Comparative sequence analysis of 16S rRNA genes was used to determine
the phylogenetic relationship of the genus Cristispira to other spiroc
hetes. Since Cristispira organisms cannot presently be grown in vitro,
16S rRNA genes were amplified directly from bacterial DNA isolated fr
om Cristispira a cell-laden crystalline styles of the oyster Crassostr
ea virginica. The amplified products were then cloned into Escherichia
coli plasmids. Sequence comparisons of the gene coding for 16S rRNA (
rDNA) insert of one clone, designated CP1, indicated that it was spiro
chetal. The sequence of the 16S rDNA insert of another clone was mycop
lasmal. The CP1 sequence possessed most of the individual base signatu
res that are unique to 16S rRNA (or rDNA) sequences of known spirochet
es. CP1 branched deeply among other spirochetal genera within the fami
ly Spirochaetaceae, and accordingly, it represents a separate genus wi
thin this family. A fluorescently labeled DNA probe designed from the
CP1 sequence was used for in situ hybridization experiments to verify
that the sequence obtained was derived from the observed Cristispira c
ells.