Thus far, the evolutionary history of 1w4 has been elucidated. The work in this section concentrates on determining which of the nine mutations are required for manifestation of colony and cell dimorphism.
5.3.4.1 Reconstruction of carB alleles in various genetic backgrounds 5.3.4.1.1 Construction of pUIC3-carBwt and pUIC3-carBmut
To test the mechanistic role of the carB mutation in phenotypic switching, the C2020T and wild type carB alleles (named carBmutand carBwt, respectively) were constructed in various backgrounds. Firstly, separate PCRs were performed using SBW25 (carBwt) and 1w4 (carBmut) genomic DNA (see section 2.2.2.1). For each, a DNA fragment of ~1 kb was amplified using the primer pair CarB3f/CarB4r (61˚C annealing temperature, 1 minute extension time). PCR fragments were purified and cloned into pCR8/GW/TOPO for sequencing (see section 2.2.3.1). For both alleles, a clone containing a mutation-free PCR fragment was selected, the inserts retrieved by BglII digestion, cloned into pUIC3 and the resulting construct used to transform chemically competent E. coli cells (see section 2.2.3.2.3). Each set of E. coli transformants was screened for incorporation of the construct. The resulting constructs, pUIC3-carBwtand pUIC3-carBmut, were used to perform carB allelic exchanges in various genetic backgrounds, according to the methods outlined in sections 2.2.6.2 and 2.2.7.
C2020T
carA carB greA 5263 1 kb
L ATP Olg L ATP
5.3.4.1.2 Re-construction of carBmut in 1s4 and carBwt in 1w4
To determine if the carB mutation was the sole mutational cause of switching in 1w4, the carBmut allele was constructed in the 1s4 background, and the carBwt allele was constructed in the 1w4 background. The resulting genotypes, 1s4-carBmut and 1w4-
carBwt, were assayed for colony morphology, capsule production and calcofluor binding (Figure 5.9). The phenotype of the re-constructed switcher was identical to that of 1w4; 1s4-carBmut showed colony and capsule dimorphism, and ACP production. The phenotype of the re-constructed ancestor was identical to that of 1s4; 1w4-carBwt produced monomorphic colonies and cells, and synthesized ACP. These results demonstrate that the carB mutation is necessary and sufficient to cause colony and cell- level dimorphism in the 1s4 genetic background.
Figure 5.9: Phenotypic characterisation of 1s4-carBmut and 1w4-carBwt. Construction of the carB
mutation in 1s4 results in the switcher phenotype, while construction of the wild type carB allele in 1w4 results in a non-switching phenotype: (top: colony morphology on KB agar at 48 hours (scale bar indicates ~3 mm); middle: light microscope images (x40) of India ink counter-stained cells (scale bar indicates ~5 µm); bottom: fluorescence microscope images (x40 or x100) showing calcofluor binding.
1w4 1s4 1s4-carBmut 1w4-carBwt
Tr Op Op
5.3.4.1.3 Re-construction of carBmut in SBW25 and 1w0
Next, the dependence of switching on the first eight mutations in the line one series was investigated. The carBmut allele was artificially reconstructed in two more distant 1w4 ancestors: SBW25 (no mutations) and 1w0 (mwsR G2778A mutation), resulting in SBW25-carBmut and 1w0-carBmut, respectively. Introduction of the carBmut allele into both of these backgrounds resulted in a dimorphic phenotype similar to that of 1w4 (Figure 5.10). However, the dimorphic colony morphologies of the constructed genotypes were slightly different from those produced by 1w4; SBW25-carBmut produced smooth and opaque colonies, while 1w0-carBmut produced wrinkly and opaque colonies. Unlike 1w4 and 1w0-carBmut, SBW25-carBmut did not bind significant amounts of calcofluor, demonstrating that the carB mutation does not cause ACP biosynthesis.
Figure 5.10: Phenotypic characterisation of the artificially constructed genotypes SBW25-carBmut
and 1w0-carBmut. Construction of the mutant carB allele in both genetic background resulted in
dimorphism at the colony and cell levels. Top row: colony morphology on KB agar at 48 hours (scale bar indicates ~3 mm). Middle row: light microscope images (x40 magnification) showing India ink counter- staining of cells grown in overnight cultures (scale bar indicates ~5 µm). Bottom row: fluorescence microscope images (x40 or x100 magnification) showing calcofluor binding. See text for details.
1w4 SBW25 SBW25-carBmut 1w0-carBmut Sm Op Op Wr Op Tr
5.3.4.1.4 Capsule counting assays for carB allelic reconstructions
According to the method outlined in section 2.2.12.4, a capsule counting assay was performed for SBW25, 1s4, 1w4, 1s4-carBmut, 1w4-carBwt and SBW25-carBmut (Appendix A3.1). Presented below in Figure 5.11 and Table 5.4, the results of this assay provide a number of revelations. Firstly, there is no significant difference between the proportion of capsulated cells in 1w4 and 1s4-carBmut populations (P=0.278), verifying that the carB mutation is the sole cause of the capsulated cell phenotype. This observation was further supported by the fact that no significant difference was found between the proportion of capsulated cells in 1s4 and 1w4-carBwt populations (P=1.00). Secondly, the proportion of capsulated cells produced in SBW25-carBmut populations was significantly greater than in 1s4 populations (P=7.71 x 10-6), but significantly less than in 1w4 populations (P=7.84 x 10-3). This suggested that although the carB mutation is alone sufficient to cause the capsule switching phenotype, at least some of the additional eight mutations in the 1w4 background contribute to capsule expression.
Figure 5.11: Relative proportion of capsulated cells in SBW25, 1s4, 1w4, 1s4-carBmut, 1w4-carBwt
and SBW25-carBmut cultures. Each bar represents the mean of five replicates (error bars indicate one
Comparison of population means
Genotype Mean ± SE
Vs. genotype 2 P-value 95 % C.I.
SBW25 0.0016 ± 0.000748 - - - 1s4 0.0016 ± 0.000748 SBW25 1.00 - 1w4 0.0844 ± 0.00643 1s4 1.32 x 10-6*** 0.0677, 0.0977 1s4-carBmut 0.0760 ± 0.00323 1w4 0.278 - 1w4 1.32 x 10-6*** 0.0677, 0.0977 1w4-carBwt 0.00160 ± 0.00748 1s4 1.00 - 1w4 7.84 x 10-3** -0.0101, -0.483 SBW25-carBmut 0.0552 ± 0.00524 1s4 7.71 x 10-6*** 0.0414, 0.0658
Table 5.4: Relative proportion of capsulated cells in SBW25, 1s4, 1w4, 1s4-carBmut, 1w4-carBwt and
SBW25-carBmut cultures. Mean and standard error (SE) of five replicates are given for each genotype.
P-values generated from two-sample t-tests were used to compare the indicated population means (vs.=versus). Where appropriate, an estimation of the size of the difference is given as a 95 % confidence interval (C.I.). Mean, standard error and P-values given to three significant figures.