Introduction>

Hemerythrin (1HMO) in a protein mainly found in marine invertebrates such as lobsters, crabs, shrimps etc. This protein is capable of binding iron without the need to possess a heme group. Its active site is characterised by presence of five histidines and two carboxylate ligands which coordinate two iron atoms (Stenkamp et al., 1985) (Fig. 1). Upon the incorporation of iron, this protein can bind surrounding oxygen and change its colour from the colourless state into violet-pink. It is a relatively small protein, with strongly conserved sequence and structure. Using hemerythrin, we hypothesised that we would be able to detect an increase in iron concertation within the cell, through observing the colour change of expressed hemerythrin.

Fig. 1 – Active site of hemerythrins comprising of five histidine residues and two glutamate residues. One of the incorporated irons can get bind oxygen and change the colour of the protein from colourless to violet/pink.

The hemerythrin we have initially chosen for our project comes from marine organism Themiste dyscritum (Loehr et al., 1978) (TdHr). Although it is an attractive protein to use in our experiment, marine invertebrates are eukaryotic organisms and expression of these hemerythrins in bacterial cells is somewhat challenging. Through the literature research, we were able to find hemerythrin-like bacterial proteins, which share similarities in sequence and function. These proteins can be found in organisms such as methane-oxidising Methylococcus capsulatus (McHr) (Kao et al., 2008) and sulfate-reducing Desulfovibrio vulgarisi (Dcr-Hr) (Xiong et al., 2000).

Sequence comparison among our three chosen hemerythrins showed conservation of 5 histidine residues required for iron binding, as well as some other crucial domains. All of the proteins were previously documented to show a colour change upon oxidation, therefore can be considered suitable for the project. However, visibility, speed of the colour change and dependence on iron concentrations can differ among these proteins, which would need to be tested in our experiments.

Results

All hemerythrin genes were successfully cloned under control of constitutive or inducible promoter to aid us with characterisation of our proteins. Presence of the cloned gene has been confirmed by plasmid isolation and restriction digest. However, none of the cells have shown to be expressing our proteins. TO BE CONTINUED AFTER I COLLECT SOME MORE DATA.

The Theory

Our Fur repressor/RyhB reporter system is based upon the way that the cell normally detects, and then controls, its intracellular iron levels. This homeostasis is necessary as, although iron is vital for the cell to function, it is also potentially dangerous due to the Fenton reaction, which produces dangerous reactive oxygen species (ROS) under aerobic conditions (Andrews et al., 2003).

To combat this the cell downregulates genes needed for import of iron, such as those that produce and uptake siderophores, with the Ferric uptake regulator (Fur) repressor. When bound to Fe2+ Fur is able to bind a region of DNA known as the Fur box, repressing the genes downstream. However, in low iron conditions Fur is no longer able to bind to Fe2+, which causes a change in structure that renders it unable to bind to DNA, de-repressing the genes under its control and hence increasing iron uptake (Andrews et al., 2003).

Fur has also been noted to positively regulate certain genes under high iron conditions. These genes include proteins that require iron, those that store it, and some that help to deal with its negative effects. One of these genes is sodB, which encodes a superoxide dismutase that is able to detoxify the ROS produced by iron via the Fenton reaction under aerobic conditions. (Andrews et al., 2003)

This positive regulation is actually done indirectly, via a small regulatory RNA (srRNA) known as RyhB. This is able to bind to complementary sequences found on the mRNA of certain genes, such as sodB, and inhibit their translation. ryhB is located directly downstream of a Fur box, meaning that it is downregulated in high iron conditions, causing genes that are negatively regulated by RyhB to be effectively upregulated in high iron conditions when ryhB is repressed (Massé and Gottesman, 2002).

In order to exploit this, we have included the RyhB binding sequence from sodB in the 5’ untranslated region (5’ UTR) of superfolder green fluorescent protein (sfGFP). We hypothesised that when RyhB was expressed then translation of sfGFP would be inhibited, and hence sfGFP would be downregulated in low iron conditions when ryhB was not being repressed. Ultimately this would lead to increased fluorescence at high intracellular iron concentrations, but decreased fluorescence when these concentrations were lower.

Clonning Diagrams

Our reporter system contains a RyhB binding sequence in the 5’ UTR of sfGFP and is present under the control of two promoters of different strengths, a medium and a strong. These constructs were cloned into both pBluescript KS(+) and pSB1C3 plasmids and then transformed into our siderophore production mutant, JC28, along with our wild type W3110 for use as a control (figure 1).

Figure 1: (top-left) Map of pSB1C3 plasmid containing RyhB-GFP insert. (top-right) Map of pBluescript KS(+) plasmid containing RyhB-GFP insert. (C and D) Agarose gel electrophoresis picture of a restriction digest with SpeI and EcoRI restriction enzymes of either our insert containing pSB1C3 (bottom-left) or pBluescript KS(+) (bottom-right) stock. These plasmids were then used to transform our JC28 mutant and our W3110 wild type control.

Fluorescence change when grown at different iron concentrations

As an initial test, we wanted to see if fluorescence changed in response to growth in different iron concentrations. We grew our mutant and wild type strains containing pBluescript KS(+)-RyhB-GFP construct in defined medium with either 5μM or 100μM FeCl3 for low or high iron conditions respectively. We then measured the OD600 and the fluorescence of our cells at 535nm, which we adjusted according to each samples respective OD (figure 2). We were unable to detect any significant correlation between fluorescence of the sample and iron concentration in the growth medium, suggesting that our RyhB-GFP reporter system is not affected by changes in extracellular iron concentrations.

Fluorescence change in response to addition of iron

Figure 2: Fluorescence of JC28 mutants or W3110 wild types transformed with RyhB-GFP constructs under the control of medium or strong promoters. Measurements were taken at a single time point after growth overnight in either low iron containing medium (5μM FeCl3) or high iron containing medium (100μM FeCl3).

Figure 3: Change in fluorescence after 90 minutes following addition of FeCl3 to a final concentration of 100μM (A) or no addition of FeCl3 (B). Significance is conveyed with * <0.05, **<0.01, *** <0.001, **** <0.0001.