Sinorhizobium Meliloti Soil Bacterium
1.1 Sinorhizobium meliloti
Sinorhizobium meliloti is a free-living gram-negative soil bacterium and is best known for its ability to induce the formation of nitrogen-fixing nodules on the roots of leguminous plants such as Medicago, Melilotus and Trigonella sp. These nodules differentiate to form bacteroids, which contain nitrogenase, an iron-containing enzyme that are capable of reducing atmospheric nitrogen to ammonia. The plant in turn supplies the bacteria with various nutrients(Chao, T. C. et al., 2004). Because nitrogenase and other iron-containing proteins make up such a large portion of the protein mass of both plant and bacteria cells, the amount of iron available to bacterial symbionts is restricted.
1.2 Iron and Iron transport
Iron is an essential micronutrient for bacterial growth. Cells rely on iron for a wide range of metabolic and signalling functions (Wandersman, C., & Delepelaire, P., 2004). Iron is the fourth most abundant element on earth; under physiological conditions - that is, in the presence of oxygen and at neutral pH (Stintzi, A. et al., 2000)- its bioavailability is extremely limited because of the rapid oxidation of Fe2+ (ferrous iron) to Fe3+ (ferric iron) and the subsequent formation of insoluble hydroxides(Faraldo-Gómez, J. D., & Sansom, M. S. P., 2003). To solubilize iron and acquire levels adequate for growth, bacteria frequently secrete siderophores. These catechol, hydroxyamate, or carboxylate compounds bind ferric iron with high affinity and maintain it in a soluble state whence it can be brought into the cell by high-affinity active transport systems(Kadner, R. J., 2005). These complexes are often too large to diffuse through the protein pores in the outer-membrane. Therefore these high-affinity active transport systems generally rely on receptor proteins at the bacterial surface that bind specifically to iron-siderophore complexes (Faraldo-Gómez, J. D., & Sansom, M. S. P., 2003).
Sinorhizobium meliloti expresses high specific outer membrane receptors that recognize iron bound to their own siderophores as well as receptors specific for siderophores produced by other microorganisms such as fungi. An outer membrane receptor has two distinct functional domains: a 22-stranded antiparallel β-barrel, and an N-terminal globular domain, which fold inside the β-barrel, acting as a channel gate or plug (Stintzi, A. et al., 2000). The plug domain consists of a mixed four-stranded β-sheet extending from the periplasm to the iron-siderophore binding site, which blocks the entry of siderophores through the outer membrane. The plug domain is connected to the barrel wall by extensive hydrogen bonding (Ferguson, A.D. et al., 1998). When the iron-siderophore complex binds to an extracellular pocket within the β-barrel, conformational changes take place on the extracellular side of the plug. The α-helix at the N-terminal of the plug unwinds causing further conformational changes to the structure on the periplasmic side of the outer membrane. These conformational changes signal to initiate TonB interaction.
Iron-siderophore complex uptake and transport across the outer membrane is an energy dependent process. There is no energy source within the outer membrane to aid in the uptake and transport of iron. The TonB complex consists of three proteins TonB, ExbB and ExbD which deliver the energy provided by the electrochemical charge gradient or proton motive force of the cytoplasmic membrane in order to transport the iron-siderophore complex through the outer membrane receptors (Rudolph, G. et al., 2006).
Once in the periplasmic space, the iron-siderophore complex binds to its cognate periplasmic binding protein and is then actively transported across the cytoplasmic membrane by an ATP-transporter system (Stintzi, A. et al., 2000).
Plug β-Barrel 2 3 Periplasmic space Cytoplasm 1
|
Figure 1: Siderophore-mediated iron transport in gram-negative bacteria. (1) The iron-siderophore complex (light blue and red) is transported by an outer membrane receptor protein (yellow) embedded in the outer membrane (green), which consists of a β-barrel (yellow) and plug domain (grey). The binding of the iron-siderophore complex to the outer membrane receptor causes conformational changes to the plug domain. This conformational change signals to initiate TonB interaction (Light blue, red and yellow). Interaction of the TonB protein results in the release of the iron-siderophore complex into the periplasm. (2) Once inside the periplasmic space, the iron-siderophore complex is transported by periplasmic binding proteins (orange). The periplasmic binding proteins transport it to the inner membrane (pink) where it binds to an ABC transporter (dark blue). (3) The ABC transporter transports the iron-siderophore complex across the inner membrane into the cytoplasm where the iron (red) is released from the siderophore (light blue) and the siderophore recycled.
These ABC-type permeases consist of a periplasmic binding protein and an inner membrane complex energized by an ATPase (Köster, A., 2001). Once the iron-siderophore complex is inside the cell, iron is released from the siderophore and then the siderophore is recycled.
1.3 Project Outline
The aim of this project was to investigate iron-regulated gene expression in Sinorhizobium meliloti by quantitative real-time PCR and further investigate the outer membrane recepter protein FhuA and its expression levels in the presence of iron and ferrichrome.
In this study we investigated the iron-regulated gene expression of two outer membrane receptor proteins of Sinorhizobium meliloti, rhtA and FhuA. Rhizobactin 1021, which is a citrate-based dihydroxamate type siderophore, is the only siderophore produced and secreted by Sinorhizobium meliloti 2011. It is transported by the outer-membrane receptor protein rhtA.
Rhizobactin 1021 Periplasmic space Ferrichrome Outer membrane RhtA FhuA1 |
Figure 2: A model of Hydroxamate Siderophore Utilisation in S. meliloti. Rhizobactin 1021 binds to its outer membrane receptor protein RhtA in yellow and ferrichrome binds to its outer membrane receptor protein FhuA in red. Both iron-siderophore complexes are transported via these outer membrane receptor proteins from the outer membrane in green into the periplasmic space where they are bound by periplasmic proteins and transported to the inner membrane.
It has been shown that Sinorhizobium meliloti 2011 is capable of utilising siderophores produced by other microorganisms. FhuA is the outer membrane receptor protein for ferrichrome. Ferrichrome is a hydroxamate-type siderophore produced by fungi including Ustilago sphaerogena, which chelates Fe3+ using its nitrogen atoms of thiazoline and oxazoline rings.
Quantitative real-time PCR is the gold standard method for quantifying levels of gene expression. Its power resides in the ability to detect the amount of PCR product at every cycle of the PCR, using fluorescence. Real-time PCR (qPCR) uses fluorescent reporter dyes to combine the amplification and detection steps of the PCR reaction in a single tube format (Nolan, T. et al., 2006). There are several approaches employed to detect PCR products. In this study, SYBR Green was the detection chemistry chosen. SYBR Green is a fluorescent dye that binds only to double-stranded DNA. Fluorescence is emitted proportionally to the amount of double-stranded DNA.
The primary aim was to grow Sinorhizobium meliloti 2011 under iron-replete and iron-deplete conditions and to carry out real-time PCR (qPCR). These outer membrane receptor proteins are generally only induced under iron-deplete conditions and are not present under iron-replete conditions. Hopefully upon carrying out real-time PCR (qPCR) this would enable us to show that these outer membrane receptor proteins are iron-regulated. Then in order to see the effect of a siderophore on the outer membrane receptor protein FhuA, Sinorhizobium meliloti 2011 was grown under iron-deplete conditions in the presence of ferrichrome.
We provide a professional essay writing service that thousands of our customers use as an effective way of improving their grades, improving their research and saving them lots of time.
