Haem and xenosiderophore mediated iron acquisition by sinorhizobium meliloti and pseudomonas aeruginosa
Keogh, Damien (2008) Haem and xenosiderophore mediated iron acquisition by sinorhizobium meliloti and pseudomonas aeruginosa. PhD thesis, Dublin City University.
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The bacterial cell membrane is the principal barrier for the acquisition of essential nutrients such as iron. Although critical to the survival of bacteria, iron is extremely limited in the environment due to the formation of insoluble hydroxides at physiological pH. To surmount these obstacles bacteria have evolved several mechanisms of iron acquisition. Sinorhizobium meliloti 2011, the endosymbiont of Medicago sativa, is capable of utilising several xenosiderophores and the haem compounds haemoglobin and haemin to overcome conditions of iron limitation. The high demand for iron in the nodule makes the study of S. meliloti 2011 biologically significant.
Two putative hydroxamate siderophore outer membrane receptors were identified on the S. meliloti 2011 chromosome. Insertional inactivation indicated that FhuA1 and FhuA2 function in the utilisation of ferrichrome and ferrioxamine B respectively. Analysis of the region directly downstream of FhuA2 resulted in the identification of a ferrioxamine B specific ferric iron reductase, fhuF (smc01658), and a periplasmic siderophore binding protein, fhuP (smc01659). FhuP was found to function with the inner membrane haem permease and ATPase, HmuU and HmuV respectively, to effect ferrichrome and ferrioxamine B utilisation. Insertions in hmuPSTUV resulted in a reduction in haem utilisation suggesting the presence of a secondary haem utlilisation system. Heterologous expression of HmuTUV but not HmuUV in an E. coli dppF mutant restored haem utilisation suggesting that all three proteins are required for haem utilisation and that HmuUV can function with two separate periplasmic binding proteins. Orthologues of HmuUV were cloned from Rhizobium leguminosarum bv. vicae 3841 and found to restore ferrichrome, ferrioxamine B and haem utlilisation in a S. meliloti hmuU mutant.
Previously it was demonstrated that Pseudomonas aeruginosa PAO1 is capable of utilising the xenosiderophores ferrichrome, ferrioxamine B and schizokinen. An inner membrane transporter, FoxB, was identified by heterologous expression in a S. meliloti siderophore transport deficient background. The expression and purification of full-length recombinant His-tagged FoxB was achieved.
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