The serotyping of Azospirillum spp. by cell-gold immunoblotting

FEMS Microbiology'Letters 96 (1992) 115-118 © 1992 Federation of European MicrobiologicalSocieties0378-1097/92/$05.00 Published by Elsevier

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The serotyping of Azospirillum spp. by cell-gold immunoblotting V i a d i m i r A. Bogatyrev, L e v A. D y k m a n , Larisa Yu. M a t o r a and Boris I. S c h w a r t s b u r d Institute of Biochemistry and Physiologyof Plants and Microorganisms. Academy of Sciences o)"Russia. Saratol'. Russia Received5 May 1992 Revision received 15 June 1992 Accepted 15 June Iqq2 Key words: Azospirillum; Serotyping; lmmunoblotting; Colloidal gold

1. S U M M A R Y Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interaeted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.

2. I N T R O D U C T I O N Bacteria of the genus Azospirillum live in association with plants and promote plant growth [1,2]. Several species of Azospirilhtm have been identified [3-5], and the study of systematics of Azospirillum is in progress. Correspondence to: V. Bogatyrev, Institute of Biochemistry and Physiology of Plants and Microorganisms, Academy of Sciences of Russia, Prospect Entuziastov 13. Saratov 410015, Russia.

Immunological methods are widely used in bacterial s~stematics and serotyping. Dot-blot immune-overlay as,ays (visualization of antigens attached to immunobilizing matrices) were shown to be useful in the analysis of isolated antigen [6] as well as whole bacterial cells [7,8]. Therefore, these methods could be used for bacterial serotyping. Dot-blot immune-overlay assays usually involve the use of isotopes or enzymes as markers [9]. The use of colloidal gold as a marker of isolated antigens for immunoblotting has been successfully demonstrated [10,11]. The colloidal gold technique simplified the procedure of labelling relative to immunoenzyrne assays, and the sensitivity of gold markers in some cases is higher than that of enzyme markers [11-13]. In this work we demonstrate the use of immunoblotting of bacterial cells with colloidal gold markers to serotype bacteria. 3. M A T E R I A L S A N D M E T H O D S 3.1. Bacterial strains and growth conditions Bacterial strains used are listed in Table 1. All strains were obtained via the culture collection (our institute).

116 Table I Bacterial strains used Strain

Source or reference A. brasilense So 7 J. D6bereiner SO7 (S-variant) [15] Sp 107 J. D/ibereiner Sp 245 J. DBbereiner JM 125 A2 J. Milam S 27 A.N. Labiri KR 77 C. EImerich UQ 1794 J. Beringer Sp Br 14 J. DBbereiner A. lipoferum Sp 59b J. D6bereiner RG 20a J. D6bereiner The cells were grown in defined malate medium [14] overnight at 30°C on a rotary shaker. The medium for growth of A. lipoferum was supplemented with biotin. The cells were harvested, washed twice and then suspended in 20 mM Tris- HCI, 150 mM NaCl buffer, pH 7.2.

3.2. Chemicals Nitrocellulose sheets (0.45 /zm) were purchased from Millipore; BSA (bovine serum albumin) type V from Sigma; protein A from Pharmacia. All other chemicals were of analytical grade.

3.3. Antibodies Rabbits were immunized with whole Azospirillure cells treated with 2% glutaraldehyde. Immunoglobulin G fraction was obtained as previously described [16]. Colloidal gold particles (10 nm in diameter) were obtained using tetrachlorauric acid reduction by sodium citrate [!2]. Conjugation of colloidal gold with protein A was carried out as described previously [12].

3.5. lmmunogold staining Nitrocellulose strips were prepared as described above; the strips were preliminarily incubated with primary antibodies ( 1 0 - 5 0 / ~ g / m l ) for l h and then washed with 0.1% BSA-Tris, and then incubated for 2 h with a gold-protein A conjugate, diluted to ODs20 = 0.5. After incubation and washing with 0.1% BSA-Tris, the strips were air-dried.

3.6. Preparation of a cell membrane fraction Cells were incubated in 50 mM Tris. HCI, 20 mM EDTA, 0.1% phenylmethylsulfonyl fluoride buffer, pH 8.0, disrupted by passing through a French press and treated with DNAase and RNAase. Unbroken cells were removed by centrifugation at 100000 × g, 60 min. The pellet was resuspended in the above buffer and washed under the same conditions.

3. 7. Two-dimensional immunoelectrophoresis SDS-polyacrylamide gel electrophoresis of the crude membrane fraction was carried out according to Hitchcock [17] on a 7.5% gel. After electrophoresis, gel lanes were cut into strips, some of which were stained with Coomassie brilliant blue; the remaining strips were washed in distilled water and then transferred to electrode buffer used for electrophoresis in a second dimension (35 mM Tris-HCI, 15 mM barbital, 75 mM glycine, 1% Triton X-100, pH 8.6). The washed strip was placed on the agarose slab gel containing antibodies and electrophoresis was carried out in a second dimension as described [181.

4. R E S U L T S A N D D I S C U S S I O N

3. 4. Spotting of cells on nitrocellulose. One ~1 drops of a cell suspension (4 × 109 cells per ml) were spotted on dry nitrocellulose paper strips (3.0 ×0.5 cm) with a Hamilton microsyringe. After spotting, the strips were allowed to dry for 15 min at 60°C. Remaining protein-binding sites on the strips were saturated by incubating them with a solution of 1% w / v BSA in 20 mM Tris-buffered saline, pH 8.2, for 20 min at room temperature.

4.1. Dot-blot immune-overlay assay The results of a dot-blot analysis of whole

Azospirillum cells using homologous antibodies visualized by colloidal gold protein A conjugate (indirect labelling) are shown in Fig. 1. Antibodies showed species and strain specificity. Distinct cross-reactions were revealed for A. brasilense strains sp. 245 and sp. 107. Both strains have been isolated from wheat roots: one (sp. 245) in

117

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B

C

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•

D "

E

F

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dramatic difference in the carbohydrate content of the cell surface of the variant (I.M. Skvortsov, personal communication). More detailed serological dot-blot analysis of ten Azospirillum strains is summarized in Table 2. lnterspecies cross-reactions between antibodies raised against A. brasilense sp. 7 and A. lipoferum R G 20a cells were very weak. T h e same weak serological interactions of $27 and KR77 strains with antibodies raised against Sp 107 and Sp 245 cells were seen. Antibodies raised against JM125A2 strain showed weak interaction with B r l 4 cells. The results presented in Fig. 1 and in Table 2 are in good agreement with the previous data obtained by immunofluorescence and agglutination assays [20]. T h e results presented in Fig. 1 were reproduced u n d e r conditions where the cells were h e a t e d at 100*C for 1 h before spotting on nitrocellulose. Thermostability of antigen determinants indicates their carbohydrate r a t h e r than protein origin. This suggestion was confirmed by immunoelectrophoresis of cell m e m b r a n e antigenie fractions.

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Fig. 1. Dot-blot immune overlay assay of whole bacterial cells (A-F) by homologous antibodies (1-6) visualized by colloidal gold protein A conjugate. A. lipoferum sp. 59b (A, I), A. brasilense Sp 107 (B, 2), A. brasilense JM 125A2 (C, 3), A. brasilense Sp 7 (S-variant) (D, 4), A. brasilense Sp 7 (E, 5). A, brasilense Sp 245 (F, 6). soil conditions and the other one (sp. 107) in laboratory conditions [19]. Further investigation could possibly demonstrate the identity of these strains. T h e absence of a complete cross-reaction between A. brasilense sp. 7 stra;.n and its S-variant (Fig. 1) is not clear. Most likely, it is due to the

4.2. lmmunoelectrophoresis o f antigen fractions The results of immunoelectrophoresis of memb r a n e fractions from A. brasilense Sp 7 are pre-

Table 2 Dot-blot analysis of cross-reactions within Azospirillum spp. staining by indirect Protein A - Gold method Strains

A. brasilense Sp7 Sp 107 Sp 245 JM 125 A2 S 27 KR 77 UQ 1794 Sp Br 14 A. lipoferum Sp 59b R g 20a

Antibodies to: A. brasilense Sp 7 +++

+

Sp 107

Sp 245

+++ +++

+++ +++

+ +

+ +

+

+ + + = strong staining, + = weak staining, - = absence of staining.

JM 125 A2

A. lil~ferum Sp 59b

118 vice, to S.Yu. Shchyogolev a n d N.G. Khlebtsov for their s u p p o r t and interest in this work. W e thank J. D 6 b e r e i n e r , J. Beringer, C. Eimerich, J. Milam and A.N. Labiri for strains provided.

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REFERENCES ! 14kD

Fig. 2. Two-dimensional immunoeleetrophoresis of A. hrasi/ense Sp 7 membrane fraction. A. Separation in a first dimension in a SDS-polyacrylamide gel. B. Separation in a second dimension in an agarose gel with antibodies.

s e n t e d in Fig. 2. A n t i g e n s w e r e evident in a starting z o n e o f the ' s e p a r a t i n g ' polyacrylamide gel as two peaks o f precipitation in an agarose gel after s e p a r a t i o n in a s e c o n d d i m e n s i o n . N o p r o teins w e r e s e e n in this starting z o n e o f a polyacrylamide gel whilst p r o t e i n s w e r e p r e s e r v e d in a g r e a t a m o u n t in o t h e r p a r t s o f the gel (Fig. 2). T h e s a m e gel was s t a i n e d with Alcyane blue, a specific dye for polysaccharides. This dye revealed polysaccharides in t h e starting z o n e o f a polyacrylamide gel (data not shown). T h e r e f o r e , antibodies o b t a i n e d by t h e proced u r e d e s c r i b e d in MATERIALS; AND METHODS r e c o g n i z e d c a r b o h y d r a t e antigens o f the cell surface. In earlier work by D e Polli et ai. [20], t h e immunization p r o c e d u r e p r o d u c e d a n t i b o d i e s to c a r b o h y d r a t e as well as to p r o t e i n antigens. T h a t is why n u m e r o u s cross-reactions b e t w e e n differe n t strains, e l i m i n a t e d by heating, w e r e f o u n d [20]. T o summarize, we have shown that cell-gold i m m u n o b l o t t i n g can b e successfully u s e d to identify Azospirillum strains. T h e t e c h n i q u e s provides significant ramifications for taxonomic and ecological experiments.

ACKNOWLEDGEMENTS W e are grateful to I.B. Zhulin a n d M.S. J o h n son for r e a d i n g t h e m a n u s c r i p t and helpful ad-

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