Abstract/Summary

Background: Swimming motility, an important trait for successful root colonization, by Pseudomonas fluorescens SBW25 requires flagella, expression of which is activated in a hierarchical manner by the master regulator FleQ. A non-motile, aflagellate strain, AR2 ΔfleQviscB had been shown to reacquire flagella driven motility through 2 step mutation of a related two-component regulator, the NtrBC nitrogen sensor:regulator. Overexpressed NtrC-P is assumed to activate flagella expression. NtrBC responds to nitrogen limitation by upregulating expression of operons involved in nitrogen assimilation, including glnAntrBC [encoding: glutamine synthetase (GS), NtrBC] and glnKamtB (encoding PII and AmtB ammonium channel). Aims: To investigate the influence of different nitrogen sources and different motility phenotypes on the evolution pathway and probability/frequency of re-establishment of swimming motility in aflagellate P. fluorescens SBW25 ΔfleQ strains. Research hypothesis: The physiological status of NtrBC under nitrogen limitation will increase the probability of identification of evolved swimming isolates of P. fluorescens SBW25 ΔfleQ strains carrying mutations in ntrBC. Also, as mutation of ntrBC has global effects and impact on cell fitness, it was hypothesized that other enhancer binding proteins (EBP) might rescue loss of fleQ, particularly in nitrogen replete conditions. 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 timelapse 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-turnHelix (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 indication of 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. Delayed recovery of these mutants from ammonium/glucose plates may be due to a combination of low transcription of ntrBC and uncontrolled assimilation of ammonia due to dysregulation of the Ntr regulon. Details of the mechanism by which mutated NtrBC compensates for loss of the FleQ regulator remain to be fully elucidated and will provide a fuller picture of the evolution pathway. Understanding details of crosstalk between these critical regulons, flagella regulon, and the Ntr system are likely to enhance understanding of the ecology of this important rhizosphere associated bacterium and may have downstream implications in the application of P. fluorescens as biopesticide or biofertiliser.