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Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

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Science  27 Feb 2015:
Vol. 347, Issue 6225, pp. 1014-1017
DOI: 10.1126/science.1259145

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  1. Fig. 1 Phenotypic assays of motility variants.

    (A) Surface-spreading motility assays of ancestral (AR2) and evolved slow-spreading (AR2S) and fast-spreading (AR2F) mutants, after 27 hours. (B) Electron microscopy confirms the presence of a flagellum in AR2F but fails to confirm presence in AR2S. (C) Mean (n = 4) cell doublings per hour (±1 SEM, error bars). Strains were grown in differing nitrogen environments: in 10 mM glutamine (Gln), glutamate (Glu), and ammonium (NH4) as the sole nitrogen source or in high-nutrient lysogeny broth (LB) (AR2: F3,12 = 13.460, P < 0.001; AR2S: F3,12 = 72.674, P < 0.001; AR2F: F3,12 = 52.538, P < 0.001). There were also differences between the doubling rate of strains within each nitrogen medium [Glu: F2,9 = 12.654, P = 0.002; NH4: F2,9 = 40.529, P < 0.001], with the exception of Gln (F2,9 = 3.703, P = 0.067).

  2. Fig. 2 Heat map of microarray expression profiles for all evolved and ancestral motility variants.

    Heat maps show where there is significant (P ≤ 0.05) fold change of ≥2 in genes selected based on gene ontology terms for (A) bacterial-type flagellum (24 genes) and (B) nitrogen compound transport (146 genes) for all strains. The gradation of colors reflects normalized raw signal values across the entire array. Genes are ordered according to chromosomal position to enable clearer visualization of co-regulated gene clusters. Full transcriptome analysis is reported in the supplementary materials.

  3. Fig. 3 Full-chain multi-template 3D models of protein structures of slow- and fast-spreading motility variants.

    Slow-spreading variants can follow either the direct regulatory route through mutation of NtrB or GlnK (A) or the physiological route through mutation of GlnA, causing overactivation of NtrB (B). Both routes are predicted to lead to hyperphosphorylation of NtrC. Fast-spreading variants all show mutational changes to NtrC (C), which has a similar global structure to FleQ (D). The color scheme represents the variation in models, which correlates with local (per-residue) quality and disorder. Regions colored in blue and green represent low local variability in structure, whereas those in red show high local variability (see Table 1 and table S2 for mutation details). § denotes all mutations mapped onto SBW25 WT protein structures for illustrative purposes; * indicates a truncated domain.

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