The role of ChpA in Pseudomonas aeruginosa motility

Andrew James Leech (2004). The role of ChpA in Pseudomonas aeruginosa motility PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

       
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Author Andrew James Leech
Thesis Title The role of ChpA in Pseudomonas aeruginosa motility
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Dr John Mattick
Total pages 187
Collection year 2004
Language eng
Subjects L
270200 Genetics
780105 Biological sciences
Formatted abstract

ChpA is a Pseudomonas aeruginosa signal transduction protein that was originally identified in a transposon library for non-twitching mutants and has subsequently been identified in whole-genome virulence screens, but as yet the precise role of this protein in these processes remains to be determined. Analysis of the protein sequence revealed that ChpA possesses 9 potential sites of phosphorylation, including 6 histidine phosphotransfer domains (HPt), a novel serine and threonine phosphotransfer domain (SPt, TPt) and a C-terminal CheY-like domain. As such, ChpA is probably the most complex signal transduction protein yet described in nature. 

 

A genome database search revealed that several other bacteria, predominantly p and γ-Proteobacteria, also possess genes homologous to chpA, and together these genes encode what appears to represent a distinct sub-class of CheA-like histidine kinases, hereafter referred to as multi-HPt CheA/CheY hybrid proteins. Phylogenetic analysis of the multi-HPt CheA/CheY hybrid proteins showed that the HPt-like domains can be grouped into distinct clusters that probably reflect the evolutionary development of the protein class. Interestingly, the most ancient (inferred from clustering nearest to the cyanobacterial outgroup) and most conserved HPt-like domains of ChpA both participate in the regulation of twitching motility. This suggests that this sub-class of proteins is likely to have evolved with twitching motility as its primary function, possibly acquiring additional HPt-like domains to either integrate additional signalling inputs into the regulation of twitching motility or to coordinate twitching motility with the expression or function of other virulence determinants. Analysis of HPt domain active site substitutions in multi-HPt domain CheA/CheY hybrid proteins suggests that there may be a selective advantage, or a particular function, conferred by threonine substitutions of histidine residues, possibly reflecting the use of different phosphotransfer chemistries. 

 

The encoded chemotaxis-like protein domain structure, the surrounding chromosomal gene organisation and the identification in a non-twitching screen all suggest that chpA is involved in the regulation of twitching motility, but to date this has not been definitively proven by complementation of the phenotype. Therefore, a range of chpA expression vectors and ChpA phosphotransfer domain point mutants were generated, that together constitute an invaluable resource for the elucidation of the role of ChpA in motility and virulence in P. aeruginosa. The involvement of ChpA in regulating twitching motility was then confirmed by complementation. The results also showed that ChpA function is dosage sensitive, and that ChpA is expressed to relatively low levels off its native promoter. The HPt2, HPt3 and CheY domains of ChpA were identified as the specific domains that participate in the regulation of twitching motility, with further characterisation of the phosphotransfer domain point mutants showing that ChpA is not responsible for regulating twitching motility at the level of pilA transcription. A model for the phosphotransfer circuitry that regulates twitching motility is therefore proposed that integrates the findings of this thesis with what is known about phosphotransfer relays in chemosensory pathways. 

 

The ChpA point mutants displayed several distinct phenotypes under conditions conducive to swarming. However, because the observed motility displayed a complete dependence on type IV pili it is not entirely clear whether the motility is swarming (flagella-mediated) or a more complex manifestation of a retractile motility (fimbriae-mediated). Regardless of this, the swarm assay appears to be a more sensitive tool to investigate and analyse the perturbations and complexities of the ChpA point mutants than the standard twitching stab assay, as is evidenced by the five different motility phenotypes displayed. Of particular importance is the identification of a motility phenotype for the SPt point mutant that has tentatively been linked to aberrant rhamnolipid levels. This is then the first report to show functionality for a bacterial serine phosphotransfer domain in the context of a CheA-like protein kinase, and possibly constitutes evidence that a function of ChpA is to coordinate regulation of auxiliary virulence determinants with motility. 

Keyword Pseudomonas aeruginosa
Bacteria -- Motility

 
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Created: Fri, 24 Aug 2007, 18:34:34 EST