Pathways and enzymes of polyamine synthesis.

Surface motility of W3110-LR. (A) Surface motility of parental and derivative strains lacking the major subunits of the flagella (FliC), pili (FimA), or both. (B) Swimming motility with and without 0.5% glucose. (C) TEM images of cells of W3110-LR and W3110-LR ΔfliC ΔfimA taken from the movement’s edge directly from surface motility plates.

Genetics of surface motility. (A) PDSM of mutants with defects in polyamine anabolic genes. (B) Diameter of surface movement of polyamine mutants after 36 hours. Error bars represent standard deviations for three independent replicates. Statistical analysis was performed using the Dunnett test of significance: *, P<0.05; **, P<0.01; ***, P<0.001. All assays were performed in triplicate and representative images are shown. In this figure the ΔspeB mutant was IM26.

Nutritional supplementation of ΔspeB mutant. (A) Putrescine and spermidine supplementation of wild type and ΔspeB strains. (B) Diameter of surface movement of polyamine mutants after 36 hours. Error bars represent standard deviations for three independent replicates. Statistical analysis was performed using the Sadik test of significance: *, P<0.05; **, P<0.01; ***, P<0.001. All assays were performed in triplicates and representative images are shown. In this figure the ΔspeB mutant was IM26.

Putrescine transport and surface motility. (A) Representative images of surface motility for strains defective in putrescine transport genes. (B) Diameter of surface movement of polyamine mutants after 36 hours. Error bars represent standard deviations for three independent replicates. Statistical analysis was performed using the Dunnett test of significance: *, P<0.05; **, P<0.01; ***, P<0.001. All assays were performed in triplicate and representative images are shown.

Putrescine catabolism and surface motility. (A) Pathways and enzymes of putrescine catabolism. (B) Motility diameter of putrescine catabolic mutants after 36 hours. Error bars represent standard deviations for three independent replicates. Statistical analysis was performed using the Dunnett test of significance: *, P<0.05; **, P<0.01; ***, P<0.001. All assays were performed in triplicates and representative images are shown. (C) Effect of loss of speE on the patA puuA catabolic double mutant.

Surface motility and pili expression in W3110 and ΔspeB. (A) W3110 ΔspeB surface motility with 0.0, 0.1, 1.0, and 4.0 mM exogenous putrescine. All assays were performed in triplicate. Representative images are shown. (B) Average diameter of 3 separate surface motility plates. The parental strain without putrescine is shown for reference. Significance was determined by comparing the diameters of the ΔspeB mutants in the different concentrations of putrescine compared to the parental W3110. One way ANOVA was used with Dunnett hypothesis testing to determine p values, ***, p <0.001. (C) Reverse transcriptase-quantitative PCR using primers targeting the fimA gene from cells grown in 0.0, 0.1, 1.0, and 4.0 mM of supplemented putrescine. Double deltas were generated by normalizing the parental and the ΔspeB mutant RNA libraries to rpoD then comparing fimA expression. Two-way ANOVA was used to determine significance followed by Sadik hypothesis testing. ***, p<0.001. (D) Enzyme linked immunosorbent assays using antibodies targeting pili (FimA) from cells grown with 0.0, 0.1, 1.0, and 4.0 mM of supplemented putrescine. Two-way ANOVA was used to determine significance followed by FDR adjusting. ***, p<0.001. In this figure the ΔspeB mutant was J15.

Transmission electron micrographs of ΔspeB mutant cells after surface motility. Representative images are shown and cells were removed from motility plates with the following exogenous putrescine concentrations: (A) none, (B) 0.1 mM, (C) 1.0 mM, and (D) 4.0 mM. No pili were observed with 0 and 0.1 mM putrescine. Optimal pili production was observed with 1.0 mM putrescine. Shorter and less pili were observed with 4.0 mM. The arrows in (D) point to apparent pili fragments. The bar represents one micron. In this figure the ΔspeB mutant was J15.

Visual representation of the results from transcriptomic sequencing of W3110 and the ΔspeB mutant’s gene expression in media with and without 1.0 mM putrescine. (A) Multidimensional scaling plot of the parental and ΔspeB mutant transcriptomes. When grown with 1.0 mM putrescine (red), the ΔspeB mutant transcriptome (triangles) are nearly identical to the parental transcriptomes when grown without and with putrescine (blue and red circles, respectively). When grown without putrescine, the ΔspeB mutant transcriptome (blue triangles) is greatly skewed from transcriptomes of the parental strain and the ΔspeB mutant grown with putrescine. (B) Heatmap of the top 100 most variable genes further demonstrates the distinctiveness of the ΔspeB mutant grown without putrescine and the similarities of the ΔspeB mutant transcriptome when grown with 1.0 mM putrescine supplementation and the parental grown with or without putrescine. In this figure the ΔspeB mutant was J15.

Transcriptomic sequencing of genes for the fim region and regulators that control fim gene expression. (A) Expression of the fim genes in the parental W3110 and the speB mutant with and without putrescine supplementation. Values were calculated by dividing the individual replicates’ counts per million (CPM) value for each gene by the mean CPM of that gene in W3110 grown without putrescine and multiplying by 100 to yield the percent expression. Arrows below the genes signify the known operons: fimB and fimE belong to single gene operons, while fimAICDFGH belong to one operon. One way ANOVA was used to determine significance using Dunnett hypothesis testing. **, p <0.01; ***, p <0.001. (B) Expression of some regulators known to affect fim gene expression. Values were calculated as described in (A). ***, p <0.001. (C) Surface motility of W3110 and three regulator mutants (Δhns, ΔihfA, and Δlrp). A gene found to be significantly different by this transcriptomic analysis (hns) was confirmed to be important in surface motility. In this figure the ΔspeB mutant was J15.

Differentially expressed genes that are proposed to contribute to putrescine homeostasis. A positive number means more transcripts in the ΔspeB (lower putrescine) strain.

The deduced diversion of metabolism in a speB mutant away from energy metabolism toward putrescine synthesis compared to the parental strain. The effects of low putrescine are shown. Genes and processes (transport and transcriptional regulators) in red have fewer transcripts, while those in black or blue have more. The dashed lines represent proposed reduction in metabolic flux because of fewer transcripts from genes coding for the enzymes involved. Operons are shown except for the larger operons or regulons. For example, pur is meant to represent the unlinked genes that code for enzymes of purine synthesis. Table 1 or Supplemental Table 1 should be consulted for specific genes and the quantitative change in transcripts.

Strains