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Pakistan Journal of Biological Sciences

Year: 2005 | Volume: 8 | Issue: 10 | Page No.: 1361-1367
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Research Article

Molecular Characterization of the hrpN Gene of Erwinia (Pantoea) stewartii, a Bacterium that Causes Vascular Wilt and Leaf Blight of Corn

Musharaf Ahmad, Doris R. Majerczak and David L. Coplin

ABSTRACT

The collinear hybridization of the portions of wts cluster of E. stewartii with the hrp cluster of E. amylovora suggested that the two clusters were related and that the wts and hrp genes had a common function in pathogenicity. So, we used hrpNEa as a probe and located a 1.8 kbp Hind III fragment within the wts cluster of E. stewartii. This gene was designated as hrpNEs. Sequencing of this gene revealed a 1,146 bp open reading frame with a typical ribosome-binding site located 6 bases upstream of an ATG translational initiation codon. A promoter sequence, hrp box, was found 74 bp upstream of the start of the hrpN open reading frame. The hrpN ORF encodes a 382 amino acid polypeptide and has a predicted molecular mass of 43 kDa and a PI of 4.28. The polypeptide is rich in glycine, has only three tyrosines and lacks cysteine. The protein is highly hydrophilic and does not show any transmembrane domains. To confirm that the ORF of hrpNEs produces harpinEs, the gene was over-expressed in E. coli using T 7 system. The estimated molecular mass of this protein, as determined by SDS-PAGE, was 44 kDa which agrees with the sequence data.
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