Dataset Information

Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

ABSTRACT: An Escherichia coli clone expressing activity on DNase agar was obtained by cloning chromosomal DNA of Aeromonas hydrophila JMP636 into plasmid pUC19. Examination (of the clone's nuclease activity on a sodium dodecyl sulfate (SDS)-polyacrylamide gel containing DNA as a substrate revealed an activity band at approximately 100 kDa. Subsequently, subcloning localized the gene, designated nucH, to a 3.6-kb DNA fragment (pJP9521). Southern blotting of the nucH gene against chromosomal DNA of JMP636 confirmed that it had originated from this strain and demonstrated that it was present in a single copy, although additional faint bands were also detected. Analysis of the subclone using in vivo transcription and translation revealed only a single polypeptide of approximately 110 kDa. Sequencing of pJP9521 predicted an open reading frame of 3,213 bp encoding a protein of 1,070 amino acids and having a molecular mass of 114 kDa. Comparison of the deduced nucleotide sequence and the NucH predicted protein sequence with relevant databases indicated that no known homologs have previously been identified. A signal sequence was predicted from these data, and cellular fractionation of a nucH clone in E. coli indicated that the protein was able to be processed to the periplasm. An activity similar in size was detected in an extracellular protein sample of JMP636, while inactivation of the nucH gene resulted in loss of this activity band. By native SDS-polyacrylamide gel electrophoresis, NucH substrate specificity, cofactor requirements, and sensitivity to denaturing agents were assessed.

SUBMITTER: Dodd HN

PROVIDER: S-EPMC232655 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

Dodd H N HN Pemberton J M JM

Journal of bacteriology 19960701 13

An Escherichia coli clone expressing activity on DNase agar was obtained by cloning chromosomal DNA of Aeromonas hydrophila JMP636 into plasmid pUC19. Examination (of the clone's nuclease activity on a sodium dodecyl sulfate (SDS)-polyacrylamide gel containing DNA as a substrate revealed an activity band at approximately 100 kDa. Subsequently, subcloning localized the gene, designated nucH, to a 3.6-kb DNA fragment (pJP9521). Southern blotting of the nucH gene against chromosomal DNA of JMP636 c ...[more]

PMID: 8682799

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Project description:The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.

Project description:By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Dataset Information

Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

Publications

Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

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