Genotyping bacterial and fungal pathogens using sequence variation in the gene for the CCA-adding enzyme

Abstract

BackgroundTo allow an immediate treatment of an infection with suitable antibiotics and bactericides or fungicides, there is an urgent need for fast and precise identification of the causative human pathogens. Methods based on DNA sequence comparison like 16S rRNA analysis have become standard tools for pathogen verification. However, the distinction of closely related organisms remains a challenging task. To overcome such limitations, we identified a new genomic target sequence located in the single copy gene for tRNA nucleotidyltransferase fulfilling the requirements for a ubiquitous, yet highly specific DNA marker. In the present study, we demonstrate that this sequence marker has a higher discriminating potential than commonly used genotyping markers in pro- as well as eukaryotes, underscoring its applicability as an excellent diagnostic tool in infectology.ResultsBased on phylogenetic analyses, a region within the gene for tRNA nucleotidyltransferase (CCA-adding enzyme) was identified as highly heterogeneous. As prominent examples for pro- and eukaryotic pathogens, several Vibrio and Aspergillus species were used for genotyping and identification in a multiplex PCR approach followed by gel electrophoresis and fluorescence-based product detection. Compared to rRNA analysis, the selected gene region of the tRNA nucleotidyltransferase revealed a seven to 30-fold higher distinction potential between closely related Vibrio or Aspergillus species, respectively. The obtained data exhibit a superb genome specificity in the diagnostic analysis. Even in the presence of a 1,000-fold excess of human genomic DNA, no unspecific amplicons were produced.ConclusionsThese results indicate that a relatively short segment of the coding region for tRNA nucleotidyltransferase has a higher discriminatory potential than most established diagnostic DNA markers. Besides identifying microbial pathogens in infections, further possible applications of this new marker are food hygiene controls or metagenome analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0670-2) contains supplementary material, which is available to authorized users.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

“…In the single nucleotide-binding pocket (motif D; EDxxR), conserved amino acids serve as a polymerization template, forming Watson-Crick-like hydrogen bonds with the incoming nucleotides [10]. The specificity switch from CTP to ATP binding is mediated by a flexible loop acting as a lever to adjust the relative orientation of the templating side chains for interaction with either CTP or ATP [22][23][24][25][26]. Deletions, as well as most point mutations in this region, have a strong inhibitory effect on the incorporation of the terminal A residue, underlining its importance for a complete CCA synthesis.…”