SEQUENCE VARIATIONS IN SMALL-SUBUNIT RIBOSOMAL-RNAS OF HARTMANNELLA-VERMIFORMIS AND THEIR PHYLOGENETIC IMPLICATIONS

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic rel ationships that may be important for the understanding of pathogenicit y in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phyloge ny of Hartmannella vermiformis, a free-living amoeba that can harbor t he etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide seque nces were determined for nuclear srDNA from three strains of H. vermif ormis isolated from the United Kingdom, Germany, and the United States , These sequences then were compared with a sequence previously obtain ed for a North American isolate by J. H. Gunderson and M. L. Sogin. Th e four genes are 1,840 bp long, with an average GC content of 49.6%. S equence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 mono typic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA se condary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Crypt omonas. All ditypic sites were consistent with a separation between Eu ropean and North American strains of Hartmannella, but results of othe r tests of this relationship were statistically inconclusive.