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Title: Molecular genetic analysis of yncD and yncE genes in Escherichia coli
Author: Baba Dikwa, Aisha.
ISNI:       0000 0001 3434 6702
Awarding Body: University of Reading
Current Institution: University of Reading
Date of Award: 2008
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Nearly all bacteria require iron for growth as it is utilised in many major biological processes. Despite its indispensability, iron causes problems of toxicity, poor solubility and poor bioavailability. Highly efficient iron acquisition and storage systems are used to overcome these problems. Gram-negative bacteria take up iron and haem complexes via specific outer-membrane (OM) receptors. Translocation across the OM is dependent on the cytosolic membrane potential and the energy transducing TonB-ExbB-ExbD system. Iron-complexes thus delivered to the periplasm are in tum translocated across the cytoplasmic membrane (CM) to the cytosol via binding-protein dependent ATP-binding cassette (ABC) transporters. DNA array analysis of global iron-dependent gene regulation in Escherichia coli K-12 has revealed several novel Fe-repressed genes likely to specify new components of iron uptake. Such genes include the yncD and yncE genes. yncD and yncE are divergently orientated in the E. coli genome being separated by 241 bp of non-coding DNA. yncD encodes a possible TonBdependent outer-membrane ferric-siderophore receptor and, like yncE, is conserved in other E. coli strains and Salmonella. YncD appears to possess all the structural features of typical TonBdependent OM proteins, including an N-terminal signal sequence directing export across the CM and a C-terminal TonB box. However, the yncD gene is, at best, only weakly Fur and iron regulated. In contrast, the yncE gene is strongly Fur and iron regulated (up to 18 fold) suggesting that the intergenic Fur-box like sequence acts upon the yncE promoter. yncE encodes a predicted periplasmic protein of unknown function. Close homologues of Y ncE are multi-domain proteins from archaebacteria and are annotated as cell surface antigens/proteins, although their precise functions are unclear. In the research described in this thesis, Y ncE has been over-produced and purified, and antibodies have been successfully raised. Western blotting has confirmed that YncE levels are iron and Fur regulated, and that levels are maximal in the early/mid logarithmic phase of growth. Analysis of subcellular fractions show that Y ncE is mostly located in the periplasm, as anticipated. The N-terminus of isolated YncE is processed consistent with its periplasmic location. Difference spectroscopy indicated that YncE-His6 bind haems in an approximate ratio of 1:1, and BiAcore analysis confirmed haem binding with a high affinity (KD value of -6 nM). However, spectroscopic analysis of native (tag free) Y ncE did not show any clear interaction with haem, thus indicating that YncE-His6 binds haem via its His tag. Studies with strains containing yncD-or yncE-lacZ transcriptional fusions confirmed that expression of yncE is growth-phase dependent, with maximal expression in the exponential phase. yncE transcription was observed to be induced by Bip as expected. On the other hand, although yncD transcription had a similar growth-phase dependency, the overall degree of expression was far less (-20 fold). Furthermore, Bip was not found to induce expression, indicating thatyncD, in contrast to yncE, is not Fe2 + -Fur repressed. Crystals of YncE-His6 were obtained. The crystals are monoclinic, space group P2., a=69.7A, b=108.8IA, c=85.37A and 13=104.96°. Complete data sets for the native (2.0 A) and two isomorphous derivatives (Au and Ag, 2.6A and 3.oA, respectively) allowed the structure to be solved. Model building and refinement gave a seven bladed B-propeller structure for Y ncE. Superimposition of the methanol dehydrogenase (MDH; a PQQ containing B-propeller protein) and YncE structures showed that similar residues are located in the two proteins function in MDH as pyrroloquinoline quinone (PQQ) ligands. Y ncE appears to support binding of a PQQ-like cofactor in that it has a similar hydrophobic edge (Phe62) and several positively charged residues all residing on the top of the B-propeller domain (ArgI14, Lys199, His285, Asn287, Lys303). In addition, it was possible to test whether YncD is required for transport of PQQ into the periplasm for glucose dehydrogenase (GCD) activity which is required for glucose-dependent growth in the absence of the glucose transport systems. The phenotypic characterisation of a yncD null mutation in a glucose/mannose transport negative background (ptsG and manZ) , clearly showed that Y ncD is not required for PQQ enhanced growth of ptsG and ptsG manZ strains on glucose. This clearly shows that YncD is not involved in the acquisition of extracellular PQQ (at least not for GCD utilisation) as was suggested. Also, no PQQ binding to YncE could be detected by BiAcore analysis. This is consistent with no role for YncD in PQQ delivery.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available