Evolutionary Relationships among Members of the Genus Chlamydia Based on 16S Ribosomal DNA Analysis
BERTIL PETTERSSON,1 ANNALENA ANDERSSON,1 THOMAS LEITNER,2 ØRJAN OLSVIK,3 MATHIAS UHLEN,1 CHRISTOPHER STOREY,4 AND CAROLYN M. BLACK5*
1Department of Biochemistry and Biotechnology, The Royal Institute of Technology, Stockholm, Sweden
2Theoretical Biology and Biophysics, Group T-10, Los Alamos National Laboratory, Los Alamos, New Mexico
3Department of Medical Microbiology, School of Medicine, University of Tromsø, Tromsø, Norway
4Department of Microbiology, University of Leeds, Leeds, United Kingdom
5National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C.
pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal
small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing
of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene,
indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was
found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between
their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar
to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore,
the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the
other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an
attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by
analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family
Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster.
The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former
cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of
C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering
and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic
cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16
branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine.
Furthermore, they also share less reliable features like normal elementary body morphology and plasmid
content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain
N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C.
trachomatis being the more recently evolved species within the genus Chlamydia.