As 1w4 capsule switching appears to result from a decrease in flux through the CP- arginine and/or CP-UTP pathway(s), it was postulated that addition of the relevant nutrients to the medium would alleviate the metabolic stress and abolish switching. As illustrated in Figure 6.1, Gram-negative bacteria are able to take up arginine and uracil from the extracellular environment, via salvage pathways encoded by arcD (arginine transport), uraA and upp (involved in uracil transport and conversion to UMP, respectively). As homologs of these transporters exist in SBW25 (arcD: Pflu4890,
uraA: Pflu0898 and upp: Pflu0899, with 46 %, 40 % and 70 % amino acid similarity to their respective E. coli counterparts), it was probable that these nutrients could also be salvaged in P. fluorescens.
1w4
1w4-ΔcarB
1w4-ΔrecA
6.3.4.1 Effects of arginine and uracil on 1w4 phenotypic switching
Colony dimorphism, cell morphology in liquid culture and calcofluor binding by 1w4 were investigated on four types of medium: KB, KB+arginine, KB+uracil, KB+arginine+uracil (Figure 6.7). In the case of cell morphology, overnight pre-cultures were required in order to acclimatize cells to environmental conditions (see section 2.2.12). Addition of uracil (or arginine and uracil) completely abolished colony dimorphism, and greatly reduced the frequency of capsulated cells. Contrastingly, the addition of arginine alone did not alter colony or cell dimorphism. ACP-production was not noticeably altered by addition of either arginine or uracil.
Figure 6.7: Investigation of the effects of arginine and/or uracil addition on the phenotype of 1w4.
Colony morphology at 48 hours (top row, scale bar indicates ~3 mm), cell morphology and capsule production in overnight cultures (middle row; India ink stained cells under x40 light microscope, scale bar indicates ~10 µm), and calcofluor binding (bottom row; x63 fluorescence microscope images). Brightness/contrast of some images altered in iPhoto.
Subsequently, the effects of arginine and/or uracil addition on capsule expression were quantified. A capsule counting assay was performed on 1w4 cultures containing increasing amounts of arginine and/or uracil (see section 2.2.12.4). Presented in Figure
6.8 and Appendix A4.3, the results demonstrate that the addition of 2 mM arginine had no significant effect on capsule expression (P=0.499), while the addition of uracil or both arginine and uracil significantly reduced capsule expression (P<0.001). Together, these results indicate that a deficiency in the CP-UTP biochemical pathway is responsible for 1w4 colony dimorphism.
Figure 6.8: Effect of addition of increasing amounts of uracil and/or arginine on capsule expression
in 1w4 populations. The addition of arginine (red) did not alter capsule expression, while the addition of
uracil (blue) or uracil and arginine (green) drastically reduced capsule expression.Data points are mean values of five replicates, and error bars indicate one standard error.
6.3.4.2 Effects of uracil on capsule expression in re-evolved switcher genotypes In order to assess the generality of the conclusions in section 6.4.3.1, the effects of uracil addition on capsule expression in the other switching genotypes evolved from 1s4 were quantified. A capsule counting assay was performed on 1s4, 1w4 and each of the six additional switchers in KB and KB+2mM uracil cultures, with pre-cultures (see section 2.2.12.4). As depicted in Figure 6.9, the addition of uracil significantly reduced capsule production in all genotypes containing a carB mutation (P<0.01, see Appendix A4.4). The addition of uracil had no effect on the level of capsule expression in 1s4 or the pyrH switcher, 1w4-reN1.4 (P=0.771 and 0.778, respectively). This result is not particularly surprising, given that pathway-blocking pyrH mutation of 1w4-reN1.4
occurs downstream of UMP; addition of uracil (which is converted into UMP) would not alleviate flux reduction in this genotype (see Figure 6.1). Collectively, these results lend further support to the hypothesis that switching occurs as a result of a deficiency in the CP-UTP biochemical pathway.
Figure 6.9: Relative proportions of capsulated cells in indicated switcher populations grown in KB
and KB+2 mM uracil cultures. Names of the re-evolved switcher genotypes are shortened; ‘1w4-re’ was
removed from the beginning of each. Addition of uracil significantly reduced capsule expression in all genotypes except 1s4 and 1w4-reN1.4. Bars represent mean values of five replicates, and error bars indicate one standard error. Stars indicate level of statistical significance (*<0.05, **<0.01, ***<0.001).
6.3.4.2.1 Investigation of an indirect role for arginine in 1w4 phenotypic switching
The experiments above suggest that arginine is not involved in phenotypic switching. However, as KB medium contains a significant amount of arginine (in tryptone), arginine may play an indirect role in switching; uptake of arginine and conversion to CP
via the arc pathway may contribute to the 1w4 phenotype (see Figure 6.1). In order to investigate this possibility, an artificial blockage was created in the 1w4arc pathway by deleting arcB (Pflu4892). The arcB gene encodes catabolic ornithine carbamoyl transferase (OCT), which converts citrulline into CP and ornithine (anabolic OCT, encoded by argF (Pflu1146), catalyzes the forward reaction) (Legrain et al., 1977).
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To construct 1w4-ΔarcB, a similar protocol to that described for the carB and recA
deletion strains was used (see section 6.3.3), with the following modifications. For the SOE-PCR, two separate DNA fragments of 570 bp and 490 bp were amplified using primers pairs ArcBKO-1/-2 and ArcBKO-3/-4 (˚C annealing temperature, 30 seconds extension time). These were then combined and used as a template for the second round of PCR that amplified the ~1 kb deletion fragment using primers ArcBKO-1/-4 (58˚C annealing temperature, 1 minute extension time). Phenotypic characterization of 1w4-
ΔarcB included examination of calcofluor binding, colony and cell morphology (Figure 6.10). Deletion of arcB did not alter 1w4 colony dimorphism, capsular phenotype or ability to bind calcofluor. A quantitative capsule counting assay (see section 2.2.11.4) demonstrated that 1w4-ΔarcB did not produce a significantly different proportion of capsulated cells to 1w4 (P=0.723, see Appendix A4.2). Thus, arginine catabolism via
the arc pathway does not appear to play any role in 1w4 phenotypic switching.
Figure 6.10: Phenotypic characterisation of 1w4-∆arcB, a genotype constructed by deletion of arcB
(Pflu4892) from 1w4. Like 1w4, 1w4-∆arcB produced two distinct types of colonies on KB agar (48
hours, scale bar indicates ~3 mm and applies to both genotypes; A) and cap-/cap+ cells (x40 light microscope images, scale bar indicates ~10 µm and applies to both genotypes; B). Additionally, 1w4-
∆arcB retained the ability to bind calcofluor (x63 fluorescence microscope images, C). Images are of comparable magnification between genotypes. Brightness/contrast of some images altered in iPhoto.
1w4
1w4-∆arcB