Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation.
The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNA and differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.
Yeates
Christine
C
Advanced Wastewater Management Centre, The University of Queensland, St Lucia, 4072, Australia.
Saunders
Aaron M
AM
Advanced Wastewater Management Centre, The University of Queensland, St Lucia, 4072, Australia.
Crocetti
Gregory R
GR
Advanced Wastewater Management Centre, The University of Queensland, St Lucia, 4072, Australia.
Blackall
Linda L
LL
Advanced Wastewater Management Centre, The University of Queensland, St Lucia, 4072, Australia.
D020562:Betaproteobacteria / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D020563:Gammaproteobacteria / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S