Browsing by Author "Crampton, Michael Craig"

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    The development of a flagellin surface display expression system in the gram-positive bacterium, Bacillus halodurans Alk36
    (2007) Crampton, Michael Craig; Reid, Sharon J; Louw, Maureen E; Berger, E
    This study relates to the development of an alkaliphilic, thermo-tolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium harbors the endogenous genetic background to over-produce flagellin protein continuously. In order to harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene which codes for flagellin was inactivated on the chromosome giving rise to the B. halodurans BhFC0l mutant. This strain was non-motile as determined on motility plates and confirmed by PCR analysis. Motility was, however, restored through complementation of the expression vector carrying a functional hag gene. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions in order to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-l clade C gpl20 epitope were respectively incorporated into both of the permissive sites as in-frame fusions and found to be successfully displayed on the cell surface. The poly-His peptide was shown to be functional through metal binding and affinity purification studies. The display of the HIV-1 subtype C gp 120 V3 loop was also shown to be functional through immunological studies using peptide specific antibodies. Surface display of the poly-His and HIV-l epitope was shown to have improved metal binding and enhanced expression levels of the chimeric flagellin when the peptides were insel1ed at amino acid position 180 (pSECNC6). This specific site is the only insertion point that falls within the re-defined variable domain of the FliC protein from B. halodurans Alk36.
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    Physiological and genetic evidence for an OmpB signal transduction system in Erwinia chrysanthemi
    (1996) Crampton, Michael Craig; Qhobela, Molapo
    In order for bacteria to survive in their environment they must continuely sense signals such as, presence of host organisms, chemical concentrations, or variationsin other physiological parameters. Many bacteria sense their environment through the use of a two component regulatory systems. These systems usually employ the use of two different proteins, a sensor protein and its cognate response regulator. Some bacteria can survive fluctuations in medium osmolarity through the use of a two component signal transduction system. In Escherichia coli and Salmonella typhimurium this two component system includes the EnvZ sensor protein and its cognate response regulator, OmpR. The two genes that code for these proteins are envZ and ompR genes respectively. The two genes together form the ompB operonrespectively. This operon regulates the expression of two outer membrane proteins, OmpF and OmpC in response to medium osmolarity in E. coli.Erwinia chrysanthemi has been found to be sensitive to desication. Proliferation of soft rot, caused by this organism, has also been associated with irrigation. E.chrysanthemi has also been observed to respond to changes in medium osmolarity. Evidence of an ompB operon was thus sought. Outer membrane proteins were isolated using sodium lauroylsarcosine. Three major outer membrane proteins were isolated, namely Ompl (37.5 kd), Omp2 (35.5 kd) and Omp3 (34.5 kd). Increase in medium osmolarity resulted in an increase in expression of Omp3, while Ompl was suppressed. This lends support to the presence of an ompB like signal transduction system in E. chrysanthemi. Growth temperature was shown to have no effect on the expression of the major OMP. Similarly, culture growth phase had no effect on major OMP expression. However, two induced OMP were present from mid log phase onwards.