Transcriptional dynamics during Rhodococcus erythropolis infection with phage WC1

Abstract

Background: Belonging to the Actinobacteria phylum, members of the Rhodococcus genus thrive in soil, water, and even intracellularly. While most species are non-pathogenic, several cause respiratory disease in animals and, more rarely, in humans. Over 100 phages that infect Rhodococcus species have been isolated but despite their importance for Rhodococcus ecology and biotechnology applications, little is known regarding the molecular genetic interactions between phage and host during infection. To address this need, we report RNA-Seq analysis of a novel Rhodococcus erythopolis phage, WC1, analyzing both the phage and host transcriptome at various stages throughout the infection process.

Results: By five minutes post-infection WC1 showed upregulation of a CAS-4 family exonuclease, putative immunity repressor, an anti-restriction protein, while the host showed strong upregulation of DNA replication, SOS repair, and ribosomal protein genes. By 30 min post-infection, WC1 DNA synthesis genes were strongly upregulated while the host showed increased expression of transcriptional and translational machinery and downregulation of genes involved in carbon, energy, and lipid metabolism pathways. By 60 min WC1 strongly upregulated structural genes while the host showed a dramatic disruption of metal ion homeostasis. There was significant expression of both host and phage non-coding genes at all time points. While host gene expression declined over the course of infection, our results indicate that phage may exert more selective control, preserving the host's regulatory mechanisms to create an environment conducive for virion production.

Conclusions: The Rhodococcus genus is well recognized for its ability to synthesize valuable compounds, particularly steroids, as well as its capacity to degrade a wide range of harmful environmental pollutants. A detailed understanding of these phage-host interactions and gene expression is not only essential for understanding the ecology of this important genus, but will also facilitate development of phage-mediated strategies for bioremediation as well as biocontrol in industrial processes and biomedical applications. Given the current lack of detailed global gene expression studies on any Rhodococcus species, our study addresses a pressing need to identify tools and genes, such as F6 and rpf, that can enhance the capacity of Rhodococcus species for bioremediation, biosynthesis and pathogen control.

Keywords: Rhodococcus; Phage; RNA-Seq; Transcriptome; WC1.

Fig. 1

A: Phage titer during host infection. Grey lines and circles represent individual replicate experiments. The blue line shows the average. B: Percentage of reads at each time point mapped to host versus phage genomes. Solid circles show averages of two replicates and the x labels show individual replicates – in most cases these are close to the mean and thus do not appear

Fig. 2

Temporal and spatial patterns of transcriptional activation in WC1. (A) Phage expression patterns over time. Parallel coordinate plots (left) show relative counts of phage transcripts for Early, Middle, and Late genes. The counts are transcripts per million (TPM) normalized internally for each gene, i.e. with respect to the lowest expression level for that individual gene. The heatmap (right) shows log TPM averaged at each time point and subsequently standardized. The standardization is performed by column. (B) Genomic locations of early, middle, and late genes in WC1. Colors used in the genome map correspond to those in A

Fig. 3

Differentially expressed (DE) genes over the time course of infection. A-D: Volcano plots for each time point compared to baseline. Each point on the volcano plot is based on 2 replicates. Vertical lines indicate Log₂ Fold Change of 0.58 and − 0.58, which corresponds to a fold change of 1.5. The horizontal line indicates p of 0.05. E: Counts of up and down-regulated host genes as compared to the 0 min baseline. Genes considered DE had FC greater than 1.5 and p-values less than 0.05

Fig. 4

Transcribed intergenic non-coding regions in WC1. The y axis shows reads normalized per million aligned transcripts in each individual library and then averaged for each set of replicates at each genomic location

Fig. 5

Enriched KEGG pathways during time course of infection as compared to baseline. The direction of the arrows indicate if the DE genes were up-regulated or down-regulated, and the numeric values indicate the number of genes. Only DE genes with absolute log₂ fold change greater than or less than − 0.58 were included in the analysis to reflect a fold-change of 1.5

Fig. 6

Enriched GO categories during time course of infection as compared to baseline by ontology. All categories indicated were up-regulated. The numbers inside the circles indicate the median gene count for each category. Categories were obtained as described in methods; the full list of terms in each category and individual gene counts for each term are provided in Supplementary Table S6. Only DE genes with absolute log₂ fold change greater than or less than − 0.58 were included in the analysis to reflect a fold-change of 1.5