4. Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
4.4 Characterization of the uppS promoter
Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
74 thereby allowing RNApol progress between binding events. The level of repression can be
controlled by the concentration of guided dCas9 proteins resulting in the occupation time of the targeted location. We used CRISPRi to knockdown RNA transcription of frr (Peters et al. 2016).
To ensure high efficiency knock-down and to reduce the possibility of interacting with an internal promoter within frr, which could drive uppS expression, the guide RNA was targeted at the 5’ end of frr. We expected that if uppS and frr were found on the same transcript, knocking down frr transcripts should result in a proportionally similar knockdown of uppS transcripts. This however was not the case, as we saw that knocking down frr transcripts resulted in no correlated change in uppS transcript abundance (Figure 4.8). This shows that the expression of uppS and subsequently the downstream cell envelope genes are independent of the expression being driven from the
upstream promoters.
RNA was extracted from Bacillus subtilis mutant expressing dCas9 with a sgRNA targeting frr and grown in LB with varying concentrations of xylose and subjected to qPCR.
Higher concentrations of xylose represent an increased knockdown of frr. To see how many uppS transcripts contained the frr coding sequence both frr and uppS transcript levels were measured with qPCR. Error bars represent the standard error between the replicates. Fold changes are relative to the transcript abundances measured in the 0.01% xylose condition. qPCR levels were normalized to the constitutively expressed genes recA and gyrB.
Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
75 alone did not show promoter activity. Combining it with increasingly sized parts of the UTR of
uppS did not restore activity until the full 180bp was present. Due to the cloning method used, there was a 40bp between the 40bp and the 140bp fragment (Figure 4.9) and we believe this resulted in a reduction in activity (Figure 4.9). This could suggest that the promoter actually lies in the region 160-120 bp upstream of uppS and was incomplete in the 140bp fragment and therefore also truncated in the 40bp part explaining why there was no detectable activity from either of the promoter fusion constructs alone. Additionally, duplicating this 40bp region before the full 180bp region resulted in a reduction in activity of this putative promoter (Figure 4.9). This conflicts with the previous explanation and could suggest there is some repressive binding activity occurring from this sequence or that it is titrating away transcriptional activators.
One of the largest UTRs in the cluster in B. subtilis (130bp), exceeded only by the UTR between tsf and pyrH (146bp), lies between uppS and frr. As activity only occurs in fragments larger than this UTR this suggests the promoter may fall within the frr gene itself. It is striking that within the UTR, and therefore downstream of this putative promoter, there is a terminator with an efficiency of 60%, as mentioned previously. This means that there could be wasteful transcription initiation which is terminated shortly after beginning. However, these shorter transcripts may not form correct hairpin structures and allow transcription through this terminator. This may explain why the terminator was measured with only 60% efficiency despite us seeing little to no readthrough with the CRISPRi experiments (4.8). The presence of the terminator following the TSS start site may explain why we were unable to detect a TSS with the 5’ RACE method as RNA secondary structures can reduce the efficiency of reverse transcription or later polyadenylation, thereby resulting in no primer binding site for subsequent PCR amplification and detection.
38 - Figure 4.9 - Schematic of promoter fusion constructs
Construction of different promoter fusions to identify the active transcription region driving uppS expression. Each bar represents the length of the different tested promoter fusion constructs under their respective derived sequence. Grey lines represent inactive constructs, red lines represent active constructs and the faded-red constructs represent active constructs, however with weaker activity. Thin lines represent a 60bp dummy sequence that is introduced due to the cloning strategy and contains synthetic transcriptionally inactive DNA sequence used to connect the different 5’ UTR regions.
Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
76 39 - Figure 4.10 - Reporter activity of uppS 5’ UTR truncations
Different truncations of sequence upstream of uppS were placed before the LUX reporter and integrated into the Bacillus subtilis genome. The length is measured from the 10bp upstream of the uppS start codon.
Measurement was taken during exponential phase in LB media. Error bars represent the standard deviation between the replicates (n=3). 140-180 represents that only the sequence difference between the 180bp and 140bp fragments was used in the promoter fusion construct.
PuppS is transcriptionally upregulated in low amino acid conditions
Now that we identified the promoter which independently controls uppS we wanted to understand how this promoter was regulated. Given its tight genomic association with ribosomal genes and the need for increased lipid II carriers at higher growth rates we would expect the promoter to be upregulated under faster growth conditions. We measured the activity of the promoter in fast and slow growth conditions, respectively LB and MOPS media which in our plate reader promote doubling times of ~23 and ~78 minutes. Surprisingly, we found that opposite to what we expected, the activity of PuppS doubled in the slow growth conditions (Figure 4.11). To confirm this relationship, we tested an intermediate growth rate using MOPS media supplemented with amino acids (doubling time ~60 minutes), however, unexpectedly we saw the same level of reporter activity as with the much faster LB media (Figure 4.11). Supplementation of any of the individual amino acids used in the 6 amino acid mixture (methionine, histidine, arginine, proline, threonine) or any other amino acids we tested resulted in similar activity levels seen in the LB media (tryptophan the other amino acid is always present in the media). We did see reduced activity from supplementation of tyrosine and threonine but these both had detrimental effects on the growth rate of the cell. We cannot confirm the stimulus is slow growth in this case as between the MOPS media and LB media, there are many other nutritional differences. Given that we saw similar reductions
Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
77 upon the supplementation of all tested amino acids, a likely reason would be the nitrogen
scavenging from the amino acids directly. Promoter fusion reporter assays measure expression activity of a nucleotide sequence, however, this could be derived from both transcriptional regulation (the promoter) and post transcriptional regulation (such as proteins binding to the 5’UTR). Therefore, we performed qPCR analysis of our exponentially grown 180bp-PuppS strain in both MOPS media with and without the amino supplementation to measure if the transcript levels of the first gene in the luciferase operon increased. We saw a similar two-fold increase in the MOPS media condition, suggesting that regulation of the uppS promoter occurs by a transcriptional mechanism (Figure 4.12). As uppS is a key gene in the production of peptidoglycan and wall teichoic acids, we hypothesized that this additional promoter allows the cell to respond to perturb ants of these pathways by increasing production of cell wall intermediaries through the expression of uppS. Preliminary experiments, testing sub-lethal concentrations of four different cell wall targeting antibiotics which trigger the σM response, bacitracin, nisin, ramoplanin and tunicamycin, showed no effect towards the activity of the promoter.
40 - Figure 4.11 - PuppS reporter fusion activity under different conditions
The 180bp PuppS fragment activity was measured under different media conditions. The measurement was taken during exponential phase. Glucose and tryptophan were added to all MOPS media conditions. MOPS media (6AA) include methionine, histidine, arginine, proline, threonine in the media. The other amino acid conditions are each individually added to basic MOPS media. Error bars represent the standard deviation between the replicates (n=3).
Perturbation of a conserved translation and cell envelope synthesis associated gene cluster
78 RNA was extracted from B. subtilis containing the PuppS-lux
reporter grown in MOPS media with and without amino acid supplementation and subjected to qPCR. Error bars represent the standard error between the replicates. Fold change is relative to the transcript abundances in the 6 amino acid condition. qPCR levels were normalized to the constitutively expressed genes recA and gyrB.