Genomic and transcriptomic profiling of phoenix colonies

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium responsible for numerous human infections. Previously, novel antibiotic tolerant variants known as phoenix colonies as well as variants similar to viable but non-culturable (VBNC) colonies were identified in response to high concentrations of aminoglycosides. In this study, the mechanisms behind phoenix colony and VBNC-like colony emergence were further explored using both whole genome sequencing and RNA sequencing. Phoenix colonies were found to have a single nucleotide polymorphism (SNP) in the PA4673 gene, which is predicted to encode a GTP-binding protein. No SNPs were identified within VBNC-like colonies compared to the founder population. RNA sequencing did not detect change in expression of PA4673 but revealed multiple differentially expressed genes that may play a role in phoenix colony emergence. One of these differentially expressed genes, PA3626, encodes for a tRNA pseudouridine synthase which when knocked out led to a complete lack of phoenix colonies. Although not immediately clear whether the identified genes in this study may have interactions which have not yet been recognized, they may contribute to the understanding of how phoenix colonies are able to emerge and survive in the presence of antibiotic exposure.

Figure 1

Isolation of P. aeruginosa colony variants for assaying the genome and gene expression. (A) Diagram of the experimental methods for obtaining variant colony emergence for P. aeruginosa PAO1 with tobramycin. (B) In vitro imaging system (IVIS) images of representative phoenix colony plates show areas of bacterial sampling. Seventy-two hours post tobramycin bead placement, samples were taken from the edge of the zone of clearance (ZOC) where phoenix colonies would be expected to arise the following day after continued incubation. Ninety-six hours post tobramycin bead placement, samples were taken from phoenix colonies, VBNC-like colonies, and the outer background lawn. Additionally, samples were taken from bacterial lawns grown for 72 h that had not been exposed to antibiotics to be used as comparative controls. Red indicates high levels of metabolic activity, blue indicates low levels of metabolic activity, black indicates no metabolic activity.

Figure 2

Protein structure of wild-type and mutant PA4673. Protein structures were derived from the amino acid sequences of wild-type and mutant PA4673 using Phyre2. The red box indicates the structural change caused by the SNP. Functional domain identification in both PA4673 isoforms indicates that they likely function as GTP-binding proteins. As the SNP causes an amino acid change in the exterior portion of the predicted protein structure, it may lead to alteration of a binding site or protein function.

Figure 3

Multi-dimensional scaling (MDS) plot showing clustering of isolates. An MDS plot was generated using biological coefficients of variation (BCV) from transcript counts for each isolate sample. Samples from each group cluster well together aside from the Edge samples, and the phoenix colonies and VBNC-like colonies overlap considerably. Control lawn, Edge, and Outer samples cluster on their own and separately from the Phoenix and VBNC-like samples.

Figure 4

Volcano plots of DEGs. (A) Comparison of phoenix colony transcript counts to control lawn transcript counts showed 63 DEGs. (B) Comparison of VBNC-like colony transcript counts to control lawn transcript counts showed 90 DEGs. (A, B) Red dots indicate gene transcripts which were significantly upregulated (greater than twofold change and corrected p-value of 0.05 or less). Blue dots indicate gene transcripts which were significantly downregulated (greater than twofold change and corrected p-value of 0.05 or less). Black dots indicate genes with no-significant difference in transcription.

Figure 5

Complementation of PA3626 shows rescue of the phoenix phenotype. Phoenix colony counts were obtained for wild-type P. aeruginosa PAO1, the PA3626 transposon mutant, and the PA3626 complementation strain. After complementation of the transposon mutant, phoenix colony counts returned to wild-type levels, further confirming the importance of the tRNA pseudouridine synthase D in the emergence of phoenix colonies. *p < 0.05, **p < 0.01, n = 3.