The 16S rRNA(or ribosomal small subunit rRNA) has been the workhorse molecule for bacterial phylogenetic analysis. It provide important information about the relationships among major bacterial lineages. This molecule provided the first definitive genetic information that was used to separate the members of Orientia from the lineage that leads to the genus Rickettsia (Stothard and Fuerst, 1994; Ohashi, et al., 1995).
The first sequences of the 16S rRNA gene from Rickettsia (now Orientia) tsutsugamushi were reported in 1994 (Stothard and Fuerst, 1994), including the deposition into the international DNA databases of the 16S rRNA sequences of the Gilliam, Karp and Kato isolates.
Phylogenetic relationships among the 16S rRNA genes of members of the rickettsiaseae based on maximum parsimony.
Figure from Stothard 1995 Ph.D. thesis
Subsequently, independent depositions for the same three isolates were made, together with sequences from the three additional isolates, Kawasaki, Kuroki and Shimokoshi, all of which were used as evidence for the splitting of Orientia as a separate genus from Rickettsia (Ohashi, et al., 1995).
Relationships among the 16S rRNA gene sequences of six isolates of Orientia (from Ohashi et al., 1995)
In the years following these original determinations, the number of sequences of the 16S rRNA gene in the international DNA databases have increased up to 65 in August 2017. Sequences range in size from 216 bp up to 1513 bp, with 52 of the sequence exceeding 1300 bp in length.
Although the gene is of vital importance in the identification of the position of Orientia within the alphaproteobacteria and the rickettsiaceae, the gene actually has only marginal information regarding differences between isolates within Orientia. Most isolates show less than 10 nucleotide differences from other isolates (see figure below), and the general pattern of subclades within the genus is difficult to observed with any significance. Other genes, or whole genome sequences are increasingly more important for our understanding of the patterns of evolution within Orientia.
DIFFERENTIATION OF THE 16S rRNA GENE WITHIN O. tsutsugamushi
The complete 16S rRNA gene sequences (length 1531 nucleotides) of 50 isolates of O. tsutsugamushi can now be compared. The number of nucleotide differences estimated between each pair of isolates was determined and is shown in the figure below. Most comparisons show differences below 0.5% (8 differences); these represent 57% of pairwise comparisons. Further, 91% of comparisons are below 1% difference (15 differences). The Shimokoshi isolate accounted for all but one of the comparisons with differences between 22 and 27 nucleotides. The most divergent sequence isolate was from the genome sequence of the AFSC4 isolate. This sequence appears aberrant, accounting for all pairwise differences greater than 27, most of the differences occurring at sites with a nucleotide unique to AFSC4. The differences in the sequence of this isolate would normally be attributed to sequencing errors, but this remains to be determined.
