Modelo de estrés oxidativo

In vitro, YonO displays efficient RNA polymerase activity. This, in combination with its

expression during SPβ induction and apparent essentiality strongly suggests YonO is an SPβ specific RNAP functioning during the induction of the prophage.

Whilst YonO appears to be essential for SPβ development, the genes it transcribes remain unidentified. To address this, total transcriptome analysis (RNAseq) was performed on WT and ΔyonO strains. Comparison of the WT and ΔyonO

transcriptomes during bacteriophage induction would reveal the genes under the control of YonO. Wildtype and ΔyonO strains were grown to mid-log phase (0.5 OD600) and SPβ was induced with Mitomycin C. Western blot data revealed YonO is present in WT cells 60 minutes after induction. Additionally, SPβ induction was observed 60

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minutes after Mitomycin C treatment in previously published microarray data (Goranov et al., 2006). Therefore, cells were harvested 60 minutes after treatment with Mitomycin C. In parallel, wildtype and ΔyonO cells without treatment were harvested along with biological replicates for all samples. Total RNA was extracted from the cells and subjected to next generation sequencing. The commercial company PrimBio performed all subsequent handling of total RNA and RNA sequencing. Total RNA contains rRNA which strongly skews RNAseq due to its abundance. Therefore, 16S and 23S rRNA was depleted prior to library preparation. Library preparation involved the ligation of adaptors and sample specific barcodes to the RNA followed by reverse transcription and purification of cDNA. Sequencing primers hybridise to the adaptor sequences whilst barcode sequences allow for multiplexing, the simultaneous sequencing of multiple samples. The sequencing reads were aligned to the B. subtilis reference genome and quality controlled via read metrics such as mapping quality and alignment score. For each annotated gene, a normalised expression value was

calculated. The expression of each gene in the two different strains (wildtype and

ΔyonO) and each condition (+/- Mitomycin C treatment) was compared, giving fold

changes in expression. This data was provided by PrimBio.

Comparing the changes in gene expression of the wild type strain, with and without Mitomycin C treatment, the wide ranging impact of Mitomycin C could be seen (Figure 6-3). Primarily, Mitomycin C treatment induces the SOS response through DNA damage (Goranov et al., 2006). One aspect of the SOS response is the activation of the

nucleotide excision repair machinery, comprised of UvrA, UvrB and UvrC (reviewed in Lenhart et al, 2012). Mitomycin C treatment led to an approximate 9 fold increase in the transcription of the uvrA and uvrB genes. A 4 fold increase in uvrC was observed. The pattern of lower induction of uvrC compared to uvrA and uvrB correlates with previous transcriptomic data (Au et al., 2005).

The prominent effect of Mitomycin C treatment is the induction of resident prophages PBSX, SPβ and the ICEbs1 mobile element through activation of the SOS response (Goranov et al., 2006, Auchtung et al., 2005). The RNAseq data correlated well with previously published data (Goranov et al., 2006, Seaman et al., 1964, Warner et al., 1977). Upon Mitomycin C treatment, expression of PBSX, SPβ and ICEbs1 genes was greatly increased as shown in Figure 6-3. Despite possessing homology to the late

operon of PBSX, the SKIN element was not induced by Mitomycin C. This observation is consistent with a previously reported absence of β-galactosidase activity in Mitomycin C treated cells with lacZ fusions to the SKIN late operon (Krogh et al., 1996). The 3 genes upstream of ICEbs1 displaying increased expression in response to Mitomycin C correspond to ybfE, ybfG and ybfG. The proteins encoded by these genes are

uncharacterised but bioinformatics have shown that they contain peptidoglycan binding and hydrolase domains.

Looking closer at the induction of SPβ in response to Mitomycin C, RNAseq revealed that genes of Cluster II and III underwent increased expression whilst Cluster I did not. No Cluster I genes exhibited a significant fold change in expression when WT cells treated with and without Mitomycin C were compared. Referring to the BaSysBio database supported this observation (Nicolas et al., 2012). SunT, a representative gene of Cluster I, had equal expression levels in cells treated with Mitomycin C for 45

minutes and the negative control.

Figure 6-3: Fold changes of expression across the B. subtilis genome in response to Mitomycin C. The fold changes in expression of individual genes in WT cells, with and without Mitomycin C treatment (blue diamond) were plotted. The KEGG BSU accession number for each gene was used to plot along the X axis, showing the fold changes across the genome. The fold changes of expression between WT and ΔyonO cells treated with Mitomycin C were plotted in parallel (red square). Negative values

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correspond to a decrease in expression. The regions along the genome corresponding to PBSX, SPβ and ICEbs1 have been indicated.

To determine which genes are under the control of YonO, expression levels of wildtype and ΔyonO cells treated with Mitomycin C were compared. There were no fold

changes in Cluster I and III expression (Figure 6-3). The RNAseq revealed a large disparity between the wildtype and ΔyonO strains with regards to the expression of Cluster II genes after Mitomycin C treatment, with comparisons of expression levels revealing large fold changes. The fold changes were negative; indicating Cluster II genes were not expressed in the induced ΔyonO strain and suggesting that YonO is responsible for the transcription of Cluster II. Furthermore, the overall strength of YonO expression appears to be greater than that of the endogenous, host RNAP. This conclusion was arrived at by comparing the normalised expression values generated from the RNAseq data. Using the expression of rpoC (gene encoding for β’ subunit) as an example of a gene highly expressed by the host RNAP, this gene has a normalised expression value of 0.56. This is in stark contrast to the expression value of the yomI gene of SPβ Cluster II, which has a normalised expression value of 9.9. It is appreciated the rpoC expression value was generated from cells treated with Mitomycin C. Cells treated by Mitomycin C are subjected to DNA damage and undergoing the SOS

response. Therefore, additional work will be required to accurately compare the in vivo expression strength of YonO to other RNAPs such as the bacterial RNAP and T7 RNAP.

yonO is predicted to be the middle gene in an operon comprised of yonP, yonO and yonN (Figure 6-4) based on transcription profiles constructed from the high resolution

BaSysBio microarray data (Nicolas et al., 2012). In the transcription profiles, sudden increases and decreases of transcription, referred to as upshifts and downshifts, were used to define transcription units. yonP, yonO and yonN were shown to be on the same transcription unit which indicates they belong to a single operon, the expression of which would be driven by a single promoter upstream of yonN (Figure 6-4). All three genes display greatly increased expression upon Mitomycin C treatment in the

wildtype cells. However, in ΔyonO cells treated with Mitomycin C, only yonP

expression was comparable to wild type cells. There was no expression of yonO and

yonN expression was reduced. This observation supports the prediction these three

replaced with the kanamycin resistance cassette, which explains the complete absence of yonO expression in this strain. The kanamycin resistance cassette likely disrupts the transcription of the downstream yonN gene which would account for the reduction in

yonN expression in the ΔyonO strain. yonO belonging to an operon with yonP, in

combination with the observation that yonP expression is not lessened in the ΔyonO strain, suggests yonO is transcribed by the B. subtilis RNAP during SPβ development.

Figure 6-4: Transcription profile of yonO and adjacent regions of SPβ. Data retrieved from BaSysBio database. Top: yonO and the adjacent Cluster II genes are shown in the GenBank annotation of the wildtype B. subtilis genome. Middle: Transcription profiles generated from microarray array data with a resolution of 22 bases. Profiles for both DNA strands are shown. The individual traces represent the transcription profiles for individual environmental conditions. Bottom: Transcription units were defined by sudden upshifts and downshifts of transcription, as observed in the transcription profiles. Upshifts and downs shifts are depicted by red triangles and red squares, respectively. Transcription units above the genome (black line) are those on the plus strand. Transcription units on the – strand are below the genome. yonP, yonO and

yonN exist on a single transcription unit started by upshift 1672. Downstream, yonI is

present in a transcription unit on the minus strand with the corresponding upshift 1673 indicated by a green arrow. yonI is the sole ORF of the transcription unit and is therefore not expressed as part of any operon on either strand of DNA.

The alignment of wildtype and ΔyonO RNAseq sequencing reads to the Cluster II region on the SPβ genome was used to generate a coverage map which can be seen in Figure 6-5. The coverage map revealed that the Cluster II genes downstream of yonN were not transcribed in Mitomycin C treated ΔyonO cells. This is in contrast to induced wildtype cells, in which Cluster II genes were expressed. Therefore, the RNAseq indicates YonO is responsible for the transcription of Cluster II genes. Cluster II is suggested to contain late genes which are expressed towards the final stages of SPβ development, including lytic enzymes and bacteriophage structural proteins. Despite

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being unrelated to any known examples, it appears YonO is similar to other bacteriophage encoded RNAPs in that it transcribes late genes.

Figure 6-5: Coverage map of RNAseq reads aligned to the annotated SPβ Cluster II DNA. The sequencing reads from wildtype (WT) and ΔyonO cells treated with

Mitomycin C were aligned to the annotated B. subtilis genome. The red underlining of annotated genes indicates those belonging to Cluster II. Differences between the reference sequence and read sequences are shown in red (thymine), blue (cytidine), yellow (guanidine) and green (adenine). The number of reads was plotted onto a logarithmic scale.