Supplementary Figures

Figure S4.1: Average nucleotide identity of the YeO:3 strain Y1 with other Y. enterocolitica and Y. pseudotuberculosis strains. Nucleotide sequence of the YeO:3 strain Y1 was compared with the Y. enterocolitica serotype O:3 strains Y11 and 1203, and Y. pseudotuberculosis strains YPIII and IP32953. The average nucleotide identity with other strains is indicated in percentage using the pyANI (https://github.com/widdowquinn/pyani) and MUMmer (Kutz et al., 2004) software.

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Figure S4.2: Global identification of mRNA transcriptional start sites (TSSs). (A) Schematic overview of the identified TSS: mTSS for mRNAs, lmTSS for leader less transcripts, sTSS and asTSS for the start site of small trans-acting regulatory RNAs and antisense RNAs. (B) Sequence conservation at the TSSs. Sequence logo computed from 1299 unaligned TSS of YeO:8 strain 8081v and 1076 unaligned TSS regions (TSS is located at position +1) showing nucleotide conservation around the TSSs. The initial nucleotides of transcripts (position +1 to +3) are dominated by purines.

(C) Detected conserved sequence motifs in the -10 promoter region (Pribnow Box). (D) The distribution and frequency of the length of 5’-UTRs is given for all mRNAs of Y1 and 8081v, which start upstream of the annotated TSS. More than 40% of all 5’-UTRs are 20-60 nt in length.

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Figure S4.3: Identification of ncRNAs of YeO:3 Y1 and YeO:8 8081v. Visualization of RNA-seq based cDNA sequencing reads of the Y. enterocolitica sRNA Ysr021 (A, left panel), Ysr060 (B, left panel), and Ysr143 (C, left panel) mapped to the 8081v and Y1 using the IGV genome browser.

Differential expression of the trans-encoded sRNAs Ysr012 (A, right panel), Ysr060 (B, right panel), and Ysr143 (C, right panel) determined by qRT-PCR are shown. For qRT-PCR three independent cultures of YeO:3 Y1 and YeO:8 8081v were grown in LB medium to exponential or stationary growth phase at 25°C or 37°C. qRT-PCR was performed in technical duplicates with DNA-free total RNA (primers are listed in Table S1). The gyrB gene was used for normalization and relative gene expression changes were calculated according to Pfaffl 2001. Black bars: real-time qRT-PCR for Y1;

grew bars: real-time qRT-PCR for 8081v.

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Figure S4.4: Comparison of gene expression changes obtained by RNA-seq and real-time qRT-PCR. Relative gene expression changes were examined for selected genes in response to temperature or growth phase. Three independent cultures of YeO:3 Y1 and YeO:8 8081v were grown in LB medium to exponential or stationary growth phase at 25°C or 37°C. qRT-PCR was performed in technical duplicates with DNA-free total RNA (primers are listed in Table S1). The gyrB gene was used for normalization and relative gene expression changes were calculated according to Pfaffl 2001.

Black bars: real-time qRT-PCR; grew bars: RNA-seq.

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Figure S4.5: Gene expression analysis of stress adaption genes and regulators of YeO:3 and YeO:8. Heatmaps of transcripts encoding stress adaptation genes (A) or regulators (B) which are enriched (red) and depleted (blue) in strain YeO:3 Y1 compared to YeO:8 8081v. Values represent the log₂ fold change of indicated conditions (adjusted p-value ≤0.05).

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Figure S4.6: Promoter region of the ystA gene in different Y. enterocolitica strains. A sequence comparison is shown of the ystA promoter region of the YeO:3 strains Y1, Y11, 1203 and the YeO:8 strain 8081v. The coding region is marked with a blue box, and the AT-rich regions with a red box. The identified transcriptional start site is indicated by a broken arrow, and the putative -10 and -35 region are marked with a bar.

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4.8 Acknowledgement

We thank Dr. M. Fenner for helpful discussions. We also thank Bettina Elxnat, Nicole Heyer, Tanja Krause and Simone Severitt for excellent technical assistance.

4.9 Funding

This work was supported by the Helmholtz Association and the Leibniz Association, C.

Schmuehl was supported within the Ph.D. program ’Animal and Zoonotic Infections’ of the University of Veterinary Medicine Hannover by a Lichtenberg fellowship 'Niedersächsische Ministerium für Wissenschaft und Kultur (MWK)'. P. Dersch is supported by the German Center of Infection Research (DZIF).

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5 Discussion

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5 Discussion

RNA-sequencing has been well established and improved in recent years. This method provides an unbiased high-throughput sequencing approach that can capture global transcriptional responses in a strand-specific manner. It has been successfully applied to several bacterial pathogens, such as Pseudomonas aeruginosa, Campylobacter jejuni, Vibrio cholerae and Yersinia pseudotuberculosis. (Butcher and Stintzi, 2013; Dötsch et al., 2012;

Mandlik et al., 2011; Nuss et al., 2015).

Here, RNA-sequencing was used to compare the transcriptional profiles of Y. enterocolitica serotypes O:8 (YeO:8) and O:3 (YeO:3). YeO:8 strain 8081v is a well characterized representative of this serotype, which most knowledge about Y. enterocolitica is derived from. YeO:3 strain Y1 is a recent human stool isolate from an outbreak in Germany. The strains were grown under different in vitro conditions (25°C and 37°C, exponential and stationary growth phase) to obtain sequencing samples. These conditions are commonly used to mimic different temperatures and nutritional environments that enteropathogenic Yersinia encounter during their lifecycle. Additionally, the transcriptional profile of Y. pseudotuberucolis strain IP32953 grown at these conditions was compared to bacteria from in vivo samples 3 days post infection from murine Peyer’s Patches. As a result, this study provides novel in-depth information on the transcriptional landscape and the repertoire of novel regulatory and sensory RNAs of enteropathogenic Yersinia as well as global maps of TSS for both Yersinia species.

Im Dokument Mechanisms of niche adaption by Yersinia (Seite 96-109)