SDS-PAGE of Whole-Cell Proteins and Random Amplified Polymorphic DNA (RAPD) Analyses ofLeptotrichiaIsolates

Abstract

In a polyphasic approach to improve the taxonomy of Leptotrichia we have recently demonstrated heterogeneity in the enzymatic/biochemical reactions and cellular fatty acid content of Leptotrichia isolates. In the present study, SDS-PAGE of whole-cell proteins and random amplified polymorphic DNA (RAPD) analyses were used to further characterize and distinguish these isolates of which 57 had been assigned as Leptotrichia buccalis and two as ‘ Leptotrichia pseudobuccalis ’. The phylogenetically closely related Sebaldella termitidis T and Fusobacterium perfoetens T were included for comparison. Two major protein bands at approximately 30–40 and 70–80 kDa, respectively, were characteristic for almost all Leptotrichia strains examined as well as for S. termitidis T while F. perfoetens T exhibited only the 70–80 kDa major band. Numerical data analysis of the gel patterns obtained with SDS-PAGE assigned most of the strains into 12 clusters at 70% similarity. On RAPD gels with the primer OPA 3 most of the strains shared only one band at approximately 0.40–0.50 kb. With OPA 5 almost all the strains showed two major bands at approximately 0.40 and 0.85 kb, respectively. Numerical analysis of RAPD gels at 70% similarity assigned most strains into ten clusters when the OPA 3 had been used and into 14 clusters with OPA 5. Accordingly, both SDS-PAGE and RAPD demonstrated considerable heterogeneity among Leptotrichia isolates. OPA 5 provided better strain distinction than did OPA 3. Keywords: Leptotrichia , cellular proteins, SDS-PAGE, RAPD.