Electrophoresis zyxwvutsrqponml 1998, 19, 2621-2624 zyxwvutsrqpon Electrophoresis of Bradyrhizobium polysaccharides 262 1 Ana R. Diaz-Marrero' M6nica Santamaria' Ana Poveda2 Jesus Jimknez-Barbero3 Javier Corzo' 'Departamento de Bioquimica y Biologia Molecular, Universidad de La Laguna, Tenerife, Spain 2Servicio Interdepartamental de Investigacih, Universidad Authoma de Madrid, Madrid, Spain 31nstituto de Quimica Orghnica, CSIC, Madrid, Spain Electrophoretic behavior and size distribution of the acidic polysaccharides produced by the bacteria Bradyrhizobium (Chamaecytisus) strain BGA-1 and Bradyrhizobium japonicum USDA 110 The electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteria Bradyrhizobium (Chamaecytisus) strain BGAl and Bradyrhizobium japonicum USDAl 10 has been studied. Both polysaccharides were polydisperse, producing a ladder-like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced by B. japonicum USDAl10 was wider and with larger molecules than that of the polysaccharide produced by strain BGAl zyxwvut . The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris-borate; in Tris-glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate (SDS). 1 Introduction The number of papers in which polysaccharides (other than lipopolysaccharides) are characterized by electrophoresis are scarce except for some acidic polysaccharides of intermediate size. Other than structural considerations, a possible reason for the little use of electrophoresis in the analysis of polysaccharides could be the lack of problems in which it could really be useful; in some way, polysaccharide electrophoresisis zyxwvuts an answer searching for a question. In this paper we describe the electrophoretic behavior of exopoly- saccharides from Bradyrhizobium (Chamaecytisus) and the use of PAGE to search for size differences between the former and the exopolysaccharide (EPS) produced by Bradyrhizobium japonicum USDA-110 (EPS-USDA1 10). The acidic polysaccharide produced by the bacterium Bradyrhizobium sp. (Chamaecytisus) BGA-1 (EPS-BGAI) is of interest because it precipitated with hydrolyzed metallic cations of the form Men(OH),2+ [l]. It is formed by the repetition of the pentasaccharide unit: +3-[a-~- Galp-(1+6)]-cl-~-Glcp-( 1-+3)-P-~-Glcp-( zyxwvut 1 +3)-a-~-Galp- A-( 1+3)-a-~-Manp-( 1 +). The galactose residue is sub- stituted in a nonstoichometric way by 0-methyl and acetyl groups [2]. B. japonicum USDA 110 produces a poly- saccharide (EPS-USDAl10) that is composed of the same repeating unit [3] as EPS-BGA1, but it is more acetylated [ 11. Nothing is known about the electrophoretic behavior of EPS-USDA110, but a sample of EPS-BGAl showed a ladder-like pattern in PAGE that was proposed as proof of the polydisperse nature of this molecule [4]. However, the published electrophoretic profile of the EPS-BGA1 shows a nonresolved smear above the ladder pattern [4]. Note that the chargelmass ratio of EPS-BGA1 (only one acidic group Correspondence: Javier Corzo, Departamento de Bioquimica y Biologia Molecular, Universidad de La Laguna, 38206 Tenerife, Spain (E-mail: fcorzo@ull.es) Abbreviations: EPS-BGAl, exopolysaccharide produced by Bradyrhi- zobium (Chamaecytisus) strain BGA- 1 ; EPS-USDAl10, exopolysacchar- ide produced by Bradyrhizobium japonicum strain USDA 110 Keywords: Acidic polysaccharides / Exopolysaccharides / Soil bacteria zyxwvuts I Polyacrylamide gel electrophoresis by pentasaccharide repeating unit) is far less than that of Rhizobium capsular polysaccharides [5], polyuronates [6,7] or glycosylaminoglycans [8] in which the separations according the size have been previously demonstrated. Furthermore, the composition of EPS-BGA1 published in [4] was slightly different than that corresponding to its structure [2]; it could thus be possible that a contaminating capsular polysaccharide of the type described in Rhizobium meliloti [9] was responsible for the ladder-like pattern. This is possible because Rhizobium capsular polysaccharides are polydisperse molecules with one negative charge on each disaccharide-repeatingunit and they produced a ladder-like pattern in polyacrylamide gels after their previous fixation with Alcian blue [9]. Our first goal is to prove that highly purified EPS-BGA1 is polydisperse and that PAGE separates their molecules according to their size and produces a ladder-like pattern as described for other acidic polysaccharides. 2 Materials and methods 2.1 Polysaccharide purification and gel filtration The EPS were obtained from 7-day-old culture broth of B. japonicum USDAl 10 and B. (Chamaecytisus) BGAl grown in mannitol-yeast extract medium [lo]. The "EPS purification procedure by ethanol precipitation of the culture media and further ion-exchange chromatography in DEAE-Sephacel of the crude polysaccharide and removal of acetyl groups by hydrolysis in KOH have been previously described [ 11. Protein and nucleic acid contam- ination were investigated by absorbance measurements between 240 and 300 nm of the purified EPS at a concentration of zyxw 5 mg/mL in water. The presence of contaminating polysaccharides or lipopolysaccharides and the result of the deacetylation were checked by 'H-NMR spectroscopy of EPS (14 mgimL in D20) in a Brucker zy AMX 400 spectrometer (Bruker Analytik, Ettlingen, Germany). No NMR peaks other than those corresponding to the known structure of EPS-BGA1 were found. EPS-BGA1 was chromatography 0 WILEY-VCH Verlag GmbH, 69451 Weinheim, 1998 0173-0835/98/1515-2621 $17.50+.50/0