From a networks perspective, the Type I fimbriae gene network poses an interesting question. As previously discussed in Figure 31, Type I fimbriation in S.
typhimurium is controlled by two-interlocked positive feedback loops. These loops likely
encode an AND gate in the network where only when activating signals feed into both PfimY and PfimZ promoters will fimbriation take place. Despite the presence of coupled-
positive feedback, we demonstrated that the network does not exhibit switch-like transition from the “off” to the “on” state. Rather, the transition of the PfimA promoter
from the “off” to the “on” state was like a rheostat. With this understanding of the network, we were interested in answering the following questions about the fimbriae network.
168 Firstly, what are the precise signals that are fed into the PfimY and the PfimZ
promoters that control fimbriae gene expression in S. typhimurium. Type I fimbriae are known to be assembled in static, highly aerobic conditions in liquid media (Duguid et al., 1966a). To investigate which promoter are these signals fed through, we monitored the PfimY and PfimZ promoter activity in several environmental conditions in a ∆fimYZ mutant.
Our results indicate that the two different promoters likely integrate two distinct environmental signals leading to expression of genes responsible for Type I fimbriae. We demonstrate that high oxygen signal is fed through the PfimY promoter as its activity
was 3-fold higher in high oxygen conditions than in anaerobic conditions, while there was no appreciable increase in the PfimZ promoter in the presence or absence of oxygen
in a ∆fimYZ mutant. Similarly, we also monitored PfimY and PfimZ promoter activity
following growth on solid media vs. liquid cultures. Our results demonstrate that while both, PfimY and PfimZ promoters are upregulated in liquid media as compared to solid
media, the increase in PfimZ promoter activity is much more as compared to the increase
in the PfimY promoter activity. These results suggest that information about growth on
soild/liquid media is fed to the Type I fimbrial circuit primarily at the level of PfimZ
promoter (Figure 46). Therefore, in this manner, the network integrates different signals leading to fimbriation under the most appropriate conditions.
Secondly, we were also interested in investigating the dynamics of the system at both population average and a single-cell level by changing the architecture of the network. Specifically, we wanted to strengthen the positive feedback in the network
169 and monitor if that leads to a change in qualitative behavior of the network response at a single-cell level. To reprogram the fim system where the positive feedback is strengthened, we switched the PfimY and the PfimZ promoters on the chromosome. The
motivation behind doing this was that in wild type arrangement, FimZ primarily activates PfimY promoter and FimY is an activator of the PfimZ promoter. Therefore, in the
resulting strain PfimY::PfimZ PfimZ::PfimY, both FimY and FimZ will be free to feedback on the
respective promoters driving their expression.
We first checked the gene expression dynamics of the PfimA promoter in the
PfimY::PfimZ PfimZ::PfimY strain and compared it with wild type and a ∆fimW mutant (Figure
47). Our results show that the PfimA promoter activity in the reprogrammed strain is
stronger as compared to the wild type. In fact, the expression levels were very similar to those observed in the ∆fimW mutant. This demonstrates that our hypothesis of switching the promoters to strengthen the system was correct. Along with strengthening positive feedback in the reprogrammed strain, we note that we are also strengthening the FimY-FimW negative feedback loop. To remove this negative feedback loop, we removed FimW from the reprogrammed strain. The resulting strain was called PfimY::PfimZ PfimZ::PfimY ∆fimW. In this strain, the PfimA promoter activity was
about twice as stronger than the wild type and about 50% stronger than the reprogrammed PfimY::PfimZ PfimZ::PfimY strain. Therefore, we demonstrate that the fim
circuit employs interlocked positive and negative feedback loops to generate the wild type response of the system.
170 We have previously shown that the Type I fimbrial gene expression, despite encoding coupled feedback loops, does not exhibit transient heterogeneity as cells transition from the “off” to the “on” state. To examine the response of the reprogrammed fim network, we monitored PfimA promoter activity in the wild type, the reprogrammed PfimY::PfimZ PfimZ::PfimY mutant, and the PfimY::PfimZ PfimZ::PfimY ∆fimW
mutant. Our results demonstrate that in wild type and the PfimY::PfimZ PfimZ::PfimY strain,
the transition of the cells from the “off” to “on” state was homogeneous and there was no heterogeneity in the population. However, in the PfimY::PfimZ PfimZ::PfimY ∆fimW strain,
we see a switch-like transition of PfimA gene expression as cells transition from the “off”
to “on” state (Figure 48). These results demonstrate that the natural Type I fimbrial gene circuit encodes coupled positive and negative feedback loops. The coupled positive feedback loop between FimY and FimZ acts to integrate different environmental and cellular signals and the FimY and FimW negative loop prevents a run-away reaction in terms of PfimA gene expression. By tuning the strength of the positive feedback loops (by
reprogramming the network) and by eliminating the negative feedback loop (∆fimW), we demonstrate that we can change the qualitative gene expression pattern across the population, as cells transition from the “off” to the “on” state.
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