Molecular-genetic characterisation of arginine auxotrophy in the opportunistic human pathogen, Pseudomonas aeruginosa

Pseudomonas aeruginosa (Pa) is a predominant pathogen in the airways of people with cystic fibrosis (CF). Pa undergoes several evolutionary adaptations that allow it to thrive in the CF airway environment, including the emergence of amino acid auxotrophs. Arginine auxotrophs arise at high frequency in CF, and arginine is known to play an essential physiological role in Pa, so I chose to investigate the possible molecular basis of this further. I found that a transposon (Tn) mutant containing a disrupted argA gene was auxotrophic for arginine, but readily accrued secondary mutations enabling the mutant to grow, even in the absence of this amino acid. Whole genome sequencing revealed that one of these bypass mutants (denoted hereafter, AMB128) contained a single nucleotide polymorphism (SNP) in the ambA gene. Complementation analysis confirmed that the SNP in ambA was responsible for the arginine auxotrophy bypass, and further investigation established that the SNP conferred change-of-function rather than loss-of-function to the AmbA protein. Detailed proteomic profiling of AMB128 revealed up-regulation of certain peptides derived from the enzyme, N-acetyl glutamate synthase (NAGS) encoded by argA. This was unexpected because the argA gene in AMB128 was disrupted by a Tn insertion (and this insertion was confirmed by inspection of the whole genome sequence). This up-regulation was found to be likely mediated by an outward-facing promoter in the Tn sequence, driving expression of a truncated regulatory domain and functional catalytic GNAT domain in the NAGS. The truncated regulatory domain was anticipated to disrupt feedback inhibition of the enzyme. Counter-intuitively, the Tn insertion may therefore elevate the level(s) of certain intermediates in this arginine biosynthetic pathway. The proteomic analysis also provided insight into the likely cellular response to the accumulation of these metabolic intermediates. Proteins involved in polyamine catabolism and transport of small molecules were up-regulated in AMB128, whereas many proteins involved in denitrification were down-regulated. In summary, this work demonstrates remarkable metabolic plasticity of Pa by highlighting one mechanism by which the organism can adapt when faced with dysregulated arginine biosynthesis. In this regard, it is worth noting that the ambA gene in a selection of other spontaneous argA::Tn bypass mutants did not contain any SNPs, indicating that this is just one of (likely) many possible evolutionary “solutions” to the problem.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Mohan, Veena

Advisor dc:contributor.advisor

• Spring, David

• Pseudomonas aeruginosa
• amino acid
• auxotrophy
• N-acetyl glutamate synthase
• Whole genome sequencing
• Proteomics

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c841ca4c-4680-48ca-b138-e0770689e8d5/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-7355-2824

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/322281