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1. Work Report
Chijioke Nsofor
October 15, 2021

2. Genetic mechanisms of high-level β-lactam-
resistance in the mutants derived from OS-MRSA
mecA positive Oxacillin-susceptible S. aureus (OS-MRSA)
• MRSA
• Oxacillin MIC ≧4 μg/ml or the mecA gene positive
• Cefoxitin MIC ≧8 μg/ml
• “mecA positive Oxacillin-susceptible S. aureus” or “Oxacillin-
susceptible MRSA (OS-MRSA)”
• Oxacillin MIC of ≦2 μg/ml but the mecA gene positive
• OS-MRSA have the potential to become highly resistant if exposed to
b-lactams.

5. Mutations of Oxacillin resistant mutants (Watanabe sensei data)
parent N-129 N-125 N-96 N-43 N-92 N-130 N-131 N-69 N-127 N-115
Oxacillin 0.75 1.5 4 4 4 4 4 4 4 6 6
Mutation-1
FruB RpoB JMUB217_0994 RpiA RpoC HprT FbaA RpoC FruB FruB
Gly39fs His481Asp Ala179Val Ala64Glu Ala756Asp Gly156Ser Gly21Asp Pro358Leu Asp161Gly Ala211Glu
Mutation-2
JMUB217_0285 JMUB217_0808
Lys191fs Gln46*
Mutation-3
JMUB217_0808
Gln46*
N-71 N-78 N-34 N-73 N-37 N-45 N-32 N-128 N-52 N-116 N-75
Oxacillin 8 12 12 16 24 24 24 24 32 64 >256
Mutation-1
RpoB RpoC RpoC RpoC RpoC RpoC RpoB GuaA RpoB RpoB RpoC
Asn577Tyr Arg739Cys Phe619_Asn620insHis Glu773Lys Val488Gly Gly950Arg Val1016Glu Ile249fs Gln645His His929Pro Gly498Asp
Mutation-2
RpoC RpoB
Lys673Asn His1042Gln
Mutation-3
Fructose
Metabolism
Purine/Pyrimidine
Metabolism
RpoB
RpoC
Transporter
Hypothetical
protein

6. Construction of Complementation Strains of
Oxacillin Resistant OS-MRSA Mutants
• Competent cell preparation
(N9, N43, N52, N75) using RN4220
as a positive control
• Electroporation
• Allelic exchange with pIMAY

7. Competent cell preparation and Electroporation
Based on the Monk et al mBio. 2012 Protocol
• After 48 hrs incubation no colony was observed
E. coli type for plasmid propagation DH5α
Media for competent cell preparation BHI (6ml in 15 ml tube)
1st wash buffer for competent cell
preparation
Autoclaved ice cold water
2nd wash buffer for competent cell
preparation
Autoclaved ice cold 10% glycerol
Electroporation Buffer 10% glycerol/500mM sucrose
Recovery (media, temp., time) BHI + 10% sucrose, 37°C, 1h

8. Transformation with BL21 Competent Cell
Successful
transformation with the
BL21 Strain
ICE
Heat block
LB Plate with CP10
370C Incubator
Plasmid

9. Competent cell preparation and Electroporation
Based on the Sato'o et al. et al 2018 Protocol
E. coli type for plasmid propagation BL21
Media for competent cell preparation B2low (20ml in 50 ml tube)
1st wash buffer for competent cell
preparation
Autoclaved ice cold 1.5 M NaCl
2nd wash buffer for competent cell
preparation
Autoclaved ice cold 10% glycerol
Electroporation Buffer 10% glycerol/500mM sucrose
Recovery (media, temp., time) B2, 30 °C, 5 h
1245-RN4220 1237- RN4220
Transformed colonies were observed only in the RN4220 Strains

10. Ethanol Precipitation of Plasmid DNA
Plasmid+5M NaCl+
100%EtOH
-300C Freezer
15000rpm for 10min
Air dry for 5mn.
Strain ID Plasmid conc.
1235 90.7ng/ul
1237 134.6ng/ul
1239 103.2ng/ul
1243 118.1ng/ul
1245 152.5ng/ul
1235 1237 1239 1243 1245

11. Transformants of the N9, N43, N52, N75 and RN4220 strains
transformed with pIMAY isolated from E. coli BL21
1239-RpiA-N43
1245-clpX-N9
1237-RpoC-N75
1243-relA-N9
1235-RpoB-N52
N9, N43, N52 and N75 Strains
1243-relA-N9
1235-RpoB-N52
1243-RN4220
1235-RN4220

12. TO DO
• Confirm the transformants
• Proceed with the construction of the complementation strains