Cloning, characterization and expression of A. thaliana G protein α-subunit gene for structural studies

A combined PCR-cloning strategy was implemented to express GPAl for structural studies. To begin with the presence of GPAl, which İs the ?-subunit of heterotrimeric G proteins from Arabidopsis thaliana, was checked with restriction enzyme digestion and sequence analysis. PCR was done on the pCIT857 vector including the coding sequence of GPAl. Appropriate primers were designed with and without the restriction enzyme sites to clone the GPAl into both subcloning and expression vectors. Once the above objective was analysed, three different parallel approaches were used to clone-GPAl from the pCIT857 vector into the expression vectors. In the firstapproach, GPAl was subcloned into several vectors including pGEM® -T Easy (Promega), pCR® II- TOPO (Invitrogen), pCR® -XL-TOPO (Invitrogen) vectors. Then GPAl derived from these vectors were cloned into expression vectors containing PGEX-4T2 (Amersham Pharmacia), pGFPuv (Clonetech), pETM-11 and pETM-30 (EMBL, Heidelberg). In the second approach, GPAl from the pCIT857 vector was directly cloned into the expression vectors. Different fusion partners of expressed GPAl, GST in pGEX-4T2 and GFPuv in pGFPuv vector and His-taq in pETM vectors, were used to yield the most efficient expression of GPAl, that would form the basis of subsequent structural characterisation experiments.In the third approach, GPAl from pCIT was directly cloned into the expression vectors including pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. Of the three approaches, this one proved most convenient as the number of steps and manipulations was limited in comparisons to those above. During all subcloning and cloning steps, different strains of E. coli including XLlBlue, TOP10 and TOP10 F' were used as hosts for subcloning and BL21(DE3), BL21(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS and BL21-CodonPlus® (DE3)-RIL were used for expression. The rationale being that different strains often perform differently.To summarise GPAl was successfully cloned into 3'MCS of pGFPuv, pCR® T7/NT TOPO (Invitrogen) and pTrcHis® TOPO (Invitrogen) vectors. In particular the verification was based upon the restriction enzyme digestion and sequencing data. Also the recombinant protein was expressed both using different IPTG concentrations and temperatures. But the expression was not detected by SDS-PAGE analysis. Recombinant protein is currently being expressed and further work is in progress to improve the properties of bacterial expression system

The Role of Bacteriocins in Mediating Interactions of Bacterial Isolates from Cystic Fibrosis Patients

Cystic Fibrosis (CF) is a common autosomal genetic disorder in Caucasian populations. CF is caused by mutations in the cftr gene, which encodes the CF transmembrane conductance regulator (CFTR). CFTR regulates chloride and sodium ion transport across the epithelial cells lining the exocrine organs. Mutations in the cftr result in a failure to mediate chloride transport, which leads to dehydration of the mucus layer surrounding the epithelial cells. The mucus coating in the lung epithelia provides a favorable environment for invasion and growth of several opportunistic bacterial pathogens resulting in life threatening respiratory infections in CF patients. Pseudomonas aeruginosa(Pa) and Burkholderia cepacia complex (Bcc) are associated with chronic lung infections and are responsible for much of the mortality in CF. Little is known about interactions between these two, often co-infecting, species. When in competition, it is not known whether Bcc replaces the resident Pa or if the two species co-exist in the CF lung. Bacteriocins are potent toxins produced by bacteria. They have a quite narrow killing range in comparison to antibiotics and have been implicated in intra-specific and inter-specific bacterial competition brought on by limited nutrients or niche space. Both Pa and Bcc produce bacteriocins known as pyocins and cepaciacins, respectively. More than 90% of Pa strains examined to date produce one or more of three pyocin types: R, F, and S. A limited number of phenotypic surveys suggest that approximately 30% of Bcc also produce bacteriocins. The goals of my thesis study were to determine if clinical strains of Pa and Bcc produce bacteriocins and to determine whether these toxins play a role in mediating intra- and inter-specific bacterial interactions in the CF lung. The final goal was to identify novel bacteriocins from clinical Pa and Bcc strains. First, I designed a phenotypic bacteriocin survey to evaluate bacteriocin production in 66 clinical Pa (38) and Bcc (28) strains procured from CF patients. This study revealed that 97% of Pa strains and 68% of Bcc strains produce bacteriocin-like inhibitory activity. Further phenotypic and molecular based assays showed that the source of inhibition is different for Pa and Bcc. In Pa, much of the inhibitory activity is due to the well known S- and RF-type pyocins. S-and RF pyocins were the source of within species inhibitory activity while RF pyocins were primarily implicated in the between species inhibitory activity of Pa strains. In contrast, Bcc inhibition appeared to be due to novel inhibitory agents. Finally, I constructed genome libraries of B. multivorans, B. dolosa, and B. cenocepacia to screen for genes responsible for the inhibitory activity previously described in Bcc. ~10,000 clones/genome were screened, resulting in fifteen clones with the anticipated inhibition phenotype. Of these fifteen, only five clones had stable inhibitory activity. These clones encoded proteins involved in various metabolic pathways including bacterial apoptosis, amino acid biosynthesis, sugar metabolism, and degradation of aromatic compounds. Surprisingly, none of Bcc clones possessed typical bacteriocin-like genes. These data suggest that, in contrast to all bacterial species examined in a similar fashion to date, Bcc may not produce bacteriocins. Instead, Bcc may be using novel molecular strategies to mediate intra- and inter-specific bacterial interactions