DESARROLLO DE LAS ACTIVIDADES.

3.8.1

Introduction

Biochemical characterisation of L. plantarum NC8 Δpts18CBA was accomplished using both a

plate assay (section 2.2.7.3) and liquid culture assay (section 2.2.7.2). Prior to characterisation of L. plantarum NC8 Δpts18CBA, the glycocin F minimum inhibition concentration (MIC) (section 2.2.7.1) was determined. This was done so that the response to glycocin F at either end of the inhibitory scale could be measured for L. plantarum NC8 Δpts18CBA.

3.8.2

Aims

 To determine a glycocin F MIC for L. plantarum NC8.

 To characterise the effect glycocin F has on L. plantarum NC8 Δpts18CBA.

3.8.3

Results and discussion

From triplicate glycocin F assays carried out in liquid culture (section 2.2.7.1), the MIC for L. plantarum NC8 was determined to be 50 nM (Table 3.3 and Figure 3.22). This concentration was used in liquid culture glycocin F sensitivity assays (sections 2.2.7.2 and 2.2.7.3). Plate

assays comparing L. plantarum NC8 Δpts18CBA and wild-type NC8 as indicator strain showed

that at similar concentrations of glycocin F, the amount of clearing was significantly less for the knockout strain (Figure 3.23). However due to the heavy bacterial inoculum and lengthy incubation time, the results from the plate assays were not as convincing as those seen for the naturally selected glycocin F resistant L. plantarum mutants (Figure 3.3). Time constraints did

not allow troubleshooting. Liquid culture glycocin F assays using L. plantarum NC8

Δpts18CBA as the targeted strain showed that at the MIC, inhibition of growth decreased from 93 % to 50 %, and from 95 % to 66 % at 1 µM glycocin F convincingly demonstrating that

knocking out pts18CBA confers a degree of glycocin F resistance to L. plantarum NC8 (Figure

3.24).

These glycocin F sensitivity assay results are consistent with the results obtained for the Group H glycocin F resistant mutants (Table 3.6), characterised in section 3.2. Mutations shown to

disrupt PTS18CBA conferred complete resistance to glycocin F for L. plantarum ATCC 8014

and a very high level of glycocin F resistance for L. plantarum subsp. plantarum ATCC 14917

strongly indicating that PTS18CBA is the physiological receptor of glycocin F. As seen for glycocin F resistant L. plantarum subsp. plantarum ATCC 14917 mutants (Figure 3.4, Figure 3.5 and Figure 3.3a), complete resistance was not obtained upon knocking out PTS18CBA,

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Figure 3.22 Liquid glycocin F assay of L. plantarum NC8 for MIC determination

The mean OD600nm from triplicate assays. Vertical error bars represent one standard deviation

from the mean. Legend to the right indicates the concentration of glycocin F added at time point 0.

Figure 3.23 L. plantarum solid glycocin F assays

a) MRS agar plates from solid glycocin F assay of L. plantarum NC8 (WT) and L. plantarum

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Figure 3.24 Liquid glycocin F assay of L. plantarum NC8 Δpts18CBA

From duplicate liquid glycocin F assays of L. plantarum NC8 (WT) and L. plantarum NC8 Δpts18CBA (KO). a) Bar graphs show the mean OD600nm after 400 minutes from the growth

curves shown in (b). Error bars represent the standard deviation. Statistical significance (P- value) between untreated and treated and between 50 nM treated and 1 µM treated samples was determined using the Holm-Sidak test (values in green). Statistical significance (P-value) of treated mutant isolates to their respective counterpart treated WT control was determined using the Holm multiple comparison test (values in blue). b) Three cultures were inoculated at an OD600nm of 0.05, two were treated with either 50 nM or 1 µM glycocin F and the other left

untreated. The OD600nm was measured every 30 seconds using a Cary 300 UV-Visible

spectrophotometer and every 20 minutes the mean OD600nm of duplicate cuvettes was plotted.

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suggesting that at least in these two strains PTS18CBA may not be the only receptor. The other possible receptor identified (section 1.4, page 18) was the gene product of pts22CBA.

Alignments of PTS22CBA, from L. plantarum NC8, L. plantarum ATCC 8014 and L.

plantarum subsp. plantarum ATCC 14917, showed no amino acid differences in the EIIC domain (Appendix 17). Extending this search to the promoter region of pts22CBA showed no

differences in sequences between L.plantarum NC8 and L. plantarum subsp. plantarum ATCC

14917. However in L. plantarum ATCC 8014 three nucleotides were found to differ compared

to the other two strains (Figure 3.25). The -35, -10 and RBS were identified, based on consensus sequence similarity, though the differences were not directly located within these elements. How the differences in this region affect the expression of pts22CBA needs to be experimentally verified and may eventually explain the increased sensitivity to glycocin F observed by L. plantarum subsp. plantarum ATCC 14917. It may be that these changes may prevent expression of pts22CBA so that PTS18CBA is left as the only receptor for glycocin F in the membrane. The question remains, just how does glycocin F induce its bacteriostatic effect through this GlcNAc- specific PTS transporter. This is discussed in chapter 4.

Figure 3.25

pts22CBA nucleotide alignment

The nucleotide region upstream from pts22CBA from (2) L. plantarum ATCC subsp. plantarum

ATCC 14917 (14917), (1) L. plantarum ATCC 8014 (8014) and (3) L. plantarum NC8 (NC8),

were aligned. The coloured nucleotides in (1) are the differences identified in L. plantarum

ATCC 8014. The yellow bar is the pts22CBA coding sequence with the predicted translation above. Promoter elements are highlighted by silver bars.

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