Erwinia carotovora extracellular proteases: characterization and role in soft rot

Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene encoding a Prt, prt1, was cloned from cosmid pCA7 containing Ecc genomic DNA into plasmid pSK1. Escherichia coli transformed with pSK1 or pSK23, a subclone of pSK1, produced intracellularly a 38 kDa Prt with the same pI (4.8) as the secreted Ecc Prt. Prt1 activity produced by E.coli/pSK23 was inhibited by phenanthroline, which inhibits Zn-metalloproteases, but not by Ecc intracellular proteins. Analysis of deletion mutants indicated a 1.2 kb region necessary for Prt1 production. Sequencing of the pSK1 insert revealed a 1,041 bp open reading frame (ORF1) corresponding to the prtl region. ORF1 encodes a putative polypeptide of 347 amino acids with a total molecular mass of 38.8 kDa. The location of the prt1 promoter was determined to be 173 to 1,173 bp upstream from ORF1 by constructing transcriptional fusions to lacZ in plasmid pCD267. Primer extension revealed the start of prt1 mRNA 205 bp upstream of ORF1. The deduced amino acid sequence of the prt1 was compared to other proteases; it is similar to several bacterial Zn-metalloproteases. Prt1 production by Ecc was not observed during growth in rich broth; however, Northern analysis showed prt] mRNA accumulation in Ecc grown in planta. The role of prt1 in soft rot was determined by constructing a Prt1-deficient Ecc; prt1 insertionally inactivated by a kanamycin resistance gene was used to replace wildtype prt11 in the Ecc genome by homologous recombination. This mutant (L-957) had approximately 60 to 80% reduced Prt activity suggesting the presence of a second Prt (Prt2). Prt2 was purified from Ecc culture supernatant. This protease, also a metalloprotease, has a molecular mass of 45 kDa and pI of 4.8. Its amino terminal sequence had Significant sequence identity to metalloproteases from Erwinia chrysanthemi and Serratia marcescens, but not to Prt1. Further, unlike Prt1i, Prt2 was inhibited by Ecc intracellular proteins. The effect of proteases in potato tuber maceration was measured using L-957 and L-763, a Tn5 transposon mutant constructed previously. L-763 had no extracellular protease activity and may have been mutated in a regulatory region. Both mutants macerated significantly less tuber tissue than the wildtype Ecc. Reduced maceration of L-957 and L-763 was correlated with slower in planta growth. This suggests Prt1 production provides a nutritional advantage for Ecc growth on potato.