Characterization of pNI10 plasmid in Pseudomonas, and the construction of an improved Escherichia and Pseudomonas shuttle vector, pNUK73

The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2003-05, Vol.61 (3), p.240-246
Main Authors: ITOH, N, KAWANAMI, T, NITTA, C, IWATA, N, USAMI, S, ABE, Y, KOIDE, Y
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacteria
Bacterial genetics
Bacterial Proteins - genetics
Base Sequence
Biological and medical sciences
Biotechnology
Cloning
Construction
E coli
Enzymes
Escherichia - genetics
Escherichia coli
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation, Bacterial
Genes, Bacterial - genetics
Genetic aspects
Genetic vectors
Genetic Vectors - genetics
Gram-negative bacteria
Molecular Sequence Data
Open Reading Frames - genetics
Physiological aspects
Plasmids
Proteins
Pseudomonas
Pseudomonas - genetics
Pseudomonas fluorescens
R&D
Research & development
Sequence Alignment
Studies
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Summary:The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-002-1195-1