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Comparative performance of enterobacterial repetitive intragenic consensus-polymerase chain reaction and lipopolysaccharide electrophoresis for the identification of Bradyrhizobium sp. (Lotus) strains
FEMS Microbiology Ecology 28 (1999) 163^168
Comparative performance of enterobacterial repetitive intragenic consensus-polymerase chain reaction and lipopolysaccharide electrophoresis for the identi¢cation of Bradyrhizobium sp. (Lotus) strains M. Santamar|èa a , F. Agius b , J. Monza b , A.M. Gutieèrrez-Navarro c , J. Corzo a; * a b
Departamento de Bioqu|èmica y Biolog|èa Molecular, Universidad de La Laguna, 38206 Tenerife, Spain Laboratorio de Bioqu|èmica, Facultad de Agronom|èa, Avda. Garzoèn 780, CP 12900, Montevideo, Uruguay c Departamento de Microbiolog|èa y Biolog|èa Celular, Universidad de La Laguna, 38206 Tenerife, Spain Received 17 June 1998; received in revised form 16 September 1998; accepted 14 October 1998
Abstract We compared two methods for typing bacterial strains : electrophoretic lipopolysaccharide profiling and genomic fingerprinting by enterobacterial repetitive intragenic consensus-polymerase chain reaction. Our aim was to assess the relative utility of these techniques for identification of bradyrhizobia. A collection of Uruguayan Bradyrhizobium strains isolated from Lotus subbiflorus was selected to test both techniques in terms of their discriminating ability and ease of use. Both techniques were found to be equally discriminating and they classified the samples in the same way, although each method ascribed two strains to different groups. Genomic profiling of some strains required previous DNA purification, whereas this was found to be unnecessary for others. Lipopolysaccharide profiling was found to be easier and cheaper to perform, but was not useful for determination of the genetic relationship among the strains. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bradyrhizobium (Lotus); Lipopolysaccharide; PCR ¢ngerprint; Strain identi¢cation
1. Introduction Ecological studies of natural populations of rhizobia require fast and reliable techniques for strain identi¢cation. To be useful in ¢eld studies, an identi¢cation technique for a given population must pro-
* Corresponding author. Tel.: +34 (22) 318-356; Fax: +34 (22) 318354; E-mail: [email protected]
duce the maximum number of di¡erent groups in a reproducible way [1] and must be independent of any previous manipulation of the strains [2]. Serological methods have been used for strain identi¢cation, but they are limited since they do not yield information on strains that do not react with a given set of antibodies, and therefore these strains are arti¢cially grouped. The electrophoresis of whole cell proteins avoids such arti¢cial grouping and has been used as a discriminating tool for strain identi¢cation [3^6], but the resulting pro¢les are
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rather complex and di¤cult to compare. More clearcut and easily compared patterns are produced by the electrophoresis of lipopolysaccharides (LPS) and these patterns have been used successfully for the di¡erentiation of rhizobia isolated from diverse sources [1,7,8]. A further advantage of LPS typing is that it is a fast and reliable technique that can be also applied to extracts from intact nodules, avoiding the necessity of bacterial isolation and cultivation [9]. A di¡erent type of approach, which is increasingly used, is based on DNA ¢ngerprinting. It has been shown that DNA primers corresponding to the enterobacterial repetitive intragenic consensus (ERIC) sequence [10], coupled with the polymerase chain reaction (PCR) DNA ampli¢cation method (ERICPCR) can be used to ¢ngerprint the genomes of different Rhizobium species [10^15] and Bradyrhizobium japonicum [16]. This technique has also been used successfully with nodule extracts [11,14]. The increasing use of these PCR-based techniques is due to their high discriminating power, their ease of standardisation and their suitability for computer-assisted analysis of the resulting patterns, which allows the elucidation of genetic relationships among the strains used [11]. Each strain identi¢cation procedure has its own advantages and drawbacks, but there is no published comparison between ERIC-PCR and LPS pro¢ling in terms of discriminating ability or ease of use. Such a comparison was the aim of this work. As a sample for the comparative studies, we used a collection of Bradyrhizobium strains isolated from Lotus subbi£orus that had previously been described and characterised with regard to growth, and by using serology and whole cell protein pro¢ling [4].
2. Materials and methods 2.1. Bacterial strains and culture condition Bradyrhizobium sp. (Lotus) strain NZP2309 was obtained from the Department of Microbiology, University of New South Wales, Kensington, Australia. Bradyrhizobium sp. (Lotus) strains Ls31, Ls11, Ls22, Ls81, Ls82, Ls42, Ls71, Ls7, Ls4, LsIB3 and LsIB4 were obtained from di¡erent areas in Uruguay
[4]. Bacteria were maintained on yeast-extract mannitol (YEM) medium [17]. 2.2. Electrophoresis of lipopolysaccharides The samples for electrophoresis were prepared by the Hitchcock and Brown method [18], with the following modi¢cations: the bacteria were grown at 28³C in yeast extract^mannitol medium containing (g l31 distilled water): mannitol, 1.0; yeast extract, 0.8; K2 HPO4 , 0.6; MgSO4 W7H2 O, 0.2; and NaCl, 0.2. After 5 days of incubation, the cultures were centrifuged at 10 000Ug for 20 min and the cell pellets were washed with 0.85% NaCl. The bacterial pellets were stored at 380³C until use. Bacteria were suspended in distilled water to an optical density (at 600 nm) of 1.0; 1 ml of the bacterial suspension was centrifuged at 13 000Ug for 5 min and the pellet was resuspended in 500 Wl of lysis bu¡er: 2% (w/v) SDS, 5% (w/v) dithiothreitol, 10% (v/v) glycerol, 0.017% (w/v) bromphenol blue in 0.5 M Tris bu¡er, pH 8.0. The samples were heated at 100³C for 10 min; after which, 20 Wl of a solution (2.5 Wg Wl31 ) of proteinase K (Boehringer) in lysis bu¡er was added and the mixtures were incubated under agitation at 60³C for 1 h. The samples were clari¢ed by centrifugation and the supernatants were stored at 320³C until use. Electrophoresis was carried out by the discontinuous method of Laemmli [19] in 0.5-mm-thick polyacrylamide slab gels. To study the e¡ect of di¡ering gel pore sizes in the LPS pro¢les, we used gels from 10% (w/v) to 17.5% polyacrylamide. The gels were stained by the periodic acid-silver method of Tsai and Frasch [20]. The stained gels were scanned using a Pharmacia-LKB Ultroscan XL densitometer and the software used for gel analysis and comparison was Pharmacia-LKB Analysis GelScan XL. The comparison of the pro¢les involved: (1) pro¢le normalisation to a ¢xed length; and (2) direct comparison of pro¢les by superimposing the normalised densitometric tracings. Two lanes were considered as identical when both positions and the relative intensities of all bands were the same. The comparison of pro¢les by methods based on presence^absence of bands and the subsequent construction of a similarity matrix is unsuitable for LPS, due to the fact that the bands are not mutually independent [9].
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2.3. Genomic DNA ¢ngerprinting
3. Results
DNA isolation from lysed cells was performed using the method described by Tichy and Simon [21]. When direct DNA ampli¢cation was not possible, DNA was puri¢ed using the Wizard DNA Genomic puri¢cation method (Promega, Madison, WI) with the following modi¢cations: 600 Wl of TEN8 (100 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS and 0.1 mg ml31 proteinase K in 20 mM Tris (pH 8.0)) was added to 1 ml of bacterial culture and incubated at 55³C for 15^30 min. Then 3 Wl of RNase solution kit (Promega) was added to the lysed cell suspension and DNA was isolated following the manufacturer's instructions. Repetitive-PCR ¢ngerprinting was performed using the ERIC consensus, ERIC 1R and ERIC 2 primers [10]. The PCR reaction was performed in a 10-Wl reaction mixture containing 50 mM KCl, 1.0 mM MgCl2 , 10 mM Tris-HCl pH 8.5, 0.5 U Taq (Gibco BRL), 250 WM of each dNTP and 10 pmol of each primer. In all reactions three serial dilutions of DNA from 1 to 10 ng were used. The cycling pro¢le for ERIC-PCR was as follows: one initial denaturation cycle of 5 min at 95³C, 35 cycles of denaturation at 94³C for 1 min, annealing at 52³C for 2 min, extension at 72³C for 1 min and ¢nal extension at 72³C for 5 min. All ampli¢cations were carried out using a Thermolyne model Amplitron II thermocycler. Ampli¢ed products were separated by electrophoresis on 6% polyacrylamide gels in a Mini-PROTEAN II apparatus with a 1-mm spacer and an 18-tooth comb (Bio-Rad, Hercules, CA, USA) and visualised by silver nitrate staining, as previously described [22]. The densitometry analyses and comparison of the pro¢les was done in the same way as for LPS, but a similarity matrix was constructed by using the bands from each lane whose area percentage was higher than 5% of the whole lane area. The similarity coef¢cient used was the Jaccard's coe¤cient The clustering of the pro¢les and resulting dendrogram were performed by using an unweighted pair group (UPGMA) algorithm (program MVSP, W.W. Kovach, University College of Wales, Aberystwyth, UK).
LPS pro¢les did not change among di¡erent cultures of each strain. The faster migrating (smaller) bands were better resolved in gels of high acrylamide concentration, whereas the slower bands were well resolved in more porous gels. Therefore, the LPS pro¢les of each strain were dependent on the acrylamide concentration. The best resolving power, in terms of the discernible number of bands in each lane, was obtained with gels of intermediate (12.5 or 15%) acrylamide. However, irrespective of the acrylamide concentration used, the grouping of the strains by their LPS pro¢les was the same in the range of 10^17.5% acrylamide (data not shown). Fig. 1 shows the electrophoretic LPS pro¢les obtained using 15% acrylamide gels. The isolates, serologically similar to strain Ls31 (strains Ls11, Ls22, Ls31, Ls81, Ls82, Ls42 and Ls71, [4]), produced similar patterns, characterised by the presence of tightly clustered bands of intermediate mobility. However, it was possible to di¡erentiate pro¢les of three types: (1) from the strains Ls11, Ls22, Ls31, Ls81 and Ls82; (2) produced by strain Ls42, that was very
Fig. 1. Silver stained lipopolysaccharide electrophoretic pro¢les from the Bradyrhizobium (Lotus) strains studied on a 15% polyacrylamide gel.
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similar to the former group but with a very prominent band migrating faster than the main cluster; and (3) produced by strain Ls71, whose pattern showed only a faint cluster and a group of intense bands. Strain LsIB3, which does not belong to the same serogroup [4], produced a similar pattern, but di¡erent from that produced by strains of serogroup 31. The Uruguayan isolates Ls7, Ls4 and LsIB4 and the New Zealander isolate NZP2309 produced individual patterns. Results shown in Fig. 2 were repeated three times with serial dilutions of DNA and the pro¢le for each strain was identical in the di¡erent experiments. DNA ampli¢cation directly from the lysates of colonies of the strains serologically related to strain LS31 was successful and we obtained reproducible patterns without previous DNA puri¢cation. By contrast, DNA of strains Ls7, Ls4, LsIB3, LsIB4 and NZP2037 could not be ampli¢ed from lysed cells, probably due to the presence of PCR-reaction inhibitors in these strains. DNA puri¢cation was required for reproducible PCR-¢ngerprinting of these strains. The resulting dendrogram is shown in Fig. 3. ERICPCR separated the strains belonging to serogroup 31 into three groups: Group A (strains Ls31, Ls11 and Ls22), Group B (strains Ls81, Ls82. and Ls42), and Group C (strain Ls71). It is worthwhile noting that
Fig. 2. Genomic ¢ngerprints from whole cell lysates of Bradyrhizobium sp. (Lotus) strains. DNA ampli¢cation and electrophoresis conditions were as described in Section 2.
Fig. 3. Dendrogram constructed with the data from Fig. 2. The gel was analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for constructing a presence^absence matrix. The similarities were calculated using the Jaccard's coe¤cient, and the clustering was done by using the unweighted pair group method. All calculations were done by using the MVSP (Warren W. Kovach, University College of Wales, Aberystwyth, UK) program for multivariate analysis.
strain IB3, which does not belong to serogroup 31, has LPS and ERIC-PCR pro¢les related to strains of that serogroup.
4. Discussion Both LPS pro¢ling and ERIC-PCR ¢ngerprinting were able to resolve more clusters in the samples studied than any other technique previously applied to this group of bradyrhizobia [4]. For instance, both methods were able to clearly di¡erentiate between strains LsIB3 and LsIB4, which appeared to be identical by serology and by whole cell protein pro¢ling [4]. Both techniques were also able to divide serogroup 31 (which was found to be homogeneous by protein pro¢ling) into three subgroups. However, in this case, there was a discrepancy between these two high-resolution techniques. By LPS pro¢ling, strains Ls81 and Ls82 were identical to strains Ls11, Ls22 and Ls31, whereas by ERIC-PCR they belonged to the same group as strain Ls42. By both procedures, strain Ls71 was clearly di¡erent from the other components of the serogroup 31. In conclusion, both methods showed a similar discriminating power, and were able to resolve the sample into the same number of groups, although two groups had slightly di¡erent compositions. In terms of ease of use, LPS pro¢ling was faster,
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cheaper and simpler than ERIC-PCR ¢ngerprinting. LPS pro¢ling was also more reliable, in the sense that all strains could be processed in the same way. It has been proposed that LPS pro¢les change when bacteria are grown in di¡erent media, or in the bacteroids. However, we have shown that the LPS pro¢les are the same for free cultured bacteria and for bacteroids in a group of Bradyrhizobium isolates [9]. As LPS pro¢ling does not require any ampli¢cation step, it has the additional advantage that it is less sensitive than ERIC-PCR to the presence of contaminants in the sample that could produce spurious bands. However, LPS pro¢ling has an important limitation, since the relative similarity between LPS electrophoretic patterns did not allow us to draw conclusions about the genetic relationships between the strains compared. This limitation is due to the fact that the LPS pattern depends on the LPS biosynthetic enzymes of the bacteria, and there is no proof of correlation between the type of enzymes for LPS synthesis and the structure of the bacterial genome. LPS pro¢ling is a good tool for strain identi¢cation, but in the absence of other evidence, one should not draw the conclusion that two strains with similar LPS patterns are more closely related to each other than to other strains with di¡erent patterns. Therefore, LPS pro¢ling is not useful for population genetics studies, nor for evolutionary and phylogenetic studies. On the other hand, ERIC-PCR was sometimes cumbersome, since it required a further DNA isolation step for some strains. This problem could be speci¢c for Bradyrhizobium strains, because it has not been reported for other rhizobia [13^15]. In conclusion, both techniques are discriminatory, and in some cases complementary. For example, whereas LPS pro¢ling permits good discrimination of Bradyrhizobium strains, it is less useful for Rhizobium species since generally their LPS lack the distinctive ladder-like pattern of the former [1]. ERICPCR pro¢ling works very well with isolated cells or extracts from nodules produced by Rhizobium [13,14], whereas a universal protocol for Bradyrhizobium seems to require an additional DNA puri¢cation step, precluding its application to direct nodule typing. Furthermore, preliminary studies carried out in our laboratory showed that ERIC-PCR from Lotus nodules was unsuccessful, probably due to interference from polyphenols, polysaccharides or tan-
167
nins of plant origin [23] (data not shown). The Bradyrhizobium sample studied here is too small to draw universal conclusions, but we suggest that, taking into account the advantages and limitations of each technique, a reasonable strategy for the study of bradyrhizobial populations involving numerous isolates could be: (1) LPS pro¢ling for fast grouping of the isolates; and (2) genomic ¢ngerprinting of a representative of each LPS group to obtain the desired information about the genetic relationships among them.
Acknowledgments The work of M.S., A.M.G.N. and J.C. was supported by a grant of the Canary Island Government. The work of F.A. and J.M. was supported by CSIC Project 205 and PEDECIBA. We thank Prof. Meleèndez-Hevia for his facilities in the densitometric analysis.
References [1] Santamaria, M., Corzo, J., Leon-Barrios, M. and GutierrezNavarro, A.M. (1997) Characterisation and di¡erentiation of indigenous rhizobia isolated from Canarian shrub legumes of agricultural and ecological interest. Plant Soil 190, 143^152. [2] Barnet, Y.M. (1991) Ecology of legume root-nodule bacteria. In: Biology and Biochemistry of Nitrogen Fixation (Dilworth, M.J. and Glenn, A.R., Eds.), pp. 199^228. Elsevier, Amsterdam. [3] Monza, J., Fabiano, E. and Arias, A. (1992) Characterization of an indigenous population of rhizobia nodulating Lotus corniculatus. Soil Biol. Biochem. 24, 241^247. [4] Irisarri, P., Milnitsky, F., Monza, J. and Bedmar, E.J. (1996) Characterization of rhizobia nodulating Lotus subbi£orus from Uruguayan soils. Plant Soil 180, 39^47. [5] Noel, K.D. and Brill, W.J. (1980) Diversity and dynamics of indigenous Rhizobium japonicum populations. Appl. Environ. Microbiol. 40, 931^938. [6] Kamicker, B.J. and Brill, W.J. (1986) Identi¢cation of Bradyrhizobium japonicum nodule isolates from Wisconsin soybean farms. Appl. Environ. Microbiol. 51, 487^492. [7] De Maagd, R.A., Van Rossum, C. and Lugtenberg, B.J.J. (1988) Recognition of individual strains of fast-growing rhizobia by using pro¢les of membrane proteins and lipopolysaccharides. J. Bacteriol. 170, 3782^3785. [8] Lindstroëm, K. and Zahran, H.H. (1993) Lipopolysaccharide patterns in SDS-PAGE of rhizobia that nodulate leguminous trees. FEMS Microbiol. Lett. 107, 327^330.
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[9] Santamaria, M., Gutierrez-Navarro, A.M. and Corzo, J. (1998) Lipopolysaccharide pro¢les from nodules as strains markers of Bradyrhizobium nodulating wild legumes. Appl. Environ. Microbiol. 64, 902^906. [10] De Bruijn, F.J. (1992) Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain reaction to ¢ngerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol. 58, 2180^2187. [11] Schneider, M. and de Bruijn, F.J. (1996) Rep-PCR mediated genomic ¢ngerprinting of rhizobia and computed-assisted phylogenetic pattern analysis. World J. Microbiol. Biotech. 12, 163^174. [12] Selenska-Pobell, S., Gigova, L. and Petrova, N. (1995) Strainspeci¢c ¢ngerprinting of Rhizobium galegae generated by PCR with arbitrary and repetitive primers. J. Appl. Bacteriol. 79, 425^431. [13] Labes, G., Ulrich, A. and Lentzsch, P. (1996) In£uence of bovine slurry deposition on the structure of nodulating Rhizobium leguminosarum bv. viciae soil populations in natural habitat. Appl. Environ. Microbiol. 62, 1717^1722. [14] Nick, G. and Lindstroëm, K. (1994) Use of repetitive sequences and the polymerase chain reaction to ¢ngerprint the genomic DNA of Rhizobium galegae strains and to identify the DNA obtained by sonicating the liquid cultures and root nodules. System. Appl. Microbiol. 17, 265^273. [15] Agius, F., Sanguinetti, C. and Monza, J. (1997) Strain-speci¢c ¢ngerprints of Rhizobium loti generated by PCR with arbitrary and repetitive sequences. FEMS Microbiol. Ecol. 24, 87^94.
[16] Judd, A.K., Schneider, M., Sadowsky, M. and de Bruijn, F. (1993) Use repetitive sequence and polymerase chain reaction technique to classify genetically related Bradyrhizobium japonicum serocluster 123 strain. Appl. Environ. Microbiol. 59, 1702^1708. [17] Vincent, J.M. (1970) A Manual for the Practical Study of Root-Nodule Bacteria, pp. 1^13. Blackwell, Oxford. [18] Hitchcock, P.J. and Brown, T.M. (1983) Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154, 269^ 277. [19] Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680^ 685. [20] Tsai, C.M. and Frasch, C.E. (1982) A sensitive silver staining for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115^119. [21] Tichy, H.V. and Simon, R. (1993) Alternative PCR-¢ngerprinting protocols. In: Molecular Ecology of Rhizosphere Bacteria (Dowling, D.N., Boesten, B. and O'Gara, F., Eds.), pp. 14^21. University College, Cork. [22] Sanginetti, C., Diaz Nieto, E. and Simson, A.J.G. (1994) Rapid silver staining and recovery of PCR products separated on acrylamide gels. BioTechniques 17, 915^918. [23] Porebski, S., Bayley, G. and Baum, R. (1997) Modi¢cation of a CTAB-DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 15, 8^15.
FEMSEC 987 4-2-99
Comparative performance of enterobacterial repetitive intragenic consensus-polymerase chain reaction and lipopolysaccharide electrophoresis for the identi¢cation of Bradyrhizobium sp. (Lotus) strains M. Santamar|èa a , F. Agius b , J. Monza b , A.M. Gutieèrrez-Navarro c , J. Corzo a; * a b
Departamento de Bioqu|èmica y Biolog|èa Molecular, Universidad de La Laguna, 38206 Tenerife, Spain Laboratorio de Bioqu|èmica, Facultad de Agronom|èa, Avda. Garzoèn 780, CP 12900, Montevideo, Uruguay c Departamento de Microbiolog|èa y Biolog|èa Celular, Universidad de La Laguna, 38206 Tenerife, Spain Received 17 June 1998; received in revised form 16 September 1998; accepted 14 October 1998
Abstract We compared two methods for typing bacterial strains : electrophoretic lipopolysaccharide profiling and genomic fingerprinting by enterobacterial repetitive intragenic consensus-polymerase chain reaction. Our aim was to assess the relative utility of these techniques for identification of bradyrhizobia. A collection of Uruguayan Bradyrhizobium strains isolated from Lotus subbiflorus was selected to test both techniques in terms of their discriminating ability and ease of use. Both techniques were found to be equally discriminating and they classified the samples in the same way, although each method ascribed two strains to different groups. Genomic profiling of some strains required previous DNA purification, whereas this was found to be unnecessary for others. Lipopolysaccharide profiling was found to be easier and cheaper to perform, but was not useful for determination of the genetic relationship among the strains. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bradyrhizobium (Lotus); Lipopolysaccharide; PCR ¢ngerprint; Strain identi¢cation
1. Introduction Ecological studies of natural populations of rhizobia require fast and reliable techniques for strain identi¢cation. To be useful in ¢eld studies, an identi¢cation technique for a given population must pro-
* Corresponding author. Tel.: +34 (22) 318-356; Fax: +34 (22) 318354; E-mail: [email protected]
duce the maximum number of di¡erent groups in a reproducible way [1] and must be independent of any previous manipulation of the strains [2]. Serological methods have been used for strain identi¢cation, but they are limited since they do not yield information on strains that do not react with a given set of antibodies, and therefore these strains are arti¢cially grouped. The electrophoresis of whole cell proteins avoids such arti¢cial grouping and has been used as a discriminating tool for strain identi¢cation [3^6], but the resulting pro¢les are
0168-6496 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 1 0 1 - 9
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rather complex and di¤cult to compare. More clearcut and easily compared patterns are produced by the electrophoresis of lipopolysaccharides (LPS) and these patterns have been used successfully for the di¡erentiation of rhizobia isolated from diverse sources [1,7,8]. A further advantage of LPS typing is that it is a fast and reliable technique that can be also applied to extracts from intact nodules, avoiding the necessity of bacterial isolation and cultivation [9]. A di¡erent type of approach, which is increasingly used, is based on DNA ¢ngerprinting. It has been shown that DNA primers corresponding to the enterobacterial repetitive intragenic consensus (ERIC) sequence [10], coupled with the polymerase chain reaction (PCR) DNA ampli¢cation method (ERICPCR) can be used to ¢ngerprint the genomes of different Rhizobium species [10^15] and Bradyrhizobium japonicum [16]. This technique has also been used successfully with nodule extracts [11,14]. The increasing use of these PCR-based techniques is due to their high discriminating power, their ease of standardisation and their suitability for computer-assisted analysis of the resulting patterns, which allows the elucidation of genetic relationships among the strains used [11]. Each strain identi¢cation procedure has its own advantages and drawbacks, but there is no published comparison between ERIC-PCR and LPS pro¢ling in terms of discriminating ability or ease of use. Such a comparison was the aim of this work. As a sample for the comparative studies, we used a collection of Bradyrhizobium strains isolated from Lotus subbi£orus that had previously been described and characterised with regard to growth, and by using serology and whole cell protein pro¢ling [4].
2. Materials and methods 2.1. Bacterial strains and culture condition Bradyrhizobium sp. (Lotus) strain NZP2309 was obtained from the Department of Microbiology, University of New South Wales, Kensington, Australia. Bradyrhizobium sp. (Lotus) strains Ls31, Ls11, Ls22, Ls81, Ls82, Ls42, Ls71, Ls7, Ls4, LsIB3 and LsIB4 were obtained from di¡erent areas in Uruguay
[4]. Bacteria were maintained on yeast-extract mannitol (YEM) medium [17]. 2.2. Electrophoresis of lipopolysaccharides The samples for electrophoresis were prepared by the Hitchcock and Brown method [18], with the following modi¢cations: the bacteria were grown at 28³C in yeast extract^mannitol medium containing (g l31 distilled water): mannitol, 1.0; yeast extract, 0.8; K2 HPO4 , 0.6; MgSO4 W7H2 O, 0.2; and NaCl, 0.2. After 5 days of incubation, the cultures were centrifuged at 10 000Ug for 20 min and the cell pellets were washed with 0.85% NaCl. The bacterial pellets were stored at 380³C until use. Bacteria were suspended in distilled water to an optical density (at 600 nm) of 1.0; 1 ml of the bacterial suspension was centrifuged at 13 000Ug for 5 min and the pellet was resuspended in 500 Wl of lysis bu¡er: 2% (w/v) SDS, 5% (w/v) dithiothreitol, 10% (v/v) glycerol, 0.017% (w/v) bromphenol blue in 0.5 M Tris bu¡er, pH 8.0. The samples were heated at 100³C for 10 min; after which, 20 Wl of a solution (2.5 Wg Wl31 ) of proteinase K (Boehringer) in lysis bu¡er was added and the mixtures were incubated under agitation at 60³C for 1 h. The samples were clari¢ed by centrifugation and the supernatants were stored at 320³C until use. Electrophoresis was carried out by the discontinuous method of Laemmli [19] in 0.5-mm-thick polyacrylamide slab gels. To study the e¡ect of di¡ering gel pore sizes in the LPS pro¢les, we used gels from 10% (w/v) to 17.5% polyacrylamide. The gels were stained by the periodic acid-silver method of Tsai and Frasch [20]. The stained gels were scanned using a Pharmacia-LKB Ultroscan XL densitometer and the software used for gel analysis and comparison was Pharmacia-LKB Analysis GelScan XL. The comparison of the pro¢les involved: (1) pro¢le normalisation to a ¢xed length; and (2) direct comparison of pro¢les by superimposing the normalised densitometric tracings. Two lanes were considered as identical when both positions and the relative intensities of all bands were the same. The comparison of pro¢les by methods based on presence^absence of bands and the subsequent construction of a similarity matrix is unsuitable for LPS, due to the fact that the bands are not mutually independent [9].
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2.3. Genomic DNA ¢ngerprinting
3. Results
DNA isolation from lysed cells was performed using the method described by Tichy and Simon [21]. When direct DNA ampli¢cation was not possible, DNA was puri¢ed using the Wizard DNA Genomic puri¢cation method (Promega, Madison, WI) with the following modi¢cations: 600 Wl of TEN8 (100 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS and 0.1 mg ml31 proteinase K in 20 mM Tris (pH 8.0)) was added to 1 ml of bacterial culture and incubated at 55³C for 15^30 min. Then 3 Wl of RNase solution kit (Promega) was added to the lysed cell suspension and DNA was isolated following the manufacturer's instructions. Repetitive-PCR ¢ngerprinting was performed using the ERIC consensus, ERIC 1R and ERIC 2 primers [10]. The PCR reaction was performed in a 10-Wl reaction mixture containing 50 mM KCl, 1.0 mM MgCl2 , 10 mM Tris-HCl pH 8.5, 0.5 U Taq (Gibco BRL), 250 WM of each dNTP and 10 pmol of each primer. In all reactions three serial dilutions of DNA from 1 to 10 ng were used. The cycling pro¢le for ERIC-PCR was as follows: one initial denaturation cycle of 5 min at 95³C, 35 cycles of denaturation at 94³C for 1 min, annealing at 52³C for 2 min, extension at 72³C for 1 min and ¢nal extension at 72³C for 5 min. All ampli¢cations were carried out using a Thermolyne model Amplitron II thermocycler. Ampli¢ed products were separated by electrophoresis on 6% polyacrylamide gels in a Mini-PROTEAN II apparatus with a 1-mm spacer and an 18-tooth comb (Bio-Rad, Hercules, CA, USA) and visualised by silver nitrate staining, as previously described [22]. The densitometry analyses and comparison of the pro¢les was done in the same way as for LPS, but a similarity matrix was constructed by using the bands from each lane whose area percentage was higher than 5% of the whole lane area. The similarity coef¢cient used was the Jaccard's coe¤cient The clustering of the pro¢les and resulting dendrogram were performed by using an unweighted pair group (UPGMA) algorithm (program MVSP, W.W. Kovach, University College of Wales, Aberystwyth, UK).
LPS pro¢les did not change among di¡erent cultures of each strain. The faster migrating (smaller) bands were better resolved in gels of high acrylamide concentration, whereas the slower bands were well resolved in more porous gels. Therefore, the LPS pro¢les of each strain were dependent on the acrylamide concentration. The best resolving power, in terms of the discernible number of bands in each lane, was obtained with gels of intermediate (12.5 or 15%) acrylamide. However, irrespective of the acrylamide concentration used, the grouping of the strains by their LPS pro¢les was the same in the range of 10^17.5% acrylamide (data not shown). Fig. 1 shows the electrophoretic LPS pro¢les obtained using 15% acrylamide gels. The isolates, serologically similar to strain Ls31 (strains Ls11, Ls22, Ls31, Ls81, Ls82, Ls42 and Ls71, [4]), produced similar patterns, characterised by the presence of tightly clustered bands of intermediate mobility. However, it was possible to di¡erentiate pro¢les of three types: (1) from the strains Ls11, Ls22, Ls31, Ls81 and Ls82; (2) produced by strain Ls42, that was very
Fig. 1. Silver stained lipopolysaccharide electrophoretic pro¢les from the Bradyrhizobium (Lotus) strains studied on a 15% polyacrylamide gel.
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similar to the former group but with a very prominent band migrating faster than the main cluster; and (3) produced by strain Ls71, whose pattern showed only a faint cluster and a group of intense bands. Strain LsIB3, which does not belong to the same serogroup [4], produced a similar pattern, but di¡erent from that produced by strains of serogroup 31. The Uruguayan isolates Ls7, Ls4 and LsIB4 and the New Zealander isolate NZP2309 produced individual patterns. Results shown in Fig. 2 were repeated three times with serial dilutions of DNA and the pro¢le for each strain was identical in the di¡erent experiments. DNA ampli¢cation directly from the lysates of colonies of the strains serologically related to strain LS31 was successful and we obtained reproducible patterns without previous DNA puri¢cation. By contrast, DNA of strains Ls7, Ls4, LsIB3, LsIB4 and NZP2037 could not be ampli¢ed from lysed cells, probably due to the presence of PCR-reaction inhibitors in these strains. DNA puri¢cation was required for reproducible PCR-¢ngerprinting of these strains. The resulting dendrogram is shown in Fig. 3. ERICPCR separated the strains belonging to serogroup 31 into three groups: Group A (strains Ls31, Ls11 and Ls22), Group B (strains Ls81, Ls82. and Ls42), and Group C (strain Ls71). It is worthwhile noting that
Fig. 2. Genomic ¢ngerprints from whole cell lysates of Bradyrhizobium sp. (Lotus) strains. DNA ampli¢cation and electrophoresis conditions were as described in Section 2.
Fig. 3. Dendrogram constructed with the data from Fig. 2. The gel was analyzed by densitometry and the bands whose areas were greater than 5% of the whole lane area were used for constructing a presence^absence matrix. The similarities were calculated using the Jaccard's coe¤cient, and the clustering was done by using the unweighted pair group method. All calculations were done by using the MVSP (Warren W. Kovach, University College of Wales, Aberystwyth, UK) program for multivariate analysis.
strain IB3, which does not belong to serogroup 31, has LPS and ERIC-PCR pro¢les related to strains of that serogroup.
4. Discussion Both LPS pro¢ling and ERIC-PCR ¢ngerprinting were able to resolve more clusters in the samples studied than any other technique previously applied to this group of bradyrhizobia [4]. For instance, both methods were able to clearly di¡erentiate between strains LsIB3 and LsIB4, which appeared to be identical by serology and by whole cell protein pro¢ling [4]. Both techniques were also able to divide serogroup 31 (which was found to be homogeneous by protein pro¢ling) into three subgroups. However, in this case, there was a discrepancy between these two high-resolution techniques. By LPS pro¢ling, strains Ls81 and Ls82 were identical to strains Ls11, Ls22 and Ls31, whereas by ERIC-PCR they belonged to the same group as strain Ls42. By both procedures, strain Ls71 was clearly di¡erent from the other components of the serogroup 31. In conclusion, both methods showed a similar discriminating power, and were able to resolve the sample into the same number of groups, although two groups had slightly di¡erent compositions. In terms of ease of use, LPS pro¢ling was faster,
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cheaper and simpler than ERIC-PCR ¢ngerprinting. LPS pro¢ling was also more reliable, in the sense that all strains could be processed in the same way. It has been proposed that LPS pro¢les change when bacteria are grown in di¡erent media, or in the bacteroids. However, we have shown that the LPS pro¢les are the same for free cultured bacteria and for bacteroids in a group of Bradyrhizobium isolates [9]. As LPS pro¢ling does not require any ampli¢cation step, it has the additional advantage that it is less sensitive than ERIC-PCR to the presence of contaminants in the sample that could produce spurious bands. However, LPS pro¢ling has an important limitation, since the relative similarity between LPS electrophoretic patterns did not allow us to draw conclusions about the genetic relationships between the strains compared. This limitation is due to the fact that the LPS pattern depends on the LPS biosynthetic enzymes of the bacteria, and there is no proof of correlation between the type of enzymes for LPS synthesis and the structure of the bacterial genome. LPS pro¢ling is a good tool for strain identi¢cation, but in the absence of other evidence, one should not draw the conclusion that two strains with similar LPS patterns are more closely related to each other than to other strains with di¡erent patterns. Therefore, LPS pro¢ling is not useful for population genetics studies, nor for evolutionary and phylogenetic studies. On the other hand, ERIC-PCR was sometimes cumbersome, since it required a further DNA isolation step for some strains. This problem could be speci¢c for Bradyrhizobium strains, because it has not been reported for other rhizobia [13^15]. In conclusion, both techniques are discriminatory, and in some cases complementary. For example, whereas LPS pro¢ling permits good discrimination of Bradyrhizobium strains, it is less useful for Rhizobium species since generally their LPS lack the distinctive ladder-like pattern of the former [1]. ERICPCR pro¢ling works very well with isolated cells or extracts from nodules produced by Rhizobium [13,14], whereas a universal protocol for Bradyrhizobium seems to require an additional DNA puri¢cation step, precluding its application to direct nodule typing. Furthermore, preliminary studies carried out in our laboratory showed that ERIC-PCR from Lotus nodules was unsuccessful, probably due to interference from polyphenols, polysaccharides or tan-
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nins of plant origin [23] (data not shown). The Bradyrhizobium sample studied here is too small to draw universal conclusions, but we suggest that, taking into account the advantages and limitations of each technique, a reasonable strategy for the study of bradyrhizobial populations involving numerous isolates could be: (1) LPS pro¢ling for fast grouping of the isolates; and (2) genomic ¢ngerprinting of a representative of each LPS group to obtain the desired information about the genetic relationships among them.
Acknowledgments The work of M.S., A.M.G.N. and J.C. was supported by a grant of the Canary Island Government. The work of F.A. and J.M. was supported by CSIC Project 205 and PEDECIBA. We thank Prof. Meleèndez-Hevia for his facilities in the densitometric analysis.
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