Site-Specific Inactivation of Bacterial Genes Involved in Plant Rotting

ABSTRACT

The soft rot bacterium Erwinia carotovora subspecies carotovora strain EC14 contains multiple pectate lyase genes. Genes pel9.5 and pel10.5, coding for pectate lyases (pI 9.5 and 10.5), were cloned on a plasmid, transformed into Escherichia coli, and expressed catalytically active products. The isoenzymes were subcloned separately and sequenced. Along most of their lengths, the genes are homologous to each other and to the pelBC family of Erwinia extracellular pectate lyase genes. However, the amino acid sequence of the Pel10.5 carboxy-terminus diverges markedly from the PelBC consensus. We constructed hybrid genes and their products were found to be pectolytic. The pel9.5 and pel10.5 genes were mutagenized by marker exchange mutagenesis. A double mutant, pel9.5⁻pel10.5⁻, was obtained by mobilization of pLA115 into the pel10.5::kan mutant and exchange recombination by the above method. Hybridization of EcoRI digests of the mutant chromosomal DNAs with pel9.5-, pel10.5-, kan-, and tet-specific probes gave profiles consistent with single-site insertions of the kan and tet genes in the corresponding pel genes. In planta accumulation of Pel9.5 and Pel10.5 mRNAs way induced within 3 to 4 hr after inoculation, reaching maxima between 6 and 12 hr with Pel10.5 peaking at 6 hr and Pel9.5 at 9 hr. The induction of the two genes was regulated differentially and the level of their mRNAs was higher during compatible than during incompatible conditions. The three mutants, obtained here, especially the double mutant, have diminished virulence compared to the wild type.