Mutations within the catalytic motif of DNA adenine methyltransferase (Dam) of Aeromonas hydrophila cause the virulence of the dam-overproducing strain to revert to that of the wild-type phenotype

Tatiana E. Erova, Amin A. Fadl, Jian Sha, Bijay K. Khajanchi, Lakshmi L. Pillai, Elena V. Kozlova, Ashok Chopra

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/dam D/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam- overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.

Original languageEnglish (US)
Pages (from-to)5763-5772
Number of pages10
JournalInfection and Immunity
Volume74
Issue number10
DOIs
StatePublished - Oct 2006

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Catalytic DNA
Aeromonas hydrophila
Methyltransferases
Adenine
Virulence
Phenotype
Mutation
DNA
Escherichia coli
Lethal Dose 50
Genes
Plasmids
Peptide Hydrolases
Bacteria
Quorum Sensing
Enterotoxins
Lactones
Aspartic Acid
Alanine
Tyrosine

ASJC Scopus subject areas

  • Immunology

Cite this

Mutations within the catalytic motif of DNA adenine methyltransferase (Dam) of Aeromonas hydrophila cause the virulence of the dam-overproducing strain to revert to that of the wild-type phenotype. / Erova, Tatiana E.; Fadl, Amin A.; Sha, Jian; Khajanchi, Bijay K.; Pillai, Lakshmi L.; Kozlova, Elena V.; Chopra, Ashok.

In: Infection and Immunity, Vol. 74, No. 10, 10.2006, p. 5763-5772.

Research output: Contribution to journalArticle

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abstract = "In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/dam D/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100{\%} of the animals dying within 2 to 3 days with two 50{\%} lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam- overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.",
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T1 - Mutations within the catalytic motif of DNA adenine methyltransferase (Dam) of Aeromonas hydrophila cause the virulence of the dam-overproducing strain to revert to that of the wild-type phenotype

AU - Erova, Tatiana E.

AU - Fadl, Amin A.

AU - Sha, Jian

AU - Khajanchi, Bijay K.

AU - Pillai, Lakshmi L.

AU - Kozlova, Elena V.

AU - Chopra, Ashok

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N2 - In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/dam D/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam- overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.

AB - In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of Aeromonas hydrophila virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/dam D/A, pBAD/damY/A, and pBAD/damAhSSU (with the native dam gene) recombinant plasmids into the Escherichia coli GM33 (dam-deficient) strain. Genomic DNA (gDNA) isolated from either of the E. coli GM33 strains harboring the pBAD vector with the mutated dam gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of E. coli GM33 strain containing the pBAD/damAhSSU plasmid, indicating nonmethylation of E. coli gDNA with mutated Dam. Overproduction of mutated Dam in A. hydrophila resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native dam gene was overexpressed, with its levels returning to that of the control strain when the dam gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD50s) of the WT bacterium. Importantly, immunization of mice with a native-Dam- overproducing strain at the same LD50 did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.

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