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Nitrogen Limitation and Evolution of Swimming Motility in Aflagellate Mutant Strains of Pseudomonas fluorescens SBW25
EP30901
Poster Title: Nitrogen Limitation and Evolution of Swimming Motility in Aflagellate Mutant Strains of Pseudomonas fluorescens SBW25
Submitted on 06 Nov 2019
Author(s): Astrid E. Altamirano-Junqueira
Affiliations: University of Reading
This poster was presented at 42nd Senior Technical Meeting (American Chemical Society). Puerto RIco Section. Scientific approaches to celebrate the International Year of the Periodic Table. 09 November 2019. MAYAGUEZ, PUERTO RICO
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Poster Information
Abstract: Methods: Evolution of swimming motility in the aflagellate strains, SBW25ΔfleQ (Fla-, Visc+), AR1 (Fla- , Visc-), and AR2 (Fla-, Visc-) was performed in 0.25 % M9 –glucose agar medium with glutamine, glutamate (nitrogen limiting) or ammonium (nitrogen replete) as nitrogen source. Colony spreading phenotypes of evolved isolates on minimal and rich media were monitored and quantitated by time lapse photography. Mutations were identified by targeted sequencing of segments of ntrB, ntrC and the entire glnK gene or by whole genome sequencing (WGS).
Results: As predicted, swimming motility of the sessile strains, AR1 and AR2, evolved later with ammonium as N-source (mean = 5.53 days, SD = 0.61 days, n = 19) compared to glutamine (mean = 2.88 days, SD = 0.89 days, n = 17) or glutamate (mean = 2.94 days, SD = 0.80 days, n = 17). Notably, SBW25ΔfleQ which spreads with spidery-like tendrils over the surface of the agar also evolved swimming motility thus enhancing migration within the liquid phase and access to nutrients within the agar. Irrespective of parent strain or N-source all evolved mutants had acquired a mutation in NtrB, primarily T97P within the PAS domain, or a D228A/N mutation known to interfere with PII interaction, thus increasing NtrC-P levels by decreasing phosphatase activity. Despite impaired growth in ammonium, none of the evolved mutants possessed anticipated secondary mutations in the Helix-turn-Helix (HTH) domain of NtrC that might moderate the unregulated Ntr response. Mutation in AlgP histone-like protein may have been selected in response to a different stress, but could be an indicationof involvement of an alternate EBP. Possession of an unrelated mutation in a predicted ammonium transporter PFLU_RS08590, correlated with improved growth on ammonium and deserves further investigation.
Conclusions: Mutation in NtrB is the primary evolutionary pathway for re-establishment of swimming motility in P. fluorescens ΔfleQ strains irrespective of nitrogen status.
Summary: As predicted, swimming motility of the sessile strains, AR1 and AR2, evolved later with ammonium as N-source compared to glutamine or glutamate. All evolved mutants had acquired a mutation in NtrB (T97P). Mutation in NtrB is the primary evolutionary pathway for re-establishment of swimming motility in non-flagellate mutant strains.References: 1. Altamirano Junqueira, A. E. E.( Evolution of swimming motility in aflagellate strains of Pseudomonas fluorescens SBW25. PhD thesis, University of Reading. ( http://centaur.reading.ac.uk/85599).

2. Science 347: 6225 pp. 1014 107
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