Transcriptomic Profiling of Yersinia pseudotuberculosis Reveals Reprogramming of the Crp Regulon by Temperature and Uncovers Crp as a Master Regulator of Small RNAs

Abstract

One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated ‘acetate switch’, which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.

Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis

“…This number includes the 1151 previously reported transcriptional start sites (TSSs) (28) and 5′ ends of 498 transcripts identified by manual revision of pilot RNAseq experiments (SI Appendix). By using the MicrobesOnline resource (29), we identified 1,046 monocistronic RNAs and 568 polycistronic operons on the chromosome and 14 monocistronic and 14 polycistronic operons on plasmid pYV.…”

“…For two genes (trxA and manX), fusions to a short and long UTR were constructed due to two alternative TSSs (28). In the bgaB reporter gene system (33), transcription from the P BAD promoter was induced by arabinose, and β-galactosidase activity was measured at 25°C, 37°C, and 42°C.…”

“…The signals corresponding to the full-length transcript (FL) and the toeprint (toe, corresponding to the position +14 to +16 from the AUG) are indicated. described previously (28), with some modifications to enrich RNA fragments deriving from nuclease cleavage events, as described in SI Appendix.…”

“…Temperature and nutrient changes experienced during the early stage of infection, when the bacterium enters a warmblooded host, are the major cues of a complex gene expression cascade inducing not only virulence factors but also a metabolic switch to adapt to the host environment (27). A recent transcriptome study revealed 324 differentially regulated Y. pseudotuberculosis genes when cells were grown at 25°C or 37°C (28). Apart from the expected arsenal of virulence-associated genes, almost half of the temperature-regulated genes are linked to metabolic functions including enzymes of the energy metabolism, β-oxidation of fatty acids, and sugar and amino acid transporters.…”

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

“…The NCBI SRA database was screened with the key word "Yersinia," followed by a manual check for each data set. Only two experiments were found for transcriptome sequencing (RNAseq) analysis of Yersinia, both for Y. pseudotuberculosis YPIII cultured at different temperatures (25,26, and 37°C), analyzed during different growth phases (exponential and stationary phases), or mutated at an important regulatory gene (crp gene) (43,44). The raw reads were downloaded and reanalyzed for chromosomally encoded LRR protein expression.…”