Goralski, T. D. | bioRxiv Science

bioRxiv2020

barCoder: a tool to generate unique, orthogonal genetic tags for qPCR detection

BackgroundTracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains. Recent work has shown that genomic insertion of short identification tags, called "barcodes" here, allows detection of chromosomally tagged strains by real-time PCR. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. ResultsHere we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode "modules" that consist of binding sites for primers and probes and appropriate spacer sequences. Each module is designed to yield optimal PCR amplification and unique identification. Optimal amplification includes metrics such as ideal Tm and G+C-content, appropriate spacing, and minimal stem-loop formation; unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases, such as NCBI. We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki, a simulant for the potential biowarfare agent Bacillus anthracis, and three each for other potential target organisms with variable G+C content. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. ConclusionsThe barCoder algorithm facilitates the generation of barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.

bioinformatics↗

bioRxiv2018

A new mechanism for ribosome rescue can recruit RF1 or RF2 to non-stop ribosomes

Bacterial ribosomes frequently translate to the 3 end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued using trans-translation. In some species, genes required for trans-translation are essential, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type cells but not in cells that lack trans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues non-stop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.\n\nImportanceFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identifies a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

microbiology↗