Utilization of tmRNA sequences for bacterial identification.
BACKGROUND
Ribosomal RNA molecules are widely used for phylogenetic and in situ identification of bacteria. Nevertheless, their use to distinguish microorganisms within a species is often restricted by the high degree of sequence conservation and limited probe accessibility to the target in fluorescence in situ hybridization (FISH). To overcome these limitations, we examined the use of tmRNA for in situ identification. In E. coli, this stable 363 nucleotides long RNA is encoded by the ssrA gene, which is involved in the degradation of truncated proteins.


RESULTS
Conserved sequences at the 5'- and 3'-ends of tmRNA genes were used to design universal primers that could amplify the internal part of ssrA from Gram-positive bacteria having low G+C content, i.e. genera Bacillus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Listeria, Streptococcus and Staphylococcus. Sequence analysis of tmRNAs showed that this molecule can be used for phylogenetic assignment of bacteria. Compared to 16S rRNA, the tmRNA nucleotide sequences of some bacteria, for example Listeria, display considerable divergence between species. Using E. coli as an example, we have shown that bacteria can be specifically visualized by FISH with tmRNA targeted probes.


CONCLUSIONS
Features of tmRNA, including its presence in phylogenetically distant bacteria, conserved regions at gene extremities and a potential to serve as target for FISH, make this molecule a possible candidate for identification of bacteria.
Schönhuber
W
W
Lehrstuhl für Mikrobielle Okologie, Universität Konstanz, Fach M654, Universitätsstrasse 10, D-78457 Konstanz, Germany. wilhelm.schoenhuber@uni-konstanz.de
Le Bourhis
G
G
Tremblay
J
J
Amann
R
R
Kulakauskas
S
S
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence / Q000235:genetics; D019143:Evolution, Molecular; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence; D013294:Lactococcus lactis / Q000235:genetics / Q000302:isolation & purification; D010802:Phylogeny; D015347:RNA Probes / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D017423:Sequence Analysis, RNA; D013045:Species Specificity
D015347:RNA Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger; C096222:tmRNA; D012343:RNA, Transfer