Vibrio cholerae contains numerous outer membrane proteins, however, they vary in their abundance and immunogenicity (1, 2). Manning et al. (3) have previously described the molecular cloning of the gene for a 22kDa outer membrane protein which is produced in minor amounts under normal laboratory conditions. The function of this protein is at present unknown, but it is very immunogenic and may correspond to one of the major immunogenic proteins detected by Western blot analysis using convalescent human sera (4). The structural gene, ompW, for the 22kDa protein was previously localized by transposon mutagenesis of plasmid pPM440, a pBR322 clone of partially Sau3A cleaved V. cholerae DNA (3). Analysis of the various TN] 725 derivatives of pPM440 suggested that the 22kDa protein was synthesized as a precursor of about 24kDa, consistent with its outer membrane location. The nucleotide sequence determined here was derived using subclones of pPM440 in M13 mpl8 and mpl9, as well as by supercoil sequencing of the TN] 725 insertions using oligodeoxynucleotide primers (5' GCTGTCACGAGAA-CACCGTT 3' and 5' CTTACGGATGCCCGGAAA 3') specific for the two ends of the transposon. The nucleotide sequence is shown in Fig. 1. The translated open reading frame corresponds to a 23.474kDa precursor protein with a 21.447kDa mature form. The signal peptide and cleavage site agree well with the rules for such sequences, and the direction of transcription is consistent with the polarity effects of TN] 725 insertions close to the beginning of the gene. The potential transcriptional promoter and terminator sequences have been detected. Both the-35 and-10 regions are not ideal as might be expected from the low level of expression of the protein under laboratory conditions, perhaps implying that the gene is subject to regulation. The potential terminator structure detected after the termination codon is also relatively weak. This sequence provides the basis for further studies, in particular, for site-directed mutagenesis of the V. cholerae chromosome to construct isogenic pairs of strains for assessing the role of the protein in pathogenesis.

The complete nucleotide sequence of the 4746bp HindIII fragment encoding the genes for the biosynthesis and assembly of CS3 pili has been determined. By site-directed mutagenesis in conjunction with analysis of the plasmid-encoded proteins in minicells, the actual reading frames for the various products have been determined. This demonstrated that the genes for four of the proteins (63 kD, 48 kD, 33 kD, and 20 kD in size) are encoded entirely within the same open reading frame as a fifth protein (104 kD). However, for synthesis of this latter protein, suppression or readthrough of an internal amber codon is required. Termination at this codon is also necessary for synthesis of the former proteins. Two further proteins are also encoded within the HindIII fragment: a 27 kD precursor of a periplasmic protein and the 17.5kD precursor of the major CS3 fimbrial subunit.

One-sentence summary: Repression of miR159 in tobacco triggers a constitutive defense response that confers immunity to Phytophthora parasitica.

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