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C142A
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mutant shows reduced reductase activity compared to the wild type enzyme
C142S
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mutant shows reduced reductase activity compared to the wild type enzyme
C145A
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mutant shows reduced reductase activity compared to the wild type enzyme
C145S
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mutant shows reduced reductase activity compared to the wild type enzyme
A164G/R183F
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the mutant shows reduced activity but is still able to dimerize though with an increase in intermediary forms
A164G/V182E
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the mutant shows increased activity compared to the wild type enzyme
A164G/V182E/R183F
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the mutant shows reduced activity compared to the wild type enzyme but is still able to dimerize though with an increase in intermediary forms
C140S
no activity with substrate CHLI-1 ATPase
C120A/C220A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C122A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C14A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C14A/C120A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C14A/C120A/C220A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C14A/C220A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C220A
site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
C122A
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site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
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C14A
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site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
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C14A/C120A
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site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
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C14A/C220A
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site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
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C220A
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site-directed mutagenesis, the mutant retains NADPH oxidase activity, but with reduced activity compared to the wild-type enzyme
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TR-GCCS
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mutant with C-terminal sequence of GCCS
TR-SCCS
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mutant with C-terminal sequence of SCCS
C489S
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mutant is incapable of reducing thioredoxin and can only be reduced to the 2-electron-state of enzyme
C489S/C490S
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mutant is incapable of reducing thioredoxin and can only be reduced to the 2-electron-state of enzyme
C490S
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mutant is incapable of reducing thioredoxin and can only be reduced to the 2-electron-state of enzyme
E469A
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the mutant retains 28% of the wild type activity
E469Q
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the mutant retains 35% of the wild type activity
E470A
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the mutant retains 70% of the wild type activity
H106F
catalytic activity drops considerably yet pH-profile does not reveal differences
H106N
catalytic activity drops considerably yet pH-profile does not reveal differences
H106Q
catalytic activity drops considerably yet pH-profile does not reveal differences
C135S/C32S
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via the active disulfide centers a subunit complex of tightly bound enzyme, C135 and C138, and thioredoxin, C32 and C35, is formed, exchange of one cysteine for one serine in each protein by site-directed mutagenesis, conformation analysis
C135S/C35S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C136S
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site-directed mutagenesis, reduced activity
C138S/C35S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C139S
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site-directed mutagenesis, reduced activity
C535S
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site-directed mutagenesis, changed conformation
C73S
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recombinant, His-tagged
TrxR-16
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truncated form of TrxR missing the last 16 C-terminal amino acids, without thioredoxin-reducing activity
TrxR-16 K29R
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without thioredoxin-reducing activity
TrxR-16 K29R/H108Y
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without thioredoxin-reducing activity
TrxR-16 K29R/H108Y/A119N/V478E
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without thioredoxin-reducing activity
U498C
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1.4fold higher GSSG-reducing activity compared to the TrxR-16 enzyme
C145S
active site cysteine residue. Kinetic data
C148S
residue Cys148 probably performs an initial nucleophilic attack on the active site disulfide in thioredoxin disulfide. Kinetic data
Delta42-47
FAD-domain mutant. Kinetic data
G222D/A223G/G224E
NADPH-domain mutant. Kinetic data
G225R/G226D
NADPH-domain mutant. Kinetic data
G225R/G226D/P227V
NADPH-domain mutant. Kinetic data
M43A
mutant in a loop of the FAD-binding domain, strongly affects the interaction with thioredoxin. Kinetic data
N139A
NADPH-domain mutant. Kinetic data
N45A/D46A
FAD-domain mutant. Kinetic data
R140A
NADPH-domain mutant. Kinetic data
W42A
mutant in a loop of the FAD-binding domain, strongly affects the interaction with thioredoxin. Kinetic data
W42A/M43A
mutant in a loop of the FAD-binding domain, strongly affects the interaction with thioredoxin
Sec489C
pH-optimum shifts from pH 7.0 to 8.0
U489C
barely detectable activity towards thioredoxin and hydrogen peroxide
C535S
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construction of a homodimer and heterodimer, the latter containing 1 mutant and 1 wild-type subunit, activity is reduced by 56 and 92%, respectively
C535S/C88A
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double mutant, construction of a homodimer and a heterodimer, the latter containing 1 mutant and 1 wild-type subunit, activity is reduced by 89 and 95%, respectively
C88S
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site-directed mutagenesis, no activity
C93A
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site-directed mutagenesis, no activity
H509A
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site-directed mutagenesis, reduced activity
H509Q
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site-directed mutagenesis, reduced activity
C146S
no activity against insulin disulfide, no DTNB-reducing activity
C35S
strong activity as high as that of a wild type against insulin disulfide, 98% of residual activity. The activity of C35S against the reduction of DTNB was not decreased
C146S
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no activity against insulin disulfide, no DTNB-reducing activity
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C35S
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strong activity as high as that of a wild type against insulin disulfide, 98% of residual activity. The activity of C35S against the reduction of DTNB was not decreased
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C458S
inactive mutant of SCCS
C458S/C475T
inactive mutant of SCCS
C475T
inactive mutant of SCCS
SCCS
mutant with flanking serine residues introduced into the C-terminal tetrapeptide of the wild type enzyme, less than 0.5% activity of the wild type enzyme
SeC498C
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antisense technique, exchange in the catalytic active selenosulfide at the C-terminus, resulting in higher pH-optimum, 100fold lower turnover number, 10fold lower Km-value, no activity with H2O2
SeC498G
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antisense technique, reduced activity
SeC498S
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antisense technique, reduced activity
U498C
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specific activity of 50% of wild-type enzyme
Y116F
the mutant protein is not soluble
K137A
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the mutation does not alter enzyme activity
C57S
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inactive mutant containing a redox-active [Fe4S4]3+/2+ center, can be reduced by dithionite
C87A
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inactive mutant containing a redox-inactive [Fe4S4]2+ cluster
C53S
site-directed mutagenesis, no activity since the redox cycle system is abolished
C135S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C135S
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exchange of 1 cysteine in the active center disulfide, 1 cysteine at position 138 is remaining
C138S
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C138S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C138S
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exchange of 1 cysteine in the active center disulfide, 1 cysteine at position 135 is remaining, very low activity
C32S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C32S
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exchange of 1 cysteine in the active center disulfide, 1 cysteine at position 35 is remaining, low activity
C35S
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fluorescence spectroscopic investigation of the interaction with the flavin group
C35S
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exchange of 1 cysteine in the active center disulfide, 1 cysteine at position 32 is remaining
C88A
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site-directed mutagenesis, no activity
C88A
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construction of a homodimer and a heterodimer, the latter containing 1 mutant and 1 wild-type subunit, activity is reduced by 4 and 90%
Y116I
the mutation decreases sensitivity to inhibition by cisplatin and lowers catalytic efficiency in reduction of thioredoxin compared to the wild type enzyme
Y116I
the mutation decreases sensitivity to inhibition by cisplatin, lowers catalytic efficiency in reduction of thioredoxin, and increases turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate compared to the wild type enzyme
C147A
significantly reduced activity, decrease in FAD content
C147A
the mutant exhibits a marginal NADH oxidase activity with FAD canonically bound to the enzyme
C147A
in the active site of the C147A mutant, which exhibits a marginal NADH oxidase activity, the FAD is canonically bound to the enzyme
G10A
site-directed mutagenesis, the mutation completely abolishes cofactor binding activity as glycine 10 is the first glycine residue in a putative Rossman fold domain (GxGxxG), which is characteristic of cofactor binding enzymes and presumed to function in the reduction of oxidized bacillithiol disulfide (BSSB). The YpdA G10A protein is unable to consume NADPH or NADH. Carbohydrate metabolism is mostly down regulated in the mutant, reduced fitness of the ypdA mutant in the competitive fitness assays with the wild-type in chemical defined medium
G10A
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site-directed mutagenesis, the mutation completely abolishes cofactor binding activity as glycine 10 is the first glycine residue in a putative Rossman fold domain (GxGxxG), which is characteristic of cofactor binding enzymes and presumed to function in the reduction of oxidized bacillithiol disulfide (BSSB). The YpdA G10A protein is unable to consume NADPH or NADH. Carbohydrate metabolism is mostly down regulated in the mutant, reduced fitness of the ypdA mutant in the competitive fitness assays with the wild-type in chemical defined medium
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G10A
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site-directed mutagenesis, the mutation completely abolishes cofactor binding activity as glycine 10 is the first glycine residue in a putative Rossman fold domain (GxGxxG), which is characteristic of cofactor binding enzymes and presumed to function in the reduction of oxidized bacillithiol disulfide (BSSB). The YpdA G10A protein is unable to consume NADPH or NADH. Carbohydrate metabolism is mostly down regulated in the mutant, reduced fitness of the ypdA mutant in the competitive fitness assays with the wild-type in chemical defined medium
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additional information
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where insertion of alanine between the redox-active Cys residues of the C-terminal redox center has very little effect on DTNB reductase activity
additional information
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His-tagged transcript specific mutants, varied N-terminus, 1 null-mutant
additional information
truncated enzyme (missing residues CCS from the C-terminus) so that Ser488 is the C-terminal amino acid shows no activity with thioredoxin disulfide + NADPH
additional information
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truncated enzyme (missing residues CCS from the C-terminus) so that Ser488 is the C-terminal amino acid shows no activity with thioredoxin disulfide + NADPH
additional information
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When the C-terminus of DmTR is changed from a carboxylate to either a thiocarboxylate or a hydroxamic acid, the result is a mutant enzyme with an about 1.7fold increase in activity with thioredoxin. Alanine insertion mutants (DmTR-SCACS and DmTR-SCAACS) show activity with thioredoxin that is greatly reduced compared to that of wild-type DmTR. Increasing the ring size of the Cys-Cys dyad results in a 150-300fold loss in kcat, while the Km is affected little. The 5,5'-dithiobis(2-nitrobenzoic acid) reductase activity of DmTR is also increased when the negative charge at the C-terminus is either neutralized by converting the carboxylate to a neutral hydroxamic acid or modulated by conversion to a thiocarboxylate. Similar to the Sec-containing mammalian enzyme, the truncated DmTR mutant also shows very high 5,5'-dithiobis(2-nitrobenzoic acid) reductase activity, as do the alanine insertion mutants
additional information
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additional information
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trxB gene is combined to chimeric enzyme NT-TrR by exchanging the N-terminus of Escherichia coli with the N-terminus of Salmonella typhimurium AhpF gene protein, which encodes a protein with about 35% homology in the N-terminal region, 2 other mutants are constructed in the same way but with double mutation C129S/C132S and C342S/C345S, the first in the Escherichia coli part and the latter in the Salmonella part of the chimeric protein, activity corressponding to organism wild-type giving the N-terminal part, except for C342S/C345S chimeric mutant who has no activity
additional information
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transposon mutagenesis
additional information
expression as a truncated form without both the plastid-targetingpeptide and Trx domain
additional information
expression as a truncated form without both the plastid-targetingpeptide and Trx domain
additional information
expression as a truncated form without both the plastid-targetingpeptide and Trx domain
additional information
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expression as a truncated form without both the plastid-targetingpeptide and Trx domain
additional information
the mutant in the which Sec and Cys residues are switched (TR-GUCG), shows activity similar to that of the Sec489Cys mutant (TR-GCCG). Replacement of the Cys-Sec dyad with a Sec-Sec dyad (TR-GUUG) results in a mutant enzyme with very low catalytic activity. Even if the kcat values are normalized for selenium content, the TR-GUCG and TR-GUUG mutants have catalytic activity 90fold and 185fold lower, respectively, than that of the wild-type enzyme. The mutants in which alanine residues are inserted to increase the ring size (TR-GCAUG and TR-GCAAUG) show only a modest decrease in catalytic activity, 6fold and 4fold, respectively
additional information
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the mutant in the which Sec and Cys residues are switched (TR-GUCG), shows activity similar to that of the Sec489Cys mutant (TR-GCCG). Replacement of the Cys-Sec dyad with a Sec-Sec dyad (TR-GUUG) results in a mutant enzyme with very low catalytic activity. Even if the kcat values are normalized for selenium content, the TR-GUCG and TR-GUUG mutants have catalytic activity 90fold and 185fold lower, respectively, than that of the wild-type enzyme. The mutants in which alanine residues are inserted to increase the ring size (TR-GCAUG and TR-GCAAUG) show only a modest decrease in catalytic activity, 6fold and 4fold, respectively
additional information
the truncated mutant enzyme (missing residues CUG from the C-terminus) so that Gly521 is the C-terminal amino acid shows no activity with thioredoxin disulfide + NADPH
additional information
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the truncated mutant enzyme (missing residues CUG from the C-terminus) so that Gly521 is the C-terminal amino acid shows no activity with thioredoxin disulfide + NADPH
additional information
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thioredoxin is cut off the native fusion protein thioredoxin-thioredoxin reductase resulting in enhanced activity
additional information
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truncated enzyme mutant without catalytic active C-terminus
additional information
mutation of gene ypdA by replacing the ORF with a kanamycin cassette through homologous recombination in an SH1000 strain corrected for BSH production, and construction of the complement strain by replacing the kanamycin cassette with the native ypdA gene, phenotypes, overview. No obvious growth defect of the mutant vs. the parent or the complemented mutant in TSB is detected. Fitness defects might be masked by other metabolic pathways in rich media that neutralize the effect of stress. Accordingly, the fitness of the ypdA mutant is evaluated by a competition assay which has been shown to be useful for teasing out subtle fitness defects between closely related strains. Despite identical growth kinetics of the mutant vs. the parent in complex media such as TSB and CDM, overnight competition assays reveal that the wild-type strain out-competes the ypdA mutant in competitive growth in CDM, while the competitive index does not change in TSB media. These results suggest that while the ypdA mutation has a limited effect on growth in complex media in vitro, the mutant exhibits a fitness defect vs. the wild-type when grown competitively in chemically defined medium. Cells overexpressing YpdA are able to survive better than cells with just the empty vector, and this difference in survival is abolished when oxidative burst of PMNs is blocked by DPI
additional information
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mutation of gene ypdA by replacing the ORF with a kanamycin cassette through homologous recombination in an SH1000 strain corrected for BSH production, and construction of the complement strain by replacing the kanamycin cassette with the native ypdA gene, phenotypes, overview. No obvious growth defect of the mutant vs. the parent or the complemented mutant in TSB is detected. Fitness defects might be masked by other metabolic pathways in rich media that neutralize the effect of stress. Accordingly, the fitness of the ypdA mutant is evaluated by a competition assay which has been shown to be useful for teasing out subtle fitness defects between closely related strains. Despite identical growth kinetics of the mutant vs. the parent in complex media such as TSB and CDM, overnight competition assays reveal that the wild-type strain out-competes the ypdA mutant in competitive growth in CDM, while the competitive index does not change in TSB media. These results suggest that while the ypdA mutation has a limited effect on growth in complex media in vitro, the mutant exhibits a fitness defect vs. the wild-type when grown competitively in chemically defined medium. Cells overexpressing YpdA are able to survive better than cells with just the empty vector, and this difference in survival is abolished when oxidative burst of PMNs is blocked by DPI
additional information
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mutation of gene ypdA by replacing the ORF with a kanamycin cassette through homologous recombination in an SH1000 strain corrected for BSH production, and construction of the complement strain by replacing the kanamycin cassette with the native ypdA gene, phenotypes, overview. No obvious growth defect of the mutant vs. the parent or the complemented mutant in TSB is detected. Fitness defects might be masked by other metabolic pathways in rich media that neutralize the effect of stress. Accordingly, the fitness of the ypdA mutant is evaluated by a competition assay which has been shown to be useful for teasing out subtle fitness defects between closely related strains. Despite identical growth kinetics of the mutant vs. the parent in complex media such as TSB and CDM, overnight competition assays reveal that the wild-type strain out-competes the ypdA mutant in competitive growth in CDM, while the competitive index does not change in TSB media. These results suggest that while the ypdA mutation has a limited effect on growth in complex media in vitro, the mutant exhibits a fitness defect vs. the wild-type when grown competitively in chemically defined medium. Cells overexpressing YpdA are able to survive better than cells with just the empty vector, and this difference in survival is abolished when oxidative burst of PMNs is blocked by DPI
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additional information
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mutation of gene ypdA by replacing the ORF with a kanamycin cassette through homologous recombination in an SH1000 strain corrected for BSH production, and construction of the complement strain by replacing the kanamycin cassette with the native ypdA gene, phenotypes, overview. No obvious growth defect of the mutant vs. the parent or the complemented mutant in TSB is detected. Fitness defects might be masked by other metabolic pathways in rich media that neutralize the effect of stress. Accordingly, the fitness of the ypdA mutant is evaluated by a competition assay which has been shown to be useful for teasing out subtle fitness defects between closely related strains. Despite identical growth kinetics of the mutant vs. the parent in complex media such as TSB and CDM, overnight competition assays reveal that the wild-type strain out-competes the ypdA mutant in competitive growth in CDM, while the competitive index does not change in TSB media. These results suggest that while the ypdA mutation has a limited effect on growth in complex media in vitro, the mutant exhibits a fitness defect vs. the wild-type when grown competitively in chemically defined medium. Cells overexpressing YpdA are able to survive better than cells with just the empty vector, and this difference in survival is abolished when oxidative burst of PMNs is blocked by DPI
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