Ph.D. - Botanical Sciences (Plant Pathology - Cell, Molecular, and Neurosciences)

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    Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv. phaseolicola and sequence analysis of the phtE locus
    ( 1994) Zhang, Yuanxin
    Phaseolotoxin, Nδ(N'-sulfo-diaminophosphinyl)ornithylalanylhomoarginine, produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25 kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production to Tox- mutants. Tn5 mutagenesis of pHK120 and marker exchange of pHK120:: Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing strains for toxin production that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation). Pair complementation analysis of nine marker-exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCPI7, a clone reported previously (Peet et al, 1986, J. Bacteriol. 166:1096-1105) by another laboratory to contain some of the phaseolotoxin genes and the resistant-OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects. The sequence of phtE, the largest locus identified in pHK120, revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction. In order to decide whether all six ORFs are in an operon, RT-PCR was performed using RNA extracted from a transconjugant of 4612-1 containing pHK120. Expected fragments (with correct sizes and ability to hybridize to specific probes) were generated by RT-PCR using the RNA as the template. Each fragment overlapped the continuous, and the contig of them covered ORFI through ORF6, indicating that all six ORFs are transcribed in a single message, i.e. phtE locus is an operon. A primer extension experiment demonstrated that the expression of this operon is not temperature regulated because the same primer-extended product was generated using RNA from the wild-type strain at both 18°C and 28°C. A comparison of the sequences of the putative ORFs with the sequences of known genes in the data bases revealed that ORF3, encoding a 395 aa protein, has about 55% homology with the acetylornithine aminotransferase gene from E. coli and ornithine aminotransferase genes from other organisms. These data suggest that ORF3 encodes a protein required for the biosynthesis of ornithine, a constituent of phaseolotoxin. ORF6, encoding a 359 aa protein, has 40% homology with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803, suggesting that the gene product of ORF6 may be involved in the secretion of phaseolotoxin. The functions of the remaining ORFs are not known because computer searches did not discover significant homologous gene sequences for the rest of the ORFs in the data base. However, all of the deduced amino acid sequences of these ORFs contain one to several hydrophobic regions, suggesting that the products encodes by these ORFs may be membrane-associated.