Journal: Applied and Environmental Microbiology

Article Title: Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869

doi: 10.1128/AEM.02225-18

Figure Lengend Snippet: Effects of Y489C point mutation and deletion of ponA on electrotransformation efficiency. (A) Function of PonA in transglycosylation and transpeptidation of cell wall synthesis. (B) Electrotransformation efficiency and susceptibility to electroporation of the wild-type (WT), ponAY489C, and ΔponA strains in the absence of cell wall-weakening agents. Competent cells were prepared using LBG medium without cell wall-weakening agents. The cells used for each electrotransformation were approximately 108. After electroporation and recovery, cells were spread on selective LBG plates supplemented with 25 µg/ml kanamycin to determine the electrotransformation efficiency and LBG plates without kanamycin to determine the susceptibility to electroporation. (C) Growth curves of wild-type (WT), ponAY489C, and ΔponA strains. Cells were cultured in LBG medium with an initial OD600 value of 0.2. The cell density was determined every 3 hours. (D) Electrotransformation efficiency of wild-type (WT), ponAY489C, and ΔponA strains in the presence of cell wall-weakening agents. Competent cells were prepared using NCM medium supplemented with glycine, threonine, INH and Tween 80 according to the protocol described previously (12). Error bars indicate standard deviations from three parallel experiments. Asterisks indicate significant changes in electrotransformation efficiency and susceptibility to electroporation based on a comparison between the mutants and the wild-type strain. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s two-tailed t test). NS indicates nonsignificant change between the wild-type strain and the mutant based on Student’s two-tailed t test (P > 0.05).

Article Snippet: The primers used for genetic manipulation are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Strain or plasmid Relevant genotype or description Source or reference Strains E. coli DH5α F − supE44 Δ lacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 CWBiotech C. glutamicum ATCC 13869 Wild-type C. glutamicum ATCC SL4 Mutant strain derived from ATCC 13869 Lab stock fli A63V mutant ATCC 13869 derivative with A63V mutation of fli This work ponA Y489C mutant ATCC 13869 derivative with Y489C mutation of ponA This work ddl G256S mutant ATCC 13869 derivative with G256S mutation of ddl This work murJ P373S-T882I mutant ATCC 13869 derivative with P373S and T882I mutations of murJ This work emb A962T mutant ATCC 13869 derivative with A962T mutation of emb This work ufaA R90K mutant ATCC 13869 derivative with R90K mutation of ufaA This work fadD4 G32S mutant ATCC 13869 derivative with G32S mutation of fadD4 This work otsB V69I mutant ATCC 13869 derivative with V69I mutation of otsB This work ponA Y489C ddl G256S mutant ATCC 13869 derivative with both Y489C mutation of ponA and G256S mutation of ddl This work Δ ponA mutant ATCC 13869 with ponA deletion This work Δ ponA (pEC-gfp-ponA) mutant Δ ponA derivative harboring pEC-gfp-ponA This work Δ ponA (pEC-gfp-ponA Y489C ) mutant Δ ponA derivative harboring pEC-gfp-ponA Y489C This work Plasmid pK18mobsacB Vector for allelic exchange in C. glutamicum , pK18 oriV E. coli sacB lacZα (Kan r ) 44 pEC-XK99E E. coli - C. glutamicum shuttle expression vector (Kan r ) 13 pTRCmob-egfp pTRCmob derivative carrying gfp 39 pK-fli A63V pK18mobsacB harboring a 1.9-kb homologous fragment for fli A63V construction This work pK-ponA Y489C pK18mobsacB harboring a 2.3-kb homologous fragment for ponA Y489C construction This work pK-ddl G256S pK18mobsacB harboring a 2.0-kb homologous fragment for ddl G256S construction This work pK-murJ P373S,T882I pK18mobsacB harboring a 3.8-kb homologous fragment for murJ P373,T882I construction This work pK-emb A962T pK18mobsacB harboring a 2.4-kb homologous fragment for emb A962T construction This work pK-ufaA R90K pK18mobsacB harboring a 2.4-kb homologous fragment for ufaA R90K construction This work pK-fadD4 G32S pK18mobsacB harboring a 2.1-kb homologous fragment for fadD4 G32S construction This work pK-otsB V69I pK18mobsacB harboring a 1.6-kb homologous fragment for otsB V69I construction This work pK-ΔponA pK18mobsacB harboring a 2.1-kb homologous fragment for ponA deletion This work pEC-gfp-ponA pEC-XK99E derivative containing gfp-ponA under P trc promoter This work pEC-gfp-ponA Y489C pEC-XK99E derivative containing gfp-ponA Y489C under P trc promoter This work Open in a separate window a ATCC, American Type Culture Collection.

Techniques: Mutagenesis, Electroporation, Cell Culture, Comparison, Two Tailed Test

Journal: Applied and Environmental Microbiology

Article Title: Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869

doi: 10.1128/AEM.02225-18

Figure Lengend Snippet: Glutamate fermentation by the WT strain and the ponAY489C mutant. (A) Glutamate fermentation in shake flasks. Strains were cultivated in biotin-rich or biotin-poor fermentation medium supplemented with 80 g/liter glucose at 30°C and 220 rpm. OD600, glucose consumption, and glutamate production were detected after 24 h of fermentation. Error bars indicate standard deviations from the results from three parallel experiments. NS indicates a nonsignificant change based on Student’s two-tailed t test (P > 0.05). (B) Glutamate fermentation in 5-liter bioreactors. Strains were cultivated in biotin-poor fermentation medium supplemented with 140 g/liter glucose. Samples were picked periodically, and the OD600, glucose consumption, and glutamate production were detected. The data shown are the average and standard deviations of the results from three parallel determinations.

Article Snippet: The primers used for genetic manipulation are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Strain or plasmid Relevant genotype or description Source or reference Strains E. coli DH5α F − supE44 Δ lacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 CWBiotech C. glutamicum ATCC 13869 Wild-type C. glutamicum ATCC SL4 Mutant strain derived from ATCC 13869 Lab stock fli A63V mutant ATCC 13869 derivative with A63V mutation of fli This work ponA Y489C mutant ATCC 13869 derivative with Y489C mutation of ponA This work ddl G256S mutant ATCC 13869 derivative with G256S mutation of ddl This work murJ P373S-T882I mutant ATCC 13869 derivative with P373S and T882I mutations of murJ This work emb A962T mutant ATCC 13869 derivative with A962T mutation of emb This work ufaA R90K mutant ATCC 13869 derivative with R90K mutation of ufaA This work fadD4 G32S mutant ATCC 13869 derivative with G32S mutation of fadD4 This work otsB V69I mutant ATCC 13869 derivative with V69I mutation of otsB This work ponA Y489C ddl G256S mutant ATCC 13869 derivative with both Y489C mutation of ponA and G256S mutation of ddl This work Δ ponA mutant ATCC 13869 with ponA deletion This work Δ ponA (pEC-gfp-ponA) mutant Δ ponA derivative harboring pEC-gfp-ponA This work Δ ponA (pEC-gfp-ponA Y489C ) mutant Δ ponA derivative harboring pEC-gfp-ponA Y489C This work Plasmid pK18mobsacB Vector for allelic exchange in C. glutamicum , pK18 oriV E. coli sacB lacZα (Kan r ) 44 pEC-XK99E E. coli - C. glutamicum shuttle expression vector (Kan r ) 13 pTRCmob-egfp pTRCmob derivative carrying gfp 39 pK-fli A63V pK18mobsacB harboring a 1.9-kb homologous fragment for fli A63V construction This work pK-ponA Y489C pK18mobsacB harboring a 2.3-kb homologous fragment for ponA Y489C construction This work pK-ddl G256S pK18mobsacB harboring a 2.0-kb homologous fragment for ddl G256S construction This work pK-murJ P373S,T882I pK18mobsacB harboring a 3.8-kb homologous fragment for murJ P373,T882I construction This work pK-emb A962T pK18mobsacB harboring a 2.4-kb homologous fragment for emb A962T construction This work pK-ufaA R90K pK18mobsacB harboring a 2.4-kb homologous fragment for ufaA R90K construction This work pK-fadD4 G32S pK18mobsacB harboring a 2.1-kb homologous fragment for fadD4 G32S construction This work pK-otsB V69I pK18mobsacB harboring a 1.6-kb homologous fragment for otsB V69I construction This work pK-ΔponA pK18mobsacB harboring a 2.1-kb homologous fragment for ponA deletion This work pEC-gfp-ponA pEC-XK99E derivative containing gfp-ponA under P trc promoter This work pEC-gfp-ponA Y489C pEC-XK99E derivative containing gfp-ponA Y489C under P trc promoter This work Open in a separate window a ATCC, American Type Culture Collection.

Techniques: Mutagenesis, Two Tailed Test

Journal: Applied and Environmental Microbiology

Article Title: Mutations in Peptidoglycan Synthesis Gene ponA Improve Electrotransformation Efficiency of Corynebacterium glutamicum ATCC 13869

doi: 10.1128/AEM.02225-18

Figure Lengend Snippet: Bacterial strains and plasmids used in this study a

Article Snippet: The primers used for genetic manipulation are listed in . table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Strain or plasmid Relevant genotype or description Source or reference Strains E. coli DH5α F − supE44 Δ lacU169 (φ80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 CWBiotech C. glutamicum ATCC 13869 Wild-type C. glutamicum ATCC SL4 Mutant strain derived from ATCC 13869 Lab stock fli A63V mutant ATCC 13869 derivative with A63V mutation of fli This work ponA Y489C mutant ATCC 13869 derivative with Y489C mutation of ponA This work ddl G256S mutant ATCC 13869 derivative with G256S mutation of ddl This work murJ P373S-T882I mutant ATCC 13869 derivative with P373S and T882I mutations of murJ This work emb A962T mutant ATCC 13869 derivative with A962T mutation of emb This work ufaA R90K mutant ATCC 13869 derivative with R90K mutation of ufaA This work fadD4 G32S mutant ATCC 13869 derivative with G32S mutation of fadD4 This work otsB V69I mutant ATCC 13869 derivative with V69I mutation of otsB This work ponA Y489C ddl G256S mutant ATCC 13869 derivative with both Y489C mutation of ponA and G256S mutation of ddl This work Δ ponA mutant ATCC 13869 with ponA deletion This work Δ ponA (pEC-gfp-ponA) mutant Δ ponA derivative harboring pEC-gfp-ponA This work Δ ponA (pEC-gfp-ponA Y489C ) mutant Δ ponA derivative harboring pEC-gfp-ponA Y489C This work Plasmid pK18mobsacB Vector for allelic exchange in C. glutamicum , pK18 oriV E. coli sacB lacZα (Kan r ) 44 pEC-XK99E E. coli - C. glutamicum shuttle expression vector (Kan r ) 13 pTRCmob-egfp pTRCmob derivative carrying gfp 39 pK-fli A63V pK18mobsacB harboring a 1.9-kb homologous fragment for fli A63V construction This work pK-ponA Y489C pK18mobsacB harboring a 2.3-kb homologous fragment for ponA Y489C construction This work pK-ddl G256S pK18mobsacB harboring a 2.0-kb homologous fragment for ddl G256S construction This work pK-murJ P373S,T882I pK18mobsacB harboring a 3.8-kb homologous fragment for murJ P373,T882I construction This work pK-emb A962T pK18mobsacB harboring a 2.4-kb homologous fragment for emb A962T construction This work pK-ufaA R90K pK18mobsacB harboring a 2.4-kb homologous fragment for ufaA R90K construction This work pK-fadD4 G32S pK18mobsacB harboring a 2.1-kb homologous fragment for fadD4 G32S construction This work pK-otsB V69I pK18mobsacB harboring a 1.6-kb homologous fragment for otsB V69I construction This work pK-ΔponA pK18mobsacB harboring a 2.1-kb homologous fragment for ponA deletion This work pEC-gfp-ponA pEC-XK99E derivative containing gfp-ponA under P trc promoter This work pEC-gfp-ponA Y489C pEC-XK99E derivative containing gfp-ponA Y489C under P trc promoter This work Open in a separate window a ATCC, American Type Culture Collection.

Techniques: Plasmid Preparation, Mutagenesis, Derivative Assay, Expressing