FUR system

Iron is an essential element in the development of E. coli, but also, it can be toxic and E. coli can be killed, if iron is absorbed in high quantity. Using the ferric-uptake regulator protein (Fur), bacteria developed an advanced system to regulate their iron homeostasis.

FUR protein (Ferric Uptake Regulator)

The Fur protein is a transcriptional repressor for more than 90 genes involved, in majority, in iron homeostasis^1,2. It plays an important role in the control of the intracellular concentration of iron in E. coli.

Fur acts as a positive repressor in presence of ferrous ion (Fe²⁺), its co-repressor. Then, Fe²⁺ binds to the Fur protein (one ferrous ion per subunit of Fur). This interaction leads to a structural modification and induces the dimerization of Fur and Fe²⁺. Then the homodimeric Fur-Fe²⁺ complex will bind to the DNA on a Fur binding site and inhibit the mRNA transcription. In absence of Fe²⁺, a disinhibiting effect occurs and mRNA can be transcribed.

Figure 1: Regulation by the Fur transcriptional factor. In absence of iron mRNA can be transcribed. In presence of iron, Fur binds the DNA, on the Fur binding site and mRNA expression is inhibited.

Fur is found at a level of 5000 copies in E. coli during exponential growth and it can be up to 10’000 copies in stationary phase. Note that this high number of Fur protein is essential to E. coli during its development. As explain previously, Fur regulates the expression of gene involved in the iron absorption, so it avoids an over absorption of iron which could be really toxic and kill E. coli.

FUR binding site architecture

Figure 2: Consensus palindromic sequence of Fur binding site.

The Fur Binding site also named “Fur box” or “iron box” is localized between -35 and -10 site at the promoters of Fur-repressed genes. As shown in the Figure 2, the Fur binding site is composed of 19-base pair that are organized as a palindromic consensus sequence. It must be noted that this consensus sequence is not an exact sequence. Others sequences of Fur binding sites can be found into E. coli’s genome.

Often, Fur binding site is repeated (as shown in the Figures 3 and 4). It means that several homodimer of Fur-Fe²⁺ can bind among the DNA. Then Fur binding sites can be localized in a larger region than between -35 and -10 of the promoter region (defined previously). The affinity of these Fur binding site to Fur-Fe²⁺ is different. In a first time the complex Fur-Fe²⁺ binds to the 1^st Fur binding site, which presents a higher affinity for its complex. Then, others Fur-Fe²⁺ bind to the following Fur binding sites which present a weaker affinity. A polymer of Fur protein is formed among the DNA which allows a stronger inhibition of the expression of the gene downstream these Fur binding sites.

Figure 3: Repeated Fur binding site sequences.

Figure 4: Repeated Fur binding site sequences on a double strand DNA.

Natural inverter system

It had been shown that Fur could be involved in a positive regulation of several genes of E. coli (fntA, bfr, acnA, fumA, sdh operon, sodB). In fact E. coli uses an inverter system mediated by a small RNA named RhyB.
RhyB is a 90 nucleotids long sRNA which is regulated by the Fur transcriptional factor, then it is expressed at low concentration of iron³.

Figure 5: Regulation by the Fur transcriptional and the RhyB sRNA, acting as an inverter. In absence of iron mRNA is binded by the RhyB and then it is degradated. In presence of iron, Fur binds the DNA, the RhyB expression is inhibited and the mRNA can be transcribed.

As shown in Figure 5, and if we apply it to the sodB gene expression system. In iron starvation, Fur-Fe²⁺ cannot be formed and RyhB transcription is disinhibited. RyhB is expressed in the intracellular environment and it can binds to sodB mRNA which contains RyhB’s target sequence. Once RyhB binds to SodB mRNA, RNA degrading enzymes, such as RNaseE and RNase III, are recruited and the new formed complex is digested.
However, if the iron concentration is high enough, Fur-Fe²⁺ complex is formed and it can inhibit the RyhB transcription. SodB is not repressed and can be synthesized³. That is why Fur is described as an activator transcriptional factor in such a case.

Siderophores

In lack of iron, bacteria produced siderophores, small molecules, that can catch any atom of iron in the environment to feed the bacteria. There is different types of siderophores, depending on their structure and their interaction with iron:

• Catecholate,
• phenolate,
• hydroxymate,
• carboxylate types.

We focus more specifically on enterobactins which are catecholate siderophore (composed with catechol = bi-phenol) produced by E. coli. Enterobactins present a very high affinity for ferric iron K = 10⁵² M^-1. This number is greater than the affinity of heme B and EDTA metal chelator for iron, respectively K = 10³⁹ M⁻¹ and 10²⁵ M⁻¹. De facto, enterobactins are one of the most powerfull siderophore known: it can extract iron from hemic source and even from air!

Figure 7: Structure of Enterobactin molecule

Ferric iron Fe³⁺ established hydrogen bound with the alcohol groupment of enterobactins.

Figure 8: Hydrogen bounds with Ferric Iron
(from Raymond, K. N. 2003; link to the pdf).

Enterobactin pathway is under the regulation of the Fur System in E.coli. When there is a lack of iron in the environment, genes of enterobactin pathway are expressed.
In our project, we focus on the 6 enzymes (EntA, EntB, EntC, EntD, EntE and EntF) of it biosynthesis from chorismic acid but there are other genes involved in this pathway as FepA that export enterobactin out of the bacteria.
The biosynthesis pathway of enterobactin is the following:

Note: The last step of the biosynthesis remains unclear.

Constructions

Our goal is to lower the iron absorption from the intestins to the blood by using an iron chelating bacteria, Iron Coli. The objective of the labwork is to design an iron-sensible promoter with FUR and overexpress the enterobactin synthesis pathway in the presence of iron.

Before going in the detail of our construction, we needed a iron marker in E. coli. Thus, we selected the FUR protein which negatively regulates the downstream genes. It is used in the iron regulation of the bacterium and smoothly shuts down the production ot siderophores, natural chelators of iron in the environment in iron depletion situation. When the concentration of iron exceeds 10⁶, the FUR proteins binds the ion, dimerizes and its inhibitory mechanism is activated. Below this threshold, the FUR protein's inhibition is suppressed and indirectly activates the downstream genes.

Figure 6: Construction..

Conclusion

In our project, we have design a system that could produce enterobactins from chorimsic acid, under the regulation of an iron iron sensor with an inverter. Those, when the amount in the environment (ie intestine) is high, our Iron Coli will produce lot of siderophores that will chelate the iron and hence reduce the amount that could be absorbed by the patient.
We also model enterobactin production of our Iron Coli and the potential efficiency of our treatment to cure iron overload.