Cloning of genes necessary for survival of Erwinia chrysanthemi in plant tissue

Erwinia chrysanthemi is an important plant pathogenic bacterium that causes soft-rot disease in many plants. The pectin-degrading enzymes secreted by the bacterium appear to be the pathogenic agents. These enzymes degrade the pectinaceous component of the plant cell wall into monomers, saturated and unsaturated dimers, trimers and oligomers of galacturonic acid that are taken into the bacterial cell to be used as carbon and energy sources and for pectolytic enzyme induction. This indicates that the transport of these compounds is a vital step in the pathogenicity of the bacterium.

The uptake system for the dimer molecuIe,digalacturonic acid (dGA), in E. chrysanthemi EC 16 was studied and shown to be a separate system from the galacturonic acid (GA) uptake system. Two dGA uptake mutants, T5 and T9, were constructed from E. chrysanthemi EC 16 by transposon mutagenesis. Inverse Polymerase Chain Reaction (IPCR) was used to isolate the genes disrupted by the transposon Tn5 insertion. The 1-kb IPCR fragment obtained was sequenced after digesting with HaelU to remove the primer from one end, and the sequence was searched in GenBank and found to be 53% homologous to H. influenzae citrate lyase beta chain based on the deduced amino acid sequence. The 5' end of exwr was used to probe the EC 16 genomic library to identify genes vrith common regulatory sequences to exuT and presumably dgaT. A nucleotide sequence of 735 bp was identified which contained the partial sequence for both exuT and uxaCA operons. This region contained upstream sequences of exuT from E. chrysanthemi EC 16 that have not been previously identified. Additionally, a highly conserved cAMPCRP binding site is also present in this region.