Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of Klebsiella Strains

Abstract

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-,ul samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains ofKlebsieUla pneumoniae and 24 strains ofKlebsieUla oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.

Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis–nitrocellulose blotting

“…The aim of the present work was to examine if the MLEE method that we originally developed for K. pneumoniae and K. oxytoca [7] was only applicable to the study of these bacteria, or could be proposed whatever the bac-terial species considered. Thus, to assess the general suitability of the present method, we analyzed a set of widely diverse bacterial species.…”

“…Bacterial strains included in this study belonged to the Gram-positive aerobic species Staphylococcus aureus, Streptococcus pneumoniae [8], and Streptococcus agalactiae, the Gram-negative aerobic species Klebsiella pneumoniae and K. oxytoca [7], the Gram-positive anaerobic species Clostridium bifermentans and C. sordellii [9], and the Gram-negative anaerobic species Prevotella bivia (Table 1).…”

“…Protein extracts were prepared from bacterial cells as previously described [7]. Brie£y, washed cells were collected by centrifugation and were disrupted by sonication ¢ve times for 20 s at 0³C (after preliminary treatment with lysostaphin for 45 min at 37³C in the case of S. aureus).…”

“…The MLEE method used in this study, initially developed for Klebsiella sp. strains [7], was further evaluated for the species mentioned above: thin horizontal slab gels (1 mm thick) contained 0.8% (w/v) agarose, 6% (w/v) polyacrylamide, 1.6% (w/v) N,NP-methylene-bisacrylamide in gel bu¡er (37.5 mM Tris^30 mM glycine, pH 8.7), and were polymerized by 0.03% (v/v) TEMED and 0.012% (w/v) ammonium persulfate. Agarose (indubiose A37HAA) was purchased from IBF Biotechnics (Villeneuve la Garenne, France) and the other gel components from Eastman Kodak Co. (Rochester, NY, USA).…”

“…The need for an alternative method allowing multiple enzyme analysis from single electrophoretic gels was previously pointed out, but the method proposed, based on the application of indicator agarose gels on separation polyacrylamide gels, was only evaluated for Staphylococcus aureus strains [6]. Studying genetic relationships within the genus Klebsiella, we described another method based on the combination of polyacrylamide^agarose gel electrophoresis with serial electrophoretic transfers onto nitrocellulose sheets, which allows the simultaneous analysis of several enzymes from a single separating gel [7]. The aim of the present work was to establish that this time-saving method is suitable for the analysis of enzyme polymorphism in a wide variety of bacteria.…”