Team:Sheffield/project/science/CAS

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CAS PLATES

Introduction

The ability of E. coli cells to scavenge iron, through secretion of high affinity molecules called siderophores (Fig. 1), is crucial for their survival(1). The strain used in our project, JC28, is deficient in enterobactin production (2) leading to decreased intracellular iron levels in an iron-restrictive environment. To assay the levels of siderophore production in our strain, we used the CAS (Chrome Azurol S) plate assay (3)(4). In this assay, a blue dye, chrome azurol S, binds with high affinity to iron, producing a blue coloured-agar plate. Only very high-affinity molecules, such as siderophores, are capable of removing iron from the CAS dye and transporting it into the bacterial cell. This creates a visible orange/yellow halo, indicating the production of siderophores. This method can therefore be used to characterise and confirm the enterobactin-deficiency of strain JC28.

Fig. 1 – Siderophores are small organic molecules binding iron with high affinity. Enterobactin is a siderophore produced by E. coli and binds iron with K D =10 -52 M -1 (5)

The Experiment

To demonstrate the deficiency in siderophore production by JC28 we plated 10 µl of O/N cultures directly onto the pre-made CAS plates (Fig. 2A). As a positive controls we used strain W3110, which is the wild type background for strain JC28 (Fig. 2B) and the TOP10 strain (Fig. 2E). The system was further validated using supernatant of a tonB mutant of Burkholderia cenocepacia, which secretes siderophores but is deficient in their uptake, thus producing a siderophore-rich medium (Fig. 2F). As another positive control we plated 10 µl of concentrated EDTA (10-50mM). EDTA exhibits a high affinity to iron and shows a colour change after chelation of iron from the CAS dye (Fig. 2C). As a negative control we used 10 µl of our medium used for growing our strains (Fig. 2D).

Experiment were repeated three times (Fig. 3) and each replica showed similar results, with JC28 forming a small or no halo, compared to TOP10 and W3110 forming a large visible halos. The supernatant of ΔtonB caused the formation of a large halo around the colony. EDTA (50xTAE buffer used) also shows chelation of iron ions from the CAS dye, whereas the negative control using medium showed no halo and no colony growth.

In conclusion, the CAS assay worked and we could confirm that JC28 produces less siderophores compared to its wild type strain W3110. This is important, since the inability or hindrance for JC28 to take up iron is required for our following reporter system.

Fig.2 - CAS plate (blue) with yellow halos showing production of siderephores, with a - JC28, b -W3110, c- 10xTAEd, d -water, e-Top10(WT), f-siderephores

References

  1. De Serrano LO, Camper AK, Richards AM. An overview of siderophores for iron acquisition in microorganisms living in the extreme. BioMetals [Internet]. 2016 Aug [cited 2016 Oct 2];29(4):551–71. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27457587

  2. Cao J, Woodhall MR, Alvarez J, Cartron ML, Andrews SC. EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E. coli O157:H7. Mol Microbiol [Internet]. 2007 Aug [cited 2016 Oct 2];65(4):857–75. Available from: http://doi.wiley.com/10.1111/j.1365-2958.2007.05802.x
  3. Milagres AMF, Machuca A, Napoleão D. Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay. J Microbiol Methods. 1999;37(1):1–6.
  4. Shin SH, Lim Y, Lee SE, Yang NW, Rhee JH. CAS agar diffusion assay for the measurement of siderophores in biological fluids. J Microbiol Methods. 2001;44(1):89–95.
  5. Carrano CJ, Raymond KN. Ferric Ion Sequestering Agents. 2. Kinetics and Mechanism of Iron Removal from Transferrin by Enterobactin and Synthetic Tricatechols. J Am Chem Soc. 1978;101:18(32):1–4.