The Cystine Binding Protein (BspA) of Lactobacillus fermentum BR11
Hung, Jacky (2005) The Cystine Binding Protein (BspA) of Lactobacillus fermentum BR11. Masters by Research thesis, Queensland University of Technology.
BspA was first identified on the basis of being the major constituent of 5 M LiCl washes of whole Lactobacillus fermentum BR11 cells. The bspA gene is encoded within a putative ATP-binding cassette (ABC) transport operon, and sequence analysis revealed that it is a member of the family III solute binding proteins. Unlike the majority of solute binding proteins from Gram-positive bacteria, BspA is not tethered to a lipid anchor in the cell membrane, and hence is not a lipoprotein. Extraction of BspA with concentrated salt solutions such as 5 M LiCl is consistent with the notion that electrostatic interactions are responsible for securing it to the L. fermentum BR11 cell.
L. fermentum PNG201 is a BspA negative mutant strain created by disrupting bspA. This strain was shown to be incapable of cystine uptake. Thus, the genetic and biochemical evidence strongly suggests BspA is a cystine binding protein of an ABC transporter. Measurement of the binding affinity between BspA and L-cystine has confirmed high affinity binding (dissociation constant is 0.2 µM), and high specificity (over 100-fold excess of non-target amino acids did not disrupt BspA / L-cystine binding). In addition, collagen did not appear to affect BspA/cystine binding, indicating extracellular matrix (ECM) binding capacity noted by other researchers may be unrelated to amino acid binding.
An interesting phenotypic characteristic of L. fermentum PNG201 is its apparent increased sensitivity to oxygen and the superoxide-generating chemical - paraquat compared to the parent L. fermentum BR11 strain. Catalase supplemented aerobic cultures of L. fermentum BR11, and L. fermentum PNG201 were protected from oxidative stress, suggesting hydrogen peroxide is responsible for the observed oxidative stress. It was found that addition of cystine to aerobic cultures of L. fermentum BR11 or L. fermentum PNG201 protected both strains from oxidative stress, with L. fermentum BR11 able to utilize smaller concentrations of cystine compared to L. fermentum PNG201. Detection of hydrogen peroxide in aerobic cultures of L. fermentum BR11 and L. fermentum PNG201 confirmed the production of hydrogen peroxide is responsible for causing oxidative stress. The BspA mutant strain L. fermentum PNG201 consistently produced more hydrogen peroxide per optical density compared with the wild type, indicating it overproduced hydrogen peroxide. When 0.4 mM hydrogen peroxide has been accumulated by growing cell cultures, both L. fermentum BR11 and L. fermentum PNG201 enters stationary phase, suggesting both strains have a similar sensitivity to hydrogen peroxide.
Small epitopes from the HIV gp41 protein and the Chlamydia psittaci major outer membrane protein have been successfully displayed on the cell surface of L. fermentum BR11 as fusion proteins to the BspA molecule. However, the capability of BspA in exporting larger polypeptides has not been tested. In this study, the large extracellular enzyme - glucosyltransferase (GtfJ) from Streptococcus salivarius ATCC 25975 was fused to BspA to demonstrate that this expression system is capable of exporting large functional enzymes to the cell surface of L. fermentum BR11. The native GtfJ is 160kDa in size and also contained an export signal, which was deleted in the cloning process and replaced with BspA, resulting in a fusion protein of 175kDa. Export of the BspA/GtfJ fusion protein is dependant entirely on BspA's export signal. Recombinant enzyme expression and glucosyltransferase activity were detected by measuring the glucan formed by sonicated cell extracts in acrylamide gels. Enzyme activity measurements on whole cells has revealed the recombinant Lactobacillus was incorporating 20-40 nmol of sucrose-derived-glucose into glucan per ml of cell culture per OD unit, which is comparable to activity levels exhibited by the native bacteria that expressed this enzyme. Comparison of GtfJ enzyme activity between whole cells and sonicated cell extracts of recombinant L. fermentum confirmed the extracellular location of BspA/GtfJ as enzyme activity was essentially identical.
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|Item Type:||QUT Thesis (Masters by Research)|
|Supervisor:||Giffard, Philip, Hafner, Louise, & Walsh, Terence|
|Keywords:||Lactobacillus, ABC uptake system, cystine, affinity constant, oxidative stress, hydrogen sulfide, fusion protein, glucosyltransferase.|
|Divisions:||Past > QUT Faculties & Divisions > Faculty of Science and Technology|
Past > Schools > School of Life Sciences
|Department:||Faculty of Science|
|Institution:||Queensland University of Technology|
|Copyright Owner:||Copyright Jacky Hung|
|Deposited On:||03 Dec 2008 13:57|
|Last Modified:||29 Oct 2011 05:43|
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