anabaena-free Azolla ferns were used for plating experiments, no bacteria was isolated from the squashed fronds. The isolated bacteria grew slowly as aerobic, Gram-negative, short rods on TY, NA (3 days) and BMM (5 days) plates. In order to characterize the isolated bacteria an identification system AP! 20NE was used. Tested bacteria appeared to have an urease, ~8-glucosidase and ,8-galactosidase activity. All the other conventional tests were negative. The assimilation tests revealed that the isolated bacteria were able to assimilate all substrates except caprate, adipate and phenyl-acetate. Running 3 independent tests of AP! 20NE system identical results were obtained, showing unequivocally that the isolated bacterium was an Agrobacterium strain. This strains was designated AFSR-1. Several other laboratories have already reported isolation of bacteria such as Pseudomonas [5], Caulobacter [7] or Arthrobacter [8,9] from various Azolla species. Because our results contradict those previously reported, we attempted to characterize further the AFSR-1 strain. The common biochemical test for the production of 3-ketolactose was employed. This test is routinely used for characterization of the Agrobaeterium strains [20]. The AFSR-I strain did not produce a 3-ketolactose, therefore it should be placed into the biovar 2 cluster of agrobacteria as suggested by Kersters and DeLey [26]. Since the applicability of small subunit ribosomal R N A (16 S RNA) sequences for bacterial classification is now well accepted [27-30], we used this method to characterize the AFSR-] strain. Fig. 1 shows results of hybridization of the r R N A gene probe to total DNAs isolated from the AFSR-1 and three other bacterial strains. AFSR-1 and Agrobacterium tumefaciens C58 strains con-
44.7
lID
| ~"
Q
a
~
~ 0.55
Fig. 1. Autoradiogram of 32P-labelled rRNA gene probe hybridized to ~otal DNAs extracted from: (1) Agrobacterium radiobacter AFSR-I; (2) Arthrobacter strain ATCCS010; (3) symbiotic Anabaena azollae isolated from Azolla microphylla fern. and (4) Agrobacteriumtumefaciens strain C58. All DNA samples were digested with EcoRl. Sizesate shown in kilobase pairs (kb).
tain D N A fragments which hybridize to the probe and are identical in electrophoretic mobility. Hybridizing fragments from Arthrobacter are of different size, indicating that the AFSR-1 is more closely related to A. tumefaciens than to Arthrobacter. Therefore, the rRNA hybridization analysis could not detect small taxonomic differences between AFSR-1 and C58 strains, but detected gross differences, such as those between Arthrobacter and agrobacteria. It has been suggested that bacteria in the Azolla cavities protect Anabaena azollae by diminishing intracavity ammonium accumulation [31]. In order to check this hypothesis the AFSR-1 strain was grown in the presence of different concentrations of ammonium sulphate. The experiment showed that the AFSR-I strain was able to grow normally
A
B
C
pD-
ch~"
Fig. 2. Plasmid visualizationand DNA/DNA hybridization in the Agrobacterium radiobacter strain AFSR-I. (A) Horizontal gel electrophoresisof AgrobacteriumradiobacterAFSR-I lysate showing the presence of a 100 Mdal indigenous plasmid (p). (B) Corresponding autoradiogram showing hybridization of AFSR-1 plasmid DNA with a Rhizobium nodL probe. (C) Plasmid profile of the AFSR-I postconjugant. The presence of the native plasmid (p) as well as the pLAFR3 (pL-3) vector was demonstrated by using a modified Eckhardt method [21]. ch, chromosomal DNA. Details concerning DNA probes are presented in MATERIALSANDMETHODS.
in medium containing 400 ppm. Thus the AFSR-1 strain could indeed utilize excess N H ~ nitrogen inside the cavity and thereby could protect synthesis of the Anabaena azollae nitrogenase complex. Using an in-gel lysis electrophoretic method [22] we found that the AFSR-1 strain possesses an indigenous plasmid of molecular weight of 100 M D a (Fig. 2A). Several heterologous D N A probes were used to seek homologous regions on the AFSR-1 plasmid DNA. One D N A probe, the Rhizobium nodL [32], hybridized to the AFSR-1 plasmid (Fig. 2B). The nodL gene, which has homology to the acetyl transferase of the E. coli lacA gene [32,33], is required for the host-specific nodulation of red clover [32]. Another Rhizobium D N A clone, a nodABC operon, hybridized weakly to the digested chromosomal D N A of the AFSR-I strain (Fig, 3B). The products of the Rhizobium nodABC operon are involved in synthesizing a low molecular weight factor which evokes early plant responses such as root hair deformations and plant
cell mitosis during the Rhizobium-legume symbiotic interactions [34]. Positive hybridization of the~e Rhizobium D N A sequences to the AFSR-1 gehome has important implications in studying the evolutionary origins of these genes. Moreover, if such genes are functional in the AFSR-1 strain, they may effect the development of the AzollaAnabaena symbiosis or help the bacterium to persist in the Azolla cavity. Further studies are needed to determine whether this bacterium has any effect on the Azolla-Anabaena symbiosis. It is possible that a bacterium which lives in Azolla leaf cavities could be exploited as a vehicle for introducing genes and gene products into the host plant. We explored this by attempting to transfer a broad host range plasmid p L A F R 3 [18,19] into the AFSR-1 strain. The transfer was done by triparentai mating using a helper plasmid pRK2013 [17]. Putative postconjugants were screened on B M M plates containing 4 / t g / m l of tetracycline. The tetracycline resistant postconjugants, which were purified three times on the same medium, appeared at a low frequency of 1 - 2 × 10-6 per recipient cells. The postconjugants
A
B kb ,i~' -,,7.2 ....
~'~4.3
Fig, 3, Hybridization of the Rhizobium nodABC gene probe to the genomic DNA of AFSR-I. (A) AFSR-I genomic DNA digested with Hindlll and separated in a 0.8?0 agarose gel; (B) Corresponding autoradiogram of 32P-labelled Rhizobium nodABC DNA clone hybridized to the total DNA of AFSR-1. Sizes are given in kilobase pairs (kb). Details concerning DNA probes are presented in MATERIALSAHDMETHODS.
59 tested were found to contain b o t h the indigenous a n d the p L A F R 3 plasmid (Fig. 2C), as s h o w n by the in-gel lysis electrophoretic method. The successful plasmid transfer into the AFSR-1 strain indicates that it should be possible to deliver cloned genes into this strain, a n d potentially to deliver gene p r o d u c t s (e.g., for insecticide production) to Azolla cavities.
ACKNOWLEDGEMENTS W e are grateful to D r P. H e b b a r for his help with the muititest system, to Drs. M. Sugit~,a, M. Djordjevic and J. W e i n m a n for the D N A probes, a n d to Professor Liu C h u n g - C h u for providing Anabaena-free Azolla cultures. W.S. wishes to t h a n k Prof. B. G u n n i n g and Dr. S. L e t h a m for the o p p o r t u n i t y to w o r k in the Plant Cell Biology G r o u p , RSBS, at A N U , and he also thanks the N o r t h e r n Territory University Council for study leave and financial s u p p o r t . This work and o u r collaboration with the N a t i o n a l Azoila Research C e n t r e in F u z h o u w a s s u p p o r t e d by the Australian C e n t r e for International Agricultural Research, project 8501.
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