Members of the important disease causing bacterial generas, Chlamydia and Chlamydophila, are characterised by a complex developmental cycle which is comprehensively described by microscopy. The inability to use standard genetic techniques for this obligate intracellular bacterium, however, means that significant gaps in our understanding of the molecular mechanisms used to control growth and development of Chlamydia still exist. The current study investigated the function of bacterial guanosine triphosphatases (GTPases), components of the organism's limited signal transduction arsenal, in regulatory control of the chlamydial development cycle. Initial analysis of the gene transcription of chlamydial GTPases and other predicted signal transduction genes using real time RT-PCR, in a Chlamydophila pneumoniae A-03 tryptophan depletion model of persistence, revealed significant differential expression of genes in response to the addition of interferon gamma (IFN-γ). Predicted chlamydial GTPase encoding genes, ychF, yhbZ and yphC, associated with ribosome function amongst other processes were strongly up-regulated, while hflX was down-regulated in the persistent cultures. Analysis of an additional model of Cp. pneumoniae persistence, induced by limitation of host cell iron, revealed that ychF, yhbZ and yphC were also up-regulated in the persistent cultures. This study provided the most comprehensive analysis of Cp. pneumoniae gene transcription to date and suggest that chlamydial GTPases serve a role in generation of the persistent chlamydial phenotype. Cloning and expression of Cp. pneumoniae and Cp. abortus yhbZ, including demonstration of in vitro GTPase activity, indicates that this chlamydial gene encodes a member of the universally conserved and essential bacterial Obg subfamily of GTPases. Evidence is building that members of this latter family of bacterial GTPases are important regulators of bacterial growth and morphological differentiation in developmentally complex bacteria. Over-expression of chlamydial YhbZ subfamily GTPases in Escherichia coli revealed inhibition of bacterial growth and disruption of cell division and chromosome functions leading to the generation of elongated cells with limited chromosome segregation, as described for Obg subfamily members from E. coli and other bacteria. Although more analysis is required, we suggest a novel mechanism of chlamydial Obg GTPase regulation involving sensing of host cell GTP/GDP pools to control secondary differentiation of reticulate bodies (RBs) back to elementary bodies (EBs). Analysis of the chlamydial complement of bacterial GTPases was extended to HflX, a previously uncharacterised and only predicted GTPase conserved in bacteria. HflX sequence analysis revealed conservation of G motifs responsible for nucleotide binding and hydrolysis (G1, G3, G4) and protein interaction (G2), although the latter was unique to HflX subfamily GTPases. Recombinant Cp. pneumoniae HflX displays GTPase activity with nucleotide specificity for GTP. We tested Cp. pneumoniae HflX function by over-expression in E. coli which led to inhibition of growth in E. coli and elongation of cells with normal chromosome partitioning. This phenotype was the probable result of disruption of a stage in cell division subsequent to chromosome segregation. This present study provides the first evidence to show that bacterial HflX is a GTPase and suggests a regulatory role in bacterial cell cycle control.
| Identifer | oai:union.ndltd.org:ADTP/265239 |
| Date | January 2006 |
| Creators | Polkinghorne, Adam |
| Publisher | Queensland University of Technology |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
| Rights | Copyright Adam Polkinghorne |
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