Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted ami...

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Bibliographic Details
Published in:International microbiology 2004-03, Vol.7 (1), p.27-34
Main Authors: Park, Jeong Eun, Schlegel, Hans-Günter, Rhie, Ho Gun, Lee, Ho Sa
Format: Article
Language:English
Subjects:
Achromobacter xylosoxidans
Base Sequence
Cloning, Molecular - methods
Cobalt - pharmacology
DNA Primers
DNA, Bacterial - genetics
Drug Resistance, Bacterial - genetics
Escherichia coli
Genotype
Hafnia - genetics
Hafnia alvei
Molecular Sequence Data
Nickel - pharmacology
Operon - genetics
Plasmids - genetics
Restriction Mapping
Transcription, Genetic - genetics
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Summary:The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription.
ISSN:1139-6709