Complementation of TK005 (sco5204), TK006 (sco4847) and TK008 (sco4934)

To confirm that the changes in the phenotypes observed in the TK005 (sco5204), TK006 (sco4847) and TK008 (sco4934) strains were directly linked to the disruption of sco5204, sco4847 and sco4934 respectively, it was decided to attempt to complement the mutants. The wild type copies of sco5204, sco4847 and sco4934 respectively were amplified by PCR and cloned into the NdeI restriction site of the shuttle vector pIJ10257 (as described in Materials and Methods). The new constructs were confirmed by restriction digests with BamHI and EcoRI to confirm the presence of the insert (Figure 6.13). The new constructs and expected digest fragments are summarised in Table 6.3. All fragment sizes in the restriction digests were as expected. Sequencing of the plasmids confirmed the presence of the correct insert. The constructs were introduced into E. coli ET12567(pUZ8002) and then into the S. coelicolor glycoprotein deficient mutants as described in the Materials and Methods. The empty pIJ10257 vector was introduced into TK005 (sco5204), TK006 (sco4847) and TK008 (sco4934) to serve as a negative control for the complementation.

The shuttle vector pIJ10257 enables the constitutive expression of genes under the ermEp* promoter (Hong et al. 2005). The plasmid encodes the phiBT1 phage integrase and attP site which enables the site specific integration of the vector into the phiBT1 attB site in the S. coelicolor genome (Gregory et al. 2003). Genomic DNA was isolated from the exconjugates and the presence of the integrated plasmids were confirmed by PCR. To confirm the presence of the empty pIJ10257 vector in the TK005 (sco5204), TK006 (sco4847) and TK008 (sco4934) strains respectively, PCR primers were designed in the S. coelicolor genome upstream of sco4848 and in the pIJ10257 vector upstream of the ermE*p site (Figure 6.14.A). To confirm the presence of pTAK29 in TK005 (sco5204) PCR primers were designed on the vector backbone and in the sco5204 gene (Figure 6.14.B). To confirm the presence of pTAK30 and pTAK32 in TK006 (sco4847) and TK008 (sco4934) respectively, PCR primers were designed on the vector backbone flanking the sco4847 and sco4934 genes respectively (Figure 6.14.C-D).

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Figure 6.13 Restriction digests to confirm the correct construction of pTAK29, pTAK30 and pTAK32. A. Digestion of pTAK29 (pIJ10257 with sco5204) constructs with BamHI-HF and

EcoRI-HF. B. Digestion of pTAK30 (pIJ10257 with sco4847), pTAK32 (pIJ10257 with sco4934) and pIJ10257 constructs with BamHI-HF and EcoRI-HF.

Table 6.3 New constructs generated by cloning sco5204 (pTAK29), sco4847 (pTAK30) and

sco4934 (pTAK32) respectively into pIJ10257.

Name Construct Function BamHI-HF

fragments (bp) EcoRI-HF fragments (bp) pIJ10257 Shuttle vector; Integrates into phiBT1 attachment site (attB); constitutive ermEp* promoter in front of the MCS; Hygromycinᴿ . Cloning vector in E. coli and integrating vector in S. coelicolor 3280, 3144 2466, 3246, 712 pTAK29 sco5204 in pIJ10257 Complementation of sco5204 mutants (TK005) 4896, 1393, 3144 2466, 6255, 712 pTAK30 sco4847 in pIJ10257 Complementation of sco4847 mutants (TK006) 4604, 3144 2466, 4570, 712 pTAK32 sco4934 in pIJ10257 Complementation of sco4934 mutants (TK008) 4531, 3144 2466, 4497, 712

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Figure 6.14 PCR primer design to confirm the presence of the integrated pIJ10257 (A), pTAK29 (B), pTAK30 (C) and pTAK32 (D) constructs. OriT: origin of

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For each PCR the negative control was J1929 genomic DNA and a positive control was the plasmid that was introduced into each respective strain. The PCRs confirmed that all of the pTAK29:TK005, pTAK32:TK008, pIJ10257:TK005, pIJ10257:TK006 and pIJ10257:TK008 exconjugates screened contained the integrated plasmids. However only four of the pTAK30:TK006 exconjugates (Figure 6.15.B – lanes 2,3,8 and 9) were positive for the integrated vector. The new strains, PCR primers and expected sizes of the PCR products are summarised in Table 6.4.

To investigate if the increase in antibiotic production observed previously in the TK006 (sco4847) and TK008 (sco4934) strains (Figure 6.10) was complemented in TK013 (TK006: pTAK30) and TK010 (TK008:pTAK32) respectively, the strains were plated onto SMMS and grown for 7 days (Figure 6.16). The slight increase in antibiotic production was still observed in TK013 (Figure 6.16.A) and TK010 (Figure 6.16.B). Complementation of the lysozyme sensitivity phenotype observed previously in TK006 (sco4847) (Figure 6.12.A) was observed after the reintroduction of wild type sco4847 in the TK013 (TK006: pTAK30) strain (Figure 6.17.A). No reduction in lysozyme sensitivity was observed in the empty vector control strain TK016 (TK006:pIJ10257) as expected.

Partial complementation of the β-lactam hypersensitivity observed previously in the TK006 (sco4847) (Figure 6.11.B) and TK008 (sco4934) (Figure 6.11.C) strains was observed upon re- introduction of the wild type sco4847 and sco4934 in TK013 (TK006: pTAK30) (Figure 6.18.A) and TK010 (TK008:pTAK32) (Figure 6.18.B) respectively. Additionally, a slight reduction in vancomycin sensitivity was observed in TK013 (TK006: pTAK30). The β-lactam hypersensitivity observed previously in TK005 (sco5204) (Figure 6.11.A) was not complemented in TK012 (TK005: pTAK29) (Figure 6.18.C). However, the constitutive expression of sco5204 in TK012 (TK005: pTAK29) appeared to result in increased sensitivity to imipenem, penicillin and ampicillin. Taken together these results suggest that the phenotypes observed as a result of knocking out sco4847 and sco4934 in TK006 (sco4847) and TK008 (sco4934) respectively, are directly linked to the disruption of the respective genes. Additionally, the slightly exacerbated phenotypes observed in TK012 (TK005: pTAK29) might suggest that constitutive expression of sco5204 is damaging in S. coelicolor.

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Figure 6.15 PCRs to confirm the genomic integration of the complementation constructs.

Panels indicate: A. pTAK29 integration into TK005. B. pTAK30 integration into TK006. C. pTAK32 integration into TK008. D. pIJ10257 integration into TK005, TK006 and TK008 respectively.

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Table 6.4 New S. coelicolor strains generated by complementation of the glycoprotein deficient mutants and the expected sizes of the PCR products.

S. coelicolor

strain Genotype Description PCR primers

Expected PCR products

(bp)

J1929 N/A Parent strain

NR88, TK143 no product TK91, TK92 no product TK143, TK144 no product TK2, TK3 no product TK010 TK008: pTAK32 Complementation of sco4934 knockout TK2, TK3 1442 TK012 TK005: pTAK29 Complementation of sco5204 knockout TK91, TK92 1160 TK013 TK006: pTAK30 Complementation of sco4847 knockout TK143, TK144 1720 TK015 TK008: pIJ12057

Plasmid only controls

NR88, TK143 1456 TK016 TK006: pIJ12057 NR88, TK143 1456 TK017 TK005: pIJ12057 NR88, TK143 1456

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Figure 6.16 Antibiotic production of the complement strains TK013 (TK006:pTAK30) (A) and TK010 (TK008:pTAK32) (B) respectively. A. TK006 (sco4847), TK013 (TK006: pTAK30) and

TK016 (TK006:pIJ10257). B. TK008 (sco4934), TK012 (TK008:pTAK32) and TK015 (TK008:pIJ10257). Approximately 10⁶ spores were inoculated. Images are representative of at least two biological replicates and two technical replicates.

Figure 6.17 Lysozyme sensitivity of the complement strain TK013 (TK006:pTAK30). Spores

were adjusted to 10⁸ spores/mL and a ten-fold serial dilution was carried out to get 10⁴ spores/mL. 5 µL of each spore stock was plated onto DNA without lysozyme (left-hand panel) and with 0.25 mg/mL of lysozyme (right-hand panel). Images are representative of two biological replicates and two technical replicates. These images are the same plates as in Figure 6.12.A.

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Figure 6.18 Antibiotic sensitivity of the complement strains TK013 (TK006:pTAK30) (A), TK010 (TK008:pTAK32) (B) and TK012 (TK005:pTAK29) (C). Shown

are the diameters of growth inhibition zones from disc diffusion assays for each complement strain against the mutant, the parent strain J1929, and the glycosylation deficient strains DT1025 (pmt) and DT3017 (ppm1). Mutant strains with the integrated empty vector pIJ10257 served as negative controls for the complementations. Bars represent the mean of at least three biological replicates except for TK006, where the bars indicate the mean of two biological replicates tested three times each. Error bars indicate SEM. * indicates p < 0.05 that the observed difference between the glycoprotein deficient mutant and the complement strain has occurred by chance. The full data set is in Table A.5, A.7 and A.8. Data shown for J1929, DT1025, DT3017 and TK006 in panel A is the same as for Figure 6.11.B.

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In a further attempt to rescue the β-lactam hypersensitivity phenotype in TK005 (sco5204) it was decided to complement the strains with sco5204 under the control of its native promoter. sco5204, along with an extended upstream sequence (251 bp) to include potential regulatory elements, was amplified from J1929 genomic DNA by PCR and cloned into the HindIII site in the shuttle vector pMS82 (as described in Materials and Methods). pMS82 is an integrating vector similar to pIJ10257 without a promoter sequence (Gregory et al. 2003). The new construct, pTAK28 was confirmed by restriction digestion with PstI (expected fragments: 802 bp and 8793 bp) and BamHI (expected fragments: 1292 bp, 1904 bp and 6399 bp) (Figure 6.19.A) and sequenced to confirm the presence of the correct insert. The construct was introduced into E. coli ET12567(pUZ8002) and then into S. coelicolor TK005 (sco5204), as described in the Materials and Methods. The empty pMS82 vector was introduced into TK005 (sco5204) to serve as a negative control for the complementation. Genomic DNA was isolated from the exconjugates and the presence of the integrated plasmids were confirmed by PCR (Figure 6.19 B-C). To confirm the presence of the empty pMS82 vector in the TK005 (sco5204), PCR primers were designed in the S. coelicolor genome upstream of sco4848 and in the pMS82 vector upstream of the origin of replication (Figure 6.19.D). To confirm the presence of pTAK28 in TK005 (sco5204) PCR primers were designed on the vector backbone and in the sco5204 gene (Figure 6.19.E). The PCR products observed from both of the TK005:pTAK28 and TK005:pMS82 exconjugates were slightly smaller than the expected 3022 bp (Figure 6.19.B) and 3261 bp (Figure 6.19.C) respectively. However, the PCR product amplified from the positive control pTAK28 was similarly smaller than expected suggesting that the TK005:pTAK28 exconjugates do contain the integrated pTAK28 vector (Figure 6.19.B). The new strains were named TK009 (TK005:pTAK28) and TK018 (TK005: pMS82) respectively.

No complementation of the β-lactam hypersensitivity phenotype observed previously in the TK005 (sco5204) (Figure 6.11.A) was observed in the TK009 (TK005:pTAK28) strains, however ampicillin sensitivity was increased (Figure 6.20). Additionally, no change in rifampicin sensitivity was observed in the TK009 (TK005:pTAK28) strains. These results suggest that the changes in antibiotic sensitivity in TK005 (sco5204) compared to the parent strain J1929 may not be a direct result of a loss of sco5204.

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Figure 6.19 Restriction digest analysis of pTAK28 and confirmation of vector integration into TK005 (sco5204). A. Restriction digest analysis of pTAK28 with

PstI and BamHI-HF. B. PCR confirming pTAK28 in TK005:pTAK28 exconjugates. C. PCR confirming pMS82 in TK005:pMS82 exconjugates. D-E. PCR primer design to confirm pMS82 (D) and pTAK28 (E) in the exconjugates. OriT: origin of transfer; TraJ: oriT recognising protein; Ori: origin of replication; ermEp*: ermE promoter.

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Figure 6.20 Antibiotic sensitivity of the complement strain TK009 (TK005:pTAK28). Shown

are the diameters of growth inhibition zones from disc diffusion assays for the complement strain against the mutant, the parent strain J1929, and the glycosylation deficient strains DT1025 (pmt) and DT3017 (ppm1). The mutants strain with the integrated empty vector pIJ10257 served as negative control for the complementation. Bars represent the mean of at least three biological replicates and error bars indicate SEM. * indicates p < 0.05 that the observed difference between the glycoprotein deficient mutant and the complement strain has occurred by chance. The full data set is in Table A.8. Data for J1929, DT1025, DT3017 and TK005 is the same as shown in Figure 6.18.C.

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6.3 Investigating the importance of the glycosylated amino acid in the SCO4934